摘要
目的:探讨结直肠癌(colorectal cancer,CRC)中环状RNA(circular RNA,circRNA)RNF13(circRNF13)的表达水平及临床意义,并探索circRNF13调控微RNA(microRNA,miRNA,miR)-196b-5p表达对CRC细胞增殖的影响。方法:采用circRNA芯片从6对CRC患者癌和癌旁组织中筛选出差异表达的circRNA;实时荧光定量PCR法检测80例CRC患者癌和癌旁组织及CRC细胞和人结肠黏膜上皮细胞FHC中circRNF13和miR-196b-5p的表达水平,并分析其与患者临床病理特征的关系。构建circRNF13过表达载体(circRNF13-OE),以空载体pcDNA(+)作为阴性对照(negative control,NC),分别转染HCT116和HT29细胞;CCK-8法检测细胞的增殖能力,实时荧光定量PCR法检测细胞中miR-196b-5p的表达水平。HCT116和HT29细胞分别转染NC、circRNF13-OE、miR-196b-5p-抑制子(miR-196b-5p-inhibitor)、circRNF13-OE+miR-196b-5p-模拟物(miR-196b-5p-mimics),再行CCK-8法检测细胞的增殖能力。结果:circRNA芯片检测结果显示,circRNF13在CRC组织中的表达水平明显低于癌旁组织。实时荧光定量PCR验证结果显示,CRC组织中circRNF13表达水平显著低于癌旁组织(P <0.001)。HCT116、HT29、SW620和SW480细胞中circRNF13的表达水平均明显低于FHC细胞(P值均<0.001)。circRNF13过表达后,HCT116和HT29细胞增殖能力受到明显抑制(P值均<0.05)。CRC组织以及HCT116、HT29、SW620和SW480细胞中miR-196b-5p的表达水平较癌旁组织和FHC细胞明显升高(P值均<0.01)。CRC组织中circRNF13的表达水平与miR-196b-5p的表达水平呈负相关(P=0.000 1,r=-0.484)。CRC患者癌组织中circRNF13和miR-196b-5p的表达水平与淋巴结转移、分化程度及TNM分期有关(P值均<0.05)。与circRNF13-OE组细胞相比,circRNF13-OE+miR-196b-5p-mimics组HCT116和HT29细胞的增殖能力均明显提高(P值均<0.05)。结论:CRC组织中circRNF13的表达水平下调,上调circRNF13的表达水平可抑制CRC细胞增殖,circRNF13通过下调miR-196b-5p表达促进CRC细胞的增殖。
Objective: To investigate the expression and clinical significance of circular RNA(circRNA)RNF13(circRNF13) in colorectal carcinoma(CRC), and to explore the effect of circRNF13 on the proliferation of CRC cells by regulating the expression of microRNA(miRNA, miR)-196 b-5 p.Methods: The expression profiles of circRNAs in 6 pairs of CRC tissues and para-cancer tissues were analyzed by circRNA microarray. The expression levels of circRNF13 and miR-196 b-5 p in 80 cases of human CRC and para-cancer tissues, human CRC cells and normal colorectal epithelial FHC cells were detected by real-time fluorescent quantitative PCR. The correlations of the expressions of circRNF13 and miR-196 b-5 p with the clinicopathological features of CRC patients were analyzed. The circRNF13 over-expression recombinant plasmid(circRNF13-OE) and the empty vector pcDNA3.1(+) as the negative control(NC) were designed and synthesised, and transfected into CRC HCT116 and HT29 cells, respectively.The proliferation capacity of HCT116 and HT29 cells transfected with circRNF13-OE and NC was detected by CCK-8 assay, and the expression level of miR-196 b-5 p was detected by real-time fluorescent quantitative PCR. Furthermore, HCT116 and HT29 cells were transfected with NC, circRNF13-OE, miR-196 b-5 p inhibitor or circRNF13-OE + miR-196 b-5 p mimic, then the proliferation capacity was detected by CCK-8 assay.Results: CircRNA microarray analysis showed that circRNF13 level in CRC tissues was significantly lower than that in para-cancer tissues. The result of real-time fluorescent quantitative PCR showed that the expression of circRNF13 in CRC tissues was significantly lower than that in para-cancer tissues(P < 0.001). The expression level of circRNF13 in HCT116, HT29, SW620 and SW480 cells was lower than that in FHC cells(all P < 0.001). Over-expression of circRNF13 inhibited the proliferation of HCT116 and HT29 cells(both P < 0.05). The expression of miR-196 b-5 p in CRC tissues and HCT116, HT29, SW620 and SW480 cells was significantly higher than that
作者
余昆
李强
王伟雅
蔡昕怡
程先硕
李云峰
YU Kun;LI Qiang;WANG Weiya;CAI Xinyi;CHENG Xianshuo;LI Yunfeng(Department of Colorectal Surgery,Tird Afliated Hospital of Kunming Medical University(Yunnan Cancer Hospital),Kunming 650118,Yunnan Province,China)
出处
《肿瘤》
CAS
CSCD
北大核心
2019年第11期887-896,共10页
Tumor
基金
云南省科技计划[编号：2017FE467(-072)]
云南省卫生科技计划（编号：2016NS101)~~
