线粒体转录因子A相关的微小RNA在结肠癌中的表达和增殖调控研究

Expression and proliferative regulation of miR-204 related to mitochondrial transcription factor A A in colon cancer

目的筛选并验证可调控结肠癌细胞增殖的微小RNA（miRNA）。方法以癌基因线粒体转录因子A（TFAM）作为靶基因，通过miRwalk2．0软件筛选可能调控结肠癌增殖的miRNA。通过实时荧光定量PCR检测miRNA在30例结肠癌手术标本和3种结肠癌细胞系（SW480、HT-29和HCT116）中的表达。选取其中表达量最低的miR．204和SW480进行后续研究。双荧光素酶报告基因实验验证TFAM和miR-204的作用关系。分别应用miR-204慢病毒和anti—miR-204慢病毒上调和下调miR-204的表达，通过Westernblot检测慢病毒感染后SW480细胞中TFAM蛋白水平的变化：并采用MTT实验和Brdu实验观察慢病毒感染后SW480细胞增殖能力的变化。结果初筛获得miR-204、miR-211、miR-214、miR-381、miR-590．3p5种miRNA，其中miR-204、miR-211、miR-214和miR-381在结肠癌组织中的表达较癌旁组织明显降低（P〈0．05），在3种结肠癌细胞中的表达亦较正常结肠细胞降低（P〈0．05），其中miR-204的表达量最低；而miR-590—3p表达水平则在结肠癌组织和3种结肠癌细胞中均明显上调（P〈0．05）。结肠癌组织中miR-204表达与TFAM表达呈显著负相关（P〈0．05）。双荧光素酶报告基因实验显示TFAM与miR-204可直接结合。通过慢病毒感染上调miR-204表达后，SW480细胞中TFAM蛋白表达下降，细胞增殖能力升高（P〈O．05）；下调miR-204表达后，TFAM蛋白表达升高，细胞增殖能力下降（P〈0．05）。结论miR-204可以通过降低TFAM的蛋白表达来抑制结肠癌细胞的增殖。

Objective To screen the microRNAs involved in colon cancer proliferation and to investigate the expression and regulating function of target miRNA in colon cancer. Methods Mitochondrial transcription factor A （TFAM）, which was proved to be an oncogene to colon cancer in prior study, was used as target gene. The microRNAs involved in colon cancer proliferation were screened with miRWalk 2.0 software. The expression of screened miRNAs was examined in 30 samples of colon cancer tissue, para-cancer tissue, normal colon cell strain, and 3 colon cancer strains （SW480, HT-29, and HCTll6） by real-time PCR. MiR-204 presenting lowest expression was selected to further study in SW480 cells. Dual luciferase reporter assays was performed to examine the association of TFAM with miR-204. Anti-miR-204 lentivirus and miR-240 lentivirus were used to down- regulate and up-regulate miR-204 expression respectively. Change of TFAM protein expression in SW480 cells was detected by Western blotting, and change of SW480 cells proliferation was detected by MTY and BrdU assay after lentivirus trausfection. Results After screening, the candidate miRNAs were miR-204, miR-211, miR-214, miR-381 and miR-590-3p. Expressions of miR-204, miR-211, miR- 214 and miR-381 were lower, but miR-590-3p expression was higher, in colon cancer tissues than those in para-cancer tissues （all P〈0.05）. Meanwhile expressions of above 4 miRNAs（miR-204, miR-211, miR-214 and miR-381） were also lower, but miR-590-3p expression was higher as well, in SW480, HT-29 and HCT116 cells compared to normal colon cells （all P〈0.05）. Among above 4 miRNAs, miR-204 showed the lowest expression in both colon cancer tissues and cell lines. Expression of miR-20g was negatively correlated with TFAM expression in colon cancer tissues（P〈0.05）. Dual luciferase reporter assays revealed TFAM could be integrated with miR-20g directly, suggesting TFAM as the direct target of miR-204. After up-regulating miR-204 by lentivirus, expression of TFAM decreased and proliferation increased in SW480 cells （all P〈0.05）. After down-regulating miR-204 by lentivirus, expression of TFAM increased and proliferation decreased in SW480 cells （all P〈0.05）. Conclusion MiR-204 inhibits TFAM expression and up-regulates the proliferation of colon cancer cells SW480.