Wu Kaiming, He Yulong, Li Guanghua, Peng Jianjun
Gastrointestinal Surgery Center, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China.
Zhonghua Wei Chang Wai Ke Za Zhi. 2015 Oct;18(10):1041-6.
To screen the microRNAs involved in colon cancer proliferation and to investigate the expression and regulating function of target miRNA in colon cancer.
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 HCT116) 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 MTT and BrdU assay after lentivirus transfection.
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-204 was negatively correlated with TFAM expression in colon cancer tissues(P<0.05). Dual luciferase reporter assays revealed TFAM could be integrated with miR-204 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).
MiR-204 inhibits TFAM expression and up-regulates the proliferation of colon cancer cells SW480.
筛选参与结肠癌增殖的微小RNA，并研究靶标微小RNA在结肠癌中的表达及调控功能。
将前期研究证实的结肠癌癌基因线粒体转录因子A（TFAM）作为靶基因，利用miRWalk 2.0软件筛选参与结肠癌增殖的微小RNA。采用实时荧光定量PCR检测筛选出的微小RNA在30例结肠癌组织、癌旁组织、正常结肠细胞株及3种结肠癌细胞株（SW480、HT-29和HCT116）中的表达。选取表达量最低的miR-204在SW480细胞中进一步研究。采用双荧光素酶报告基因检测法检测TFAM与miR-204的相关性。分别用抗miR-204慢病毒和miR-240慢病毒下调和上调miR-204表达。慢病毒转染后，采用蛋白质免疫印迹法检测SW480细胞中TFAM蛋白表达变化，采用MTT法和BrdU法检测SW480细胞增殖变化。
筛选出候选微小RNA为miR-204、miR-211、miR-214、miR-381和miR-590-3p。结肠癌组织中miR-204、miR-211、miR-214和miR-381表达低于癌旁组织，miR-590-3p表达高于癌旁组织（均P<0.05）。同时，SW480、HT-29和HCT116细胞中上述4种微小RNA（miR-204、miR-211、miR-21
