1，25-二羟维生素D_3上调肺癌细胞miRNA let-7α2表达的机制研究
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摘要
microRNA (miRNA)是一类小分子非编码RNA,通常为21-25个核苷酸,可与其靶基因的3’-非翻译区互补配对,激活核蛋白复合体miRNAP,对靶mRNA进行切割或翻译抑制,从而参与调控基因表达。miRNA广泛存在于动植物细胞中,与肿瘤关系密切。多数已知的miRNA位于基因组中与肿瘤相关的区域,某些组织特异性miRNA表达图谱的改变可见于直肠结肠癌、肺癌、乳腺癌、胶质细胞瘤、垂体瘤等多种肿瘤。
肺癌是当今世界各国常见的恶性肿瘤,Takamizawa等发现肺癌患者的let-7表达显著降低,并且let-7表达水平越低,预后越差,术后生存期越短。体外组织培养实验表明,在人肺癌细胞中瞬时表达let-7可以抑制细胞的增殖。let-7在肺癌的发生发展中作为一个抑癌基因在发挥作用。let-7a2是这个家族的成员之
一,其基因定位于染色体11q24.1。
1,25-二羟维生素D3 (1,25-(OH)2D3)是维生素D的体内活性形式,能够调节细胞的分化增殖,而这种作用是通过维生素D受体(VDR)介导发挥其生物学效应的。人类VDR基因位于染色体12q13.11,基因全长约4669 bp。VDR蛋白为配体依赖性转录因子,1,25-(OH)2D3通过激活VDR,进而形成VDR/RXR/辅转录因子复合体,与靶基因启动子区域的VDR应答元件(VDRE)识别结合,影响RNA聚合酶Ⅱ介导的某些基因转录,影响细胞的生长、分化、凋亡等过程。1,25-(OH)2D3可降低肺癌细胞系的生长,促进细胞凋亡。VDR属于核转录因子,在肺癌组织中VDR mRNA的表达下调。VDR表达和功能的改变可能导致细胞对1,25-(OH)2D3的反应性变化,而改变对细胞的生长、分化、凋亡等过程的调控。
let-7a2在肺癌中作用及表达调控的机制尚未见研究报道,并且在研究let-7a2表达调控时发现,1,25-(OH)2D3可在转录水平上调let-7α2在肺癌细胞A549中的表达,其调控机制及其在肺癌中的作用很有必要进行深入研究。本课题在基因启动子及转录水平研究1,25-(OH)2D3在肺癌中对let-7α2基因表达的调控作用,并初步探讨其调控机制。在此研究中,(1)首先利用5'RACE寻找到let-7α2基因5’上游的转录起始点,进一步通过PCR扩增其5’上游包含启动子的2.8 kb片段(+336 bp至-2523 bp),构建let-7α2的2.8 kb启动子-荧光素酶报道基因质粒并检测其在肺癌A549细胞中的启动子活性。(2)A549细胞经过9-顺维甲酸(9-cis-RA)、全反式维甲酸(AT-RA)、氯化锂(LiCl)、地塞米松(Dex)、1,25-(OH)2D3处理或者分别转染let-7α2启动子-荧光素酶报道基因质粒和let-7α2靶序列-报道基因融合质粒后,经9-cis-RA、AT-RA、LiCl、Dex、1,25-(OH)2 D3处理48 h后,分别通过RT-PCR和荧光素酶活性检测,观察它们对let-7α2在A549细胞中表达的影响。(3)通过构建let-7α2基因5’上游VDR反应元件(VDRE)-绿色荧光蛋白报道基因质粒,转染A549细胞并经1,25-(OH)2 D3处理后,荧光倒置显微镜下观察绿色荧光蛋白的表达,了解1,25-(OH)2D3激活VDR转录因子并与VDRE结合,促进报告基因表达的作用。(4)通过电泳迁移率改变实验(EMSA)和染色质免疫共沉淀分析(ChIP),检测在1,25-(OH)2 D3作用下VDR与VDRE的结合活性。(5)收集18组临床肺癌标本,用RT-PCR方法检测let-7α2和VDR在肺癌组织中表达的相关性。(6)构建let-7α2真核表达质粒,并检测其在肺癌细胞A549中的表达及对A549细胞增殖凋亡的影响。
结果：(1)通过5'RACE,确定了let-7α2的转录起始点,并克隆了let-7α2基因5’上游2.8 kb启动子片段,成功构建了let-7α2启动子-荧光素酶报告基因质粒,并且在肺癌细胞A549中显示有启动子活性。(2)9-cis-RA、AT-RA和1,25-(OH)2D3能够增强let-7α2在肺癌细胞中的表达；而LiCl和转录因子CEBPα则使let-7α2在肺癌细胞中的表达降低。(3)1,25-(OH)2 D3能够增强VDRE-绿色荧光蛋白报道基因质粒中绿色荧光蛋白的表达,表明1,25-(OH)2 D3能促进VDR与let-7α2启动子区域中VDRE的结合。(4)EMSA和ChIP鉴定了let-7α2基因上游的VDRE (-1730～-1706 bp),并证实VDRE参与1,25-(OH)2D3对let-7α2基因表达的正向调控作用。(5)在肺癌组织中,let-7α2和VDR mRNA表达密切相关,二者较正常肺组织均降低的占44%(18例中8例),二者表达呈正相关的占67%(18例中12例)。(6)成功构建了let-7α2真核表达质粒,在肺腺癌细胞A549中能有效表达,并且抑制A549细胞增殖,促其凋亡。
结论：Iet-7a2与肺癌有密切关系,其在肺癌细胞A549中的表达可能受维甲酸、1,25-(OH)2D3、氯化锂和转录因子CEBPa调控。1,25-(OH)2D3通过VDR与Iet-7a2基因启动子区域中的VDR反应元件结合,发挥对Iet-7a2在肺癌中表达的正向调控作用。这一研究结果为进一步研究Iet-7a2和1,25-(OH)2 D3在肺癌中的作用奠定了基础。
microRNAs (miRNAs) are a class of small non-coding RNAs of 21 to 25 nucleotides. They bind to the 3'- untranslated regions (3'-UTRs) of target genes and then the binding event causes translational repression of the target gene or stimulates rapid degradation of the target transcript, so they play key roles in regulation of gene expression. miRNAs were found both in animals and plants, and they correlated with various cancers closely. Most of known miRNA located at genomic regions involved in cancers. Some tissue-specific changes in miRNA expression profiles can be found in colorectal cancer, lung cancer, breast cancer, glial cell tumors, pituitary tumors.
Lung cancer is the common cancer in the world. Takamizawa et al found that the expression of let-7 was frequently reduced in lung cancers both in vitro and in vivo. Furthermore, lung cancer patients with reduced let-7 expression were found to have significantly worse prognosis after potentially curative resection, but the overexpression of let-7 inhibited growth of lung cancer cells in vitro. let-7 may play a major role in human lung carcinogenesis as a tumor suppressor gene. let-7α2 is a member of let-7 family, and located on chromosome 11q24.1.
1,25-dihydroxyvitamin D3(1,25-(OH)2D3) is the active metabolite of vitamin D. It regulates cell differentiation and proliferation. The actions of 1,25-(OH)2 D3 are mediated by the nuclear vitamin D receptor (VDR). Human VDR gene maps to chromosome 12q13.11 and the full length of human VDR gene is about 4669bp. VDR is the ligand-dependent transcriptional factor.1,25-(OH)2D3 activates VDR to form complex of VDR/RXR/accessory transcription factor, then the complex binds to VDR responsive element(VDRE) of target gene to affect the cell growth, differentiation, apoptosis mediated by RNA polymeraseⅡtranscription of certain genes.1,25-(OH)2 D3 could reduce the growth of lung cancer cell lines and induce cell apoptosis. The mRNA expression of VDR was decreased in lung cancer. The change of expression and function of VDR will influence the celluar responses to 1,25-(OH)2D3, and this action will influence regulation of cell growth, differentiation and apoptosis by 1,25-(OH)2D3.
So far, little is known about the role and the regulatory mechanisms of let-7α2 expression in lung cancer. In the present study, we found that 1,25-(OH)2D3 could upregulate let-7α2 gene expression in transcriptional level in A549 cancer cells. Our work is focused on the regulatory mechanisms of let-7α2 expression by 1,25-(OH)2 D3 at transcriptional level in lung cancer. (1) Firstly,5'RACE was carried out to identify the transcriptional start site of let-7α2 gene, then a 2.8 kb fragment of its 5'flanking region(+336 bp to-2523 bp) was cloned into pGL3-basic vector to construct pGL3-p7a2 recombinant and test its promoter activity in A549 cells. (2) A series of transfection and luciferase reporter assay were carried out to test its promoter activity, as well the RT-PCR and transfection of let-7α2 target sequence-reporter plasmid to detect transcriptional level of let-7α2 gene in A549 treated with 9-cis-RA, AT-RA, LiCl, Dex or 1,25-(OH)2 D3. (3) The VDR responsive element (VDRE)-green fluorescence protein(GFP) reporter plasmid was constructed and transfected into A549 cells treated with 1,25-(OH)2 D3, then the expression of GFP was detected. 1,25-(OH)2 D3 activated VDR to bind VDRE in let-7α2 promoter region and then enhanced the expression of reporter gene. (4) EMSA and ChIP were carried out to investigate the binding activities of VDRE with transcription factor VDR. (5) The correlation between let-7α2 and VDR expression was detected in 18 lung cancer specimens by RT-PCR. (6) The recombinant eukaryotic expression plasmid of let-7α2 was constructed and expressed in lung cancer A549 cells. The effect of let-7α2 on A549 cell proliferation and apoptosis was tested by MTT and flow cytometry analysis.
The results showed that:(1) The transcriptional start site of let-7α2 was identified by 5'RACE. A 2.8 kb fragment of its 5'-flanking region was cloned into pGL3-basic vector to construct pGL3-p7a2 recombinant plasid which could represent a promoter activity. (2) The promoter activity could be enchanced by 9-cis-RA, AT-RA or 1,25-(OH)2D3 treatment and down-regulated by LiCl treatment or CEBPαtransfection. (3) The expression of GFP in recombinant plasmid of VDRE increased with the treatment of 1,25-(OH)2 D3 than that in control cells. These results indicated that 1,25-(OH)2 D3 could enhance the binding of VDRE in let-7α2 promoter region with VDR. (4) The functional VDRE upstream of let-7α2 gene (-1730 bp～-1706 bp) were identified by EMSA and ChIP, which were involved in the positive regulation of let-7α2 gene expression by 1,25-(OH)2D3. (5) The expression of let-7α2 with VDR was closely correlated in lung cancer specimens, the expression of let-7α2 and VDR mRNA were simultaneously down-expressed acocounting for about 44%(8 out of 18) in lung cancer specimens and normal lung tissue, and positive correlation of VDR and let-7α2 expression was about 67%(12 out of 18). (6) The eukaryotic expression plasmid of let-7α2 is successfully constructed and effectively expressed in A549 cells. let-7α2 could inhibit the proliferation of A549 cells and induce their apoptosis..
In summary, our research suggested that let-7α2 were related to lung cancer closely. The expression of let-7α2 was possibly regulated by retinoid acid,1,25-(OH)2 D3, lithium choride and transcriptional factor CEBPαin lung cancer cell A549. The positive regulation of let-7α2 gene by 1,25-(OH)2D3 was mediated by the binding of VDR and VDRE upstream of let-7α2 promoter. Our findings will be the foundation for further study in the action of let-7α2 and 1,25-(OH)2 D3 on lung cacer.
肺癌是当今世界各国常见的恶性肿瘤,Takamizawa等发现肺癌患者的let-7表达显著降低,并且let-7表达水平越低,预后越差,术后生存期越短。体外组织培养实验表明,在人肺癌细胞中瞬时表达let-7可以抑制细胞的增殖。let-7在肺癌的发生发展中作为一个抑癌基因在发挥作用。let-7a2是这个家族的成员之
一,其基因定位于染色体11q24.1。
1,25-二羟维生素D3 (1,25-(OH)2D3)是维生素D的体内活性形式,能够调节细胞的分化增殖,而这种作用是通过维生素D受体(VDR)介导发挥其生物学效应的。人类VDR基因位于染色体12q13.11,基因全长约4669 bp。VDR蛋白为配体依赖性转录因子,1,25-(OH)2D3通过激活VDR,进而形成VDR/RXR/辅转录因子复合体,与靶基因启动子区域的VDR应答元件(VDRE)识别结合,影响RNA聚合酶Ⅱ介导的某些基因转录,影响细胞的生长、分化、凋亡等过程。1,25-(OH)2D3可降低肺癌细胞系的生长,促进细胞凋亡。VDR属于核转录因子,在肺癌组织中VDR mRNA的表达下调。VDR表达和功能的改变可能导致细胞对1,25-(OH)2D3的反应性变化,而改变对细胞的生长、分化、凋亡等过程的调控。
let-7a2在肺癌中作用及表达调控的机制尚未见研究报道,并且在研究let-7a2表达调控时发现,1,25-(OH)2D3可在转录水平上调let-7α2在肺癌细胞A549中的表达,其调控机制及其在肺癌中的作用很有必要进行深入研究。本课题在基因启动子及转录水平研究1,25-(OH)2D3在肺癌中对let-7α2基因表达的调控作用,并初步探讨其调控机制。在此研究中,(1)首先利用5'RACE寻找到let-7α2基因5’上游的转录起始点,进一步通过PCR扩增其5’上游包含启动子的2.8 kb片段(+336 bp至-2523 bp),构建let-7α2的2.8 kb启动子-荧光素酶报道基因质粒并检测其在肺癌A549细胞中的启动子活性。(2)A549细胞经过9-顺维甲酸(9-cis-RA)、全反式维甲酸(AT-RA)、氯化锂(LiCl)、地塞米松(Dex)、1,25-(OH)2D3处理或者分别转染let-7α2启动子-荧光素酶报道基因质粒和let-7α2靶序列-报道基因融合质粒后,经9-cis-RA、AT-RA、LiCl、Dex、1,25-(OH)2 D3处理48 h后,分别通过RT-PCR和荧光素酶活性检测,观察它们对let-7α2在A549细胞中表达的影响。(3)通过构建let-7α2基因5’上游VDR反应元件(VDRE)-绿色荧光蛋白报道基因质粒,转染A549细胞并经1,25-(OH)2 D3处理后,荧光倒置显微镜下观察绿色荧光蛋白的表达,了解1,25-(OH)2D3激活VDR转录因子并与VDRE结合,促进报告基因表达的作用。(4)通过电泳迁移率改变实验(EMSA)和染色质免疫共沉淀分析(ChIP),检测在1,25-(OH)2 D3作用下VDR与VDRE的结合活性。(5)收集18组临床肺癌标本,用RT-PCR方法检测let-7α2和VDR在肺癌组织中表达的相关性。(6)构建let-7α2真核表达质粒,并检测其在肺癌细胞A549中的表达及对A549细胞增殖凋亡的影响。
结果：(1)通过5'RACE,确定了let-7α2的转录起始点,并克隆了let-7α2基因5’上游2.8 kb启动子片段,成功构建了let-7α2启动子-荧光素酶报告基因质粒,并且在肺癌细胞A549中显示有启动子活性。(2)9-cis-RA、AT-RA和1,25-(OH)2D3能够增强let-7α2在肺癌细胞中的表达；而LiCl和转录因子CEBPα则使let-7α2在肺癌细胞中的表达降低。(3)1,25-(OH)2 D3能够增强VDRE-绿色荧光蛋白报道基因质粒中绿色荧光蛋白的表达,表明1,25-(OH)2 D3能促进VDR与let-7α2启动子区域中VDRE的结合。(4)EMSA和ChIP鉴定了let-7α2基因上游的VDRE (-1730～-1706 bp),并证实VDRE参与1,25-(OH)2D3对let-7α2基因表达的正向调控作用。(5)在肺癌组织中,let-7α2和VDR mRNA表达密切相关,二者较正常肺组织均降低的占44%(18例中8例),二者表达呈正相关的占67%(18例中12例)。(6)成功构建了let-7α2真核表达质粒,在肺腺癌细胞A549中能有效表达,并且抑制A549细胞增殖,促其凋亡。
结论：Iet-7a2与肺癌有密切关系,其在肺癌细胞A549中的表达可能受维甲酸、1,25-(OH)2D3、氯化锂和转录因子CEBPa调控。1,25-(OH)2D3通过VDR与Iet-7a2基因启动子区域中的VDR反应元件结合,发挥对Iet-7a2在肺癌中表达的正向调控作用。这一研究结果为进一步研究Iet-7a2和1,25-(OH)2 D3在肺癌中的作用奠定了基础。
microRNAs (miRNAs) are a class of small non-coding RNAs of 21 to 25 nucleotides. They bind to the 3'- untranslated regions (3'-UTRs) of target genes and then the binding event causes translational repression of the target gene or stimulates rapid degradation of the target transcript, so they play key roles in regulation of gene expression. miRNAs were found both in animals and plants, and they correlated with various cancers closely. Most of known miRNA located at genomic regions involved in cancers. Some tissue-specific changes in miRNA expression profiles can be found in colorectal cancer, lung cancer, breast cancer, glial cell tumors, pituitary tumors.
Lung cancer is the common cancer in the world. Takamizawa et al found that the expression of let-7 was frequently reduced in lung cancers both in vitro and in vivo. Furthermore, lung cancer patients with reduced let-7 expression were found to have significantly worse prognosis after potentially curative resection, but the overexpression of let-7 inhibited growth of lung cancer cells in vitro. let-7 may play a major role in human lung carcinogenesis as a tumor suppressor gene. let-7α2 is a member of let-7 family, and located on chromosome 11q24.1.
1,25-dihydroxyvitamin D3(1,25-(OH)2D3) is the active metabolite of vitamin D. It regulates cell differentiation and proliferation. The actions of 1,25-(OH)2 D3 are mediated by the nuclear vitamin D receptor (VDR). Human VDR gene maps to chromosome 12q13.11 and the full length of human VDR gene is about 4669bp. VDR is the ligand-dependent transcriptional factor.1,25-(OH)2D3 activates VDR to form complex of VDR/RXR/accessory transcription factor, then the complex binds to VDR responsive element(VDRE) of target gene to affect the cell growth, differentiation, apoptosis mediated by RNA polymeraseⅡtranscription of certain genes.1,25-(OH)2 D3 could reduce the growth of lung cancer cell lines and induce cell apoptosis. The mRNA expression of VDR was decreased in lung cancer. The change of expression and function of VDR will influence the celluar responses to 1,25-(OH)2D3, and this action will influence regulation of cell growth, differentiation and apoptosis by 1,25-(OH)2D3.
So far, little is known about the role and the regulatory mechanisms of let-7α2 expression in lung cancer. In the present study, we found that 1,25-(OH)2D3 could upregulate let-7α2 gene expression in transcriptional level in A549 cancer cells. Our work is focused on the regulatory mechanisms of let-7α2 expression by 1,25-(OH)2 D3 at transcriptional level in lung cancer. (1) Firstly,5'RACE was carried out to identify the transcriptional start site of let-7α2 gene, then a 2.8 kb fragment of its 5'flanking region(+336 bp to-2523 bp) was cloned into pGL3-basic vector to construct pGL3-p7a2 recombinant and test its promoter activity in A549 cells. (2) A series of transfection and luciferase reporter assay were carried out to test its promoter activity, as well the RT-PCR and transfection of let-7α2 target sequence-reporter plasmid to detect transcriptional level of let-7α2 gene in A549 treated with 9-cis-RA, AT-RA, LiCl, Dex or 1,25-(OH)2 D3. (3) The VDR responsive element (VDRE)-green fluorescence protein(GFP) reporter plasmid was constructed and transfected into A549 cells treated with 1,25-(OH)2 D3, then the expression of GFP was detected. 1,25-(OH)2 D3 activated VDR to bind VDRE in let-7α2 promoter region and then enhanced the expression of reporter gene. (4) EMSA and ChIP were carried out to investigate the binding activities of VDRE with transcription factor VDR. (5) The correlation between let-7α2 and VDR expression was detected in 18 lung cancer specimens by RT-PCR. (6) The recombinant eukaryotic expression plasmid of let-7α2 was constructed and expressed in lung cancer A549 cells. The effect of let-7α2 on A549 cell proliferation and apoptosis was tested by MTT and flow cytometry analysis.
The results showed that:(1) The transcriptional start site of let-7α2 was identified by 5'RACE. A 2.8 kb fragment of its 5'-flanking region was cloned into pGL3-basic vector to construct pGL3-p7a2 recombinant plasid which could represent a promoter activity. (2) The promoter activity could be enchanced by 9-cis-RA, AT-RA or 1,25-(OH)2D3 treatment and down-regulated by LiCl treatment or CEBPαtransfection. (3) The expression of GFP in recombinant plasmid of VDRE increased with the treatment of 1,25-(OH)2 D3 than that in control cells. These results indicated that 1,25-(OH)2 D3 could enhance the binding of VDRE in let-7α2 promoter region with VDR. (4) The functional VDRE upstream of let-7α2 gene (-1730 bp～-1706 bp) were identified by EMSA and ChIP, which were involved in the positive regulation of let-7α2 gene expression by 1,25-(OH)2D3. (5) The expression of let-7α2 with VDR was closely correlated in lung cancer specimens, the expression of let-7α2 and VDR mRNA were simultaneously down-expressed acocounting for about 44%(8 out of 18) in lung cancer specimens and normal lung tissue, and positive correlation of VDR and let-7α2 expression was about 67%(12 out of 18). (6) The eukaryotic expression plasmid of let-7α2 is successfully constructed and effectively expressed in A549 cells. let-7α2 could inhibit the proliferation of A549 cells and induce their apoptosis..
In summary, our research suggested that let-7α2 were related to lung cancer closely. The expression of let-7α2 was possibly regulated by retinoid acid,1,25-(OH)2 D3, lithium choride and transcriptional factor CEBPαin lung cancer cell A549. The positive regulation of let-7α2 gene by 1,25-(OH)2D3 was mediated by the binding of VDR and VDRE upstream of let-7α2 promoter. Our findings will be the foundation for further study in the action of let-7α2 and 1,25-(OH)2 D3 on lung cacer.
引文
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7. Takamizawa J, Konishi H, Yanagisawa K et al. Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival. Cancer Res,2004,64:3753-56.
8. Vanden Bemd GJ, Chang GT. Vitamin D and vitamin D analogs in cancer treatment.Curr Drug Targets,2002,3(1):85-94.
9. Nakagawa K, Kawaura A, Kato S, et al.1 alpha,25-Dihydroxyvitamin D(3) is a preventive factor in the metastasis of lung cancer. Carcinogenesis,2005, 26(2):429-40.
10. Zinser GM,McEleney K,Welsh J.Characterization of mammary tumor cell lines from wild type and vitamin D3 receptor knockout mice. Mol Cell Endocrinol,2003,200(12):67-80.
11. Trump DL, Hershberger PA, Bernardi RJ, et al. Anti-tumor activity of calcitriol: pre-clinical and clinical studies. J Steroid Biochem Mol Biol,2004, 89-90(1-5):519-26.
12. Ylikomi T, Laaksi I, Rulou Y, et al. Antipoliferative action of vitamin D.Vitamins and Hormones,2002,64:357-406.
13. Jones G, Strugnell SA, DeLuca HF. Current understanding of the molecular actions of vitamin D. Physiol Rev,1998,78(4):1193-231.
14. Nakagawa K, Kawaura A, Kato S, et al. Metastatic growth of lung cancer cells is extremely reduced in Vitamin D receptor knockout mice. J Steroid Biochem Mol Biol,2004,89-90(1-5):545-547.
15. Menezes RJ, Cheney RT, Husain A,et al. Vitamin D Receptor Expression in Normal, Premalignant, and Malignant Human Lung Tissue.,2008,17:1104-1110, Cancer Epidemiol Biomarkers Prev,2008,17(5):1104-10.
16. Anderson MG, Nakane M, Ruan X, et al. Expression of VDR and CYP24A1 mRNA in human tumors. Cancer Chemother Pharmacol,2006,57(2):234-40.
17. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell, 2004,116:281-297.
18. Brueckner B, Stresemann C, Kuner R et al.The Human let-7a-3 Locus Contains an Epigenetically Regulated MicroRNA Gene with Oncogenic Function. Cancer Res,2007,67:1419-1423.
19. Zhang HH, Wang XJ, Li GX et al. Detection of let-7a microRNA by real-time PCR in gastric carcinoma. World J Gastroentero,2007,13:2883-8.
20. Lokshin A, Zhang H, Mayotte J et al. Early effects of retinoic acid on proliferation, differentiation and apoptosis in nonsmall cell lung cancer cell lines. Anticancer Res,1999,19:5251-4.
21. Choi EJ, Whang YM, Kim SJ et al. Combinational Treatment with Retinoic Acid Derivatives in Non-small Cell Lung Carcinoma In Vitro. J Korean Med Sci,2007,22:52-60.
22. Li JS, Zhu M, Tian D, et al. Effect of Lithium on Cell Proliferation and Cell Cycle in A549 Cells. Acta Med Univ Sci Technol Huazhong,2007,36:145-8.
23. Rao AS, Kremenevskaja N, Resch J et al. Lithium stimulates proliferation in cultured thyrocytes by activating Wnt/beta-catenin signaling. Eur J Endocrinol, 2005,153:929-938.
24. Hershberger PA, Modzelewski RA, Shurin ZR, Rueger RM, Trump DL, Johnson CS.1,25-Dihydroxycholecalciferol (1,25-D3) inhibits the growth of squamous cell carcinoma and downmodulates p21(Wafl/Cipl) in vitro and in vivo. Cancer Res,1999,59:2644-9.
25. Reichel H, Koeffler HP, Norman AW. The role of the vitamin D endocrine system in health and disease. New Engl J Med,1989,320:980-91.
26. Holick MF. Vitamin D deficiency. N Engl J Med,2007,357(3):266-81.
27. Samuel S, Sitrin MD.Vitamin D’S role in cell proliferation and differentiation. NutrRev,2008,66(10 Suppl 2):S116-24.
28.王英田,白雪丽,李怀臣.全反式维甲酸联合维生素D3对肺癌原代细胞化疗敏感性的影响.山东医药,2007,47(5)：9-11.
29. Calin GA, Sevignani C, Dumitru CD et al. Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. Proc Natl Acad Sci U S A.2004,101:2999-3004.
30. Calin G A, Dumitru CD, Shimizu M, et al. Frequent deletions and down-regulation of micro-RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci USA,2002,99(24):15524-29.
31. Caudy A A, Myers M, Hannon G J, et al. Fragile X-related protein and VIG associate with the RNA interference machinery. Genes Dev,2002, 16(19):2491-96.
32. Kumar M S, Erkeland S J, Pester R E, et al.Suppression of non-small cell lung tumor development by the let-7 microRNA family. Proc Natl Acad Sci USA,2008,105(10):3903-08.
33. Bartel D P. MicroRNAs genomics, biogenesis, mechanism and function. Cell, 2004,116(2):281-97.
2. Fabbri M, Croce C M, Calin G A. MicroRNAs. Cancer J,2008,14(1):1-6.
3. Zhang B, Pan X, Cobb G P, et al. microRNAs as oncogenes and tumor suppressors. Dev Biol,2007,302(1):1-12.
4. Esquela-Kerscher A, Slack FJ.Oncomirs - microRNAs with a role in cancer. Nat Rev Cancer,2006,6:259-69.
5.Akao Y, Nakagawa Y, Naoe T.let-7 MicroRNA Functions as a Potential Growth
Suppressor in Human Colon Cancer Cells. Biol Pharm Bull,2006,29:903-906.
6. Johnson SM, Grosshans H, Shingara J et al. RAS is regulated by the let-7 microRNA family. Cell,2005,120:635-47.
7. Takamizawa J, Konishi H, Yanagisawa K et al. Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival. Cancer Res,2004,64:3753-56.
8. Vanden Bemd GJ, Chang GT. Vitamin D and vitamin D analogs in cancer treatment.Curr Drug Targets,2002,3(1):85-94.
9. Nakagawa K, Kawaura A, Kato S, et al.1 alpha,25-Dihydroxyvitamin D(3) is a preventive factor in the metastasis of lung cancer. Carcinogenesis,2005, 26(2):429-40.
10. Zinser GM,McEleney K,Welsh J.Characterization of mammary tumor cell lines from wild type and vitamin D3 receptor knockout mice. Mol Cell Endocrinol,2003,200(12):67-80.
11. Trump DL, Hershberger PA, Bernardi RJ, et al. Anti-tumor activity of calcitriol: pre-clinical and clinical studies. J Steroid Biochem Mol Biol,2004, 89-90(1-5):519-26.
12. Ylikomi T, Laaksi I, Rulou Y, et al. Antipoliferative action of vitamin D.Vitamins and Hormones,2002,64:357-406.
13. Jones G, Strugnell SA, DeLuca HF. Current understanding of the molecular actions of vitamin D. Physiol Rev,1998,78(4):1193-231.
14. Nakagawa K, Kawaura A, Kato S, et al. Metastatic growth of lung cancer cells is extremely reduced in Vitamin D receptor knockout mice. J Steroid Biochem Mol Biol,2004,89-90(1-5):545-547.
15. Menezes RJ, Cheney RT, Husain A,et al. Vitamin D Receptor Expression in Normal, Premalignant, and Malignant Human Lung Tissue.,2008,17:1104-1110, Cancer Epidemiol Biomarkers Prev,2008,17(5):1104-10.
16. Anderson MG, Nakane M, Ruan X, et al. Expression of VDR and CYP24A1 mRNA in human tumors. Cancer Chemother Pharmacol,2006,57(2):234-40.
17. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell, 2004,116:281-297.
18. Brueckner B, Stresemann C, Kuner R et al.The Human let-7a-3 Locus Contains an Epigenetically Regulated MicroRNA Gene with Oncogenic Function. Cancer Res,2007,67:1419-1423.
19. Zhang HH, Wang XJ, Li GX et al. Detection of let-7a microRNA by real-time PCR in gastric carcinoma. World J Gastroentero,2007,13:2883-8.
20. Lokshin A, Zhang H, Mayotte J et al. Early effects of retinoic acid on proliferation, differentiation and apoptosis in nonsmall cell lung cancer cell lines. Anticancer Res,1999,19:5251-4.
21. Choi EJ, Whang YM, Kim SJ et al. Combinational Treatment with Retinoic Acid Derivatives in Non-small Cell Lung Carcinoma In Vitro. J Korean Med Sci,2007,22:52-60.
22. Li JS, Zhu M, Tian D, et al. Effect of Lithium on Cell Proliferation and Cell Cycle in A549 Cells. Acta Med Univ Sci Technol Huazhong,2007,36:145-8.
23. Rao AS, Kremenevskaja N, Resch J et al. Lithium stimulates proliferation in cultured thyrocytes by activating Wnt/beta-catenin signaling. Eur J Endocrinol, 2005,153:929-938.
24. Hershberger PA, Modzelewski RA, Shurin ZR, Rueger RM, Trump DL, Johnson CS.1,25-Dihydroxycholecalciferol (1,25-D3) inhibits the growth of squamous cell carcinoma and downmodulates p21(Wafl/Cipl) in vitro and in vivo. Cancer Res,1999,59:2644-9.
25. Reichel H, Koeffler HP, Norman AW. The role of the vitamin D endocrine system in health and disease. New Engl J Med,1989,320:980-91.
26. Holick MF. Vitamin D deficiency. N Engl J Med,2007,357(3):266-81.
27. Samuel S, Sitrin MD.Vitamin D’S role in cell proliferation and differentiation. NutrRev,2008,66(10 Suppl 2):S116-24.
28.王英田,白雪丽,李怀臣.全反式维甲酸联合维生素D3对肺癌原代细胞化疗敏感性的影响.山东医药,2007,47(5)：9-11.
29. Calin GA, Sevignani C, Dumitru CD et al. Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. Proc Natl Acad Sci U S A.2004,101:2999-3004.
30. Calin G A, Dumitru CD, Shimizu M, et al. Frequent deletions and down-regulation of micro-RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci USA,2002,99(24):15524-29.
31. Caudy A A, Myers M, Hannon G J, et al. Fragile X-related protein and VIG associate with the RNA interference machinery. Genes Dev,2002, 16(19):2491-96.
32. Kumar M S, Erkeland S J, Pester R E, et al.Suppression of non-small cell lung tumor development by the let-7 microRNA family. Proc Natl Acad Sci USA,2008,105(10):3903-08.
33. Bartel D P. MicroRNAs genomics, biogenesis, mechanism and function. Cell, 2004,116(2):281-97.