RNAi沉默Med19基因对人肺癌A549细胞生长、转移影响的实验研究
摘要
目的:探讨应用RNAi技术沉默Med19基因抑制人肺癌A549细胞生长、转移的效果。
方法:①以Med19己知序列为靶点,构建siRNA-Med19重组慢病毒载体。②siRNA-Med19慢病毒表达载体与pCDNA3.1(-)- Med19过表达载体共转染293T细胞,Western blot外源筛选靶点。③将siRNA-Med19慢病毒表达载体转染人肺癌A549细胞株,应用Real-time PCR和Western blot检测Med19基因的转录水平及蛋白表达;应用流式细胞术检测细胞周期及凋亡峰;应用MTT、基底膜侵袭实验、细胞克隆形成实验检测细胞增殖、侵袭能力及细胞凋亡。④将siRNA-Med19慢病毒表达载体转染人肺癌A549细胞株,复制裸鼠移植瘤模型,测量肿瘤组织体积及重量,连续病理组织切片, HE染色观察移植瘤及转移瘤结节,并计算各组转移瘤数目和转移率。
结果:①成功构建siRNA-Med19重组慢病毒载体,并经基因测序证实。②成功构建目的基因过表达载体pCDNA3.1-Med19,并与siRNA-Med19重组慢病毒载体共转染293T细胞,Western blot外源验证靶点的有效性。③Real-time PCR及Western blot结果显示,转染siRNA-Med19慢病毒表达载体组中Med19的基因转录及蛋白表达水平明显降低。流式细胞术、MTT、基底膜侵袭实验、细胞克隆形成实验结果显示:siRNA-Med19慢病毒表达载体转染组抑制细胞增殖、侵袭,检测到细胞凋亡。④成功建立裸鼠移植人肺癌模型,siRNA-Med19慢病毒表达载体转染组肿瘤体积及重量均低于对照组。连续病理组织切片观察转染siRNA-Med19慢病毒表达载体组肝脏、肺脏转移率低、转移瘤数目少。
结论:应用RNAi技术沉默Med19基因可以降低人肺癌A549细胞的转录、表达,导致细胞凋亡,抑制肿瘤的生长、侵袭及转移。
Lung cancer as a common seen malignant tumor in the world has become major reason leading to death of cancer for most of countries, and at present it is considered the biggest malignant tumor to human health and life-threatening. In 2005, statistic data showed that there were 1,350,000 new cases of lung cancer each year in the world, and there were about 1,180,000 deaths cases of lung cancer [130], i.e., someone died of lung cancer on average less than 30 seconds in the world. Lung cancer will become the first killer in the new century. Around the world, epidemiology has shown that the incidences of lung cancer showed constant rise tendency. In America, lung cancer that has surpassed the sum of casualty of three main tumors namely prostate cancer, breast cancer and colon cancer (Greenlee RT et. al, 2001) occupies the first position in cancer casualty (Black, W.C. et. al, 2007). In 1990, there were 154,000 new cases of lung cancer in the US, 146,000 people died of lung cancer; in 2000 there were 16940,000 new cases of lung cancer and 154,900 died of lung cancer [122]. In 2002, there were 172,000 new cases of lung cancer in the United States and 157,000 people died of lung cancer, the mortality rate was 90% [131]. In recent years, with China's social progress and the process of industrial modernization, which increases environmental pollution, as well as an increase in smokers caused china morbidity and mortality of lung cancer constant rise tendency. According to the New England Journal in 2005 reported [1] that Cancer had become the first cause of death in Chinese men (374.1/10 million), the third cause of death in Chinese women (214.1/10 million), mortality rate for men’s lung cancer was (96.9/10 million), mortality rate for women’s lung cancer was (46.7/10 million), both were the first cause of cancer casualty. Surgery has been regarded as one of the most important means in comprehensive treatment of lung cancer. Despite rapid advances in surgical technique and method, limiting in improved survival rate has been limmited. Clinical research of Lung cancer radiotherapy and chemotherapy is made by random comparison of different periods and different options. Researchers are looking for better radiotherapy and chemotherapy methods with high efficacy and lower toxicity to improve patients quality of life, alleviate a variety of chemotherapy toxicity, while attention has been paid to targeted therapy. It is gratifying that the therapy of gene level has achieved rapid development in recent years.
RNA polymeraseⅡis essential for eucaryote genetic transcription, transcripts all precursors mRNA and the majority small nuclear RNA (snRNA), mRNA transcription impacts directly on the follow-up translation process from mRNA to protein, and further affects the protein function exercise. The study on RNA polymeraseⅡshowed that the latter only had transcription function on the condition adding general transcription factor compound to the purified RNA polymeraseⅡ, general transcription factor compound composed with five protein factors were required by all transcription activities, their existence was basic condition to sustain the basal transcription (basal transcription), in addition to the above-mentioned general transcription factor, RNA polymeraseⅡalso needed another protein compound. The function of the protein compound is to transmit various control signals to RNA polymeraseⅡand general transcription factor. The compound is composed with nearly 20 proteins [57]. It was named Mediator (intermediary). Mediator is an important component of the RNA polymeraseⅡgeneral transcription device, the Med compound with different kinase components plays a control function for different types of gene [8]. Conformational change of Med will produce a surface to collect and assemble to PIC components in RNA polymeraseⅡand general transcription factor, this is an important aspect of the Med at the role of transcription mediation[9], and plays a key role in activation and inhibition of eukaryotic mRNA synthesis. Genetic name of Med19 in human encode is associated protein of metastasis of lung cancer, plays an important role in the cell growth. It can directly or indirectly participate and mediate a variety of signaling pathways which are highly expressed in lung cancer cell line 95D with high metastasis capacity, but lowly expressed in lung cancer cell lines 95C with low metastasis capacity. To adjust and control transcription and expression of Med19Gene’s can prevent invasion and metastasis of lung cancer. Therefore, the application of siRNA interfering Med19 gene may be one of the most effective option prospects of gene therapy for lung cancer.
Med19 gene is closely related with cell proliferation, differentiation and apoptosis. Difference expression study with DNA chip through closed antisense digonucleotides LCMR1 is adopted to investigate many signals transduction genes capable of inhibiting tumor cell proliferation, invasion, and promote tumor cell apoptosis such as tumor necrosis factor receptor (TNF RSF10), tumor necrosis factor super family (TNF SF10), serine (cysteine) protease inhibitors (SERPINB8), bone morphogenetic protein receptorⅡ(BMPR2), serine/threonine kinase 2 (STK2), serine/threonine kinase 3 (STK3), G proteinβ5 (GNB4), G protein 4 (GNG4), IL-3 byα(L3RA) and so on. Signal transduction gene which plays a very important positive role in cell proliferation, differentiation and the construction of the cytoskeleton is lowly expressed, mainly includes ras homology gene family member C (ARHC), ras homology gene family member A (ARHA). Signal transduction and gene in regulation of apoptosis and cell cycle occupies a large proportion in the differential expression gene, but three parts correlate and influence each other, and constitute an extremely complex and sophisticated regulatory network.
Initial speculation is that LCMRI gene can probably play a role in signal transduction pathway by regulating cell proliferation, differentiation, cell cycle regulation and suppression of apoptosis. We believe that the construction of siRNA-lentivirus expression vector interfere with Med19 can inhibit cell growth and proliferation, and promote apoptosis.
It has been confirmed that blocking Med19 gene can induce tumor apoptosis and growth inhibition. Therefore, cancer gene therapy regarding Med19 as target can block upstream and downstream cancer-causing factors and cancer-causing gene only by blocking a Med19 protein which is more stronger specificity and higher application value to therapy of some or suppression of cancer comparing to therapy for a simple oncogene or tumor suppressor gene and provides an ideal candidate target goal for gene therapy for malignant tumor
Choice of target genes should be the first consideration for gene therapy. As tumor recurrence and development relates to oncogene change, blocking a bridge of general transcription device–Mediator can probably be one of the most effective methods for the cancer therapy. Studies have shown that blocking Med19 can block various downstream anomalistic tumor signals leading to transcription and translation stop, consequently Med19 may have potential as the ideal molecular target for treatment of malignancies. Theoretically, blocking tumor cell Med19 gene signal transduction pathway can probably play a role in the treatment for cancers.
RNAi refers to one phenomenon which the normal biology inhibits gene expression. Once double-stranded RNA(double stranded RNA, dsRNA) homology with endogenous mRNA encoding region is introduced into cells, it can trigger a post-transcriptional control procedures to specifically identify mRNA with homologous sequences give rise to block of translation function, a phenomenon, that is known as posttranscriptional gene silencing (posttranscriptional gene silencing, PTGS). Due to RNAi with specificity and efficiency, this technology has become an important tool for the study of gene function, and can probably play a role in viral, genetic diseases and the treatment of tumor diseases. The siRNA lentivirus expression vector by application of encoding shorthairpin RNA (short hairpin RNA, shRNA) can inhibit gene expression in the human cells for steady-long time.
Med19 gene has not been fully understood about its molecular mechanism in the pathogenesis of lung cancer. This study constructs siRNA lentivirus eukaryotic expression vector of Med19 specificity and transfected into human lung cancer cell line A549, understand the reorganization lentivirus vector interference result on Med19 gene of A549 cells, as well as influence to lung cancer cell growth and proliferation, invasion and metastasis, cell cycle and apoptosis, further explores Med19 gene role in the occurrence and development of lung cancer, provides a new means and theoretical foundation for RNA interference (RNAi) molecular target gene therapy for lung cancer.
Objective: to construct siRNA-Med19 lentivirus expression vector and study its inhibitory effect on human lung cancer A549 cells in vitro and in vivo.
Method:(1) Construction of lentivirus expression vector: according to known sequence of Med19 gene mRNA in the genebank to determine the appropriate target site, synthesize DNA templates of encoding siRNA, connect Med19siRNA annealed template oligonucleotide to linearized pGCSIL-GFP expression vector, and to construct siRNA-Med19 lentivirus expression vector by sequencing and enzyme digestion;
(2) Construction of target gene over-expression vector: primer design, the use of PCR to amplify Med19 functional gene, to connect with eukaryotic over-expression vector after digestion or linearization, to construct pCDNA3.1(-)-MED19 over-expression vector by sequencing and enzyme digestion;
(3) Selection of exogenous target by Western blot: cotransfection of 293T cells with siRNA-Med19 lentivirus expression vector and pCDNA3.1(-)-MED19 over-expression vector, exogenous target selection with Western blot.
(4) Packaging and titer determination of siRNA-Med19 lentivirus vector: to pack three plasmid vector with the best target siRNA-Med19 lentivirus expression vector and pHelper 1.0 vector and vector pHelper 2.0 in 293T cells, and to demarcate virus titer.
(5) In vitro study: transfection of human lung cancer A549 cell line with siRNA-Med19 lentivirus expression vector; application of Real-time PCR and Western blot to determine transcription levels of Med19 gene and protein expression; applying flow cytometry to test cell cycle and apoptosis peak; to applying MTT, the basement membrane invasion assay and cell colony to form assay cell proliferation, invasion ability and apoptosis.
(6) In vivo study: Nude mice transplanted human lung cancer model was establish by s.c. injecting transfected A549 cells with siRNA-Med19 lentivirus expression vector followed by; measurement of tumor size and weight, and observation of the liver and lung metastasis, and tissue section and HE staining histology to apply to study transplanted tumor and metastatic tumor nodules, and calculate the number of metastatic tumor and metastatic rate in each group.
Results:(1) successful construction of siRNA-Med19 lentivirus expression vector by sequencing and enzyme digestion
(2) successful construction of target gene over-expression vector pCDNA3.1 (-)- MED19. by sequencing and enzyme digestion.
(3) co-transfection of 293T cells was performed with greater than 90% fluorescence expression of the rate. Exogenous selection by Western blot showed that target 4 # is the best target.
(4) High-titer lentivirus liquid was prepared after packaging and enrichment of siRNA-Med19 lentivirus vector. The virus titer was 2E+9 TU/ml, which was measured and calibrated in 293T cells.
(5) In vitro data revealed that siRNA-Med19 lentivirus expression vector can inhibit proliferation, invasion, and promote apoptosis of human lung cancer A549 cells. Real-time PCR and Western blot results showed that expression level of Med19 mRNA and protein of Med19 was significantly reduced after transfection of A549 cells with siRNA-Med19 lentivirus expression vector. Flow cytometry, MTT, basement membrane invasion assay, and cell colony-forming assay showed that siRNA-Med19 can significantly inhibit cell proliferation, invasion, induce apoptosis of A549 cells.
(6) We have successfully established human lung cancer A549 xenograft model in nude mice. Tumor volume and weight in animals treated with Med19 mRNA was lower than the control groups. Continuous observation of the pathological tissue revealed that liver and lung metastases were significantly lowered in animals treated with Med19 mRNAe.
Conclusion: (1) This study represents the first study to demonstrate that siRNA-Med19 lentivirus can specifically and efficiently interfere with human lung cancer A549 cells, and inhibit gene expression of Med19 leading to inhibition of proliferation and invasion and apoptosis induction of A549 cells. Med19 prompt signal pathway is in an ideal molecular target for gene therapy of human lung cancer which is expected to be one of the reasonable strategies.
(2) This study represents the first study to demonstrate that siRNA-Med19 lentivirus can effectively inhibit growth and metastasis of transplant A549 tumor mice model in vivo.
方法:①以Med19己知序列为靶点,构建siRNA-Med19重组慢病毒载体。②siRNA-Med19慢病毒表达载体与pCDNA3.1(-)- Med19过表达载体共转染293T细胞,Western blot外源筛选靶点。③将siRNA-Med19慢病毒表达载体转染人肺癌A549细胞株,应用Real-time PCR和Western blot检测Med19基因的转录水平及蛋白表达;应用流式细胞术检测细胞周期及凋亡峰;应用MTT、基底膜侵袭实验、细胞克隆形成实验检测细胞增殖、侵袭能力及细胞凋亡。④将siRNA-Med19慢病毒表达载体转染人肺癌A549细胞株,复制裸鼠移植瘤模型,测量肿瘤组织体积及重量,连续病理组织切片, HE染色观察移植瘤及转移瘤结节,并计算各组转移瘤数目和转移率。
结果:①成功构建siRNA-Med19重组慢病毒载体,并经基因测序证实。②成功构建目的基因过表达载体pCDNA3.1-Med19,并与siRNA-Med19重组慢病毒载体共转染293T细胞,Western blot外源验证靶点的有效性。③Real-time PCR及Western blot结果显示,转染siRNA-Med19慢病毒表达载体组中Med19的基因转录及蛋白表达水平明显降低。流式细胞术、MTT、基底膜侵袭实验、细胞克隆形成实验结果显示:siRNA-Med19慢病毒表达载体转染组抑制细胞增殖、侵袭,检测到细胞凋亡。④成功建立裸鼠移植人肺癌模型,siRNA-Med19慢病毒表达载体转染组肿瘤体积及重量均低于对照组。连续病理组织切片观察转染siRNA-Med19慢病毒表达载体组肝脏、肺脏转移率低、转移瘤数目少。
结论:应用RNAi技术沉默Med19基因可以降低人肺癌A549细胞的转录、表达,导致细胞凋亡,抑制肿瘤的生长、侵袭及转移。
Lung cancer as a common seen malignant tumor in the world has become major reason leading to death of cancer for most of countries, and at present it is considered the biggest malignant tumor to human health and life-threatening. In 2005, statistic data showed that there were 1,350,000 new cases of lung cancer each year in the world, and there were about 1,180,000 deaths cases of lung cancer [130], i.e., someone died of lung cancer on average less than 30 seconds in the world. Lung cancer will become the first killer in the new century. Around the world, epidemiology has shown that the incidences of lung cancer showed constant rise tendency. In America, lung cancer that has surpassed the sum of casualty of three main tumors namely prostate cancer, breast cancer and colon cancer (Greenlee RT et. al, 2001) occupies the first position in cancer casualty (Black, W.C. et. al, 2007). In 1990, there were 154,000 new cases of lung cancer in the US, 146,000 people died of lung cancer; in 2000 there were 16940,000 new cases of lung cancer and 154,900 died of lung cancer [122]. In 2002, there were 172,000 new cases of lung cancer in the United States and 157,000 people died of lung cancer, the mortality rate was 90% [131]. In recent years, with China's social progress and the process of industrial modernization, which increases environmental pollution, as well as an increase in smokers caused china morbidity and mortality of lung cancer constant rise tendency. According to the New England Journal in 2005 reported [1] that Cancer had become the first cause of death in Chinese men (374.1/10 million), the third cause of death in Chinese women (214.1/10 million), mortality rate for men’s lung cancer was (96.9/10 million), mortality rate for women’s lung cancer was (46.7/10 million), both were the first cause of cancer casualty. Surgery has been regarded as one of the most important means in comprehensive treatment of lung cancer. Despite rapid advances in surgical technique and method, limiting in improved survival rate has been limmited. Clinical research of Lung cancer radiotherapy and chemotherapy is made by random comparison of different periods and different options. Researchers are looking for better radiotherapy and chemotherapy methods with high efficacy and lower toxicity to improve patients quality of life, alleviate a variety of chemotherapy toxicity, while attention has been paid to targeted therapy. It is gratifying that the therapy of gene level has achieved rapid development in recent years.
RNA polymeraseⅡis essential for eucaryote genetic transcription, transcripts all precursors mRNA and the majority small nuclear RNA (snRNA), mRNA transcription impacts directly on the follow-up translation process from mRNA to protein, and further affects the protein function exercise. The study on RNA polymeraseⅡshowed that the latter only had transcription function on the condition adding general transcription factor compound to the purified RNA polymeraseⅡ, general transcription factor compound composed with five protein factors were required by all transcription activities, their existence was basic condition to sustain the basal transcription (basal transcription), in addition to the above-mentioned general transcription factor, RNA polymeraseⅡalso needed another protein compound. The function of the protein compound is to transmit various control signals to RNA polymeraseⅡand general transcription factor. The compound is composed with nearly 20 proteins [57]. It was named Mediator (intermediary). Mediator is an important component of the RNA polymeraseⅡgeneral transcription device, the Med compound with different kinase components plays a control function for different types of gene [8]. Conformational change of Med will produce a surface to collect and assemble to PIC components in RNA polymeraseⅡand general transcription factor, this is an important aspect of the Med at the role of transcription mediation[9], and plays a key role in activation and inhibition of eukaryotic mRNA synthesis. Genetic name of Med19 in human encode is associated protein of metastasis of lung cancer, plays an important role in the cell growth. It can directly or indirectly participate and mediate a variety of signaling pathways which are highly expressed in lung cancer cell line 95D with high metastasis capacity, but lowly expressed in lung cancer cell lines 95C with low metastasis capacity. To adjust and control transcription and expression of Med19Gene’s can prevent invasion and metastasis of lung cancer. Therefore, the application of siRNA interfering Med19 gene may be one of the most effective option prospects of gene therapy for lung cancer.
Med19 gene is closely related with cell proliferation, differentiation and apoptosis. Difference expression study with DNA chip through closed antisense digonucleotides LCMR1 is adopted to investigate many signals transduction genes capable of inhibiting tumor cell proliferation, invasion, and promote tumor cell apoptosis such as tumor necrosis factor receptor (TNF RSF10), tumor necrosis factor super family (TNF SF10), serine (cysteine) protease inhibitors (SERPINB8), bone morphogenetic protein receptorⅡ(BMPR2), serine/threonine kinase 2 (STK2), serine/threonine kinase 3 (STK3), G proteinβ5 (GNB4), G protein 4 (GNG4), IL-3 byα(L3RA) and so on. Signal transduction gene which plays a very important positive role in cell proliferation, differentiation and the construction of the cytoskeleton is lowly expressed, mainly includes ras homology gene family member C (ARHC), ras homology gene family member A (ARHA). Signal transduction and gene in regulation of apoptosis and cell cycle occupies a large proportion in the differential expression gene, but three parts correlate and influence each other, and constitute an extremely complex and sophisticated regulatory network.
Initial speculation is that LCMRI gene can probably play a role in signal transduction pathway by regulating cell proliferation, differentiation, cell cycle regulation and suppression of apoptosis. We believe that the construction of siRNA-lentivirus expression vector interfere with Med19 can inhibit cell growth and proliferation, and promote apoptosis.
It has been confirmed that blocking Med19 gene can induce tumor apoptosis and growth inhibition. Therefore, cancer gene therapy regarding Med19 as target can block upstream and downstream cancer-causing factors and cancer-causing gene only by blocking a Med19 protein which is more stronger specificity and higher application value to therapy of some or suppression of cancer comparing to therapy for a simple oncogene or tumor suppressor gene and provides an ideal candidate target goal for gene therapy for malignant tumor
Choice of target genes should be the first consideration for gene therapy. As tumor recurrence and development relates to oncogene change, blocking a bridge of general transcription device–Mediator can probably be one of the most effective methods for the cancer therapy. Studies have shown that blocking Med19 can block various downstream anomalistic tumor signals leading to transcription and translation stop, consequently Med19 may have potential as the ideal molecular target for treatment of malignancies. Theoretically, blocking tumor cell Med19 gene signal transduction pathway can probably play a role in the treatment for cancers.
RNAi refers to one phenomenon which the normal biology inhibits gene expression. Once double-stranded RNA(double stranded RNA, dsRNA) homology with endogenous mRNA encoding region is introduced into cells, it can trigger a post-transcriptional control procedures to specifically identify mRNA with homologous sequences give rise to block of translation function, a phenomenon, that is known as posttranscriptional gene silencing (posttranscriptional gene silencing, PTGS). Due to RNAi with specificity and efficiency, this technology has become an important tool for the study of gene function, and can probably play a role in viral, genetic diseases and the treatment of tumor diseases. The siRNA lentivirus expression vector by application of encoding shorthairpin RNA (short hairpin RNA, shRNA) can inhibit gene expression in the human cells for steady-long time.
Med19 gene has not been fully understood about its molecular mechanism in the pathogenesis of lung cancer. This study constructs siRNA lentivirus eukaryotic expression vector of Med19 specificity and transfected into human lung cancer cell line A549, understand the reorganization lentivirus vector interference result on Med19 gene of A549 cells, as well as influence to lung cancer cell growth and proliferation, invasion and metastasis, cell cycle and apoptosis, further explores Med19 gene role in the occurrence and development of lung cancer, provides a new means and theoretical foundation for RNA interference (RNAi) molecular target gene therapy for lung cancer.
Objective: to construct siRNA-Med19 lentivirus expression vector and study its inhibitory effect on human lung cancer A549 cells in vitro and in vivo.
Method:(1) Construction of lentivirus expression vector: according to known sequence of Med19 gene mRNA in the genebank to determine the appropriate target site, synthesize DNA templates of encoding siRNA, connect Med19siRNA annealed template oligonucleotide to linearized pGCSIL-GFP expression vector, and to construct siRNA-Med19 lentivirus expression vector by sequencing and enzyme digestion;
(2) Construction of target gene over-expression vector: primer design, the use of PCR to amplify Med19 functional gene, to connect with eukaryotic over-expression vector after digestion or linearization, to construct pCDNA3.1(-)-MED19 over-expression vector by sequencing and enzyme digestion;
(3) Selection of exogenous target by Western blot: cotransfection of 293T cells with siRNA-Med19 lentivirus expression vector and pCDNA3.1(-)-MED19 over-expression vector, exogenous target selection with Western blot.
(4) Packaging and titer determination of siRNA-Med19 lentivirus vector: to pack three plasmid vector with the best target siRNA-Med19 lentivirus expression vector and pHelper 1.0 vector and vector pHelper 2.0 in 293T cells, and to demarcate virus titer.
(5) In vitro study: transfection of human lung cancer A549 cell line with siRNA-Med19 lentivirus expression vector; application of Real-time PCR and Western blot to determine transcription levels of Med19 gene and protein expression; applying flow cytometry to test cell cycle and apoptosis peak; to applying MTT, the basement membrane invasion assay and cell colony to form assay cell proliferation, invasion ability and apoptosis.
(6) In vivo study: Nude mice transplanted human lung cancer model was establish by s.c. injecting transfected A549 cells with siRNA-Med19 lentivirus expression vector followed by; measurement of tumor size and weight, and observation of the liver and lung metastasis, and tissue section and HE staining histology to apply to study transplanted tumor and metastatic tumor nodules, and calculate the number of metastatic tumor and metastatic rate in each group.
Results:(1) successful construction of siRNA-Med19 lentivirus expression vector by sequencing and enzyme digestion
(2) successful construction of target gene over-expression vector pCDNA3.1 (-)- MED19. by sequencing and enzyme digestion.
(3) co-transfection of 293T cells was performed with greater than 90% fluorescence expression of the rate. Exogenous selection by Western blot showed that target 4 # is the best target.
(4) High-titer lentivirus liquid was prepared after packaging and enrichment of siRNA-Med19 lentivirus vector. The virus titer was 2E+9 TU/ml, which was measured and calibrated in 293T cells.
(5) In vitro data revealed that siRNA-Med19 lentivirus expression vector can inhibit proliferation, invasion, and promote apoptosis of human lung cancer A549 cells. Real-time PCR and Western blot results showed that expression level of Med19 mRNA and protein of Med19 was significantly reduced after transfection of A549 cells with siRNA-Med19 lentivirus expression vector. Flow cytometry, MTT, basement membrane invasion assay, and cell colony-forming assay showed that siRNA-Med19 can significantly inhibit cell proliferation, invasion, induce apoptosis of A549 cells.
(6) We have successfully established human lung cancer A549 xenograft model in nude mice. Tumor volume and weight in animals treated with Med19 mRNA was lower than the control groups. Continuous observation of the pathological tissue revealed that liver and lung metastases were significantly lowered in animals treated with Med19 mRNAe.
Conclusion: (1) This study represents the first study to demonstrate that siRNA-Med19 lentivirus can specifically and efficiently interfere with human lung cancer A549 cells, and inhibit gene expression of Med19 leading to inhibition of proliferation and invasion and apoptosis induction of A549 cells. Med19 prompt signal pathway is in an ideal molecular target for gene therapy of human lung cancer which is expected to be one of the reasonable strategies.
(2) This study represents the first study to demonstrate that siRNA-Med19 lentivirus can effectively inhibit growth and metastasis of transplant A549 tumor mice model in vivo.
引文
[1] He J, Gu D, Wu X, et al. Major Causes of Death among Men and Women in China[J]. N Engl J Med, 2005, 353:1124-1134.
[2]Barnes MN, Deshane JS, Rosenfeld M, et al. Gene therapy and ovarian cancer:a review[J]. Obstet Cynecol, 1997, 89:145-155.
[3]Boyer TG, Martin ME, Lees E, Ricciardi RP, and Berk AJ. Mammalian Srb/Mediator complex is targeted by adenovirus E1A protein[J]. Nature, 1999, 399: 276-279.
[4]Conaway RC, Sato S, Tomomori-Sato C, Yao T, and Conaway JW. The mammalian Mediator complex and its role in transcriptional regulation[J]. Trends Biochem Sci, 2005,30: 250-255.
[5]张郁. LCMR1反义寡核苷酸对高转移肺大细胞癌细胞系-95D细胞生物学行为的影响及LCMR1基因功能的初步研究[D].北京:中国人民解放军军医进修学院,2004.
[6]梁志欣.肺癌转移相关蛋白_LCMR1_的原核表达_多克隆抗体制备及其应用的初步研究[D].北京:中国人民解放军军医进修学院,2004.
[7]Baumli S, Hoeppner S, and Cramer P. A conserved mediator hinge revealed in the structure of the MED7.MED21 (Med7.Srb7) heterodimer[J]. J Biol Chem, 2005, 280: 18171 -18178.
[8] Dotson MR, Yuan CX, Roeder RG, et al. Structural organization of yeast and mammalian mediator complexes[J]. Proc Natl Acad Sci U S A. 2000; 97(26):14307-10.
[9]Cosma MP, Panizza S, and Nasmyth K. Cdk1 triggers association of RNA polymerase to cell cycle promoters only after recruitment of the mediator by SBF[J]. Mol Cell, 2001, 7:1213-1220.
[10]Kim YJ, Bjorklund S, Li Y, Sayre MH, and Kornberg RD. A multiprotein mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymeraseⅡ[J]. Cell, 1994, 77:599-608.
[11] Kelleher RJ, Flanagan PM, Kornberg RD. A novel mediator between activator proteins and the RNApolymeraseⅡtranscription apparatus[J]. Cell, 1990, 61(7): 1209-1215.
[12]Flaherty KM, McKay DB, Kabsch W, et al. Similarity of the three- dimensional structures of actin and the ATPase fragment of a 70-kDa heat shock cognate protein. Proc NatlAcad Sci U S A. 1991 ,88(11):5041-5045.
[13]N(?)r AM, Lemon BD, Tjian R. Transcriptional coactivator complexes[J]. Annu Rev Biochem, 2001, 70:475-501.
[14]Bjorklund S and Gustafsson CM. The yeast Mediator complex and its regulation[J]. Trends Biochem Sci, 2005, 30:240-244.
[15]van Bakel H, Huynen M, Wijmenga C. Prokaryotic diversity of the Saccharomyces cerevisiae Atx1p-mediated copper pathway[J]. 2004, 20(16):2644- 2655.
[16]Guglielmi B, Berkum N L, Klapholz B, et al. Ahigh resolution protein interaction map of the yeast Mediator complex[J]. Nucleic Acids Res, 2004, 32 (18):5379-5391.
[17]Chadick JZ and Asturias FJ. Structure of eukaryotic Mediator complexes[J]. Trends Biochem Sci, 2005, 30:264-271.
[18]Samuelsen CO, Baraznenok V, Khorosjutina O, Spahr H, Kieselbach T, Holmberg S, and Gustafsson CM. TRAP230/ARC240 and TRAP240/ARC250 Mediator subunits are functionally conserved through evolution[J]. Proc Natl Acad Sci U S A, 2003, 100: 6422-6427.
[19]Bourbon H M, Aguilera A, Ansari A,et al. Aunified nomenclature for protein subunits of mediator complexes linking transcriptional regulators to RNA polymeraseⅡ[J]. Mol Cell, 2004, 14 (5): 553-557.
[20]N(?)r AM, Taatjes DJ, Zhai W, Nogales E, Tjian R. Human CRSP interacts with RNA polymerase II CTD and adopts a specific CTD-bound conformation[J]. Genes Dev, 2002, Jun 1;16(11):1339-1344.
[21]Koleske A J, Young R A. The RNA polymeraseⅡholoenzyme and its implications for gene regulation[J]. Trends Biochem Sci, 1995, 20(6):113-116.
[22]Wang Q, Sharma D, Ren Y, Fondell JD. A coregulatory role for the TRAP-mediator complex in androgen receptor-mediated gene expression[J]. J Biol Chem, 2002, Nov 8;277(45):42852-42858. Epub 2002 Sep 5.
[23]Dotson M R, Yuan C X, Roeder R G, et al. Structural organization of yeast and mammalian mediator complexes[J]. Proc Natl Acad Sci USA, 2000, 97 (26): 14307-14310.
[24]Taatjes DJ, N??r AM, Andel F et al. Structure, function, and activator- induced conformations of the CRSP coactivator[J]. Science. 2002, 295(5557): 1058-1062.
[25]Asturias FJ, Jiang YW, Myers LC, et al. Conserved structures of mediator and RNA polymeraseⅡholoenzyme[J]. Science,1999, 283: 985-987.
[26]Andrew S. Neish SFA, et al. Factors associated with the mammalian RNA polymeraseⅡholoenzyme[J]. Nucl Acids Res, 1998, 26(3): 847-853.
[27]Yudkovsky N RJA, Hahn S. A transcription reinitiation intermediate that is stabilized by activator[J]. Nature, 2000, 324(6809): 225-229.
[28]Baidoobonso SM, Guidi BW, Myers LC. Med19(Rox3) regulates Intermodule interactions in the Saccharomyces cerevisiae mediator complex[J]. J Biol Chem, 2007, 282:5551-5559.
[29]Park J M, Kim H S, Han S J, et al. In uiuo requirement of activator-specific binding targets of mediator[J]. Mol Cell Biol, 2000, 20 (23): 8709-8719.
[30]Yudkovsky N, Ranish J A, Hahn S. Atranscription reinitiation intermediate that is stabilized by activator[J]. Nature, 2000, 408 (6809): 225-229.
[31]Bjorklund S, Gustafsson C M. The yeast Mediator complex and its regulation[J]. Trends Biochem Sci, 2005, 30 (5):240-244.
[32]Gu W, Malik S, Ito M, et al. Anovel human SRB/MED-containing cofactor complex, SMCC, involved in transcription regulation[J]. Mol Cell, 1999, 3(1): 97-108.
[33]Reeves W M, Hahn S. Activator-independent functions of the yeast mediator sin4 complex in preinitiation complex formation and transcription reinitia- tion[J]. Mol Cell Biol, 2003, 23 (1):349-358.
[34]Yoon C K, Qiu H, Govind H, et al. Simultaneous recruitment of coactivators by Gcn4p stimulates multiple Steps of Transcription in uiuo[J]. Molecular and Cellular Biology, 2005, 25 (13):5626-5638.
[35]Boube M, Faucher C, Joulia L, et al. Drosophila homologs of transcriptional mediator complex subunits are required for adult cell and segment identity specification[J]. Genes Dev. 2000, 14(22):2906-2917.
[36]Park JM, Gim BS, Kim JM, et al. Drosophila Mediator complex is broadly utilized by diverse gene-specific transcription factors at different types of core promoters[J]. Mol Cell Biol. 2001, 21(7):2312-2323.
[37]Malik S, Roeder R G. Dynamic regulation of polⅡtranscription by the mammalianMediator complex[J]. Trends Biochem Sci, 2005, 30 (5):256-263.
[38]Zhu X, Wirén M, Sinha I, Rasmussen NN, et al. Genome-wide occupancy profile of mediator and the Srb8-11 module reveals interactions with coding regions[J]. 2006, 21;22(2): 169-178.
[39]Song W TI, Qian N, Kuchin S, Carlson M. SSN genes that affect transcriptional repression in Saccharomyces cerevisiae encode SIN4, ROX3, and SRB proteins associated with RNA polymeraseII[J]. Mol Cell Biol, 1996, Jan,16(1):115-120.
[40]Evangelista CC, Jr, Rodriguez Torres AM, Limbach MP, and Zitomer RS. Rox3 and Rts1 function in the global stress response pathway in baker's yeast[J]. Genetics, 1996, 142: 1083-1093.
[41]Shu Y HS, a multicopy suppressor of hsp60-ts mutant alleles, does not encode a mitochondriallytargeted protein[J]. Mol. Cell. Biol, 1995, (15): 5618- 5626.
[42]Gustafsson CM ML, Li Y, Redd MJ, et al. Identification of Rox3 as a component of mediator and RNA polymerase II holoenzyme[J]. Biol Chem, 1997, Jan (3);272(1):48-50.
[43]Qiu H HC, Yoon S, Natarajan K, Swanson MJ, et al. An array of coactivators is required for optimal recruitment of TATA binding protein and RNA polymerase II by promoter-bound Gcn4p[J]. Mol Cell Biol, 2004, (24):4104–4117.
[44]Singh H EA, Kremer SB, Duttweiler, et al. A functional module of yeast mediator that governs the dynamic range of heat-shock gene expression[J]. Genetics, 2006, (172): 2169- 2184.
[45]Tabtiang RK and Herskowitz I. Nuclear proteins Nut1p and Nut2p cooperate to negatively regulate a Swi4p-dependent lacZ reporter gene in Saccharomyces cerevisiae[J]. Molecular and cellular biology, 1998, 18: 4707-4718.
[46]Rosenblum-Vos LS, Rhodes L, Evangelista CC, Jr, Boayke KA, and Zitomer RS. The ROX3 gene encodes an essential nuclear protein involved in CYC7 gene expression in Saccharomyces cerevisiae[J]. Molecular and cellular biology, 1991, 11:5639-5647.
[47]Kang JS, Kim SH, Hwang MS, Han SJ, Lee YC, and Kim YJ. The structural and functional organization of the yeast mediator complex[J]. J Biol Chem, 2001, 276: 42003 -42010.
[48]Takagi Y, Calero G, Komori H, Brown JA, Ehrensberger AH, Hudmon A, Asturias F,and Kornberg RD. Head module control of mediator interactions[J]. Mol Cell, 2006, 23: 355- 364.
[49]Myers L C GCM, et al. Mediator protein mutations that selectively abolish activated transcription[J]. Proc Natl Acad, 1999, (96):67-72.
[50]Lee YC, Park JM, Min S, Han SJ, and Kim YJ. An activator binding module of yeast RNA polymerase II holoenzyme[J]. Molecular and cellular biology, 1999, 19:2967-2976.
[51]Han SJ, Lee YC, Gim BS, Ryu GH, Park SJ, Lane WS, and Kim YJ. Activator-specific requirement of yeast mediator proteins for RNA polymerase II transcriptional activation[J]. Molecular and cellular biology, 1999, 19: 979- 988,.
[52]Baidoobonso SM, Guidi BW, Myers LC. Med19(Rox3) regulates Intermodule interactions in the Saccharomyces cerevisiae mediator complex[J]. JBiol Chem, 2007, 23;282(8):5551-5559.
[53]Brown TA, Evangelista C, and Trumpower BL. Regulation of nuclear genes encoding mitochondrial proteins in Saccharomyces cerevisiae[J]. Journal of bacteriology, 1995, 177: 6836-6843.
[54]Linder T, Zhu X, Baraznenok V, and Gustafsson CM. The classical srb4-138 mutant allele causes dissociation of yeast Mediator[J]. Biochemical and biophysical research communications, 2006, 349: 948-953.
[55]Sato S, Tomomori-Sato C, Banks CA, Sorokina I, Parmely TJ, Kong SE, Jin J, Cai Y, Lane WS, Brower CS, Conaway RC, and Conaway JW. Identification of mammalian Mediator subunits with similarities to yeast Mediator subunits Srb5, Srb6, Med11, and Rox3[J]. J Biol Chem, 2003, 278:15123-15127.
[56]Sato S, Tomomori-Sato C, Parmely TJ, Florens L, Zybailov B, Swanson SK, Banks CA, Jin J, Cai Y, Washburn MP, Conaway JW, and Conaway RC. A set of consensus mammalian mediator subunits identified by multidimensional protein identification technology[J]. Mol Cell, 2004, 14:685-691.
[57]Hunter T. Signaling-2000and beyond[J]. Cell, 2000, 00:113-127.
[58]Casstecker MP, Piek E, Roberts B, et al. Role of trans forming growth faeter-βsignaling in eancer[J]. Natl Cancer Inst, 2000,92(17):1388-1402.
[59]Wu G Chen YG, Ozdamar B et al. Structural basis of smad2 regulation by the Smadanchor for receptor activation[J]. Science, 2000, 287:92-97.
[60]Miyazono K. Positive and negative regulation of TGF-βsignal[J]. J Cell Sci, 2000, 131:1101-1109.
[61]Jang CW, Chen CH, Chen CC, et al. TGF-βinduces apoptosis through Smad-mediated expression of DAP-kinase[J]. Nat Cell Biol, 2002, (l):51-58.
[62]Hamm HE. The many faces of Gprotein signaling[J]. Biol Chem, 1998, 273:669-672.
[63]Hazzalin CA, Mahadevan LC. MAPK-regulated transcriPtion: aeontinuously variable gene switch[J]? Nat Rev Mol Cell Biol, 2002, 3(l):30-40.
[64]Deacon EM, al Pongraez J, Grilliths G, et al. Isoenzymes of Protein kinase C: differential involvement in apoptosis and Pathogenesis[J]. J Clin path, 1997, 50:124-131.
[65]Basu A. Involvement of Protein kinase C-delta in DNA damage-induced apoptosis[J]. J Cell Mol Med, 2003, 7(4):341-350.
[66]Susan KD. The tubulin fraternity: alPha to eta. Curr Opin Cell[J]. Curr Opin Cell Biol, 2001, 13(l):49-54.
[67]Adams J, Palobekka VJ, Ellliott PJ. Proteasome inhibition: a new strategy in caneer treatment[J]. Invest New Drugs, 2000, 18(2):109-121.
[68]Ohta T, Fukuda M. Ubiquitin and breast cancer[J]. Oneogene, 2004, 23(11): 2079- 2088.
[69]Tiliganc T, Temparis S, Combaret L. Chemotherapy inhibits skeletal musele ubiquitin-proteasome dependent proteolysis[J]. Cancer Res, 2002, 62(10):2771-2777.
[70]Yokoi S, Yasui K, Salto-Ohara F, et al. Anovel target gene, SKP2, within the SP13 amplieon that infrequently detected in small cell lung cancer[J]. Am J Pathol, 2002, 161(l): 207 -216.
[71]Sonenberg N, Dever TE. Eukaryotic translation initation factors and regulators[J]. Curr Opin Struct Biol, 2003, 13(l):56-63
[72]Rosorius O, Fries B, Stauber RH, et al. Human ribosomal Protein L5 contains defined nuclear localization and exPort signals[J]. J Biol Chem, 2000(275):12061-12066.
[73]李瑶,裘敏燕,吴超群,等.用基因表达谱芯片研究人正常肝和肝细胞癌中差异表达的基因[J].遗传学报,2000,18(5):449-452.
[74]Duman-Schee IM, Weng L, Xin S, et al. Hedgehog regulates cell growth andProliferation by induce ing cyclin D and cyclin E. Nature, 2002, 417(6886):299-304.
[75]Kim HJ,Jung WH,Kim DY, et al. Expression of cyclins in ductal hyperplasia atyPieal ductal hyperplasia and ductal earcinoma in situ of the breast[J]. Yonsei Med J, 2000,41:345-352.
[76]Myers L C, Kornberg R D. Mediator of transcriptional regulation[J]. Annu Rev Biochem, 1990, 69:729-749.
[77]Darst S A, Edwards A M, Kubalek E W, et al. Three-dimensional structure of yeast RNA polymeraseⅡat 16 ? resolution[J]. Cell, 1991, 66:121-128.
[78]Cramer P, Bushnell, D A, Komberg R D. Structural basis of transcription: RNA PolymeraseⅡat 2.8 Angstrom Resolution[J]. Science, 2001, 292:1863-1876.
[79]Gnatt A L, Cramer P, Fu J, et al. Structural basis of transcription: an RNA polymeraseⅡelongation complex at 3.3 ? resolution[J]. Science, 2001, 292: 1876-1882.
[80]Bushnell D A, Cramer P, Komberg R D. Structural basis oftranscription:α- Amanitin-RNA polymeraseⅡcocrystal at 2.8 ? resolution[J]. Proc Nat Acad Sci USA, 2002, 99:1218-1222.
[81]Marx J. Interfering with gene expression[J] . Science, 2000, 288(5470):1370-1372.
[82]Cogoni C, Romano N, Macino G.. Suppressionof gene expression by homologus transgenes [J]. Antonie Van Leeuwenhoek, 1994, 65 (3) :205-209.
[83]Guo S, Kemphues KJ. par-1, a gene required for establishing polarity in C. elehans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed[J]. Cell, 1995, 81 :611-620.
[84]Fire A. Xu S. Montgomery MK. et al. potent and specific genetic interfer- ence by double-strand RNA in Caenorhabditis elegans[J]. Nature, 998.391(6669): 806-811.
[85]Elbashir SM,Harborth J,Lendeckel W,et al.DuPlexes of 21 Nueleotide RNA interference in cultured mammalian cells[J]. NATURE, 2001,411(6945-6951): 494-498.
[86]CaPlen NJ, Parrish S, Imam F, et al. Specific inhibition of geneex Pression by small double-stranded RNAs in invertebrates and vertebrate systems[J]. Proe. Natl. Acad. Sci. USA, 2001, 98:9746-9747.
[87]Brummelkamp TR, Bemards R, and Agami R A. System for stable exPression of short interfering RNAs in mammalianc ells[J]. Science, 2002, 296(8):550-553.
[88]Browvn D, Jarvis R, Pallotta V, et al. RNA Interference in Mammalian Cell Culture: Design, Execution and Analysis of the siRNA Effect[J]. Teeh Notes, 2002, 9(l):3-5.
[89]Elbashir SM, Harborth j, weber K, et al. Analysis of gene function in Somatic mammalian cells using small interfering RNAs[J]. Methods, 2002, 26(2):199- 213.
[90]Tabara H, Sarkissian M, Kelly WG et al. The rde-1 gene, RNA interference, and transposon silencing in C.elegans[J]. Cell, 1999 Oct 15, 99(2):123-32.
[91]Nykanen A, Haley B, Zamore P D. ATP requirements and small interfering RNA structure in the RNA interference pathway[J]. Cell, 2001, 107 (3):309-321.
[92]Rand T A, Petersen S, Du F H, et al. Argonaute cleaves the anti-guide strand of siRNA during RISC activat ion [J]. Cell, 2005, 123(4):621- 629.
[93]M eister G, Tusch l T. M echanism s of gene silencing by double stranded RNA [J]. Nature, 2004, 431:343-349.
[94]Sabine B. Antisense-RNA regulation and RNA inter-ference[J]. Biochemica et Biophysica Acta, 2002, 1575:15-25.
[95]Elbashir SM,Harborth J, Weber K, et al. Analysis of gene functionin somatic mammalian cells using small interfering RNAs[J]. Methods, 2002, (26):199- 213.
[96]张明,邵宁生. RNA阻抑和基因沉默[J].军事医学科学院院刊, 2002, 26(1): 61-65.
[97]Hannon G J. RNA interference[J]. Nature, 2002, 418:244-251.
[98]Brummeikamp T R, Bernards R, A gam i R. A system fo r stable exp ression of short interfering RNAs in mammalian cells[J]. Science, 2002, 296(5667): 550-553.
[99]Zamo re P D. Ancient pathways programmed by small RNAs[J]. Science, 2002, 296(5571):1265-1269.
[100]Zilberman D, Cao X, Jacobsen SE. Argonaute4 control of locus specific siRNA accumulation and DNA and histone methylation[J]. Science, 2003, 99(5607): 716-719.
[101]Taverna SD, Coyne RS, Allis CD. Methylation of histone H3 at lysine programmed DNA elimination in tetrahymena[J]. Cell, 2002, 110(6):701-711.
[102]Grishok A, Pasquinelli AE, Conte JM, et al. Gene and mechanisms related to RNA interference regulated expression of small temporal RNAs that control C.elegants development[J]. Cell, 2001, 106:23-24.
[103]Reinhart BJ, Slack FJ, Basson M, et al. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans[J]. Nature, 2000, 403(6772):901-906.
[104]CarmellM A, Zhang L, Conk lin D S, et al. Germ line transmission of RNAi in mice[J]. Nat Struct Biol, 2003, 10(2):91-92.
[105]WESLEY S V, HELL IWELL C A, SM ITH N A, et al. Construct design for effective and high-through put gene silencing in plants[J]. Plant J, 2001, 27:581-590.
[106]Elbash ir SM, Harbo rth J, L endeckelW, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells[J]. Nature, 2001, 411(6836):494-498.
[107]Sui G, Soohoo C, A ffar E I B, et al. A DNA vetor based RNAi technology to suppress gene expression in mammalian cells [J]. Proc Natl Acid Sci, 2002, 99(8): 5515-5520.
[108]Harborth J, Elbashir SM, Tuschi T, et al. Sequence chemical, and structural variation of small interfering RNAs and short hairp in RNAs and the effect on mammalian gene silencing[J]. J D rug Dev, 2003, 13(2):83-105.
[109]Jiang M, Milner J. Selective silencing of viral gene expression in HPV-positive human cervical carcinoma cells treated with siRNA, aprimer of RNA inter ference[J]. Oncogene, 2002, (21):6041-6048.
[110]Worby C A, Simonson L N, Dixon J E. RNA interference of gene expression(RNAi ) in cultured Drosophilla cells[J]. Sci STKE, 2001, (95):PL1.
[111]Tavernarakis N, Wang SL, Dorovkov M, et al. Heritable and inducible genetic interference by double2stranded RNA encoded by transgenes[J]. Nat Genet, 2000, 24(2):180-183.
[112]Parrish S, Fleenor J, Xu S, et al. Functional anatomy of a dsRNA trigger: differential requirement for the two trigger strands in RNA interference[J]. Mol Cell, 2000, 6(5): 1077 -1087.
[113]Elbashir SM, Martinez J, Patkaniowska A, et al. Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate[J]. EMBO J, 2001, 20(23):6877-6888.
[114]Doench JG, Petersen CP, Sharp PA. siRNAs can function as miRNAs[J]. Genes Dev, 2003, 17:438-442.
[115]Eckmann CR, Kraemer B, Wickens M, et al. GLD23, a bicaudal-C homolog thatinhibits FBF to control germline sex determination in C.elegans[J]. Dev Cell, 2002, 3:697-710.
[116]Yang D, Buchhoze F, Huang Z, et al. Short RNA dup lexes p roduced by hydrolysis with Escheichia coli RnaseⅢmediate effective RNA interference in mammalian cells[J]. Proc NatlAcad Sci USA, 2002, 99(15):9942-9947.
[117]Castanotto D, Li H, Rossi J J. Functional siRNA exp ression from transfected PCR p roducts[J]. RNA, 2002, 8(11):1454-1460.
[118]Miyagishi M, Taira K. U6 promoter-driven siRNAs with four uridine 3’overhangs efficiently suppress targeted gene expression in mammalian cells[J]. Nat Biotech, 2002, 19497-19500.
[119]Maeda I, Kohara Y, Yamamoto M, et al. Large scale analysis of gene function in Caenorhabditis elegans by high throughput RNAi[J]. Curr Biol, 2001, 11(3):1712-1716.
[120]Lewis DL, Hagstrom JE, Loomis AG, et al. Efficient delivery of siRNA for inhibition of gene expression in postnatal mice[J]. Nat Genet, 2002, 32(1):107-108.
[121]XIA H B, MAO Q, PARLSON H L, et al. siRNA-mediated gene silencing in vitro and in vivo[J]. Nature Biotechnology, 2002, 20:1006-1010.
[122]HEMANNM T, FR IDMAN J S, ZILFOU J T, et al. An epiallelic series of p53 hypomorphs created by stable RNAi produces distinct tumor phenotypes in vivo [J]. Nature genetics, 2003, 33:396-400.
[123]Boutros M, Kiger A A, A rmknech t S, et al. Genome-wide RNAi analysis of growth and viability in D rosophila cells[J]. Science, 2004, 303(5659): 832-835.
[124]Ashrafi K, Chang F Y, Watts JL, et al. Genome-wide RNAi analysis of Caenorhabditis elegans fat regulatory genes[J]. Nature, 2003, 421 (6920):268-272.
[125]Hugh P C, Tomoyasu S, Chen E S, et al. Comp rehensive analysis of heteroch romat in2 and RNAi-mediated epigenetic control of the fission yeast genome[J]. Nature Genetics, 2005, 37(8):809-819.
[126]Harborth J, Elbashir SM, Bechert K, et al. Identification of essential genes in cultured mammalian cells using small interfering RNAs[J]. J Cell Sci, 2001, 114(Pt24): 4557 -4565.
[127]Paddison PJ, Silva J M, Conklin D S, et al. A resource for large-scale RNA- interference-based screens in mammals[J]. Nature, 2004, 428(6981):427-431.
[128]Capodici J, Kariko K, Weissman D. Inhibition of HIV-1 infection by small interfering RNA-mediated RNA interference[J]. J Imm unology, 2002, 169(9): 5196-5201.
[129]Novina CD, Murray MF, Dykxhoorn DM , et al. siRNA-directedinhibition of HIV-1 infection[J]. Nat Med, 2002, 8(7):681-686.
[130]MartinezM A, Gutierre ZA, Armand-U gonM, et al. Suppression of chemo -kine receptor expression by RNA interference allows for inhibition of HIV-1 replication[J]. AIDS, 2002, 16(18):2385-2390.
[131]Liu, Songkai, Asparuhova, Maria, et al. Inhibition of HIV-1 multiplica-tion by antisense U7 snRNAs and siRNAs targeting cyclophilin A[J]. Nucleic Acids Research, 2004, 32(12):3752-3759.
[132]Wolkowicz R, Nolan GP. Gene therapy progress and prospects:Novel gene therapy approaches for AIDS[J]. Gene Therapy, 2005, 12(6):467-476.
[133]Lee NS, Dohjima T, Bauer G, et al. Expression of small interfering RNAs targetd against HIV-1 retranscripts in human cells[J]. NatBiote chnol, 2002, 20:500-505.
[134]McCaffrey AP, Nakai H, Pandey K, et al. Inhibit of hepatitis B virus in mice by RNA interference[J]. Nat Biotechnol, 2003, 21:639-644.
[135]McCaff rey AP, Meuse L, Pham TT, et al. RNA interference in adult mice[J]. Nature, 2002, 418(6893):38-39.
[136]Song E , Lee SK, Wang J , et al. RNA interference targeting Fas protects mice f rom fulminant hepatitis[J]. Nat Med, 2003, 9(3):347-351.
[137]ZhangW , Singam R, Hellermann G , et al. Attenuation of dengue virus infection by adeno-associated virus-mediated siRNA delivery[J]. Genetic Vaccines and Therapy, 2004, 2:8.
[138]Ge Q, McmanusMT, Nguyen T, et al. RNA interference of influenza virus production by directly targeting mRNA for degradation and indirectly inhibiting all virvs RNA transcrip tion[J]. Proc Natl Acad Sci USA, 2003, 100(5):2718-2737.
[139]Yin JQ, Wan Y. RNA mediated gene regulation system:now and the future [J].Int J Mol Med, 2002, 10(4):355-365.
[140]Morris JC, Wang Z, Drew ME, et al. Glycolysis modulates try-panosome glycoprotein expression as revealed by an RNAi library[J]. EMBO J, 2002, 21 (17):4429-4438.
[141]Deng X, Ewton DZ, Pawlikowski B, et al. Mirk/dyrk1B is a Rho- induced kinaseactive in skeletal muscle differentiation [J]. J Biol Chem, 2003, Aug 5.
[142]Caplen NJ, Taylor J P, Statham VS, et al. Rescue of polyglu- Tamine- mediated cytotoxicity by double-st randed RNA-mediated RNA interference[J]. Hum Mol Genet, 2002, 11(2):175-184.
[143]Huang M, Page C, Reynolds RK, et al. Constitutive activation of Med19 oncogene product in human ovarian carcinoma cells[J]. Cynecol Oncol, 2000, 79(1):67-73.
[144]Wilda M, Fuchs U , Wossmann W, et al. Killing of leukemic cells with a BCR/ ABL fusion gene by RNA interference ( RNAi) [J]. Oncogene, 2002, 21 (37):5716-5724.
[145]Nagy P, Arndt-Jovin DJ, Jovin TM. Small interfering RNAs sup-press the expression of endogenous and GFP-fused epidermal growth factor receptor ( erbB1) and induce apoptosis in erbB1-overexpressing cells[J]. Exp Cell Res, 2003, 285 (1):39-49.
[146]Cioca DP, Aoki Y, Kiyosawa K. RNA interference is a functional pathway with therapeutic potential in human myeloid leukemia cell lines[J]. Cancer Gene Ther, 2003, 10(2): 125-133.
[147]Zhang L, Yang N, Mohamed-Hadley A, et al. Vector-based RNAi, a novel tool for isoform-specific knock-down of VEGF and Anti-angiogenesis gene therapy of cancer [J]. Biochem Biophys Res Commun, 2003, 303(4):1169-1178.
[148]Wu H, Hait WN, Yang JM. Small interfering RNA-induced sup- pression of MDR1 ( P-glycoprotein) restores sensitivity to mul-tidrug resistant cancer cells [J]. Cancer Res, 2003, 63(7):1515-1519.
[149] Abbas-Terki T, Blanco-Bose W, Deglon N, et al. Lentiviral mediated RNA inter-ference[J]. Hum Gene Ther, 2002, 13(18):2197-2201.
[150] Stewart SA, Dykxhoorn DM, Palliser D, et al. Lentivirus-delivered stable gene si-lencing by RNAi in primary cells[J]. RNA, 2003;9 (4):493-501.
[151] Kurreek J. Antisense teehnologies. ImProvement through novel chemieal Modifications[J]. Eur J Biochem. 2003, 270(8):1628-1644.
[152]Boutla A, Delidakis C, Livadaras I, et al. Variations of the 3’protruding ends insynthetic short interfering RNA(siRNA) tested by microinjection in Drosphilaembryos[J]. Olignucleotides, 2003, 13(5):295-301.
[153]王春光,王荣有,孙梅,赵雪俭,张兴义.沉默基因表达对肺癌细胞生长影响的实验研究[J].中国老年学杂志,2006,26 (2): 210-213.
[154]Dykxhoorn DM, Novina CD, Sharp PA. Killing the messenger: short RNAs that silence gene exp ression[J]. N at RevM ol Cell Biol, 2003, 4(6):457- 467.
[155]Reynolds A, Leake D, Boese Q, et al. Rational siRNA design for RNA interference[J]. Nat Biotechnol, 2004, 22(3):326-330.
[156]Carmell MA, Zhang L, Conklin DS, et al. Germ line transmission of RNAi in mice[J]. Nat Struct, Biol, 2003, 10(2):91-92.
[157]赵金红,杜卫华,道尔吉,等.小鼠胚胎sry基因的RNA干涉研究[J].生物化学与生物物理进展,2006,33(9):890-89.
[158]Reynolds A, Leake D, Boese Q, et al. Rational siRNA design for RNA interference. N at B iotechnol, 2004, 22(3):326 - 330.
[159]陈凌,郑祥雄. CD40发夹siRNA真核表达载体构建及其对CA46细胞CD40表达的影响[J].细胞与分子免疫学杂志,2005,21(2):163-166.
[160]陈杰斌,许从峰,虞勇,等. CVB32 VP1 siRNA对CVB3复制的抑制作用[J].细胞与分子免疫学杂志,2006,22(3):306-309.
[161]Brummelkamp TR, Bemards R, Agami R. A System for stable expression of short interfering RNAs in mammalian cells[J]. Science, 2002, 296:550-553.
[162]Brown D, Jarvis R, Pallotta V, et al. RNA interference in mammalian cell culture: design, execution and analysis of the siRNA effect[J]. Tech Notes, 2002, 9(1):3-5.
[163]Elbashir SM, Harborth J, Weber K, et al. Analysis of gene function in somatic mammalian cells using small interfering RNAs[J]. Methods, 2002, 26 (2):199-213.
[164]Hannon GJ. RNA interference[J]. Nature, 2002, 418(6894):244-251.
[165]CockreⅡAS, Kafri T. Gene delivery by lentivirus vectors[J]. Mol Biotechnol, 2007, 36(3):184-204.
[166]Abbas-TerkiT, Blanco-BoseW, Deglon N, et al. Lentiviral mediated RNA interference[J]. Hum Gene Ther, 2002, 13(18):2197-2201.
[167]Stewart SA, Dykxhoorn DM, Palliser D, et al. Lentivirus2de2livered stable gene silencing by RNAi in primary cells[J]. RNA, 2003, 9(4):493-501.
[168]Miyagishi M, Taira K. DeveloPment and aPPlication of siRNA Expressi- onvector[J]. Nucleic Res, 2002, 2(3):113-114.
[169]Hammond SM, Bernstein E, Beaeh D, et al. An RNA directed Nuclease mediatesposttranscriPtional gene silencing in DrosoPhila cells[J]. Nature, 2000, 404(5987):293-296.
[170]Lee NS, Dohjima T, Baner G, et al. EXPression of small intefering RNAs targeted against HIV 1 rev transcriPts in human cells[J]. Nature Biotechnology, 2002, 20(5):500-505.
[171]Varfolomeev EE, Ashkenazi A.Tumor necrosis factor: an apoptosis JUNKie[J]. Cell, 2004, 416(4):491-497.
[172]Miyazono K, Dijke P, Heldin CH. TGF-βsignaling by smad proteins[J]. Ady Immunol, 2000, 75(l):115-157.
[173]Massague J, Chem YG. Controlling TGF-beta signaling[J]. Genes Dev, 2000, 14(3): 624-644.
[174]Dzik WH. Apoptosis, TGF beta and transfusion-related immunosuppression: Biologic versus clinical effects[J]. Transfus Apheresis Sci. 2003, 29(2):127-129.
[175]Parkin DM,Bray F,ferlay J, et al. Global cancer statistics, 2002[J]. CA Cancer J Clin, 2005, 55(2):74-108.
[176]Parkin D M, Bary F M, Ferlay J, et al. Estimating the world cancer burden in Globocan 2000[J]. Int J Cancer, 2001, 94(13):153-156.
[177]Visser O, Belderbos J, Kwa H, et al. Improved stage-specific survival of NSCLC in Northern Holland Flevoland, the Net Herland Effect of intensified treatment or stage migration[J]? Lung Cancer, 2005, 49(Suppl2):S198 (P2316).
[178]Sanchez de Cos J, Miravet L, Nunez A, et al. Lung cancer (LC) in Spain. Last epidemiological trends concerning age, gender,smoking prevalence and histological types[J]. Lung Cancer, 2005, 49(Suppl2):S197 (P2309).
[179]Toh C, Leong S, Fong K, et al. Epidemiology of lung cancer in Singapore[J]. Lung Cancer, 2005, 49(Suppl 2):S198(P2315).
[180]Jovanovic D, Popevic S, Stevic R, et al. Changes in lung cancer presentation in 13 year interval(comparison of data bases from 1990 and 2003)[J]. Lung Cancer, 2005, 49(Suppl2):S191(P2292).
[181]Polic Vizntin M, Tripkovic I, Kukulj S, et al. The epidemiology of tracheal, bronchial and lung cancer in Croatia[J]. Lung Cancer, 2005, 49(Suppl 2):S196(P2307).
[182]Mat hur KC. Epidemiology of lung cancer in India[J]. Lung Cancer, 2005, 49(Suppl2):S385(25).
[183]M,S.M. Epidemiology of lung cancer (LC) in the underdeveloped regions of Pakistan[J]. Lung Cancer, 2005, 49(Suppl2):S193(P2296).
[184]Joshi S,BK S,Pandit N.Smoking,air pollution,and the high rates of lung cancer in developing country Nepal[J]. Lung Cancer,2005,49(Suppl 2):S191(P2291).
[185]Steeg PS. Metas tasis suppressor salter the signal trans duc-tion of cancer cells.Nat Rev Cancer,2003,3(1):55-63. [186 ]Giangreco A, Groot KR, Janes SM. Lung cancer and lung stemcells: strange bedfell ows[J]? Am J Respir Crit Care Med, 2007, 175(6):547-553.
[187]陶厚权,姚明,邹寿椿,等.血管生成抑制剂Rg3对胃癌生长和转移抑制作用的实验研究[J].中华外科杂志,2002,40(8):606.
[2]Barnes MN, Deshane JS, Rosenfeld M, et al. Gene therapy and ovarian cancer:a review[J]. Obstet Cynecol, 1997, 89:145-155.
[3]Boyer TG, Martin ME, Lees E, Ricciardi RP, and Berk AJ. Mammalian Srb/Mediator complex is targeted by adenovirus E1A protein[J]. Nature, 1999, 399: 276-279.
[4]Conaway RC, Sato S, Tomomori-Sato C, Yao T, and Conaway JW. The mammalian Mediator complex and its role in transcriptional regulation[J]. Trends Biochem Sci, 2005,30: 250-255.
[5]张郁. LCMR1反义寡核苷酸对高转移肺大细胞癌细胞系-95D细胞生物学行为的影响及LCMR1基因功能的初步研究[D].北京:中国人民解放军军医进修学院,2004.
[6]梁志欣.肺癌转移相关蛋白_LCMR1_的原核表达_多克隆抗体制备及其应用的初步研究[D].北京:中国人民解放军军医进修学院,2004.
[7]Baumli S, Hoeppner S, and Cramer P. A conserved mediator hinge revealed in the structure of the MED7.MED21 (Med7.Srb7) heterodimer[J]. J Biol Chem, 2005, 280: 18171 -18178.
[8] Dotson MR, Yuan CX, Roeder RG, et al. Structural organization of yeast and mammalian mediator complexes[J]. Proc Natl Acad Sci U S A. 2000; 97(26):14307-10.
[9]Cosma MP, Panizza S, and Nasmyth K. Cdk1 triggers association of RNA polymerase to cell cycle promoters only after recruitment of the mediator by SBF[J]. Mol Cell, 2001, 7:1213-1220.
[10]Kim YJ, Bjorklund S, Li Y, Sayre MH, and Kornberg RD. A multiprotein mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymeraseⅡ[J]. Cell, 1994, 77:599-608.
[11] Kelleher RJ, Flanagan PM, Kornberg RD. A novel mediator between activator proteins and the RNApolymeraseⅡtranscription apparatus[J]. Cell, 1990, 61(7): 1209-1215.
[12]Flaherty KM, McKay DB, Kabsch W, et al. Similarity of the three- dimensional structures of actin and the ATPase fragment of a 70-kDa heat shock cognate protein. Proc NatlAcad Sci U S A. 1991 ,88(11):5041-5045.
[13]N(?)r AM, Lemon BD, Tjian R. Transcriptional coactivator complexes[J]. Annu Rev Biochem, 2001, 70:475-501.
[14]Bjorklund S and Gustafsson CM. The yeast Mediator complex and its regulation[J]. Trends Biochem Sci, 2005, 30:240-244.
[15]van Bakel H, Huynen M, Wijmenga C. Prokaryotic diversity of the Saccharomyces cerevisiae Atx1p-mediated copper pathway[J]. 2004, 20(16):2644- 2655.
[16]Guglielmi B, Berkum N L, Klapholz B, et al. Ahigh resolution protein interaction map of the yeast Mediator complex[J]. Nucleic Acids Res, 2004, 32 (18):5379-5391.
[17]Chadick JZ and Asturias FJ. Structure of eukaryotic Mediator complexes[J]. Trends Biochem Sci, 2005, 30:264-271.
[18]Samuelsen CO, Baraznenok V, Khorosjutina O, Spahr H, Kieselbach T, Holmberg S, and Gustafsson CM. TRAP230/ARC240 and TRAP240/ARC250 Mediator subunits are functionally conserved through evolution[J]. Proc Natl Acad Sci U S A, 2003, 100: 6422-6427.
[19]Bourbon H M, Aguilera A, Ansari A,et al. Aunified nomenclature for protein subunits of mediator complexes linking transcriptional regulators to RNA polymeraseⅡ[J]. Mol Cell, 2004, 14 (5): 553-557.
[20]N(?)r AM, Taatjes DJ, Zhai W, Nogales E, Tjian R. Human CRSP interacts with RNA polymerase II CTD and adopts a specific CTD-bound conformation[J]. Genes Dev, 2002, Jun 1;16(11):1339-1344.
[21]Koleske A J, Young R A. The RNA polymeraseⅡholoenzyme and its implications for gene regulation[J]. Trends Biochem Sci, 1995, 20(6):113-116.
[22]Wang Q, Sharma D, Ren Y, Fondell JD. A coregulatory role for the TRAP-mediator complex in androgen receptor-mediated gene expression[J]. J Biol Chem, 2002, Nov 8;277(45):42852-42858. Epub 2002 Sep 5.
[23]Dotson M R, Yuan C X, Roeder R G, et al. Structural organization of yeast and mammalian mediator complexes[J]. Proc Natl Acad Sci USA, 2000, 97 (26): 14307-14310.
[24]Taatjes DJ, N??r AM, Andel F et al. Structure, function, and activator- induced conformations of the CRSP coactivator[J]. Science. 2002, 295(5557): 1058-1062.
[25]Asturias FJ, Jiang YW, Myers LC, et al. Conserved structures of mediator and RNA polymeraseⅡholoenzyme[J]. Science,1999, 283: 985-987.
[26]Andrew S. Neish SFA, et al. Factors associated with the mammalian RNA polymeraseⅡholoenzyme[J]. Nucl Acids Res, 1998, 26(3): 847-853.
[27]Yudkovsky N RJA, Hahn S. A transcription reinitiation intermediate that is stabilized by activator[J]. Nature, 2000, 324(6809): 225-229.
[28]Baidoobonso SM, Guidi BW, Myers LC. Med19(Rox3) regulates Intermodule interactions in the Saccharomyces cerevisiae mediator complex[J]. J Biol Chem, 2007, 282:5551-5559.
[29]Park J M, Kim H S, Han S J, et al. In uiuo requirement of activator-specific binding targets of mediator[J]. Mol Cell Biol, 2000, 20 (23): 8709-8719.
[30]Yudkovsky N, Ranish J A, Hahn S. Atranscription reinitiation intermediate that is stabilized by activator[J]. Nature, 2000, 408 (6809): 225-229.
[31]Bjorklund S, Gustafsson C M. The yeast Mediator complex and its regulation[J]. Trends Biochem Sci, 2005, 30 (5):240-244.
[32]Gu W, Malik S, Ito M, et al. Anovel human SRB/MED-containing cofactor complex, SMCC, involved in transcription regulation[J]. Mol Cell, 1999, 3(1): 97-108.
[33]Reeves W M, Hahn S. Activator-independent functions of the yeast mediator sin4 complex in preinitiation complex formation and transcription reinitia- tion[J]. Mol Cell Biol, 2003, 23 (1):349-358.
[34]Yoon C K, Qiu H, Govind H, et al. Simultaneous recruitment of coactivators by Gcn4p stimulates multiple Steps of Transcription in uiuo[J]. Molecular and Cellular Biology, 2005, 25 (13):5626-5638.
[35]Boube M, Faucher C, Joulia L, et al. Drosophila homologs of transcriptional mediator complex subunits are required for adult cell and segment identity specification[J]. Genes Dev. 2000, 14(22):2906-2917.
[36]Park JM, Gim BS, Kim JM, et al. Drosophila Mediator complex is broadly utilized by diverse gene-specific transcription factors at different types of core promoters[J]. Mol Cell Biol. 2001, 21(7):2312-2323.
[37]Malik S, Roeder R G. Dynamic regulation of polⅡtranscription by the mammalianMediator complex[J]. Trends Biochem Sci, 2005, 30 (5):256-263.
[38]Zhu X, Wirén M, Sinha I, Rasmussen NN, et al. Genome-wide occupancy profile of mediator and the Srb8-11 module reveals interactions with coding regions[J]. 2006, 21;22(2): 169-178.
[39]Song W TI, Qian N, Kuchin S, Carlson M. SSN genes that affect transcriptional repression in Saccharomyces cerevisiae encode SIN4, ROX3, and SRB proteins associated with RNA polymeraseII[J]. Mol Cell Biol, 1996, Jan,16(1):115-120.
[40]Evangelista CC, Jr, Rodriguez Torres AM, Limbach MP, and Zitomer RS. Rox3 and Rts1 function in the global stress response pathway in baker's yeast[J]. Genetics, 1996, 142: 1083-1093.
[41]Shu Y HS, a multicopy suppressor of hsp60-ts mutant alleles, does not encode a mitochondriallytargeted protein[J]. Mol. Cell. Biol, 1995, (15): 5618- 5626.
[42]Gustafsson CM ML, Li Y, Redd MJ, et al. Identification of Rox3 as a component of mediator and RNA polymerase II holoenzyme[J]. Biol Chem, 1997, Jan (3);272(1):48-50.
[43]Qiu H HC, Yoon S, Natarajan K, Swanson MJ, et al. An array of coactivators is required for optimal recruitment of TATA binding protein and RNA polymerase II by promoter-bound Gcn4p[J]. Mol Cell Biol, 2004, (24):4104–4117.
[44]Singh H EA, Kremer SB, Duttweiler, et al. A functional module of yeast mediator that governs the dynamic range of heat-shock gene expression[J]. Genetics, 2006, (172): 2169- 2184.
[45]Tabtiang RK and Herskowitz I. Nuclear proteins Nut1p and Nut2p cooperate to negatively regulate a Swi4p-dependent lacZ reporter gene in Saccharomyces cerevisiae[J]. Molecular and cellular biology, 1998, 18: 4707-4718.
[46]Rosenblum-Vos LS, Rhodes L, Evangelista CC, Jr, Boayke KA, and Zitomer RS. The ROX3 gene encodes an essential nuclear protein involved in CYC7 gene expression in Saccharomyces cerevisiae[J]. Molecular and cellular biology, 1991, 11:5639-5647.
[47]Kang JS, Kim SH, Hwang MS, Han SJ, Lee YC, and Kim YJ. The structural and functional organization of the yeast mediator complex[J]. J Biol Chem, 2001, 276: 42003 -42010.
[48]Takagi Y, Calero G, Komori H, Brown JA, Ehrensberger AH, Hudmon A, Asturias F,and Kornberg RD. Head module control of mediator interactions[J]. Mol Cell, 2006, 23: 355- 364.
[49]Myers L C GCM, et al. Mediator protein mutations that selectively abolish activated transcription[J]. Proc Natl Acad, 1999, (96):67-72.
[50]Lee YC, Park JM, Min S, Han SJ, and Kim YJ. An activator binding module of yeast RNA polymerase II holoenzyme[J]. Molecular and cellular biology, 1999, 19:2967-2976.
[51]Han SJ, Lee YC, Gim BS, Ryu GH, Park SJ, Lane WS, and Kim YJ. Activator-specific requirement of yeast mediator proteins for RNA polymerase II transcriptional activation[J]. Molecular and cellular biology, 1999, 19: 979- 988,.
[52]Baidoobonso SM, Guidi BW, Myers LC. Med19(Rox3) regulates Intermodule interactions in the Saccharomyces cerevisiae mediator complex[J]. JBiol Chem, 2007, 23;282(8):5551-5559.
[53]Brown TA, Evangelista C, and Trumpower BL. Regulation of nuclear genes encoding mitochondrial proteins in Saccharomyces cerevisiae[J]. Journal of bacteriology, 1995, 177: 6836-6843.
[54]Linder T, Zhu X, Baraznenok V, and Gustafsson CM. The classical srb4-138 mutant allele causes dissociation of yeast Mediator[J]. Biochemical and biophysical research communications, 2006, 349: 948-953.
[55]Sato S, Tomomori-Sato C, Banks CA, Sorokina I, Parmely TJ, Kong SE, Jin J, Cai Y, Lane WS, Brower CS, Conaway RC, and Conaway JW. Identification of mammalian Mediator subunits with similarities to yeast Mediator subunits Srb5, Srb6, Med11, and Rox3[J]. J Biol Chem, 2003, 278:15123-15127.
[56]Sato S, Tomomori-Sato C, Parmely TJ, Florens L, Zybailov B, Swanson SK, Banks CA, Jin J, Cai Y, Washburn MP, Conaway JW, and Conaway RC. A set of consensus mammalian mediator subunits identified by multidimensional protein identification technology[J]. Mol Cell, 2004, 14:685-691.
[57]Hunter T. Signaling-2000and beyond[J]. Cell, 2000, 00:113-127.
[58]Casstecker MP, Piek E, Roberts B, et al. Role of trans forming growth faeter-βsignaling in eancer[J]. Natl Cancer Inst, 2000,92(17):1388-1402.
[59]Wu G Chen YG, Ozdamar B et al. Structural basis of smad2 regulation by the Smadanchor for receptor activation[J]. Science, 2000, 287:92-97.
[60]Miyazono K. Positive and negative regulation of TGF-βsignal[J]. J Cell Sci, 2000, 131:1101-1109.
[61]Jang CW, Chen CH, Chen CC, et al. TGF-βinduces apoptosis through Smad-mediated expression of DAP-kinase[J]. Nat Cell Biol, 2002, (l):51-58.
[62]Hamm HE. The many faces of Gprotein signaling[J]. Biol Chem, 1998, 273:669-672.
[63]Hazzalin CA, Mahadevan LC. MAPK-regulated transcriPtion: aeontinuously variable gene switch[J]? Nat Rev Mol Cell Biol, 2002, 3(l):30-40.
[64]Deacon EM, al Pongraez J, Grilliths G, et al. Isoenzymes of Protein kinase C: differential involvement in apoptosis and Pathogenesis[J]. J Clin path, 1997, 50:124-131.
[65]Basu A. Involvement of Protein kinase C-delta in DNA damage-induced apoptosis[J]. J Cell Mol Med, 2003, 7(4):341-350.
[66]Susan KD. The tubulin fraternity: alPha to eta. Curr Opin Cell[J]. Curr Opin Cell Biol, 2001, 13(l):49-54.
[67]Adams J, Palobekka VJ, Ellliott PJ. Proteasome inhibition: a new strategy in caneer treatment[J]. Invest New Drugs, 2000, 18(2):109-121.
[68]Ohta T, Fukuda M. Ubiquitin and breast cancer[J]. Oneogene, 2004, 23(11): 2079- 2088.
[69]Tiliganc T, Temparis S, Combaret L. Chemotherapy inhibits skeletal musele ubiquitin-proteasome dependent proteolysis[J]. Cancer Res, 2002, 62(10):2771-2777.
[70]Yokoi S, Yasui K, Salto-Ohara F, et al. Anovel target gene, SKP2, within the SP13 amplieon that infrequently detected in small cell lung cancer[J]. Am J Pathol, 2002, 161(l): 207 -216.
[71]Sonenberg N, Dever TE. Eukaryotic translation initation factors and regulators[J]. Curr Opin Struct Biol, 2003, 13(l):56-63
[72]Rosorius O, Fries B, Stauber RH, et al. Human ribosomal Protein L5 contains defined nuclear localization and exPort signals[J]. J Biol Chem, 2000(275):12061-12066.
[73]李瑶,裘敏燕,吴超群,等.用基因表达谱芯片研究人正常肝和肝细胞癌中差异表达的基因[J].遗传学报,2000,18(5):449-452.
[74]Duman-Schee IM, Weng L, Xin S, et al. Hedgehog regulates cell growth andProliferation by induce ing cyclin D and cyclin E. Nature, 2002, 417(6886):299-304.
[75]Kim HJ,Jung WH,Kim DY, et al. Expression of cyclins in ductal hyperplasia atyPieal ductal hyperplasia and ductal earcinoma in situ of the breast[J]. Yonsei Med J, 2000,41:345-352.
[76]Myers L C, Kornberg R D. Mediator of transcriptional regulation[J]. Annu Rev Biochem, 1990, 69:729-749.
[77]Darst S A, Edwards A M, Kubalek E W, et al. Three-dimensional structure of yeast RNA polymeraseⅡat 16 ? resolution[J]. Cell, 1991, 66:121-128.
[78]Cramer P, Bushnell, D A, Komberg R D. Structural basis of transcription: RNA PolymeraseⅡat 2.8 Angstrom Resolution[J]. Science, 2001, 292:1863-1876.
[79]Gnatt A L, Cramer P, Fu J, et al. Structural basis of transcription: an RNA polymeraseⅡelongation complex at 3.3 ? resolution[J]. Science, 2001, 292: 1876-1882.
[80]Bushnell D A, Cramer P, Komberg R D. Structural basis oftranscription:α- Amanitin-RNA polymeraseⅡcocrystal at 2.8 ? resolution[J]. Proc Nat Acad Sci USA, 2002, 99:1218-1222.
[81]Marx J. Interfering with gene expression[J] . Science, 2000, 288(5470):1370-1372.
[82]Cogoni C, Romano N, Macino G.. Suppressionof gene expression by homologus transgenes [J]. Antonie Van Leeuwenhoek, 1994, 65 (3) :205-209.
[83]Guo S, Kemphues KJ. par-1, a gene required for establishing polarity in C. elehans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed[J]. Cell, 1995, 81 :611-620.
[84]Fire A. Xu S. Montgomery MK. et al. potent and specific genetic interfer- ence by double-strand RNA in Caenorhabditis elegans[J]. Nature, 998.391(6669): 806-811.
[85]Elbashir SM,Harborth J,Lendeckel W,et al.DuPlexes of 21 Nueleotide RNA interference in cultured mammalian cells[J]. NATURE, 2001,411(6945-6951): 494-498.
[86]CaPlen NJ, Parrish S, Imam F, et al. Specific inhibition of geneex Pression by small double-stranded RNAs in invertebrates and vertebrate systems[J]. Proe. Natl. Acad. Sci. USA, 2001, 98:9746-9747.
[87]Brummelkamp TR, Bemards R, and Agami R A. System for stable exPression of short interfering RNAs in mammalianc ells[J]. Science, 2002, 296(8):550-553.
[88]Browvn D, Jarvis R, Pallotta V, et al. RNA Interference in Mammalian Cell Culture: Design, Execution and Analysis of the siRNA Effect[J]. Teeh Notes, 2002, 9(l):3-5.
[89]Elbashir SM, Harborth j, weber K, et al. Analysis of gene function in Somatic mammalian cells using small interfering RNAs[J]. Methods, 2002, 26(2):199- 213.
[90]Tabara H, Sarkissian M, Kelly WG et al. The rde-1 gene, RNA interference, and transposon silencing in C.elegans[J]. Cell, 1999 Oct 15, 99(2):123-32.
[91]Nykanen A, Haley B, Zamore P D. ATP requirements and small interfering RNA structure in the RNA interference pathway[J]. Cell, 2001, 107 (3):309-321.
[92]Rand T A, Petersen S, Du F H, et al. Argonaute cleaves the anti-guide strand of siRNA during RISC activat ion [J]. Cell, 2005, 123(4):621- 629.
[93]M eister G, Tusch l T. M echanism s of gene silencing by double stranded RNA [J]. Nature, 2004, 431:343-349.
[94]Sabine B. Antisense-RNA regulation and RNA inter-ference[J]. Biochemica et Biophysica Acta, 2002, 1575:15-25.
[95]Elbashir SM,Harborth J, Weber K, et al. Analysis of gene functionin somatic mammalian cells using small interfering RNAs[J]. Methods, 2002, (26):199- 213.
[96]张明,邵宁生. RNA阻抑和基因沉默[J].军事医学科学院院刊, 2002, 26(1): 61-65.
[97]Hannon G J. RNA interference[J]. Nature, 2002, 418:244-251.
[98]Brummeikamp T R, Bernards R, A gam i R. A system fo r stable exp ression of short interfering RNAs in mammalian cells[J]. Science, 2002, 296(5667): 550-553.
[99]Zamo re P D. Ancient pathways programmed by small RNAs[J]. Science, 2002, 296(5571):1265-1269.
[100]Zilberman D, Cao X, Jacobsen SE. Argonaute4 control of locus specific siRNA accumulation and DNA and histone methylation[J]. Science, 2003, 99(5607): 716-719.
[101]Taverna SD, Coyne RS, Allis CD. Methylation of histone H3 at lysine programmed DNA elimination in tetrahymena[J]. Cell, 2002, 110(6):701-711.
[102]Grishok A, Pasquinelli AE, Conte JM, et al. Gene and mechanisms related to RNA interference regulated expression of small temporal RNAs that control C.elegants development[J]. Cell, 2001, 106:23-24.
[103]Reinhart BJ, Slack FJ, Basson M, et al. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans[J]. Nature, 2000, 403(6772):901-906.
[104]CarmellM A, Zhang L, Conk lin D S, et al. Germ line transmission of RNAi in mice[J]. Nat Struct Biol, 2003, 10(2):91-92.
[105]WESLEY S V, HELL IWELL C A, SM ITH N A, et al. Construct design for effective and high-through put gene silencing in plants[J]. Plant J, 2001, 27:581-590.
[106]Elbash ir SM, Harbo rth J, L endeckelW, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells[J]. Nature, 2001, 411(6836):494-498.
[107]Sui G, Soohoo C, A ffar E I B, et al. A DNA vetor based RNAi technology to suppress gene expression in mammalian cells [J]. Proc Natl Acid Sci, 2002, 99(8): 5515-5520.
[108]Harborth J, Elbashir SM, Tuschi T, et al. Sequence chemical, and structural variation of small interfering RNAs and short hairp in RNAs and the effect on mammalian gene silencing[J]. J D rug Dev, 2003, 13(2):83-105.
[109]Jiang M, Milner J. Selective silencing of viral gene expression in HPV-positive human cervical carcinoma cells treated with siRNA, aprimer of RNA inter ference[J]. Oncogene, 2002, (21):6041-6048.
[110]Worby C A, Simonson L N, Dixon J E. RNA interference of gene expression(RNAi ) in cultured Drosophilla cells[J]. Sci STKE, 2001, (95):PL1.
[111]Tavernarakis N, Wang SL, Dorovkov M, et al. Heritable and inducible genetic interference by double2stranded RNA encoded by transgenes[J]. Nat Genet, 2000, 24(2):180-183.
[112]Parrish S, Fleenor J, Xu S, et al. Functional anatomy of a dsRNA trigger: differential requirement for the two trigger strands in RNA interference[J]. Mol Cell, 2000, 6(5): 1077 -1087.
[113]Elbashir SM, Martinez J, Patkaniowska A, et al. Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate[J]. EMBO J, 2001, 20(23):6877-6888.
[114]Doench JG, Petersen CP, Sharp PA. siRNAs can function as miRNAs[J]. Genes Dev, 2003, 17:438-442.
[115]Eckmann CR, Kraemer B, Wickens M, et al. GLD23, a bicaudal-C homolog thatinhibits FBF to control germline sex determination in C.elegans[J]. Dev Cell, 2002, 3:697-710.
[116]Yang D, Buchhoze F, Huang Z, et al. Short RNA dup lexes p roduced by hydrolysis with Escheichia coli RnaseⅢmediate effective RNA interference in mammalian cells[J]. Proc NatlAcad Sci USA, 2002, 99(15):9942-9947.
[117]Castanotto D, Li H, Rossi J J. Functional siRNA exp ression from transfected PCR p roducts[J]. RNA, 2002, 8(11):1454-1460.
[118]Miyagishi M, Taira K. U6 promoter-driven siRNAs with four uridine 3’overhangs efficiently suppress targeted gene expression in mammalian cells[J]. Nat Biotech, 2002, 19497-19500.
[119]Maeda I, Kohara Y, Yamamoto M, et al. Large scale analysis of gene function in Caenorhabditis elegans by high throughput RNAi[J]. Curr Biol, 2001, 11(3):1712-1716.
[120]Lewis DL, Hagstrom JE, Loomis AG, et al. Efficient delivery of siRNA for inhibition of gene expression in postnatal mice[J]. Nat Genet, 2002, 32(1):107-108.
[121]XIA H B, MAO Q, PARLSON H L, et al. siRNA-mediated gene silencing in vitro and in vivo[J]. Nature Biotechnology, 2002, 20:1006-1010.
[122]HEMANNM T, FR IDMAN J S, ZILFOU J T, et al. An epiallelic series of p53 hypomorphs created by stable RNAi produces distinct tumor phenotypes in vivo [J]. Nature genetics, 2003, 33:396-400.
[123]Boutros M, Kiger A A, A rmknech t S, et al. Genome-wide RNAi analysis of growth and viability in D rosophila cells[J]. Science, 2004, 303(5659): 832-835.
[124]Ashrafi K, Chang F Y, Watts JL, et al. Genome-wide RNAi analysis of Caenorhabditis elegans fat regulatory genes[J]. Nature, 2003, 421 (6920):268-272.
[125]Hugh P C, Tomoyasu S, Chen E S, et al. Comp rehensive analysis of heteroch romat in2 and RNAi-mediated epigenetic control of the fission yeast genome[J]. Nature Genetics, 2005, 37(8):809-819.
[126]Harborth J, Elbashir SM, Bechert K, et al. Identification of essential genes in cultured mammalian cells using small interfering RNAs[J]. J Cell Sci, 2001, 114(Pt24): 4557 -4565.
[127]Paddison PJ, Silva J M, Conklin D S, et al. A resource for large-scale RNA- interference-based screens in mammals[J]. Nature, 2004, 428(6981):427-431.
[128]Capodici J, Kariko K, Weissman D. Inhibition of HIV-1 infection by small interfering RNA-mediated RNA interference[J]. J Imm unology, 2002, 169(9): 5196-5201.
[129]Novina CD, Murray MF, Dykxhoorn DM , et al. siRNA-directedinhibition of HIV-1 infection[J]. Nat Med, 2002, 8(7):681-686.
[130]MartinezM A, Gutierre ZA, Armand-U gonM, et al. Suppression of chemo -kine receptor expression by RNA interference allows for inhibition of HIV-1 replication[J]. AIDS, 2002, 16(18):2385-2390.
[131]Liu, Songkai, Asparuhova, Maria, et al. Inhibition of HIV-1 multiplica-tion by antisense U7 snRNAs and siRNAs targeting cyclophilin A[J]. Nucleic Acids Research, 2004, 32(12):3752-3759.
[132]Wolkowicz R, Nolan GP. Gene therapy progress and prospects:Novel gene therapy approaches for AIDS[J]. Gene Therapy, 2005, 12(6):467-476.
[133]Lee NS, Dohjima T, Bauer G, et al. Expression of small interfering RNAs targetd against HIV-1 retranscripts in human cells[J]. NatBiote chnol, 2002, 20:500-505.
[134]McCaffrey AP, Nakai H, Pandey K, et al. Inhibit of hepatitis B virus in mice by RNA interference[J]. Nat Biotechnol, 2003, 21:639-644.
[135]McCaff rey AP, Meuse L, Pham TT, et al. RNA interference in adult mice[J]. Nature, 2002, 418(6893):38-39.
[136]Song E , Lee SK, Wang J , et al. RNA interference targeting Fas protects mice f rom fulminant hepatitis[J]. Nat Med, 2003, 9(3):347-351.
[137]ZhangW , Singam R, Hellermann G , et al. Attenuation of dengue virus infection by adeno-associated virus-mediated siRNA delivery[J]. Genetic Vaccines and Therapy, 2004, 2:8.
[138]Ge Q, McmanusMT, Nguyen T, et al. RNA interference of influenza virus production by directly targeting mRNA for degradation and indirectly inhibiting all virvs RNA transcrip tion[J]. Proc Natl Acad Sci USA, 2003, 100(5):2718-2737.
[139]Yin JQ, Wan Y. RNA mediated gene regulation system:now and the future [J].Int J Mol Med, 2002, 10(4):355-365.
[140]Morris JC, Wang Z, Drew ME, et al. Glycolysis modulates try-panosome glycoprotein expression as revealed by an RNAi library[J]. EMBO J, 2002, 21 (17):4429-4438.
[141]Deng X, Ewton DZ, Pawlikowski B, et al. Mirk/dyrk1B is a Rho- induced kinaseactive in skeletal muscle differentiation [J]. J Biol Chem, 2003, Aug 5.
[142]Caplen NJ, Taylor J P, Statham VS, et al. Rescue of polyglu- Tamine- mediated cytotoxicity by double-st randed RNA-mediated RNA interference[J]. Hum Mol Genet, 2002, 11(2):175-184.
[143]Huang M, Page C, Reynolds RK, et al. Constitutive activation of Med19 oncogene product in human ovarian carcinoma cells[J]. Cynecol Oncol, 2000, 79(1):67-73.
[144]Wilda M, Fuchs U , Wossmann W, et al. Killing of leukemic cells with a BCR/ ABL fusion gene by RNA interference ( RNAi) [J]. Oncogene, 2002, 21 (37):5716-5724.
[145]Nagy P, Arndt-Jovin DJ, Jovin TM. Small interfering RNAs sup-press the expression of endogenous and GFP-fused epidermal growth factor receptor ( erbB1) and induce apoptosis in erbB1-overexpressing cells[J]. Exp Cell Res, 2003, 285 (1):39-49.
[146]Cioca DP, Aoki Y, Kiyosawa K. RNA interference is a functional pathway with therapeutic potential in human myeloid leukemia cell lines[J]. Cancer Gene Ther, 2003, 10(2): 125-133.
[147]Zhang L, Yang N, Mohamed-Hadley A, et al. Vector-based RNAi, a novel tool for isoform-specific knock-down of VEGF and Anti-angiogenesis gene therapy of cancer [J]. Biochem Biophys Res Commun, 2003, 303(4):1169-1178.
[148]Wu H, Hait WN, Yang JM. Small interfering RNA-induced sup- pression of MDR1 ( P-glycoprotein) restores sensitivity to mul-tidrug resistant cancer cells [J]. Cancer Res, 2003, 63(7):1515-1519.
[149] Abbas-Terki T, Blanco-Bose W, Deglon N, et al. Lentiviral mediated RNA inter-ference[J]. Hum Gene Ther, 2002, 13(18):2197-2201.
[150] Stewart SA, Dykxhoorn DM, Palliser D, et al. Lentivirus-delivered stable gene si-lencing by RNAi in primary cells[J]. RNA, 2003;9 (4):493-501.
[151] Kurreek J. Antisense teehnologies. ImProvement through novel chemieal Modifications[J]. Eur J Biochem. 2003, 270(8):1628-1644.
[152]Boutla A, Delidakis C, Livadaras I, et al. Variations of the 3’protruding ends insynthetic short interfering RNA(siRNA) tested by microinjection in Drosphilaembryos[J]. Olignucleotides, 2003, 13(5):295-301.
[153]王春光,王荣有,孙梅,赵雪俭,张兴义.沉默基因表达对肺癌细胞生长影响的实验研究[J].中国老年学杂志,2006,26 (2): 210-213.
[154]Dykxhoorn DM, Novina CD, Sharp PA. Killing the messenger: short RNAs that silence gene exp ression[J]. N at RevM ol Cell Biol, 2003, 4(6):457- 467.
[155]Reynolds A, Leake D, Boese Q, et al. Rational siRNA design for RNA interference[J]. Nat Biotechnol, 2004, 22(3):326-330.
[156]Carmell MA, Zhang L, Conklin DS, et al. Germ line transmission of RNAi in mice[J]. Nat Struct, Biol, 2003, 10(2):91-92.
[157]赵金红,杜卫华,道尔吉,等.小鼠胚胎sry基因的RNA干涉研究[J].生物化学与生物物理进展,2006,33(9):890-89.
[158]Reynolds A, Leake D, Boese Q, et al. Rational siRNA design for RNA interference. N at B iotechnol, 2004, 22(3):326 - 330.
[159]陈凌,郑祥雄. CD40发夹siRNA真核表达载体构建及其对CA46细胞CD40表达的影响[J].细胞与分子免疫学杂志,2005,21(2):163-166.
[160]陈杰斌,许从峰,虞勇,等. CVB32 VP1 siRNA对CVB3复制的抑制作用[J].细胞与分子免疫学杂志,2006,22(3):306-309.
[161]Brummelkamp TR, Bemards R, Agami R. A System for stable expression of short interfering RNAs in mammalian cells[J]. Science, 2002, 296:550-553.
[162]Brown D, Jarvis R, Pallotta V, et al. RNA interference in mammalian cell culture: design, execution and analysis of the siRNA effect[J]. Tech Notes, 2002, 9(1):3-5.
[163]Elbashir SM, Harborth J, Weber K, et al. Analysis of gene function in somatic mammalian cells using small interfering RNAs[J]. Methods, 2002, 26 (2):199-213.
[164]Hannon GJ. RNA interference[J]. Nature, 2002, 418(6894):244-251.
[165]CockreⅡAS, Kafri T. Gene delivery by lentivirus vectors[J]. Mol Biotechnol, 2007, 36(3):184-204.
[166]Abbas-TerkiT, Blanco-BoseW, Deglon N, et al. Lentiviral mediated RNA interference[J]. Hum Gene Ther, 2002, 13(18):2197-2201.
[167]Stewart SA, Dykxhoorn DM, Palliser D, et al. Lentivirus2de2livered stable gene silencing by RNAi in primary cells[J]. RNA, 2003, 9(4):493-501.
[168]Miyagishi M, Taira K. DeveloPment and aPPlication of siRNA Expressi- onvector[J]. Nucleic Res, 2002, 2(3):113-114.
[169]Hammond SM, Bernstein E, Beaeh D, et al. An RNA directed Nuclease mediatesposttranscriPtional gene silencing in DrosoPhila cells[J]. Nature, 2000, 404(5987):293-296.
[170]Lee NS, Dohjima T, Baner G, et al. EXPression of small intefering RNAs targeted against HIV 1 rev transcriPts in human cells[J]. Nature Biotechnology, 2002, 20(5):500-505.
[171]Varfolomeev EE, Ashkenazi A.Tumor necrosis factor: an apoptosis JUNKie[J]. Cell, 2004, 416(4):491-497.
[172]Miyazono K, Dijke P, Heldin CH. TGF-βsignaling by smad proteins[J]. Ady Immunol, 2000, 75(l):115-157.
[173]Massague J, Chem YG. Controlling TGF-beta signaling[J]. Genes Dev, 2000, 14(3): 624-644.
[174]Dzik WH. Apoptosis, TGF beta and transfusion-related immunosuppression: Biologic versus clinical effects[J]. Transfus Apheresis Sci. 2003, 29(2):127-129.
[175]Parkin DM,Bray F,ferlay J, et al. Global cancer statistics, 2002[J]. CA Cancer J Clin, 2005, 55(2):74-108.
[176]Parkin D M, Bary F M, Ferlay J, et al. Estimating the world cancer burden in Globocan 2000[J]. Int J Cancer, 2001, 94(13):153-156.
[177]Visser O, Belderbos J, Kwa H, et al. Improved stage-specific survival of NSCLC in Northern Holland Flevoland, the Net Herland Effect of intensified treatment or stage migration[J]? Lung Cancer, 2005, 49(Suppl2):S198 (P2316).
[178]Sanchez de Cos J, Miravet L, Nunez A, et al. Lung cancer (LC) in Spain. Last epidemiological trends concerning age, gender,smoking prevalence and histological types[J]. Lung Cancer, 2005, 49(Suppl2):S197 (P2309).
[179]Toh C, Leong S, Fong K, et al. Epidemiology of lung cancer in Singapore[J]. Lung Cancer, 2005, 49(Suppl 2):S198(P2315).
[180]Jovanovic D, Popevic S, Stevic R, et al. Changes in lung cancer presentation in 13 year interval(comparison of data bases from 1990 and 2003)[J]. Lung Cancer, 2005, 49(Suppl2):S191(P2292).
[181]Polic Vizntin M, Tripkovic I, Kukulj S, et al. The epidemiology of tracheal, bronchial and lung cancer in Croatia[J]. Lung Cancer, 2005, 49(Suppl 2):S196(P2307).
[182]Mat hur KC. Epidemiology of lung cancer in India[J]. Lung Cancer, 2005, 49(Suppl2):S385(25).
[183]M,S.M. Epidemiology of lung cancer (LC) in the underdeveloped regions of Pakistan[J]. Lung Cancer, 2005, 49(Suppl2):S193(P2296).
[184]Joshi S,BK S,Pandit N.Smoking,air pollution,and the high rates of lung cancer in developing country Nepal[J]. Lung Cancer,2005,49(Suppl 2):S191(P2291).
[185]Steeg PS. Metas tasis suppressor salter the signal trans duc-tion of cancer cells.Nat Rev Cancer,2003,3(1):55-63. [186 ]Giangreco A, Groot KR, Janes SM. Lung cancer and lung stemcells: strange bedfell ows[J]? Am J Respir Crit Care Med, 2007, 175(6):547-553.
[187]陶厚权,姚明,邹寿椿,等.血管生成抑制剂Rg3对胃癌生长和转移抑制作用的实验研究[J].中华外科杂志,2002,40(8):606.