长链非编码RNA CYTOR靶向调控miR-206对结肠癌细胞生物学行为的影响

Effect of long non-coding RNA CYTOR targeting miR-206 on biological behavior of colonic cancer cells

目的探讨长链非编码RNA(lncRNA)CYTOR靶向调控微小RNA-206(miR-206)对结肠癌细胞增殖、侵袭和迁移的影响。方法选择结肠癌患者30例,收集结肠癌组织及其配对的癌旁正常组织,采用实时荧光定量PCR(RT-qPCR)法检测CYTOR、miR-206表达,并分析结肠癌组织中CYTOR表达与miR-206表达的关系。取传2代、对数生长期、生长状态良好的结肠癌LoVo细胞,随机分为CYTOR组与对照1组、miR-206组与对照2组,分别转染CYTOR shRNA与阴性对照shRNA、miR-206 mimics与阴性对照miRNA,采用RT-qPCR法验证转染效率。收集各组转染后细胞,采用CCK-8法检测细胞增殖能力,采用Transwell小室实验检测细胞侵袭和迁移能力。通过StarBase数据库(http://starbase.sysu.edu.cn/index.php)确定CYTOR和miR-206的结合位点。将人胚肾上皮HEK-293T细胞接种于12孔板中,随机分为pMIR-CYTOR-wt+miR-206 mimics组、pMIR-CYTOR-wt+阴性对照miRNA组、pMIR-CYTOR-mut+miR-206 mimics组、pMIR-CYTOR-mut+阴性对照miRNA组,相应转染pMIR-CYTOR-wt、pMIR-CYTOR-mut、miR-206 mimics、阴性对照miRNA,采用双荧光素酶报告基因实验检测各组转染后荧光素酶活性。结果结肠癌组织CYTOR相对表达量明显高于癌旁正常组织,miR-206相对表达量明显低于癌旁正常组织(P均<0.01)。Pearson相关分析显示,结肠癌组织CYTOR相对表达量与miR-206相对表达量呈负相关关系(r=-0.599,P<0.01)。CYTOR组CYTOR相对表达量明显低于对照1组,miR-206组miR-206相对表达量明显高于对照2组(P均<0.05)。CYTOR组细胞增殖、侵袭和迁移能力均明显低于对照1组(P均<0.05);miR-206组细胞增殖、侵袭和迁移能力均明显低于对照2组(P均<0.05)。通过搜索CYTOR潜在的目标基因发现,CYTOR包含miR-206的保守目标位点。双荧光素酶报告基因实验显示,pMIR-CYTOR-wt+miR-206 mimics组荧光素酶活性明显低于pMIR-CYTOR-wt+阴性对照miRNA组、pMIR-CYTOR-mut+miR-206 mimics组、pMIR-CYTOR-mut+阴性对照miRNA组,而pMIR-CYTOR-wt+阴性对照miRNA组、pMIR-CYTOR-mut+miR-206 mimics组、pMIR-CYTOR-mut+阴性对照miRNA组两两比较P均>0.05。结论CYTOR通过靶向抑制miR-206表达促进结肠癌细胞增殖、侵袭和迁移。

Objective To investigate the effects of long non-coding RNA CYTOR targeting microRNA-206(miR-206)on the proliferation,invasion,and migration of colon cancer cells.Methods Thirty patients with colonic cancer were selected,and the colonic cancer tissues and their matched normal tissues were collected.The expression levels of CYTOR and miR-206 were detected by quantitative real-time PCR(RT-qPCR),and the relationship between the two expression levels in colonic cancer tissues was analyzed.LoVo cells of the second generation,in logarithmic phase and good growth,were randomly divided into four groups:the CYTOR knockdown group,the control group,the miR-206 group,and the control miRNA group.The transfection efficiency was verified by RT-qPCR.After the transfection,the cells were collected and proliferation was detected by CCK-8,and the cell invasion and migration were detected by Transwell experiment.The binding sites of CYTOR and miR-206 were predicted by Starbase database(http://starbase.sysu.edu.cn/index.php).HEK-293T cells were inoculated into 12-well plates and randomly divided into the pMIR-CYTOR-wt+miR-206 mimics group,pMIR-CYTOR-wt+control miRNA group,pMIR-CYTOR-mut+miR-206 mimics group,and pMIR-CYTOR-mut+control miRNA group.The luciferase activity of each group was detected by dual luciferase reporter gene test.Results The relative expression of CYTOR in the colonic cancer was significantly higher than that in the normal tissues,and miR-206 was significantly lower than that in normal tissues(P<0.01).Pearson correlation analysis showed that there was a negative correlation between the relative expression of CYTOR and miR-206(r=-0.599,P<0.01).The relative expression of CYTOR in the knockdown group was significantly lower than that in the control group,and that in the miR-206 group was significantly higher than that in the miRNA control group(P<0.05).The cell proliferation,invasion and migration abilities in the CYTOR knockdown group were significantly lower than those in the control group(all P<0.05);the cell proliferation,invasion and migration abilities in the miR-206 group was significantly lower than that in the miRNA control group(all P<0.05).CYTOR contained the conservative target site of miR-206.The dual luciferase reporter gene test showed that the luciferase activity of pMIR-CYTOR-wt+miR-206 mimics group was significantly lower than that of pMIR-CYTOR-wt+negative control miRNA group,pMIR-CYTOR-mut+miR-206 mimics group,and pMIR-CYTOR-mut+negative control miRNA group,while there were no differences between the pMIR-CYTOR-wt+negative control miRNA group,pMIR-CYTOR-mut+miR-206 mimics group,and pMIR-CYTOR-mut+negative control miRNA group.Conclusion CYTOR promotes the proliferation,invasion and migration abilities of colon cancer cells by targeting miR-206.