circ_RNF111靶向miRNA-361-5p对非小细胞肺癌细胞增殖和凋亡能力的影响

Effect of circ_RNFcirc_RNF111 targeting miRNA-miRNA-361-5p on proliferation and apoptosis ofp non-small cell lung cancer cells

目的探讨circ_RNF111靶向miRNA-361-5p对非小细胞肺癌(NSCLC)细胞增殖和凋亡能力的影响。方法取80例NSCLC患者的肿瘤组织及相应癌旁组织,采用实时荧光定量逆转录聚合酶链反应(qRT-PCR)检测circ_RNF111和miRNA-361-5p的相对表达量,并分析不同临床特征NSCLC患者肿瘤组织中circ_RNF111和miRNA-361-5p的表达情况。采用双荧光素酶报告实验检测circ_RNF111和miRNA-361-5p的靶向关系。将siNC、si-circ_RNF111、miRNA-NC、miRNA-361-5p mimic分别转染至A549细胞,分别作为si-NC组、si-circ_RNF111组、miRNA-NC组、miRNA-361-5p mimic组;将si-circ_RNF111分别与anti-miRNA-NC、miRNA-361-5p inhibitor共转染至A549细胞,分别作为si-circ_RNF111+anti-miRNA-NC组、si-circ_RNF111+miRNA-361-5p inhibitor组,采用噻唑蓝(MTT)法检测细胞增殖抑制率,克隆形成实验检测细胞集落形成数,流式细胞术检测细胞凋亡率。结果NSCLC患者肿瘤组织中circ_RNF111的相对表达量明显高于癌旁组织,miRNA-361-5p的相对表达量明显低于癌旁组织,TNM分期为Ⅲ~Ⅳ期的NSCLC患者肿瘤组织中circ_RNF111相对表达量明显高于Ⅰ~Ⅱ期患者,miRNA-361-5p相对表达量明显低于Ⅰ~Ⅱ期患者,差异均有统计学意义(P﹤0.01)。Pearson相关分析显示,circ_RNF111的表达与miRNA-361-5p的表达呈负相关(r=-0.949,P﹤0.05)。miRNA-361-5p组细胞WT-circ_RNF111相对表达量明显低于miRNA-NC组,差异有统计学意义(P﹤0.05)。si-circ_RNF111组circ_RNF111相对表达量明显低于si-NC组,miRNA-361-5p相对表达量明显高于si-NC组,差异均有统计学意义(P﹤0.01)。si-circ_RNF111组细胞增殖抑制率和细胞凋亡率均高于si-NC组,集落形成数低于si-NC组,miRNA-361-5p mimic组细胞增殖抑制率和细胞凋亡率均高于miRNA-NC组,集落形成数低于miRNA-NC组,si-circ_RNF111+miRNA-361-5p inhibitor组A549细胞增殖抑制率和细胞凋亡率均低于si-circ_RNF111+anti-miRNA-NC组,集落形成数高于si-circ_RNF111+anti-miRNA-NC组,差异均有统计学意义(P﹤0.05)。结论circ_RNF111可通过靶向负调控miRNA-361-5p的表达来抑制NSCLC细胞的增殖,并促进其凋亡。

Objective To investigate the effect of circ_RNF111 targeting miRNA-361-5p on the proliferation and apoptosis of non-small cell lung cancer(NSCLC)cells.Method The tumor tissues and corresponding paracancerous tissues of 80 NSCLC patients were collected.The relative expression levels of circ-RNF111 and miRNA-361-5p were detected by the real-time quantitative reverse transcription-polymerase chain reaction(qRT-PCR)method.The expression of circ_RNF111 and miRNA-361-5p in tumor tissue of NSCLC patients with different clinical characteristics were also analyzed.The dual-luciferase reporter assay was used to detect the targeting relationship between circ_RNF111 and miRNA-361-5p.The si-NC,si-circ_RNF111,miRNA-NC,and miRNA-361-5p mimic were transfected into A549 cells,respectively,and set as the si-NC group,si-circ_RNF111 group,miRNA-NC group,and miRNA-361-5p mimic group,respectively.The si-circ_RNF111 was co-transfected with anti-miRNA-NC and miRNA-361-5p inhibitor into A549 cells,respectively,and set as si-circ_RNF111+anti-miRNA-NC group and si-circ_RNF111+miRNA-361-5p inhibitor group,respectively.Cell proliferation inhibition rate was detected by methyl thiazolyl terazolium(MTT)assay.A colony formation assay was used to detect the number of colonies formed.The apoptosis rate was detected by flow cytometry.Result The relative expression of circ-RNF111 in tumor tissue of NSCLC patients was significantly higher than that in adjacent tissue,and the relative expression of miRNA-361-5p was significantly lower than that in adjacent tissue,and the differences were statistically significant(P<0.01).The relative expression of circ-RNF111 in tumor tissue of NSCLC patients with TNM stage III-IV was significantly higher than that of patients with TNM stage I-II,and the relative expression of miRNA-361-5p was significantly lower than that of patients with TNM stage I-II,and the differences were statistically significant(P<0.01).Pearson correlation analysis showed that circ_RNF111 was negatively correlated with the expression of miRNA-361-5p(r=-0.949,P<0.05).The relative expression of WT-circ_RNF111 in the miRNA-361-5p group was significantly lower than that in the miRNA-NC group,and the difference was statistically significant(P<0.05).The relative expression of circ_RNF111 in the si-circ_RNF111 group was significantly lower than that in the si-NC group,and the relative expression of miRNA-361-5p was significantly higher than that in the si-NC group(P<0.01).The cell proliferation inhibition and apoptosis rate in the si-circ-RNF111 group were higher than those in the si-NC group,and the number of colonies formed was lower than that in the si-NC group,and the differences were statistically significant(P<0.05).The cell proliferation inhibition and apoptosis rate in the miRNA-361-5p mimic group were higher than those in the miRNANC group,and the number of colonies formed was lower than that in the miRNA-NC group,and the differences were statistically significant(P<0.05).The proliferation inhibition and apoptosis rate of A549 cells in the si-circ-RNF111+miRNA-361-5p inhibitor group were lower than those in the si-circ-RNF111+anti-miRNA-NC group,and the number of colonies formed was higher than that in si-circ-RNF111+anti-miRNA-NC group,and the differences were statistically significant(P<0.05).Conclusion circ-RNF111 could inhibit the proliferation of NSCLC cells and promote their apoptosis by targeting miRNA-361-5p and negatively regulating its expression.