肺癌组织中HMGN5基因的表达及其在肺癌发病中作用机制的研究
摘要
肺癌是当今世界上发生率、病死率最高的恶性肿瘤之一,也是我国十大恶性肿瘤之一,其5年总生存率仅13%～15%。目前肺癌的发病率仍在上升,肺癌的治疗效果近年来仍不理想,因此新的化疗药物、分子靶向药物成为肺癌的研究热点,探讨肺癌的发病机制以寻找到有潜力的生物标记分子为肺癌的个性化治疗提供强有力的依据。多基因、多步骤参与了肺癌的发生、发展过程,因此通过研究癌变过程中异常表达的基因,寻找到具有诊断、治疗价值的基因意义重大。
RNAi为一种序列特异性的转录后的基因沉默现象,RNAi是通过将与nnRNA序列相对应的正义、反义RNA组成的21-23个双链RNA导入到靶细胞内,使细胞内特异性基因的nRNA降解,从而导致基因沉默。RNAi是一种反向遗传学技术,在后基因组时代的功能基因的研究中具有相当重要的作用。针对基因研究中的“敲除”技术,RNAi技术为“剔降”靶基因或者靶蛋白,目前已经广泛应用于信号转导途径、基因功能、肿瘤基因治疗等方面的研究,而RNAi在哺乳动物细胞上也获得了成功。RNAi因其对靶基因表达抑制的高效性、简单性使其成为基因功能研究的有效手段,RNAi可以选择性地抑制与人类疾病有关基因的表达,在基因功能研究和治疗人类疾病方面应用前景极为广阔。目前国内外研究学者已成功利用RNA干扰技术选择性地抑制或下调在肺癌发生及发展过程中起关键作用的癌基因、抑癌基因、凋亡相关基因等肺-*癌相关基因的表达,进而达到基因治疗的目的。
HMGN5是HMGN家族成员之一,与NSBPl具有高度同源,是King等人于2001年发现的一个新的基因。人HMGN5的基因全长为8600 bp,含有5个内含子和6个外显子,完全cDNA序列为1865bp,在人体内编码一种282个氨基酸的人类核蛋白-核小体结合蛋白,理论分子质量为31000。HMGN5在人和小鼠体内表达的蛋白长度并不相同,而人的HGMN5的6个外显子更短一些。
核小体与HMGN5特异性结构相结合所引起染色体结构的改变,将影响着细胞生物功能和分子转录。HMGN5特异的长酸末端使得其与连接组蛋白H1有直接的作用,负离子末端、H5正电C端(H1组蛋白的变异体)在体内外起到直接相互作用,展开染色体可能与HMGN5中含有的负电荷的长C端密切相关；HMGN5基因N端的NBD结构域,可以连接体组蛋白H1的球状结构域竞争性地与核小体结合位点相结合；这两个结构在细胞核内使得HMGN5影响染色体的结构,从而影响最终基因的转录、蛋白的表达。
虽然尚不清楚HMGN5的分子机制,但是有文献报道称IMGN5在疾病、正常细胞功能方面有潜在的关联。例如：过表达实验和基因芯片分析结果表明HMGN5可以作为一个因子诱导小鼠胚胎干细胞的分化；HMGN5在人鳞状癌细胞、前列腺癌、衰老小鼠的腺癌、高转移性MDA-MB-435HM乳腺癌细胞系中表达明显增高,实验结果表明HMGN5与肿瘤有关联性；HMGN5蛋白在肿瘤组织中表达增加属于内源性紊乱；此外HMGN5位于X染色体上,含有不成比例的大数量的基因可能与其心理功能、进化晚期出现的行为、认知功能相关联。
通过优化影响染色质反应,HMGN5不断地变化环境的细胞表型。高酸性尾端和异常的长端影响着连接染色体和组蛋白的相互作用,从而使HMGN5够更加有效地展开染色体高度有序结构。连接组蛋白与HMGN5直接的竞争为其调节染色体动态结构提供了新的机制模式。阐明IMGN5对染色体结构和功能的影响及这种竞争机制的具体模式,有待于深入地研究。在维持细胞内的染色质纤维的完整性方面IMGN5发挥着重要作用,但HMGN5的异常表达对细胞是有害的。IMGN5与肿瘤的发生有着潜在的关系。已经有报道称HMGN5在膀胱癌、前列腺肿瘤等肿瘤组织中的表达水平显著高于正常的组织,然而在肺癌中的研究还尚无明确的报道,进一步研究HMGN5基因在肺癌发展过程中的作用,通过此途径努力寻求临床治疗肺癌的突破口。
目的：探讨肺癌组织中HMGN5基因的表达水平、HMGN5与临床病理特征的相关性；构建HMGN5的shRNA重组慢病毒载体；RNA干扰技术沉默HMGN5基因对肺癌A549、H1299细胞增殖和细胞周期的影响；应用RNAi技术沉默肺癌细胞A549和H1299中的HMGN5基因,抑制HMGN5基因对裸鼠成瘤能力的影响,为肺癌的基因靶向提供理论依据。
方法：用Real-time PCR的方法检测肺癌组织内、癌旁组织中HMGN5 mRNA的表达情况,分析HMGN5表达与肺癌临床病理特征的相关性；设计靶向HMGN5基因siRNA干扰序列；将设计的序列进行合成载体构建；挑选阳性、测序正确的质粒进行病毒包装；包装的病毒感染人肺癌细胞A549、H1299,应用Real-time PCR和VVestern blot的方法验证人肺癌细胞A549、H1299的沉默效率；应用RNAi技术抑制HMGN5基因在肺癌A549、H1299细胞中的表达,HMGN5 siRNA重组慢病毒感染人肺癌细胞,应用MTT方法检测HMGN5沉默表达后人肺癌细胞增殖能力的变化；应用Brdu的方法检测沉默IMGN5表达,人肺癌细胞DNA合成速度变化；应用克隆形成实验验证HMGN5沉默后人肺癌细胞的克隆形成能力的改变；采用流式细胞仪检测细胞周期的变化,以探讨HMGN5在肺癌细胞中的生物学功能；建立人肺癌A549细胞裸鼠皮下种植瘤模型,通过观察肿瘤的大小并对瘤体进行称重,观察HMGN5基因沉默对裸鼠的体内成瘤能力的影响。
结果：Real-time PCR结果显示在肺癌组织中的表达水平(4.60±0.47)显著高于癌旁组织(1.02±0.55)(P<0.05)；HMGN5 siRNA重组慢病毒载体构建成功；Real-time PCR和Western blot结果显示,实验设计的siRNA靶点序列在人肺癌细胞A549和H1299细胞内沉默表达HMGN5的沉默效率达到80%：重组的HMGN5 siRNA慢病毒载体作用于人肺癌细胞A549、H1299细胞后,HMGN5基因的mRNA及蛋白表达水平明显降低,MTT、Brud、细胞克隆形成实验结果表明转染HMGN5-siRNA的A549、H1299细胞增殖能力、DNA复制能力、克隆形成能力明显降低,流式细胞术实验结果表明转染HMGN5-siRNA的A549、H1299细胞阻滞于Go/G1期,使细胞分裂减缓；成功建立A549裸鼠皮下移植瘤模型,体内观察实验结果表明注射HMGN5-siRNA小鼠的转移瘤质量及体积较对照组明显降低,裸鼠体内成瘤能力明显受到抑制。
结论：HMGN5在肺癌组织中高表达；成功构建了HMGN5 siRNA干扰质粒,能够抑制HMGN5基因的nnRNA和蛋白表达；HMGN5 siRNA慢病毒表达载体能够抑制肺癌细胞增殖、克隆形成能力,导致细胞阻滞于G0/G1期,使细胞分裂减缓,提示HMGN5基因参与了肺癌发展过程,HMGN5可以作为肺癌基因治疗的新靶点,为治疗肺癌提供新的思路和理论研究基础。
The incidence and mortality rates of lung cancer are extremely high in the world. The highest cancer mortality rate is one of the top ten malignant tumors, lung cancer's 5-year overall survival rate is 13% to 15%. The incidence of lung cancer is still rising incidence of lung cancer, but the treatment effect of lung cancer is still not ideal in recent years, therefore the research of lung cancer is focused on the new chemotherapy drugs, molecular targeted drugs, the application of drug therapy and probing into the pathogenesis of lung cancer in order to find potential biological marker for the personalized treatment of lung cancer. Multiple genes and multiple steps are involved in the occurrence and the developed process of lung cancer. So the process by studying the abnormal expression of cancer genes, finding a diagnostic and therapeutic gene has a great clinical significance.
As a sequence-specific post-transcriptional gene silencing, RNAi is a means which is induced 21-23 double-stranded RNA composed of sense and antisense RNA corresponding mRNA, so that mRNA of the specific genes in cells are degradated, leading to gene silencing. RNAi is a reverse genetics technology, which plays an important role in the study of functional genes in the post-genomic times. Aiming at genetic research in the "knockout" technology, RNAi technology for "ticking down" the target gene or target protein, has been widely used in signal transduction、gene function and research in cancer gene therapy,so dose in mammalian cells. In view of the RNAi technology is of great efficiency and simplicity in inhibition of target gene expression. RNAi becomes an effective means of gene function research. RNAi can selectively inhibit the expression of genes related to human disease in the study of gene function, so it has a broad prospect in the study of gene function and the treatment of human diseases. Researchers in-country and abroad have successfully used RNAi technology to selectively inhibit or down-regulate the expression of lung cancer related genes which play important roles in the development of lung cancer, such as oncogenes、anti-oncogenes and genes related to apoptosis, in order to achieve gene therapy purposes.
HMGN5 is a member of the HMGN family,it is highly homologous with the NSBP1. King has discovered HMGN5 in 2001. Human gene HMGN5 is 8600bp long and it has 5 introns and 6 exons, and its complete cDNA sequence is 1865bp long. HMGN5 encodes a kind of human nuclear protein-nucleosome-binding protein that includes 282 amino acids, and its molecular weight is 31000 bp in theory. HMGN5 expression length is different between human and mice because man's HGMN5 that consists of 6 exons which is shorter.
Although the molecular mechanism HMGN5 is not clear, but the literature reports that HMGN5 has potential relevance in disease and normal cell function. For example, over-expression experiments and gene chip analysis showed that HMGN5 can serve as a factor inducing differentiation of mouse embryonic stem cells. HMGN5 expresses highly in human squamous cancer、prostate cancer、adenocarcinoma of aging mice、highly metastatic breast cancer MDA-MB-435HM cells, so that the experimental results show that the HMGN5 is associated with the tumor. It is endogenous disorder that HMGN5 protein expresses increasedly in tumor tissue. In addition, HMGN5 in X chromosome, containing a disproportionately large number of genes may be related to mental function, as well as behavior and cognitive function in late evolutionary stage.
By optimizing the reaction of chromatin, HMGN5 constantly changes the cell phenotype of environment. Highly acidic terminal and abnormal long terminal influence the interaction between connection chromosomes and histone, so that HMGN5 can more effectively expand the highly ordered structure of chromosomes. Direct competition between connection histone and HMGN5 provides a new mechanism model for regulationing dynamic structure of chromosome. Clarifing how HMGN5 influences chromosome structure and the specific modes of this competition mechanism is needed to be studied in depth. HMGN5 plays an important role in maintaining the integrity of chromatin fibers within cells, but the abnormal expression of HMGN5 is harmful to the cells. HMGN5 and the tumor have a potential relationship. It has been reported that HMGN5's expression level in bladder cancer、prostate cancer and other tumor tissues were significantly higher than normal tissue, but studies in lung cancer has not yet been clearly reported. So further research is how HMGN5 gene affects lung cancer occurrence and development, and we can seek a breakthrough in clinical treatment of lung cancer by that way.
Objective:To investigate HMGN5 gene expression level in lung cancer tissue, and the connection with the clinical and pathological features; to build siRNA of HMGN5 in order to recombine lentiviral vectors; The influence on RNA interference silence HMGN5 gene in lung cancer cells proliferation、cell cycle and for ability of lung tumor. RNAi silencing HMGN5 genes in lung cacer cells A549 and H1299, and inhibition of HMGN5 gene working on tumor formation in nude mice, provide a theoretical basis of gene targeting therapy.
Methods:To detect HMGN5 mRNA expression in lung cancer and besides the cancer tissues by Real-time PCR method, and analyse the relativity between the expression of HMGN5 and clinicopathological features of lung cancer; Design targeted HMGN5 gene siRNA interfereNce sequence; Build synthetic vectors with designed sequences; Choose positive and correct-sequencing plasmid to package virus; Let packaged virus infect human lung cancer cells A549、H1299, and verify silencing efficiency of human lung cancer cells A549、H1299 by Real-time PCR and Western blot methods; To inhibit HMGN5 gene expression in lung cancer A549 and H1299 cells by RNAi technology, at the same time, HMGN5 siRNA recombine lentiviruses to infect human lung cancer cells, through that we can draw a conclusion what HMGN5 changes of the proliferation ability of human lung cancer cell, after detecting the silencing expression of HMGN5 by using MTT; Detect the silencing HMGN5 expression and how human lung cancer cell DNA synthesis rate changes using Brdu; After application of colony formation method validating HMGN5 silence, the change of human lung cancer cell colony formation ability was detected; Using flow cytometry test cell cycles change in order to explore HMGN5's biological functions in lung cancer cells; Tumor model with human lung cancer A549 cells subcutaneous was built in nude mice, by observing the size and weight of the tumor in order to observe the influence on lung cancer in vivo formation ability by HMGN5 gene silencing.
Results:Real-time PCR results showed that expression levels in the lung cancer tissue (4.60±0.47) was significantly higher than in adjacent tissue (1.02±0.55) (P<0.05); HMGN5 siRNA built lentiviral vector was successfully constructed. Real-time PCR and Western blot results showed that the experimental designed siRNA target sequence express silently in human lung cancer cells A549 and H1299 cells, and the silence efficiency of HMGN5 is 80%; Recombined HMGN5 siRNA lentiviral vectors have effected on human lung cancer cells A549 and H1299 cells, mRNA and protein of HMGN5 gene expression levels were significantly decreased; MTT、Brud and cell clone experimental results showed that the ability of cell proliferation, DNA replication and colony formation of transfected HMGN5-siRNA A549 and H1299 cell was significantly lower than the control group; Flow cytometry results showed that A549 and H1299 which transfected HMGN5-siRNA were arrested in G0/G1 phase, so that cell division was slow down; successfully established subcutaneous A549 nude mouse model in vivo experiment results showed that mice injected with HMGN5-siRNA's transfered tumor weight was significantly lower than the control group. Tumor formation ability of lung cancer cells in vivo was significantly inhibited.
Conclusions:HMGN5 was expressed highly in lung cancer tissue; HMGN5 siRNA interference plasmids were successfully built, and it can inhibit mRNA and protein expression of HMGN5 gene; HMGN5 siRNA lentiviral vectors can inhibit the ability of cancer cell proliferation and colony formation, leading to cell arresed in G0/G1 phase in order to slow down cell division. So it can draw a conclusion that lung cancer HMGN5 genes may have relationship with development of lung cancer. HMGN5 can be used as a new target of cancer gene therapy, which provides new ideas and theoretical research base for treatment of lung cancer.
RNAi为一种序列特异性的转录后的基因沉默现象,RNAi是通过将与nnRNA序列相对应的正义、反义RNA组成的21-23个双链RNA导入到靶细胞内,使细胞内特异性基因的nRNA降解,从而导致基因沉默。RNAi是一种反向遗传学技术,在后基因组时代的功能基因的研究中具有相当重要的作用。针对基因研究中的“敲除”技术,RNAi技术为“剔降”靶基因或者靶蛋白,目前已经广泛应用于信号转导途径、基因功能、肿瘤基因治疗等方面的研究,而RNAi在哺乳动物细胞上也获得了成功。RNAi因其对靶基因表达抑制的高效性、简单性使其成为基因功能研究的有效手段,RNAi可以选择性地抑制与人类疾病有关基因的表达,在基因功能研究和治疗人类疾病方面应用前景极为广阔。目前国内外研究学者已成功利用RNA干扰技术选择性地抑制或下调在肺癌发生及发展过程中起关键作用的癌基因、抑癌基因、凋亡相关基因等肺-*癌相关基因的表达,进而达到基因治疗的目的。
HMGN5是HMGN家族成员之一,与NSBPl具有高度同源,是King等人于2001年发现的一个新的基因。人HMGN5的基因全长为8600 bp,含有5个内含子和6个外显子,完全cDNA序列为1865bp,在人体内编码一种282个氨基酸的人类核蛋白-核小体结合蛋白,理论分子质量为31000。HMGN5在人和小鼠体内表达的蛋白长度并不相同,而人的HGMN5的6个外显子更短一些。
核小体与HMGN5特异性结构相结合所引起染色体结构的改变,将影响着细胞生物功能和分子转录。HMGN5特异的长酸末端使得其与连接组蛋白H1有直接的作用,负离子末端、H5正电C端(H1组蛋白的变异体)在体内外起到直接相互作用,展开染色体可能与HMGN5中含有的负电荷的长C端密切相关；HMGN5基因N端的NBD结构域,可以连接体组蛋白H1的球状结构域竞争性地与核小体结合位点相结合；这两个结构在细胞核内使得HMGN5影响染色体的结构,从而影响最终基因的转录、蛋白的表达。
虽然尚不清楚HMGN5的分子机制,但是有文献报道称IMGN5在疾病、正常细胞功能方面有潜在的关联。例如：过表达实验和基因芯片分析结果表明HMGN5可以作为一个因子诱导小鼠胚胎干细胞的分化；HMGN5在人鳞状癌细胞、前列腺癌、衰老小鼠的腺癌、高转移性MDA-MB-435HM乳腺癌细胞系中表达明显增高,实验结果表明HMGN5与肿瘤有关联性；HMGN5蛋白在肿瘤组织中表达增加属于内源性紊乱；此外HMGN5位于X染色体上,含有不成比例的大数量的基因可能与其心理功能、进化晚期出现的行为、认知功能相关联。
通过优化影响染色质反应,HMGN5不断地变化环境的细胞表型。高酸性尾端和异常的长端影响着连接染色体和组蛋白的相互作用,从而使HMGN5够更加有效地展开染色体高度有序结构。连接组蛋白与HMGN5直接的竞争为其调节染色体动态结构提供了新的机制模式。阐明IMGN5对染色体结构和功能的影响及这种竞争机制的具体模式,有待于深入地研究。在维持细胞内的染色质纤维的完整性方面IMGN5发挥着重要作用,但HMGN5的异常表达对细胞是有害的。IMGN5与肿瘤的发生有着潜在的关系。已经有报道称HMGN5在膀胱癌、前列腺肿瘤等肿瘤组织中的表达水平显著高于正常的组织,然而在肺癌中的研究还尚无明确的报道,进一步研究HMGN5基因在肺癌发展过程中的作用,通过此途径努力寻求临床治疗肺癌的突破口。
目的：探讨肺癌组织中HMGN5基因的表达水平、HMGN5与临床病理特征的相关性；构建HMGN5的shRNA重组慢病毒载体；RNA干扰技术沉默HMGN5基因对肺癌A549、H1299细胞增殖和细胞周期的影响；应用RNAi技术沉默肺癌细胞A549和H1299中的HMGN5基因,抑制HMGN5基因对裸鼠成瘤能力的影响,为肺癌的基因靶向提供理论依据。
方法：用Real-time PCR的方法检测肺癌组织内、癌旁组织中HMGN5 mRNA的表达情况,分析HMGN5表达与肺癌临床病理特征的相关性；设计靶向HMGN5基因siRNA干扰序列；将设计的序列进行合成载体构建；挑选阳性、测序正确的质粒进行病毒包装；包装的病毒感染人肺癌细胞A549、H1299,应用Real-time PCR和VVestern blot的方法验证人肺癌细胞A549、H1299的沉默效率；应用RNAi技术抑制HMGN5基因在肺癌A549、H1299细胞中的表达,HMGN5 siRNA重组慢病毒感染人肺癌细胞,应用MTT方法检测HMGN5沉默表达后人肺癌细胞增殖能力的变化；应用Brdu的方法检测沉默IMGN5表达,人肺癌细胞DNA合成速度变化；应用克隆形成实验验证HMGN5沉默后人肺癌细胞的克隆形成能力的改变；采用流式细胞仪检测细胞周期的变化,以探讨HMGN5在肺癌细胞中的生物学功能；建立人肺癌A549细胞裸鼠皮下种植瘤模型,通过观察肿瘤的大小并对瘤体进行称重,观察HMGN5基因沉默对裸鼠的体内成瘤能力的影响。
结果：Real-time PCR结果显示在肺癌组织中的表达水平(4.60±0.47)显著高于癌旁组织(1.02±0.55)(P<0.05)；HMGN5 siRNA重组慢病毒载体构建成功；Real-time PCR和Western blot结果显示,实验设计的siRNA靶点序列在人肺癌细胞A549和H1299细胞内沉默表达HMGN5的沉默效率达到80%：重组的HMGN5 siRNA慢病毒载体作用于人肺癌细胞A549、H1299细胞后,HMGN5基因的mRNA及蛋白表达水平明显降低,MTT、Brud、细胞克隆形成实验结果表明转染HMGN5-siRNA的A549、H1299细胞增殖能力、DNA复制能力、克隆形成能力明显降低,流式细胞术实验结果表明转染HMGN5-siRNA的A549、H1299细胞阻滞于Go/G1期,使细胞分裂减缓；成功建立A549裸鼠皮下移植瘤模型,体内观察实验结果表明注射HMGN5-siRNA小鼠的转移瘤质量及体积较对照组明显降低,裸鼠体内成瘤能力明显受到抑制。
结论：HMGN5在肺癌组织中高表达；成功构建了HMGN5 siRNA干扰质粒,能够抑制HMGN5基因的nnRNA和蛋白表达；HMGN5 siRNA慢病毒表达载体能够抑制肺癌细胞增殖、克隆形成能力,导致细胞阻滞于G0/G1期,使细胞分裂减缓,提示HMGN5基因参与了肺癌发展过程,HMGN5可以作为肺癌基因治疗的新靶点,为治疗肺癌提供新的思路和理论研究基础。
The incidence and mortality rates of lung cancer are extremely high in the world. The highest cancer mortality rate is one of the top ten malignant tumors, lung cancer's 5-year overall survival rate is 13% to 15%. The incidence of lung cancer is still rising incidence of lung cancer, but the treatment effect of lung cancer is still not ideal in recent years, therefore the research of lung cancer is focused on the new chemotherapy drugs, molecular targeted drugs, the application of drug therapy and probing into the pathogenesis of lung cancer in order to find potential biological marker for the personalized treatment of lung cancer. Multiple genes and multiple steps are involved in the occurrence and the developed process of lung cancer. So the process by studying the abnormal expression of cancer genes, finding a diagnostic and therapeutic gene has a great clinical significance.
As a sequence-specific post-transcriptional gene silencing, RNAi is a means which is induced 21-23 double-stranded RNA composed of sense and antisense RNA corresponding mRNA, so that mRNA of the specific genes in cells are degradated, leading to gene silencing. RNAi is a reverse genetics technology, which plays an important role in the study of functional genes in the post-genomic times. Aiming at genetic research in the "knockout" technology, RNAi technology for "ticking down" the target gene or target protein, has been widely used in signal transduction、gene function and research in cancer gene therapy,so dose in mammalian cells. In view of the RNAi technology is of great efficiency and simplicity in inhibition of target gene expression. RNAi becomes an effective means of gene function research. RNAi can selectively inhibit the expression of genes related to human disease in the study of gene function, so it has a broad prospect in the study of gene function and the treatment of human diseases. Researchers in-country and abroad have successfully used RNAi technology to selectively inhibit or down-regulate the expression of lung cancer related genes which play important roles in the development of lung cancer, such as oncogenes、anti-oncogenes and genes related to apoptosis, in order to achieve gene therapy purposes.
HMGN5 is a member of the HMGN family,it is highly homologous with the NSBP1. King has discovered HMGN5 in 2001. Human gene HMGN5 is 8600bp long and it has 5 introns and 6 exons, and its complete cDNA sequence is 1865bp long. HMGN5 encodes a kind of human nuclear protein-nucleosome-binding protein that includes 282 amino acids, and its molecular weight is 31000 bp in theory. HMGN5 expression length is different between human and mice because man's HGMN5 that consists of 6 exons which is shorter.
Although the molecular mechanism HMGN5 is not clear, but the literature reports that HMGN5 has potential relevance in disease and normal cell function. For example, over-expression experiments and gene chip analysis showed that HMGN5 can serve as a factor inducing differentiation of mouse embryonic stem cells. HMGN5 expresses highly in human squamous cancer、prostate cancer、adenocarcinoma of aging mice、highly metastatic breast cancer MDA-MB-435HM cells, so that the experimental results show that the HMGN5 is associated with the tumor. It is endogenous disorder that HMGN5 protein expresses increasedly in tumor tissue. In addition, HMGN5 in X chromosome, containing a disproportionately large number of genes may be related to mental function, as well as behavior and cognitive function in late evolutionary stage.
By optimizing the reaction of chromatin, HMGN5 constantly changes the cell phenotype of environment. Highly acidic terminal and abnormal long terminal influence the interaction between connection chromosomes and histone, so that HMGN5 can more effectively expand the highly ordered structure of chromosomes. Direct competition between connection histone and HMGN5 provides a new mechanism model for regulationing dynamic structure of chromosome. Clarifing how HMGN5 influences chromosome structure and the specific modes of this competition mechanism is needed to be studied in depth. HMGN5 plays an important role in maintaining the integrity of chromatin fibers within cells, but the abnormal expression of HMGN5 is harmful to the cells. HMGN5 and the tumor have a potential relationship. It has been reported that HMGN5's expression level in bladder cancer、prostate cancer and other tumor tissues were significantly higher than normal tissue, but studies in lung cancer has not yet been clearly reported. So further research is how HMGN5 gene affects lung cancer occurrence and development, and we can seek a breakthrough in clinical treatment of lung cancer by that way.
Objective:To investigate HMGN5 gene expression level in lung cancer tissue, and the connection with the clinical and pathological features; to build siRNA of HMGN5 in order to recombine lentiviral vectors; The influence on RNA interference silence HMGN5 gene in lung cancer cells proliferation、cell cycle and for ability of lung tumor. RNAi silencing HMGN5 genes in lung cacer cells A549 and H1299, and inhibition of HMGN5 gene working on tumor formation in nude mice, provide a theoretical basis of gene targeting therapy.
Methods:To detect HMGN5 mRNA expression in lung cancer and besides the cancer tissues by Real-time PCR method, and analyse the relativity between the expression of HMGN5 and clinicopathological features of lung cancer; Design targeted HMGN5 gene siRNA interfereNce sequence; Build synthetic vectors with designed sequences; Choose positive and correct-sequencing plasmid to package virus; Let packaged virus infect human lung cancer cells A549、H1299, and verify silencing efficiency of human lung cancer cells A549、H1299 by Real-time PCR and Western blot methods; To inhibit HMGN5 gene expression in lung cancer A549 and H1299 cells by RNAi technology, at the same time, HMGN5 siRNA recombine lentiviruses to infect human lung cancer cells, through that we can draw a conclusion what HMGN5 changes of the proliferation ability of human lung cancer cell, after detecting the silencing expression of HMGN5 by using MTT; Detect the silencing HMGN5 expression and how human lung cancer cell DNA synthesis rate changes using Brdu; After application of colony formation method validating HMGN5 silence, the change of human lung cancer cell colony formation ability was detected; Using flow cytometry test cell cycles change in order to explore HMGN5's biological functions in lung cancer cells; Tumor model with human lung cancer A549 cells subcutaneous was built in nude mice, by observing the size and weight of the tumor in order to observe the influence on lung cancer in vivo formation ability by HMGN5 gene silencing.
Results:Real-time PCR results showed that expression levels in the lung cancer tissue (4.60±0.47) was significantly higher than in adjacent tissue (1.02±0.55) (P<0.05); HMGN5 siRNA built lentiviral vector was successfully constructed. Real-time PCR and Western blot results showed that the experimental designed siRNA target sequence express silently in human lung cancer cells A549 and H1299 cells, and the silence efficiency of HMGN5 is 80%; Recombined HMGN5 siRNA lentiviral vectors have effected on human lung cancer cells A549 and H1299 cells, mRNA and protein of HMGN5 gene expression levels were significantly decreased; MTT、Brud and cell clone experimental results showed that the ability of cell proliferation, DNA replication and colony formation of transfected HMGN5-siRNA A549 and H1299 cell was significantly lower than the control group; Flow cytometry results showed that A549 and H1299 which transfected HMGN5-siRNA were arrested in G0/G1 phase, so that cell division was slow down; successfully established subcutaneous A549 nude mouse model in vivo experiment results showed that mice injected with HMGN5-siRNA's transfered tumor weight was significantly lower than the control group. Tumor formation ability of lung cancer cells in vivo was significantly inhibited.
Conclusions:HMGN5 was expressed highly in lung cancer tissue; HMGN5 siRNA interference plasmids were successfully built, and it can inhibit mRNA and protein expression of HMGN5 gene; HMGN5 siRNA lentiviral vectors can inhibit the ability of cancer cell proliferation and colony formation, leading to cell arresed in G0/G1 phase in order to slow down cell division. So it can draw a conclusion that lung cancer HMGN5 genes may have relationship with development of lung cancer. HMGN5 can be used as a new target of cancer gene therapy, which provides new ideas and theoretical research base for treatment of lung cancer.
引文
[1]肖湘文,周建林,周畅.高迁移率族蛋白[J].细胞生物学杂志,2006,28：501-506.
[2]Goodwin GH,Sanders C,Johns EW. A new group of chromatin-associated proteins with a high content of acidic and basic amino acids[J]. Eur J Biochem,1973,38(1):14-19.
[3]Baxevanis A D,Landsman D. The HMG-1 box protein family:classification and functional relationships[J]. Nucleic acids research,1995,23(9):1604-1613.
[4]Bustin M. Revised nomenclature for high mobility group(HMG) chromosomal proteins [J]. Trends Biochem Sci,2001,26(3):152-153.
[5]徐佳,刘志锋,姜勇.高迁移率族蛋白与真核基因表达调控[J].生物化学与生物物理进展,2005,32(5)：397-401.
[6]Reeves R. Molecular biology of HMGA proteins:hubs of nuclear function[J]. Gene,2001,277:63-81.
[7]Huth JR, Bewley CA, Nissen MS,et al. The solution structure of an HMG-I(Y)-DNA complex defines a new architectural minor groove binding motif [J]. Nat Struct Biol.1997,4(8):657-665.
[8]American Society for Microbiology [J]. Mol. Cell. Biol.,Jul 1995,15(7):3738-3747.
[9]Clio Salvagno, Giuseppina Caretti, Carol Imbriano,et al.Transcriptional Activation of the Cyclin A Gene by the Architectural Transcription Factor HMGA2[J]. Molecular and Cellular Biology,2003,23(24):9104-9116.
[10]Sgarra R, Rustighi A, Tessari MA, et al. Nuclear phosphoproteins HMGA and their relationship with chromatin structure and cancer[J]. FEBS Lett 2004,574:1-8.
[11]Abe N, Watanabe T, Sugiyama M, et al. Determination of high mobility group I(Y) expression level in colorectal neoplasias:a potential diagnostic marker[J]. Cancer Res.1999, 59:1169-1174.
[12]Nobutsugu Abel, Takashi Watanabe, Tadahiko Masaki, et al. Pancreatic Duct Cell Carcinomas Express High Levels of High Mobility Group I(Y) Proteins[J]. Cancer Res June 2000,60:3117.
[13]Melvin VS, Roemer SC, Churchill ME, et al. The C-terminal extension (CTE) of the nuclear hormone receptor DNA binding domain determines interactions and functional response to the HMGB-1/-2 co-regulatory proteins[J]. Biol.Chem.,2002,277:25115-25124.
[14]姚咏明,刘辉.对高迁移率族蛋白B1作用的新认识[J].中国危重病急救医 学,2005,17(7)：385-387.
[15]BRITTA MEYER, HUGO MURUA ESCOBAR, SVEN HAUKE, et al.Expression Pattern of the HMGB1 Gene in Sarcomas of the Dog[J]. Anticancer Research,2004,24: 707-710.
[16]王松柏,姚咏明.高迁移率族蛋白B1的细胞生物学效应及其与脓毒症的关系[J].中国危重病急救医学,2003,15(11)：701-704.
[17]Bustin M. Chromatin unfolding and activation by HMGN(3) chromosomal proteins[J]. Trends Biochem Sci,2001,26(7):431-437.
[18]Tiziana Bonaldi, Fabio Talamo, Paola Scaffidi, et al.Monocytic cells hyperac etylate chromatin protein HMGB1 to redirect it towards secretion[J]. EMBO,2003,2 2(20):5551-5560.
[19]F. Catez, D.T. Brown, T. Misteli, et al, Competition between histone H1 and HMGN proteins for chromatin binding sites[J]. EMBO Rep.,2002 (3):760-766.
[20]Adkins N L, Watts M, Georgel PT. To the 30-nm chromatin fiber and beyond[J].Biochim. Biophys. Acta,2004,1677(1-3):12-23.
[21]West KL. HMGN proteins play roles in DNA repair and gene expression in mammalian cells [J]. Biochem. Soc. Trans.,2004,32:918-919.
[22]TREMET HICK D J, HYMAN L. High mobility group protein 14 and 17 can prevent the closed packing of nucleosomes by increasing the strength of protein contacts in the linker DNA [J]. J. Biol. Chem.,1996,271(20):12009-12016.
[23]VESTNER B, BUSTIN M, GRUSS C. Stimulation of replication efficiency of a chromatin template by chromosomal protein HMG-17[J]. J. Biol. Chem.,1998,273(16): 9409-9414.
[24]HOCK R, SCHEER U, BUSTIN M. Chromosomal proteins HMG-14 and HMG-17 are released from mitotic chromosomes and imported into the nucleus by active transport [J]. J. Cell Biol.,1998,143(6):1427-1436.
[25]SPIEKER N, BEITSMA M, VANSLUIS P, et al. An integrated 5Mb physical, genetic, and radiation hybricmap of alp36.1 region implicated in neuroblastoma pathogenesis [J]. Genes Chromosomes Cancer,2000,27(2):143-152.
[26]BUSTIN M, REISCH J, EINCK L, et al. Autoantibodies to nucleosomal proteins: antibodies to HMG-17 in autoimmune diseases [J]. Science,1982,215:1245-1247.
[27]Tzioufas AG, Boumba VA, Seferiadis K, et al. Autoantibodies to HMG-17 n ucleosomal protein inpatients with scleroderma[J]. Arthritis Rheum.1993,36(7):955-961.
[28]马懿,冯云,李恒HMGN2的重组表达质粒的构建及其抗菌功能研究[J].四川生理科学杂志,2005,27(4)：147-149.
[29]李薇,张平,孔祥丽,等.人淋巴结中HMGN2蛋白的分离纯化及其亚细胞分布研究[J].生物医学工程学杂志,2010,27(4)：842-846.
[30]FENG Y, HUANG N, WU Q, et al. The helic-domain is essential for antibacterial activity of high mobility group nucleosomal binding domain 2 (HMGN2)[J]. Acta Pharmacol. Sinica.,2005,26(9):1087-1092.
[31]YOUNG ARJ, NARITA M. Oncogenic HMGA2:short or small[J]. Genes Dev, 2007,21(9):1005-1009.
[32]King LM, Francomano CA. Characterization of a human gene encoding nucleosomal binding protein NSBP1 [J]. Genomics,2001,71 (2):163-173.
[33]杨学贞,周利群,周健,等.mRNA差异显示法克隆前列腺癌相关基因[J].中华泌尿外科杂志,2003,24(8)：545-547.
[34]Mark Rochman, Cedric Malicet, Michael Bustin. HMGN5/NSBP1:A new member of the HMGN protein family that affects chromatin structure and function[J]. Biochimica et Biophysica Acta,2010,1799:86-92.
[35]M. Bustin.Regulation of DNA-dependent activities by the functional motifs of the high-mobility-group chromosomal proteins [J]. Mol. Cell. Biol.,1999(19):5237-5246.
[36]T. Ueda, F. Catez, G. Gerlitz, et al Delineation of the protein module that anchors HMGN proteins to nucleosomes in the chromatin of living cells [J]. Mol. Cell Biol., 2008(28):2872-2883.
[37]Y.V. Postnikov, D.A. Lehn, R.C. Robinson, et al. The cooperative binding of chromosomal protein HMG-14 to nucleosome cores is reduced by single point mutations in the nucleosomal binding domain [J]. Nucleic Acids Res.,1994(22):4520-4526.
[38]H. Shirakawa, D. Landsman, Y.V. Postnikov, et al, NBP-45, a novel nucleosomal binding protein with a tissue-specific and developmentally regulated expression [J]. J. Biol. Chem.,2000(275):6368-6374.
[39]M. Prymakowska-Bosak, T. Misteli, J.E. Herrera, et al. Mitotic phosphorylation prevents the binding of HMGN proteins to chromatin [J]. Mol. Cell Biol.,2001(21): 5169-5178.
[40]Y.V Postnikov, J.E. Herrera, R. Hock, et al. Clusters of nucleosomes containing chromosomal protein HMG-17 in chromatin [J]. Mol. Biol.,1997(274):454-465.
[41]S. Cherukuri, R. Hock, T. Ueda, et al. Cell cycle dependent binding of HMGN proteins to chromatin [J]. Mol. Biol. Cell,2008(19):1816-1824.
[42]M. Rochman, Y. Postnikov, S. Correll, et al. The interaction of NSBP1/HMGN5 with nucleosomes in euchromatin counteracts linker histone-mediated chromatin compaction and modulates transcription [J]. Mol. Cell,2009(35):642-656.
[43]J.J. Ward, J.S. Sodhi, L.J. McGuffin, et al. Prediction and functional analysis of native disorder in proteins from the three kingdoms of life[J]. Mol. Biol.,2004(337): 635-645.
[44]H.J. Dyson, P.E. Wright. Intrinsically unstructured proteins and their functions [J]. Nat. Rev. Mol. Cell. Biol.,2005(6):197-208.
[45]J.C. Hansen, X. Lu, E.D. Ross, et al. Intrinsic protein disorder, amino acid composition, and histone terminal domains [J]. Biol. Chem.,2006,281:1853-1856.
[46]T. Vavouri, J.I. Semple, R. Garcia-Verdugo, et al. Intrinsic protein disorder and interaction promiscuity are widely associated with dosage sensitivity [J].Cell,2009,138: 198-208.
[47]T. Furusawa, J.H. Lim, F. Catez, et al. Down-regulation of nucleosomal binding protein HMGN1 expression during embryogenesis modulates Sox9 expression in chondrocytes [J]. Mol. Cell. Biol.,2006,26:592-604.
[48]J.M. Pash, P.J. Alfonso, M. Bustin. Aberrant expression of high mobility group chromosomal protein 14 affects cellular differentiation[J]. Biol.Chem.,1993,268: 13632-13638.
[49]Y. Birger, F. Catez, T. Furusawa, et al. Increased tumorigenicity and sensitivity to ionizing radiation upon loss of chromosomal protein HMGN1[J]. Cancer Res.,2005,65: 6711-6718.
[50]H. Shirakawa, M. Rochman, T. Furusawa, et al. The nucleosomal binding protein NSBP1 is highly expressed in the placenta and modulates the expression of differentiation markers in placental Rcho-1 cells [J]. Cell Biochem.,2009,106:651-658.
[51]R.D. Phair, P. Scaffidi, C. Elbi, et al. Global nature of dynamic protein-chromatin interactions in vivo:three-dimensional genome scanning and dynamic interaction networks of chromatin proteins [J]. Mol. Cell. Biol.,2004,24:6393-6402.
[52]J.H. Lim, M. Bustin, V.V. Ogryzko, et al. Metastable macromolecular complexes containing high mobility group nucleosome-binding chromosomal proteins in HeLa nuclei [J]. Biol. Chem.,2002,277:20774-20782.
[53]L. Trieschmann, P.J. Alfonso, M.P. Crippa, et al. Incorporation of chromosomal proteins HMG-14/HMG-17 into nascent nucleosomes induces an extended chromatin conformation and enhances the utilization of active transcription complexes [J]. EMBO, 1995(14):1478-1489.
[54]Y.C. Kim, G Gerlitz, T. Furusawa, et al. Activation of ATM depends on chromatin interactions occurring before induction of DNA damage [J]. Nat. Cell Biol.,2009,11:92-96.
[55]L. Cato, K.Stott, M. Watson, et al. The interaction of HMGB1 and linker histones occurs through their acidic and basic tails [J]. Mol. Biol.,2008,384:1262-1272.
[56]Mark Rochman,Yuri Postnikov, Sarah Correll, et al. The Interaction of NSBP1/HMGN5 with Nucleosomes in Euchromatin Counteracts Linker Histone-Mediated Chromatin Compaction and Modulates Transcription [J]. Molecular Cell,2009,7(2): 642-656.
[57]T. Ueda, T. Furusawa, et al. The nucleosome binding protein HMGN3 modulates the transcription profile of pancreatic(beta) cells and affects insulin secretion [J]. Mol. Cell. Biol.,2009,29:5264-5276.
[58]R. Hock, T. Furusawa, T. Ueda, et al. HMG chromosomal proteins in development and disease [J]. Trends Cell. Biol.,2007,17:72-79.
[59]M. Pritsker, N.R. Ford, H.T. Jenq, et al. Genomewide gain-of-function genetic screen identifies functionally active genes in mouse embryonic stem cells [J]. Proc. Natl. Acad. Sci.,2006,103:6946-6951.
[60]D.Q. Li, Y.F. Hou, J. Wu, et al. Gene expression profile analysis of an isogenic tumour metastasis model reveals a functional role for oncogene AF1Q in breast cancer metastasis [J]. Eur. Cancer,2006,42:3274-3286.
[61]Lee BC, Cha K, Avraham S, et al. Microarray analysis of differentially expressed genes associated with human ovarian cancer [J]. Int. J. Oncol.,2004,24(4):847-851.
[62]Koon N, Schneider-Stock R, Sarlomo-Rikala M, et al. Molecular targets for tumour progression in gastrointestinal stromal tumours [J]. Gut.,2004,53(2):235-240.
[63]Ueda R, Iizuka Y, Yoshida K, et al. Identification of a human glioma antigen, SOX6, recognized by patients's era [J]. Oncogene,2004,23(7):1420-1427.
[64]P.P. Khil, R.D. Camerini-Otero. Molecular features and functional constraints in the evolution of the mammalian X chromosome [J]. Crit. Rev. Biochem. Mol. Biol., 2005,40:313-330.
[65]D. Nolte, J. Ramser, S. Niemann, et al. ACRC codes for a novel nuclear protein with unusual acidic repeat tract and maps to DYT3(dystonia parkinsonism) critical interval in xq 13.1 [J]. Neurogenetics,2001 (3):207-213.
[66]R. Gasa, C. Mrejen, N. Leachman, et al. Proendocrine genes coordinate the pancreatic is let differentiation program in vitro[J]. Proc.Natl.Acad.Sci.U.S.A.,2004,101: 13245-13250.
[67]H.E. MacLean, W.S. Chiu, A.J. Notini, et al. Impaired skeletal muscle development and function in male, but not female, genomic androgen receptor knockout mice [J]. FASEB J.,2008,22:2676-2689.
[68]C. Goritz, R. Thiebaut, L.H. Tessier, et al. Glia-induced neuronal differentiation by transcriptional regulation [J]. Glia,2007,55:1108-1122.
[69]Q. Lu, N. Shen, X.M. Li, et al. Genomic view of IFN-alpha response in preautoimmune NZB/W and MRL/lpr mice [J]. Genes Immun.,2007,8:590-603.
[70]Y. Chen, H. Ni, X.H. Ma, et al. Global analysis of differential luminal epithelial gene expression at mouse implantation sites[J]. Mol. Endocrinol.,2006,37:147-161.
[71]P.A.Efron, H.Tsujimoto, F.R. Bahjat,et al. Differential maturation of murine bone marrow derived dendritic cells with lipopolysaccharide and tumor necrosis factor-alpha[J]. Endotoxin.Res.,2005,11:145-160.
[72]W.Y. Tang, R. Newbold, K. Mardilovich, et al. Persistent hypomethylation in the promoter of nucleosomal binding protein 1 (Nsbp1) correlates with overexpression of Nsbp1 in mouse uterineonatally exposed to diethylstilbestrol or genistein [J]. Endocrinology,2008, 149:5922-5931.
[73]S.W. Jee, J.S. Cho, C.K. Kim, et al. Oligonucleotide-based analysis of differentially expressed genes in hippocampus of transgenic mice expressing NSE-controlled APPsw[J]. Neurochem. Res.,2006,31:1035-1044.
[74]M. Pritsker, N.R. Ford, H.T. Jenq, et al. Genomewide gain-of-function genetic screen identifies functionally active genes in mouse embryonic stem cells [J]. Proc. Natl. Acad. Sci..,2006,103:6946-6951.
[75]W.G. Beamer, K.L. Shultz, B.J. Tennent, et al. Multigenic and imprinting control of ovarian granulosa cell tumorigenesis in mice [J]. Cancer Res.,1998,58:3694-3699.
[76]H.J. Han, J. Russo,Y. Kohwi, et al. SATB1 reprogrammes gene expression to promote breast tumour growth and metastasis[J]. Nature,2008,452:187-193.
[77]J. Green, M. Ikram, J. Vyas, et al. Overexpression of the Axl tyrosine kinase receptor in cutaneous SCC-derived cell lines and tumours [J]. Cancer,2006,94:1446-1451.
[78]D.Q. Li, Y.F. Hou, J. Wu, et al. Gene expression profile analysis of an isogenic tumour metastasis model reveals a functional role for oncogene AF1Q in breast cancer metastasis[J]. Cancer,2006,42:3274-3286.
[79]F. Rizzolio, S. Bione, C. Sala, et al. Chromosomal rearrangements in Xq and premature ovarian failure:mapping of 25 new cases and review of the literature [J]. Hum. Reprod.,2006,21:1477-1483.
[80]C.Huang,L.Q.Zhou,GSong.Effect of nucleosomal binding protein 1 in androgen-independent prostatic carcinoma[J]. Zhonghua Yi Xue Za Zhi,2008,88:657-660.
[81]L.Q. Zhou, G Song, Z.S. He, et al. Effects of inhibiting nucleosomal binding proteinl on proliferation of human prostate cancer cells[J]. Zhonghua Yi Xue Za Zhi,2007,87:404-408.
[82]王建伟,周利群,杨学贞.前列腺癌细胞中NSBP1基因表达上调[J].基础医学与临床,2004,24(4)：393-397.
[83]宋刚,周利群,翁迈,等.核小体结合蛋白1在前列腺癌中的表达和意义[J].中华医学杂志,2006,86(28)：1962-1965.
[84]姚鲲,周利群,李学松.核小体结合蛋白1在膀胱癌中的表达和意义[J].中华临床医师杂志(电子版),2010,4(1)：50-54.
[85]Agresti A, Bianchi ME. HMGB proteins and gene expression [J]. Curr. Opin. Genet Dev.,2003,13(2):170-178.
[86]Trieschmann L, Alfonso PJ, Crippa MP, et al. Incorporation of chromosomal proteins HMG-14/HMG-17 into nascent nucleosomes induces an extended chromatin conformation and enhances the utilization of active transcrip tion comp lexes [J]. EMBO J., 1995,14(7):1478-1489.
[87]Aiziwa S, Nishino H, Saito K, et al. Stimulation of transcription in cultured cells by high mobility group proteinl:essential role of the acidic carboxyl-terminal region [J]. Biochem.,1994,33(49):14690-14695.
[88]Muller S, Scaffidi P, Degryse B, et al. New EMBO members'review:the double life of HMGB1 chromatin protein:architectural factor and extracellular signal [J]. EMBO J., 2001,20(16):4337-4340.
[89]Tang WY, Newbold R, Mardilovich K, et al. Persistent hypomethylation in the promoter of nucleosomal binding protein 1 (Nsbp1) correlates with overexpression of Nsbp1 in mouse uteri neonatally exposed to diethylstilbestrol or genistein [J]. Endocrinology,2008, 149(12:5922-5931.
[90]周利群,宋刚,何志嵩,等.抑制核小体结合蛋白1基因对人前列腺癌细胞LNCaP增殖的作用[J].中华医学杂志,2007,87(6)：404-408.
[91]蒋宁,周利群,姚鲲.人核小体结合蛋白1小分子干扰RNA重组慢病毒抑制非激素依赖性前列腺癌体内生长的实验研究[J].中华临床医师杂志(电子版),2010,2(4)：154-157.
[92]葛棣.肺癌基因治疗研究进展[J].中国航天工业医药.2001,6(3)：70-74
[93]陈湘琦,李志鹰,林挺岩.RNA干扰技术在肺癌基因治疗中的应用研究现状[J].中华临床医师杂志(电子版),2010,7(4)：1028-1031.
[94]Hansen HH. Treatment of advanced non-small cell lung cancer [J]. Annals of Oncology,2002,325:452-453.
[95]字应伟.肿瘤基因治疗研究进展[J].思茅师范高等专科学校校报,2005,21(3)：6-8.
[96]张利群,陈杭薇.肺癌基因治疗的动态[J].中华结核和呼吸杂志2004,27(6)：430-434.
[97]PoulsenTT, Pedersen N, Poulsen HS. Roplacenent and suicide gene therapy fox targeted treatment of lung cancer[J]. Clin Lung cancer,2005,6(4):227-223.
[98]Guo S, Kemphues K J.,A gene required for establishing polarity in C.elegans embryos, encodes a putative ser/Thr kinase that is asymmetrically distributed[J]. Cell,1995, 81:611-620.
[99]FireA,Xu S,Montgomery MK,et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J]. Natrue,1998,391:806-811.
[100]Hammond GJ.RNAi interference[J]. Nature,2002,418(6894):244-251.
[101]Pomerantz RJ.RNA interfence meets HIV-1:will silence be golden[J], Nat Med, 2002,8(7):659-660.
[102]Garzon R,Pichiorri F,Palumbo T,et al. Micro RNA fingerprints during human egakaryocytopoiesis[J]. Proc Natl Acad Sci USA,2006,103(13):5078-5083.
[103]Elbashir SM,Harborth J,Lendeckel W,et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells[J]. Nature,2001,411(6836):494-498.
[104]杨家驹,段振铃.肿瘤的基因治疗进展[J].医学综述,2005,11(10)：883-885.
[105]苗彦,陈愉生.RNA干扰在肺癌基因治疗中的应用进展[J].国际呼吸杂志,2010,30(10)：619-625.
[106]刘苏健.肺癌基因治疗的研究进展[J].山东医药,2008,48(19)：51-152.
[107]何祥梁,何东华.肺癌基因治疗研究进展[J].中国病理生理杂志,2002,18(11)：1447-1450.
[108]张鼎,陈小东.肺癌治疗的分子靶位及其相关药物的研究进展[J].国际呼吸杂志,2007,27(10)：750-753.
[109]陈湘琦,李志鹰,林挺岩.RNA干扰技术在肺癌基因治疗中的应用研究现状[J].中华临床医师杂志(电子版),2010,4(7)：1028-1031.
[110]任新玲,钱桂生,付海京,等.HER2 siRNA抑制肺癌细胞增值的实验研究[J].中华肺部疾病杂志(电子版),2007,1(1)：16-18.
[111]Yin J Q,Gao J,Shao R,et al.siRNA agents inhibition oncogene expression and attenuate human tumor cell growth[J]. Exp Ther Oncol,2003,3(4):194-204.
[112]Jiang M,Milner J.Bcl-2 constitutively suppresses p53-dependent apoptosis in colorectal cance cells[J]. Genes Dev,2003,17(7):832-837.
[113]张泳,谷仲平,周勇安,等.RNAi沉默肺癌细胞中VEGF的表达治疗肺癌机制的初步研究[J].第四军医大学学报,2009,30(19)：1490-1493.
[114]张泳,谷仲平,周勇安,等.RNAi沉默VEGF的表达以及治疗肺癌的初步研究[J].细胞与分子免疫学杂志.2009,25(4)：341-343.
[115]Krysan K,Dalwadi H,Sharma S,et al. Cyclooxygenase 2-dependent expression of surviving is critical for apoptosis resistance in non-small cell lung cancer [J]. Cancer Res,2004,64(18):6359-6362.
[116]Chao J I,Liu HE The blockage of surviving and securing expression increases the cytochalasin B-induced cell death and growth inhibition in human cancer cells[J]. Mol Pharmacol,2006,69(1):154-164.
[117]马丹,张珍祥,徐永健.CD44v3的表达对肺腺癌A549细胞侵袭行为的影响[J].中国现代医学杂志,2006,16(22)：3388-3392.
[118]钟锡明,范钰,张瑞佳.RNAi沉默PRL-1基因对肺癌细胞侵袭能力的影响[J].江苏大学学报(医学版),2008,18(6)：480-483.
[119]MacDonald NJ, Shivers WY, Narum DL, et al. Endostalnn binds fropomysin. A potential modulator of the antitumor activity of endostatin[J]. Biol Chem,2001,276(27): 25190-25196.
[120]Chandramu Chetty, Sajani S. Lakka, Praveen Bhoopathi, et al.MMP-2 alters VEGF expression via aVb3 integrin-mediated PI3K/AKT signaling in A549 lung cancer cells[J]. International Journal of Cancer,2010,127:1081-1095.
[121]Lu B,Mu Y,Cao C, et al. Survivin as a therapeutic target for rasiation sensitization in lung cancer[J].Cancer Res,2004,64(8):2840-2845.
[122]Pai S I,Lin Y Y,Macaes B, et al. Prospects of RNA interference therapy for cancer[J]. Gene Ther,2006,13(6):464-477.
[123]Song X,Liu X,Chi W, et al. Hypoxia-induced resistance to cisplatin and doxorubicin innon-small cell lung cancer is inhibited by silencing of HIF-alpha gene[J].Cancer Chemother Pharmacol,2006,58(6):776-784.
[124]Lee MW,Kim DS,Min NY, et al. Akt1 inhibition by RNA interference sensitizes humannon-small cell lung cancer cells to cisplatin[J]. Cancer,2008,122(10):2380-2384.
[125]Yonesaka K,Tamura K,Kurata T, et al. Small interfering RNA targeting surviving sensitizes lung cancer cell with mutant p53 to adriamycin[J]. Cancer,2006,118 (4):812-820.
[126]任振义,金儿,叶健.Ku80 RNAi技术增强肺癌放射敏感性的动物实验研究[J].肿瘤学杂志,2010,16(8)：614-617.
[127]Fukuoka M, Yano S, Giaccone G, et al. Multi_institutional randomized phase II trial of gefitinib for previously treated patients with advanced non-small-cell lung cancer[J]. Clin Oncol,2003,21:2237-2246.
[128]Franklin MC, Carey KD, Vajdos FF, et al. Insights into ErbB signaling from the structure of the ErbB2-pertuzurnab complex[J]. Cancer Cell,2004,5(4):317-28.
[129]Jin Z, Gao F, Flagg T, et al. Tobacco specific nit rosamine 4 (methylni trosamino)1(3-pyridyl)-1-butanone promot es functional cooperation of Bcl-2 and c-Myc through phosphorylat ion in regulating cell survival and proliferation [J].Biol Chem,2004, 279(38):40209-40219.
[130]任淑华,张伟,曲平,等.RNA干扰C-erbB-2对肺腺癌细胞calu-3增殖的影响[J].癌症,2005,24(1)：1173-1178.
[131]Poulsen TT,Pedersen N,Poulsen HS.Replacement and suicide gene therapy fortargeted treatment of lung cancer[J].Clin Lung Cancer,2005,6(4):227-236.
[132]Tkeshita F, Ochiya T.TheraPeutic potential of RNA interference againstcancer. Cancer Sei,2006,97(8):689-696.
[133]Hao DL, Liu CM, Dong WJ, etal.Knockdown of human p53 gene expression in 293-T cells by retroviral vector-mediated short hairp in RNA[J].Acta Biochim BioPhy Sin(Shanghai),2005,37(11):779-783.
[2]Goodwin GH,Sanders C,Johns EW. A new group of chromatin-associated proteins with a high content of acidic and basic amino acids[J]. Eur J Biochem,1973,38(1):14-19.
[3]Baxevanis A D,Landsman D. The HMG-1 box protein family:classification and functional relationships[J]. Nucleic acids research,1995,23(9):1604-1613.
[4]Bustin M. Revised nomenclature for high mobility group(HMG) chromosomal proteins [J]. Trends Biochem Sci,2001,26(3):152-153.
[5]徐佳,刘志锋,姜勇.高迁移率族蛋白与真核基因表达调控[J].生物化学与生物物理进展,2005,32(5)：397-401.
[6]Reeves R. Molecular biology of HMGA proteins:hubs of nuclear function[J]. Gene,2001,277:63-81.
[7]Huth JR, Bewley CA, Nissen MS,et al. The solution structure of an HMG-I(Y)-DNA complex defines a new architectural minor groove binding motif [J]. Nat Struct Biol.1997,4(8):657-665.
[8]American Society for Microbiology [J]. Mol. Cell. Biol.,Jul 1995,15(7):3738-3747.
[9]Clio Salvagno, Giuseppina Caretti, Carol Imbriano,et al.Transcriptional Activation of the Cyclin A Gene by the Architectural Transcription Factor HMGA2[J]. Molecular and Cellular Biology,2003,23(24):9104-9116.
[10]Sgarra R, Rustighi A, Tessari MA, et al. Nuclear phosphoproteins HMGA and their relationship with chromatin structure and cancer[J]. FEBS Lett 2004,574:1-8.
[11]Abe N, Watanabe T, Sugiyama M, et al. Determination of high mobility group I(Y) expression level in colorectal neoplasias:a potential diagnostic marker[J]. Cancer Res.1999, 59:1169-1174.
[12]Nobutsugu Abel, Takashi Watanabe, Tadahiko Masaki, et al. Pancreatic Duct Cell Carcinomas Express High Levels of High Mobility Group I(Y) Proteins[J]. Cancer Res June 2000,60:3117.
[13]Melvin VS, Roemer SC, Churchill ME, et al. The C-terminal extension (CTE) of the nuclear hormone receptor DNA binding domain determines interactions and functional response to the HMGB-1/-2 co-regulatory proteins[J]. Biol.Chem.,2002,277:25115-25124.
[14]姚咏明,刘辉.对高迁移率族蛋白B1作用的新认识[J].中国危重病急救医 学,2005,17(7)：385-387.
[15]BRITTA MEYER, HUGO MURUA ESCOBAR, SVEN HAUKE, et al.Expression Pattern of the HMGB1 Gene in Sarcomas of the Dog[J]. Anticancer Research,2004,24: 707-710.
[16]王松柏,姚咏明.高迁移率族蛋白B1的细胞生物学效应及其与脓毒症的关系[J].中国危重病急救医学,2003,15(11)：701-704.
[17]Bustin M. Chromatin unfolding and activation by HMGN(3) chromosomal proteins[J]. Trends Biochem Sci,2001,26(7):431-437.
[18]Tiziana Bonaldi, Fabio Talamo, Paola Scaffidi, et al.Monocytic cells hyperac etylate chromatin protein HMGB1 to redirect it towards secretion[J]. EMBO,2003,2 2(20):5551-5560.
[19]F. Catez, D.T. Brown, T. Misteli, et al, Competition between histone H1 and HMGN proteins for chromatin binding sites[J]. EMBO Rep.,2002 (3):760-766.
[20]Adkins N L, Watts M, Georgel PT. To the 30-nm chromatin fiber and beyond[J].Biochim. Biophys. Acta,2004,1677(1-3):12-23.
[21]West KL. HMGN proteins play roles in DNA repair and gene expression in mammalian cells [J]. Biochem. Soc. Trans.,2004,32:918-919.
[22]TREMET HICK D J, HYMAN L. High mobility group protein 14 and 17 can prevent the closed packing of nucleosomes by increasing the strength of protein contacts in the linker DNA [J]. J. Biol. Chem.,1996,271(20):12009-12016.
[23]VESTNER B, BUSTIN M, GRUSS C. Stimulation of replication efficiency of a chromatin template by chromosomal protein HMG-17[J]. J. Biol. Chem.,1998,273(16): 9409-9414.
[24]HOCK R, SCHEER U, BUSTIN M. Chromosomal proteins HMG-14 and HMG-17 are released from mitotic chromosomes and imported into the nucleus by active transport [J]. J. Cell Biol.,1998,143(6):1427-1436.
[25]SPIEKER N, BEITSMA M, VANSLUIS P, et al. An integrated 5Mb physical, genetic, and radiation hybricmap of alp36.1 region implicated in neuroblastoma pathogenesis [J]. Genes Chromosomes Cancer,2000,27(2):143-152.
[26]BUSTIN M, REISCH J, EINCK L, et al. Autoantibodies to nucleosomal proteins: antibodies to HMG-17 in autoimmune diseases [J]. Science,1982,215:1245-1247.
[27]Tzioufas AG, Boumba VA, Seferiadis K, et al. Autoantibodies to HMG-17 n ucleosomal protein inpatients with scleroderma[J]. Arthritis Rheum.1993,36(7):955-961.
[28]马懿,冯云,李恒HMGN2的重组表达质粒的构建及其抗菌功能研究[J].四川生理科学杂志,2005,27(4)：147-149.
[29]李薇,张平,孔祥丽,等.人淋巴结中HMGN2蛋白的分离纯化及其亚细胞分布研究[J].生物医学工程学杂志,2010,27(4)：842-846.
[30]FENG Y, HUANG N, WU Q, et al. The helic-domain is essential for antibacterial activity of high mobility group nucleosomal binding domain 2 (HMGN2)[J]. Acta Pharmacol. Sinica.,2005,26(9):1087-1092.
[31]YOUNG ARJ, NARITA M. Oncogenic HMGA2:short or small[J]. Genes Dev, 2007,21(9):1005-1009.
[32]King LM, Francomano CA. Characterization of a human gene encoding nucleosomal binding protein NSBP1 [J]. Genomics,2001,71 (2):163-173.
[33]杨学贞,周利群,周健,等.mRNA差异显示法克隆前列腺癌相关基因[J].中华泌尿外科杂志,2003,24(8)：545-547.
[34]Mark Rochman, Cedric Malicet, Michael Bustin. HMGN5/NSBP1:A new member of the HMGN protein family that affects chromatin structure and function[J]. Biochimica et Biophysica Acta,2010,1799:86-92.
[35]M. Bustin.Regulation of DNA-dependent activities by the functional motifs of the high-mobility-group chromosomal proteins [J]. Mol. Cell. Biol.,1999(19):5237-5246.
[36]T. Ueda, F. Catez, G. Gerlitz, et al Delineation of the protein module that anchors HMGN proteins to nucleosomes in the chromatin of living cells [J]. Mol. Cell Biol., 2008(28):2872-2883.
[37]Y.V. Postnikov, D.A. Lehn, R.C. Robinson, et al. The cooperative binding of chromosomal protein HMG-14 to nucleosome cores is reduced by single point mutations in the nucleosomal binding domain [J]. Nucleic Acids Res.,1994(22):4520-4526.
[38]H. Shirakawa, D. Landsman, Y.V. Postnikov, et al, NBP-45, a novel nucleosomal binding protein with a tissue-specific and developmentally regulated expression [J]. J. Biol. Chem.,2000(275):6368-6374.
[39]M. Prymakowska-Bosak, T. Misteli, J.E. Herrera, et al. Mitotic phosphorylation prevents the binding of HMGN proteins to chromatin [J]. Mol. Cell Biol.,2001(21): 5169-5178.
[40]Y.V Postnikov, J.E. Herrera, R. Hock, et al. Clusters of nucleosomes containing chromosomal protein HMG-17 in chromatin [J]. Mol. Biol.,1997(274):454-465.
[41]S. Cherukuri, R. Hock, T. Ueda, et al. Cell cycle dependent binding of HMGN proteins to chromatin [J]. Mol. Biol. Cell,2008(19):1816-1824.
[42]M. Rochman, Y. Postnikov, S. Correll, et al. The interaction of NSBP1/HMGN5 with nucleosomes in euchromatin counteracts linker histone-mediated chromatin compaction and modulates transcription [J]. Mol. Cell,2009(35):642-656.
[43]J.J. Ward, J.S. Sodhi, L.J. McGuffin, et al. Prediction and functional analysis of native disorder in proteins from the three kingdoms of life[J]. Mol. Biol.,2004(337): 635-645.
[44]H.J. Dyson, P.E. Wright. Intrinsically unstructured proteins and their functions [J]. Nat. Rev. Mol. Cell. Biol.,2005(6):197-208.
[45]J.C. Hansen, X. Lu, E.D. Ross, et al. Intrinsic protein disorder, amino acid composition, and histone terminal domains [J]. Biol. Chem.,2006,281:1853-1856.
[46]T. Vavouri, J.I. Semple, R. Garcia-Verdugo, et al. Intrinsic protein disorder and interaction promiscuity are widely associated with dosage sensitivity [J].Cell,2009,138: 198-208.
[47]T. Furusawa, J.H. Lim, F. Catez, et al. Down-regulation of nucleosomal binding protein HMGN1 expression during embryogenesis modulates Sox9 expression in chondrocytes [J]. Mol. Cell. Biol.,2006,26:592-604.
[48]J.M. Pash, P.J. Alfonso, M. Bustin. Aberrant expression of high mobility group chromosomal protein 14 affects cellular differentiation[J]. Biol.Chem.,1993,268: 13632-13638.
[49]Y. Birger, F. Catez, T. Furusawa, et al. Increased tumorigenicity and sensitivity to ionizing radiation upon loss of chromosomal protein HMGN1[J]. Cancer Res.,2005,65: 6711-6718.
[50]H. Shirakawa, M. Rochman, T. Furusawa, et al. The nucleosomal binding protein NSBP1 is highly expressed in the placenta and modulates the expression of differentiation markers in placental Rcho-1 cells [J]. Cell Biochem.,2009,106:651-658.
[51]R.D. Phair, P. Scaffidi, C. Elbi, et al. Global nature of dynamic protein-chromatin interactions in vivo:three-dimensional genome scanning and dynamic interaction networks of chromatin proteins [J]. Mol. Cell. Biol.,2004,24:6393-6402.
[52]J.H. Lim, M. Bustin, V.V. Ogryzko, et al. Metastable macromolecular complexes containing high mobility group nucleosome-binding chromosomal proteins in HeLa nuclei [J]. Biol. Chem.,2002,277:20774-20782.
[53]L. Trieschmann, P.J. Alfonso, M.P. Crippa, et al. Incorporation of chromosomal proteins HMG-14/HMG-17 into nascent nucleosomes induces an extended chromatin conformation and enhances the utilization of active transcription complexes [J]. EMBO, 1995(14):1478-1489.
[54]Y.C. Kim, G Gerlitz, T. Furusawa, et al. Activation of ATM depends on chromatin interactions occurring before induction of DNA damage [J]. Nat. Cell Biol.,2009,11:92-96.
[55]L. Cato, K.Stott, M. Watson, et al. The interaction of HMGB1 and linker histones occurs through their acidic and basic tails [J]. Mol. Biol.,2008,384:1262-1272.
[56]Mark Rochman,Yuri Postnikov, Sarah Correll, et al. The Interaction of NSBP1/HMGN5 with Nucleosomes in Euchromatin Counteracts Linker Histone-Mediated Chromatin Compaction and Modulates Transcription [J]. Molecular Cell,2009,7(2): 642-656.
[57]T. Ueda, T. Furusawa, et al. The nucleosome binding protein HMGN3 modulates the transcription profile of pancreatic(beta) cells and affects insulin secretion [J]. Mol. Cell. Biol.,2009,29:5264-5276.
[58]R. Hock, T. Furusawa, T. Ueda, et al. HMG chromosomal proteins in development and disease [J]. Trends Cell. Biol.,2007,17:72-79.
[59]M. Pritsker, N.R. Ford, H.T. Jenq, et al. Genomewide gain-of-function genetic screen identifies functionally active genes in mouse embryonic stem cells [J]. Proc. Natl. Acad. Sci.,2006,103:6946-6951.
[60]D.Q. Li, Y.F. Hou, J. Wu, et al. Gene expression profile analysis of an isogenic tumour metastasis model reveals a functional role for oncogene AF1Q in breast cancer metastasis [J]. Eur. Cancer,2006,42:3274-3286.
[61]Lee BC, Cha K, Avraham S, et al. Microarray analysis of differentially expressed genes associated with human ovarian cancer [J]. Int. J. Oncol.,2004,24(4):847-851.
[62]Koon N, Schneider-Stock R, Sarlomo-Rikala M, et al. Molecular targets for tumour progression in gastrointestinal stromal tumours [J]. Gut.,2004,53(2):235-240.
[63]Ueda R, Iizuka Y, Yoshida K, et al. Identification of a human glioma antigen, SOX6, recognized by patients's era [J]. Oncogene,2004,23(7):1420-1427.
[64]P.P. Khil, R.D. Camerini-Otero. Molecular features and functional constraints in the evolution of the mammalian X chromosome [J]. Crit. Rev. Biochem. Mol. Biol., 2005,40:313-330.
[65]D. Nolte, J. Ramser, S. Niemann, et al. ACRC codes for a novel nuclear protein with unusual acidic repeat tract and maps to DYT3(dystonia parkinsonism) critical interval in xq 13.1 [J]. Neurogenetics,2001 (3):207-213.
[66]R. Gasa, C. Mrejen, N. Leachman, et al. Proendocrine genes coordinate the pancreatic is let differentiation program in vitro[J]. Proc.Natl.Acad.Sci.U.S.A.,2004,101: 13245-13250.
[67]H.E. MacLean, W.S. Chiu, A.J. Notini, et al. Impaired skeletal muscle development and function in male, but not female, genomic androgen receptor knockout mice [J]. FASEB J.,2008,22:2676-2689.
[68]C. Goritz, R. Thiebaut, L.H. Tessier, et al. Glia-induced neuronal differentiation by transcriptional regulation [J]. Glia,2007,55:1108-1122.
[69]Q. Lu, N. Shen, X.M. Li, et al. Genomic view of IFN-alpha response in preautoimmune NZB/W and MRL/lpr mice [J]. Genes Immun.,2007,8:590-603.
[70]Y. Chen, H. Ni, X.H. Ma, et al. Global analysis of differential luminal epithelial gene expression at mouse implantation sites[J]. Mol. Endocrinol.,2006,37:147-161.
[71]P.A.Efron, H.Tsujimoto, F.R. Bahjat,et al. Differential maturation of murine bone marrow derived dendritic cells with lipopolysaccharide and tumor necrosis factor-alpha[J]. Endotoxin.Res.,2005,11:145-160.
[72]W.Y. Tang, R. Newbold, K. Mardilovich, et al. Persistent hypomethylation in the promoter of nucleosomal binding protein 1 (Nsbp1) correlates with overexpression of Nsbp1 in mouse uterineonatally exposed to diethylstilbestrol or genistein [J]. Endocrinology,2008, 149:5922-5931.
[73]S.W. Jee, J.S. Cho, C.K. Kim, et al. Oligonucleotide-based analysis of differentially expressed genes in hippocampus of transgenic mice expressing NSE-controlled APPsw[J]. Neurochem. Res.,2006,31:1035-1044.
[74]M. Pritsker, N.R. Ford, H.T. Jenq, et al. Genomewide gain-of-function genetic screen identifies functionally active genes in mouse embryonic stem cells [J]. Proc. Natl. Acad. Sci..,2006,103:6946-6951.
[75]W.G. Beamer, K.L. Shultz, B.J. Tennent, et al. Multigenic and imprinting control of ovarian granulosa cell tumorigenesis in mice [J]. Cancer Res.,1998,58:3694-3699.
[76]H.J. Han, J. Russo,Y. Kohwi, et al. SATB1 reprogrammes gene expression to promote breast tumour growth and metastasis[J]. Nature,2008,452:187-193.
[77]J. Green, M. Ikram, J. Vyas, et al. Overexpression of the Axl tyrosine kinase receptor in cutaneous SCC-derived cell lines and tumours [J]. Cancer,2006,94:1446-1451.
[78]D.Q. Li, Y.F. Hou, J. Wu, et al. Gene expression profile analysis of an isogenic tumour metastasis model reveals a functional role for oncogene AF1Q in breast cancer metastasis[J]. Cancer,2006,42:3274-3286.
[79]F. Rizzolio, S. Bione, C. Sala, et al. Chromosomal rearrangements in Xq and premature ovarian failure:mapping of 25 new cases and review of the literature [J]. Hum. Reprod.,2006,21:1477-1483.
[80]C.Huang,L.Q.Zhou,GSong.Effect of nucleosomal binding protein 1 in androgen-independent prostatic carcinoma[J]. Zhonghua Yi Xue Za Zhi,2008,88:657-660.
[81]L.Q. Zhou, G Song, Z.S. He, et al. Effects of inhibiting nucleosomal binding proteinl on proliferation of human prostate cancer cells[J]. Zhonghua Yi Xue Za Zhi,2007,87:404-408.
[82]王建伟,周利群,杨学贞.前列腺癌细胞中NSBP1基因表达上调[J].基础医学与临床,2004,24(4)：393-397.
[83]宋刚,周利群,翁迈,等.核小体结合蛋白1在前列腺癌中的表达和意义[J].中华医学杂志,2006,86(28)：1962-1965.
[84]姚鲲,周利群,李学松.核小体结合蛋白1在膀胱癌中的表达和意义[J].中华临床医师杂志(电子版),2010,4(1)：50-54.
[85]Agresti A, Bianchi ME. HMGB proteins and gene expression [J]. Curr. Opin. Genet Dev.,2003,13(2):170-178.
[86]Trieschmann L, Alfonso PJ, Crippa MP, et al. Incorporation of chromosomal proteins HMG-14/HMG-17 into nascent nucleosomes induces an extended chromatin conformation and enhances the utilization of active transcrip tion comp lexes [J]. EMBO J., 1995,14(7):1478-1489.
[87]Aiziwa S, Nishino H, Saito K, et al. Stimulation of transcription in cultured cells by high mobility group proteinl:essential role of the acidic carboxyl-terminal region [J]. Biochem.,1994,33(49):14690-14695.
[88]Muller S, Scaffidi P, Degryse B, et al. New EMBO members'review:the double life of HMGB1 chromatin protein:architectural factor and extracellular signal [J]. EMBO J., 2001,20(16):4337-4340.
[89]Tang WY, Newbold R, Mardilovich K, et al. Persistent hypomethylation in the promoter of nucleosomal binding protein 1 (Nsbp1) correlates with overexpression of Nsbp1 in mouse uteri neonatally exposed to diethylstilbestrol or genistein [J]. Endocrinology,2008, 149(12:5922-5931.
[90]周利群,宋刚,何志嵩,等.抑制核小体结合蛋白1基因对人前列腺癌细胞LNCaP增殖的作用[J].中华医学杂志,2007,87(6)：404-408.
[91]蒋宁,周利群,姚鲲.人核小体结合蛋白1小分子干扰RNA重组慢病毒抑制非激素依赖性前列腺癌体内生长的实验研究[J].中华临床医师杂志(电子版),2010,2(4)：154-157.
[92]葛棣.肺癌基因治疗研究进展[J].中国航天工业医药.2001,6(3)：70-74
[93]陈湘琦,李志鹰,林挺岩.RNA干扰技术在肺癌基因治疗中的应用研究现状[J].中华临床医师杂志(电子版),2010,7(4)：1028-1031.
[94]Hansen HH. Treatment of advanced non-small cell lung cancer [J]. Annals of Oncology,2002,325:452-453.
[95]字应伟.肿瘤基因治疗研究进展[J].思茅师范高等专科学校校报,2005,21(3)：6-8.
[96]张利群,陈杭薇.肺癌基因治疗的动态[J].中华结核和呼吸杂志2004,27(6)：430-434.
[97]PoulsenTT, Pedersen N, Poulsen HS. Roplacenent and suicide gene therapy fox targeted treatment of lung cancer[J]. Clin Lung cancer,2005,6(4):227-223.
[98]Guo S, Kemphues K J.,A gene required for establishing polarity in C.elegans embryos, encodes a putative ser/Thr kinase that is asymmetrically distributed[J]. Cell,1995, 81:611-620.
[99]FireA,Xu S,Montgomery MK,et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J]. Natrue,1998,391:806-811.
[100]Hammond GJ.RNAi interference[J]. Nature,2002,418(6894):244-251.
[101]Pomerantz RJ.RNA interfence meets HIV-1:will silence be golden[J], Nat Med, 2002,8(7):659-660.
[102]Garzon R,Pichiorri F,Palumbo T,et al. Micro RNA fingerprints during human egakaryocytopoiesis[J]. Proc Natl Acad Sci USA,2006,103(13):5078-5083.
[103]Elbashir SM,Harborth J,Lendeckel W,et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells[J]. Nature,2001,411(6836):494-498.
[104]杨家驹,段振铃.肿瘤的基因治疗进展[J].医学综述,2005,11(10)：883-885.
[105]苗彦,陈愉生.RNA干扰在肺癌基因治疗中的应用进展[J].国际呼吸杂志,2010,30(10)：619-625.
[106]刘苏健.肺癌基因治疗的研究进展[J].山东医药,2008,48(19)：51-152.
[107]何祥梁,何东华.肺癌基因治疗研究进展[J].中国病理生理杂志,2002,18(11)：1447-1450.
[108]张鼎,陈小东.肺癌治疗的分子靶位及其相关药物的研究进展[J].国际呼吸杂志,2007,27(10)：750-753.
[109]陈湘琦,李志鹰,林挺岩.RNA干扰技术在肺癌基因治疗中的应用研究现状[J].中华临床医师杂志(电子版),2010,4(7)：1028-1031.
[110]任新玲,钱桂生,付海京,等.HER2 siRNA抑制肺癌细胞增值的实验研究[J].中华肺部疾病杂志(电子版),2007,1(1)：16-18.
[111]Yin J Q,Gao J,Shao R,et al.siRNA agents inhibition oncogene expression and attenuate human tumor cell growth[J]. Exp Ther Oncol,2003,3(4):194-204.
[112]Jiang M,Milner J.Bcl-2 constitutively suppresses p53-dependent apoptosis in colorectal cance cells[J]. Genes Dev,2003,17(7):832-837.
[113]张泳,谷仲平,周勇安,等.RNAi沉默肺癌细胞中VEGF的表达治疗肺癌机制的初步研究[J].第四军医大学学报,2009,30(19)：1490-1493.
[114]张泳,谷仲平,周勇安,等.RNAi沉默VEGF的表达以及治疗肺癌的初步研究[J].细胞与分子免疫学杂志.2009,25(4)：341-343.
[115]Krysan K,Dalwadi H,Sharma S,et al. Cyclooxygenase 2-dependent expression of surviving is critical for apoptosis resistance in non-small cell lung cancer [J]. Cancer Res,2004,64(18):6359-6362.
[116]Chao J I,Liu HE The blockage of surviving and securing expression increases the cytochalasin B-induced cell death and growth inhibition in human cancer cells[J]. Mol Pharmacol,2006,69(1):154-164.
[117]马丹,张珍祥,徐永健.CD44v3的表达对肺腺癌A549细胞侵袭行为的影响[J].中国现代医学杂志,2006,16(22)：3388-3392.
[118]钟锡明,范钰,张瑞佳.RNAi沉默PRL-1基因对肺癌细胞侵袭能力的影响[J].江苏大学学报(医学版),2008,18(6)：480-483.
[119]MacDonald NJ, Shivers WY, Narum DL, et al. Endostalnn binds fropomysin. A potential modulator of the antitumor activity of endostatin[J]. Biol Chem,2001,276(27): 25190-25196.
[120]Chandramu Chetty, Sajani S. Lakka, Praveen Bhoopathi, et al.MMP-2 alters VEGF expression via aVb3 integrin-mediated PI3K/AKT signaling in A549 lung cancer cells[J]. International Journal of Cancer,2010,127:1081-1095.
[121]Lu B,Mu Y,Cao C, et al. Survivin as a therapeutic target for rasiation sensitization in lung cancer[J].Cancer Res,2004,64(8):2840-2845.
[122]Pai S I,Lin Y Y,Macaes B, et al. Prospects of RNA interference therapy for cancer[J]. Gene Ther,2006,13(6):464-477.
[123]Song X,Liu X,Chi W, et al. Hypoxia-induced resistance to cisplatin and doxorubicin innon-small cell lung cancer is inhibited by silencing of HIF-alpha gene[J].Cancer Chemother Pharmacol,2006,58(6):776-784.
[124]Lee MW,Kim DS,Min NY, et al. Akt1 inhibition by RNA interference sensitizes humannon-small cell lung cancer cells to cisplatin[J]. Cancer,2008,122(10):2380-2384.
[125]Yonesaka K,Tamura K,Kurata T, et al. Small interfering RNA targeting surviving sensitizes lung cancer cell with mutant p53 to adriamycin[J]. Cancer,2006,118 (4):812-820.
[126]任振义,金儿,叶健.Ku80 RNAi技术增强肺癌放射敏感性的动物实验研究[J].肿瘤学杂志,2010,16(8)：614-617.
[127]Fukuoka M, Yano S, Giaccone G, et al. Multi_institutional randomized phase II trial of gefitinib for previously treated patients with advanced non-small-cell lung cancer[J]. Clin Oncol,2003,21:2237-2246.
[128]Franklin MC, Carey KD, Vajdos FF, et al. Insights into ErbB signaling from the structure of the ErbB2-pertuzurnab complex[J]. Cancer Cell,2004,5(4):317-28.
[129]Jin Z, Gao F, Flagg T, et al. Tobacco specific nit rosamine 4 (methylni trosamino)1(3-pyridyl)-1-butanone promot es functional cooperation of Bcl-2 and c-Myc through phosphorylat ion in regulating cell survival and proliferation [J].Biol Chem,2004, 279(38):40209-40219.
[130]任淑华,张伟,曲平,等.RNA干扰C-erbB-2对肺腺癌细胞calu-3增殖的影响[J].癌症,2005,24(1)：1173-1178.
[131]Poulsen TT,Pedersen N,Poulsen HS.Replacement and suicide gene therapy fortargeted treatment of lung cancer[J].Clin Lung Cancer,2005,6(4):227-236.
[132]Tkeshita F, Ochiya T.TheraPeutic potential of RNA interference againstcancer. Cancer Sei,2006,97(8):689-696.
[133]Hao DL, Liu CM, Dong WJ, etal.Knockdown of human p53 gene expression in 293-T cells by retroviral vector-mediated short hairp in RNA[J].Acta Biochim BioPhy Sin(Shanghai),2005,37(11):779-783.