miR-3677-3p在胃癌组织和癌细胞中表达及靶向抑制RAP1GAP对癌细胞增殖和侵袭影响

Expression of miR-3677-3p in gastric cancer tissues and cell linesand its effect on proliferation and invasion of gastric cancer cellsby targeting Rap1 GTPase-Activating Protein 1

目的旨在揭示miR-3677-3p在胃癌组织中的表达情况,及其是否通过靶向RAP1 GTPase激活蛋白(RAP1GAP)影响胃癌细胞的增殖、凋亡和侵袭功能。方法收集2016-10-10-2018-06-08陕西省人民医院手术治疗的40例胃癌患者癌和癌旁组织(距离癌组织边缘>5 cm),qRT-PCR检测miR-3677-3p在临床组织标本及正常胃黏膜上皮细胞(GES-1)和胃癌细胞(SNU-1、AGS、HGC-27、SGC-7901)的表达情况。选取AGS细胞进行转染,分为NC inhibitor组、miR-3677-3p inhibitor组、NC mimics组、miR-3677-3p mimics组、si-NC组、si-RAP1GAP组和si-RAP1GAP+miR-3677-3p inhibitor组。采用qRT-PCR检测转染效率,CCK-8法、流式细胞仪和Transwell分别检测转染NC inhibitor、miR-3677-3p inhibitor、si-RAP1GAP+miR-3677-3p inhibitor对AGS细胞增殖、凋亡和侵袭能力的影响。各组数据经Shapiro-Wilk检验符合正态分布,2组比较采用t检验,多组比较采用单因素方差分析,组间的多重比较采用LSD-t检验。结果 miR-3677-3p在胃癌组织的相对表达量(2.443±1.006)显著高于癌旁对照组织(1.000±0.265),差异有统计学意义,t=8.941,P<0.001。miR-3677-3p在胃癌细胞株SNU-1(1.975±0.310)、AGS(2.439±0.635)、HGC-27(2.223±0.462)和SGC-7901(1.604±0.405)相对表达水平显著高于GES-1细胞(1.000±0.173),差异有统计学意义,F=8.846,P<0.001。miR-3677-3p inhibitor组细胞增殖水平(0.873±0.132)与NC inhibitor组(1.323±0.122)相比显著下降,差异有统计学意义,t=5.598,P<0.001。miR-3677-3p inhibitor组细胞凋亡比例(33.11±5.36)与NC inhibitor组(9.17±2.12)相比显著增高,差异有统计学意义,t=9.287,P<0.001。穿过Transwell小室的细胞数量miR-3677-3p inhibitor组(78.52±21.25)与NC inhibitor组(232.30±33.65)相比显著下降,差异有统计学意义,t=8.642,P<0.001。双荧光素酶报告基因结果显示miR-3677-3p能够结合RAP1GAP的3′UTR。RAP1GAP的mRNA相对表达水平miR-3677-3p inhibitor组(3.210±0.540)显著高于NC inhibitor组(1.000±0.242),差异有统计学意义,t=8.351,P<0.001,蛋白质印迹法也得出了类似结果。功能回复实验显示,si-RAP1GAP部分逆转了miR-3677-3p inhibitor对AGS细胞的增殖、凋亡和侵袭的影响。结论 miR-3677-3p在胃癌组织和细胞中高表达,其可能通过靶向抑制RAP1GAP的表达,进而调控了胃癌细胞的增殖、凋亡和侵袭。

Objective To reveal the expression and function of miR-3677-3 p in gastric cancer, and further explore its mechanism.Methods The cancer tissues and paired paracancerous tissue(at least 5 cm from the edge of the cancer tissue) of 40 patients with gastric cancer from 2016-10-10 to 2018-06-08 in Shaanxi Provincial People’s Hospital were collected, and the expression of miR-3677-3 p was detected by qRT-PCR.The expression levels of miR-3677-3 p in normal gastric epithelial cells(GES-1) and gastric cancer cells(SNU-1,AGS,HGC-27,SGC-7901) were detected by qRT-PCR.The AGS cells were transfected and grouped as follows: NC inhibitor, miR-3677-3 p inhibitor, NC mimics, miR-3677-3 p mimics, si-NC,si-RAP1 GAP,and si-RAP1 GAP+ miR-3677-3 p inhibitor.The transfection efficiency was detected by qRT-PCR.The effects of miR-3677-3 p on proliferation,apoptosis and invasion of AGS cells were detected by CCK-8,flow cytometry and Transwell,respectively.The relationship between miR-3677-3 p and RAP1 GAP was detected by dual-luciferase reporter assay,qRT-PCR and Western blotting.The functional verification was performed by rescue assay.All data were conformed to the normal distribution as determined by the Shapiro-Wilk test.The comparison between the two groups was performed by t-test.The comparison between multiple groups was performed by one-way ANOVA,and the multiple comparison between groups was performed by LSD-t test.Results The relative expression level of miR-3677-3 p in gastric cancer tissues(2.443±1.006)was significantly higher than that in the paired paracancerous tissues(1.000±0.265),t=8.941,P<0.001.The relative expression levels of miR-3677-3 p in gastric cancer cell lines:SNU-1(1.975±0.310),AGS(2.439±0.635),HGC-27(2.223±0.462)and SGC-7901(1.604±0.405)were significantly higher than those in GES-1 cells(1.000±0.173),F=8.846,P<0.001.The cell proliferation level of the miR-3677-3 p inhibitor group(0.873±0.132)were significantly lower than that of NC inhibitor group(1.323±0.122),t=5.598,P<0.001.The apoptosis rate of the miR-3677-3 p inhibitor group(33.11±5.36)was significantly more than that of the NC inhibitor group(9.17±2.12),t=9.287,P<0.001.The number of invasive cells in the miR-3677-3 p inhibitor group(78.52±21.25)was significantly lower than that in the NC inhibitor group(232.30±33.65),t=8.642,P<0.001.The dual-luciferase reporter assay showed that miR-3677-3 p could bind to the 3′UTR of RAP1 GAP.The relative mRNA expression level of RAP1 GAPin the miR-3677-3 p inhibitor group(3.210±0.540)was significantly higher than that in the NC inhibitor group(1.000±0.242),t=8.351,P<0.001.Similar results were obtained by western blot.Rescue assay showed that si-RAP1 GAP partially reversed the effects of miR-3677-3 p inhibitor on the proliferation,apoptosis and invasion of AGS cells.Conclusion miR-3677-3 p is highly expressed in gastric cancer tissues and cells,which promotes proliferation and invasion of gastric cancer cells and inhibits apoptosis of gastric cancer cells by targeting RAP1 GAPexpression.