circ_0000218通过靶向吸附miR-1182对宫颈癌HeLa细胞增殖、迁移和侵袭的影响

Effects of circ_0000218 on the proliferation,migration and invasion of cervical cancer HeLa cells by sponging miR-1182

目的探讨环状RNA 0000218(circ_0000218)是否通过靶向吸附miR-1182从而影响宫颈癌HeLa细胞增殖、迁移和侵袭。方法采用实时荧光定量PCR(RT-qPCR)技术分析43例宫颈癌患者癌组织、癌旁组织中circ_0000218和miR-1182的表达水平。根据转染序列不同分为si-NC组、si-circ_0000218组、miR-NC组、miR-1182组、pcDNA组、pcDNAcirc_0000218组、si-circ_0000218+anti-miR-NC组、si-circ_0000218+anti-miR-1182组。运用细胞计数试剂盒(CCK-8)法、Transwell实验分析circ_0000218和miR-1182表达对HeLa细胞增殖、迁移和侵袭的影响。蛋白质印迹法检测Ki-67、基质金属蛋白酶2(MMP-2)和MMP9蛋白表达。双荧光素酶报告实验和RT-qPCR分析circ_0000218和miR-1182的靶向关系。癌旁组织与宫颈癌组织比较采用配对t检验,两组间比较采用独立样本t检验进行统计学分析。结果宫颈癌组织中circ_0000218表达量高于癌旁组织(4.17±0.32比1.00±0.05),而miR-1182表达量低于癌旁组织(0.33±0.03比1.00±0.05),差异具有统计学意义(P均<0.001)。与si-NC组比较,si-circ_0000218组HeLa细胞增殖活力(0.86±0.04比0.37±0.03)、迁移数量[(86.73±7.13)个比(38.52±3.19)个]和侵袭数量[(66.80±4.95)个比(26.58±2.55)个]以及Ki-67(0.57±0.05比0.18±0.02)、MMP-2(0.74±0.07比0.28±0.03)和MMP-9蛋白表达量(0.64±0.04比0.22±0.02)降低,差异有统计学意义(P均<0.001).与miR-NC组比较,miR-1182组HeLa细胞增殖活力(0.88±0.04比0.46±0.04)、迁移数量[(89.74±5.53)个比(46.63±3.79)个]和侵袭数量[(68.03±4.34)个比(34.63±3.37)个]以及Ki-67(0.59±0.04比0.24±0.02)、MMP-2(0.76±0.05比0.33±0.03)和MMP-9蛋白表达量(0.66±0.04比0.29±0.03)降低,差异有统计学意义(P均<0.001)。circ_0000218靶向负调控miR-1182表达。与si-circ_0000218+anti-miR-NC组比较,si-circ_0000218+anti-miR-1182组HeLa细胞增殖活力(0.35±0.03比0.76±0.04)、迁移数量[(35.58±3.11)个比(77.04±4.08)个]和侵袭数量[(25.44±2.29)个比(57.61±3.47)个]以及Ki-67(0.16±0.02比0.46±0.04)、MMP-2(0.26±0.02比0.65±0.04)和MMP-9蛋白表达量(0.20±0.02比0.57±0.04)升高,差异有统计学意义(P均<0.001)。结论circ_0000218通过靶向吸附miR-1182可促进宫颈癌HeLa细胞增殖、迁移和侵袭。

Objective To explore whether circular RNA 0000218(circ_0000218)sponges miR-1182 to regulate the proliferation,migration and invasion of cervical cancer HeLa cells.Methods Expression levels of circ_0000218 and miR-1182 in the cancer tissues and paracancer tissues from 43 patients with cervical cancer were analyzed by real-time quantitative PCR(RT-qPCR).Cervical cancer cells HeLa were divided into si-NC group,si-circ_0000218 group,miR-NC group,miR-1182 group,si-circ_0000218+anti-miR-NC group,and si-circ_0000218+anti-miR-1182 group.The cell counting kit(CCK-8)method and Transwell experiment were used to analyze the effects of circ_0000218 and miR-1182 expression on the proliferation,migration and invasion of HeLa cells.Western blotting was used for detecting the expression of Ki-67,matrix metalloproteinase-2(MMP-2)and MMP9 proteins.The dual luciferase reporter assay and RT-qPCR were applied to validate the targeting relationship between circ_0000218 and miR-1182.Pared t test was used to compare the difference of the expression of circ_0000218 and miR-1182 between cancer tissues and paracancer tissues.Independenr sample t test was used to compare the difference between groups.Results The expression of circ_0000218 in the cervical cancer tissue was significantly higher than that in the adjacent tissues(4.17±0.32 vs 1.00±0.05,P<0.001),while the expression of miR-1182 was significantly lower than that in the adjacent tissues(0.33±0.03 vs 1.00±0.05,P<0.001).Compared with the si-NC group,the HeLa cell proliferation activity(0.86±0.04 vs 0.37±0.03),migration(86.73±7.13 vs 38.52±3.19)and invasion(66.80±4.95 vs 26.58±2.55),and the expression levels of Ki-67(0.57±0.05vs0.18±0.02),MMP-2(0.74±0.07vs0.28±0.03)and MMP-9(0.64±0.04 vs 0.22±0.02)proteins in the si-circ_0000218 group were significantly decreased(P<0.001).Compared with the miR-NC group,the proliferation(0.88±0.04 vs 0.46±0.04),migration(89.74±5.53 vs 46.63±3.79)and invasion(68.03±4.34 vs 34.63±3.37)of HeLa cells,and the expression of Ki-67(0.59±0.04 vs 0.24±0.02),MMP-2(0.76±0.05 vs 0.33±0.03)and MMP-9(0.66±0.04 vs 0.29±0.03)proteins in the miR-1182 group were decreased significantly.Circ_0000218 targeted and negatively regulated miR-1182 expression(P<0.001).Compared with the si-circ_0000218+anti-miR-NC group,the HeLa cell proliferation activity(0.35±0.03 vs 0.76±0.04),migration(35.58±3.11 vs 77.04±4.08)and invasion(25.44±2.29 vs 57.61±3.47),and the expression levels of Ki-67(0.16±0.02 vs 0.46±0.04),MMP-2(0.26±0.02 vs 0.65±0.04)and MMP-9(0.20±0.02 vs 0.57±0.04)proteins in the si-circ_0000218+anti-miR-1182 group were significantly increased(P<0.001).Conclusion circ_0000218 can promote the proliferation,migration and invasion of cervical cancer HeLa cells by sponging miR-1182.