摘要
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.
作者
普彦淞
李真真
段宝军
马钰
王建华
高增战
PU Yan-song;LI Zhen-zhen;DUAN Bao-jun;MA Yu;WANG Jian-hua;GAO Zeng-zhan(Shaanxi Provincial People's Hospital,Xian 710068,China)
出处
《中华肿瘤防治杂志》
CAS
北大核心
2021年第15期1144-1150,共7页
Chinese Journal of Cancer Prevention and Treatment
基金
陕西省自然科学基础研究计划(2020JQ-947,2019JQ-973,2018JM7113)
2017年陕西省创新能力支撑计划(2017KCT-28)