LncRNA LINC01963靶向miR-182-5p对胃癌细胞增殖、凋亡的影响

Effects of LncRNA LINC01963 targeting miR-182-5p on proliferation and apoptosis of gastric cancer cells

目的探讨长链非编码(Lnc)RNA LINC01963调控miR-182-5p对胃癌细胞增殖、凋亡的影响。方法实时荧光定量-聚合酶链反应(qRT-PCR)检测35例胃癌患者癌组织、癌旁组织及人正常胃黏膜细胞GES-1、胃癌细胞AGS、MGC-803、MKN-45中LINC01963及miR-182-5p表达;将AGS细胞分为CK组、si-NC组、si-LINC01963组、pIRES2组、pIRES2-LINC01963组、pIRES2-LINC01963+mimic NC组、pIRES2-LINC01963+miR-182-5p mimic组。qRT-PCR检测各组细胞中LINC01963及miR-182-5p表达;CCK-8法检测细胞增殖;流式细胞术检测细胞凋亡;Western印迹检测增殖细胞核抗原(PCNA)、B细胞淋巴瘤(Bcl)-2相关X蛋白(Bax)、Bcl-2蛋白表达;双荧光素酶实验验证LINC01963和miR-182-5p的关系。体内裸鼠成瘤实验观察肿瘤生长情况。结果在胃癌组织和细胞中,LINC01963明显低表达,miR-182-5p明显高表达,且在AGS细胞中LINC01963表达最低,miR-182-5p表达最高(P<0.05),因此,选择AGS细胞为研究对象;与si-NC组比较,si-LINC01963组LINC01963表达明显降低,miR-182-5p表达明显升高(P<0.05);与pIRES2组比较,pIRES2-LINC01963组LINC01963表达明显升高,miR-182-5p表达明显降低(P<0.05);与pIRES2-LINC01963+mimic NC组比较,pIRES2-LINC01963+miR-182-5p mimic组LINC01963表达差异不显著(P>0.05),miR-182-5p表达明显升高(P<0.05);上调LINC01963可明显抑制AGS细胞增殖及PCNA、Bcl-2蛋白表达,明显促进细胞凋亡及Bax蛋白表达,还明显抑制裸鼠体内移植瘤生长,而下调LINC01963则呈明显相反趋势(P<0.05);过表达miR-182-5p可明显减弱上调LINC01963对AGS细胞增殖的抑制作用及对细胞凋亡的促进作用,还明显减弱对裸鼠体内移植瘤生长的抑制作用(P<0.05);LINC01963与miR-182-5p存在靶向调控关系。结论过表达LINC01963可通过靶向下调miR-182-5p抑制AGS细胞增殖,促进细胞凋亡。

Objective To investigate the effects of long non-coding(Lnc)RNA LINC01963 on the proliferation and apoptosis of gastric cancer cells by regulating miR-182-5p.Methods Real time fluorescence quantitative-polymerase chain reaction(qRT-PCR)was used to detect the expressions of LINC01963 and miR-182-5p in cancer tissues,adjacent tissues from 35 gastric cancer patients,and normal gastric mucosal cells GES-1,gastric cancer cells AGS,MGC-803 and MKN-45;AGS cells were divided into CK group,si-NC group,si-LINC01963 group,pIRES2 group,pIRES2-LINC01963 group,pIRES2-LINC01963+mimic NC group and pIRES2-LINC01963+miR-182-5p mimic group.qRT PCR was used to detect the expressions of LINC01963 and miR-182-5p in each group of cells;CCK-8 method was used to detect cell proliferation;cell apoptosis was detected by flow cytometry;Western blot was used to detect the expressions of proliferating cell nuclear antigen(PCNA),B-cell lymphoma(Bcl)-2 related X protein(Bax)and Bcl-2 protein;the dual luciferase assay was used to verify the relationship between LINC01963 and miR-182-5p.Tumor growth was observed by in vivo tumor formation experiment in nude mice.Results In gastric cancer tissues and cells,LINC01963 was significantly downregulated and miR-182-5p was significantly overexpressed,LINC01963 had the lowest expression and miR-182-5p had the highest expression in AGS cells(P<0.05).Therefore,AGS cells were selected as the research object;compared with si-NC group,the expression of LINC01963 in the si-LINC01963 group was significantly reduced,while the expression of miR-182-5p was significantly increased(P<0.05);compared with pIRES2 group,the LINC01963 expression was significantly increased,miR-182-5p expression was significantly decreased in pIRES2-LINC01963 group(P<005);compared with pIRES2-LINC01963+mimic NC group,there was no significant difference in the expression of LINC01963 in the pIRES2-LINC01963+miR-182-5p mimic group(P>005),miR-182-5p expression was significantly increased(P<0.05);upregulation of LINC01963 could significantly inhibit the proliferation of AGS cells and the expressions of PCNA and Bcl-2 protein,promote cell apoptosis and Bax protein expression,and also significantly inhibit the growth of transplanted tumors in nude mice in vivo.However,downregulation of LINC01963 showed a significant opposite trend(P<0.05);overexpression of miR-182-5p could significantly weaken the inhibitory effect of up-regulated LINC01963 on AGS cell proliferation and promot cell apoptosis,while also significantly weakening its inhibitory effect on the growth of transplanted tumors in nude mice(P<0.05);LINC01963 had a targeted regulatory relationship with miR-182-5p.Conclusions Overexpression of LINC01963 could inhibit AGS cell proliferation and promote apoptosis by targeting down regulation of miR-182-5p.