长链非编码RNA C5orf66-AS1通过靶向miR-637对胃癌细胞增殖、凋亡的影响

Effects of long non-coding RNA C5orf66-AS1 on the proliferation and apoptosis of gastric cancer cells by targeting miR-637

目的:探讨长链非编码RNA C5orf66-AS1通过靶向miR-637对胃癌(GC)细胞增殖、凋亡的影响,为GC临床治疗提供依据。方法:收集32例GC病人的癌组织及其癌旁组织并体外培养人胃腺癌细胞系MKN28、MKN451、BGC823、MGC803、AGS及正常胃上皮细胞系GES-1,qRT-PCR实验检测其C5orf66-AS1和miR-637表达。将对数生长期的AGS细胞利用脂质体转染试剂进行转染并分为对照组、GV144组、GV144-C5orf66-AS1组、miR-NC组、miR-637 mimics组、GV144-C5orf66-AS1+miR-NC组、GV144-C5orf66-AS1+miR-637 mimics组。CCK-8法检测细胞增殖能力;流式细胞术分析细胞凋亡情况;Western blotting实验测定细胞周期蛋白CyclinD1、B淋巴细胞瘤-2基因(Bcl-2)及其同源蛋白Bax相对表达水平;RNA免疫共沉淀实验验证C5orf66-AS1和miR-637的作用关系。结果:与癌旁组织或正常胃上皮细胞系GES-1相比,C5orf66-AS1和miR-637在GC组织和细胞系MKN28、MKN451、BGC823、MGC803、AGS中的表达水平均降低(P<0.05),选择C5orf66-AS1表达水平最低的AGS细胞进行转染实验。转染GV144-C5orf66-AS1后,AGS细胞中C5orf66-AS1、miR-637表达水平、凋亡率及Bax蛋白表达升高,细胞活性(48、72、96 h)及CyclinD1、Bcl-2蛋白降低(P<0.05)。C5orf66-AS1和miR-637存在相互作用关系。miR-637过表达可使C5orf66-AS1过表达对AGS细胞增殖的抑制作用和凋亡的促进作用更明显(P<0.05)。结论:C5orf66-AS1和miR-637在GC组织和细胞中低表达,过表达C5orf66-AS1和上调miR-637,可协同抑制GC细胞的增殖,促进凋亡。

Objective:To investigate the effects of long non-coding RNA C5orf66-AS1 on the proliferation and apoptosis of gastric cancer(GC)cells by targeting miR-637,so as to provide a basis for the clinical treatment of GC.Methods:The cancer tissues and adjacent tissues of 32 GC patients were collected.Human gastric adenocarcinoma cell lines MKN28,MKN451,BGC823,MGC803,AGS and normal gastric epithelial cell line GES-1 were cultured in vitro.The expression of C5orf66-AS1 and miR-637 was detected by qRT-PCR.AGS cells in logarithmic growth phase were transfected with liposome transfection reagent and divided into control group,GV144 group,GV144-C5orf66-AS1 group,miR-NC group,miR-637 mimics group,GV144-C5orf66-AS1+miR-NC group,and GV144-C5orf66-AS1+miR-637 mimics group.CCK-8 method was used to detect cell proliferation ability,flow cytometry was used to analyze the apoptosis,Western blotting was used to determine the expression levels of CyclinD1,B-cell lymphoma-2 gene(Bcl-2)and its homologous protein Bax of cells.RNA immunoprecipitation experiment was used to verify the relationship between C5orf66-AS1 and miR-637.Results:Compared with adjacent tissues or normal gastric epithelial cell line GES-1,the expression levels of C5orf66-AS1 and miR-637 in GC tissues and cell lines MKN28,MKN451,BGC823,MGC803,AGS were reduced(P<0.05).The AGS cells with the lowest expression level of C5orf66-AS1 were selected for transfection experiments.After transfection of GV144-C5orf66-AS1,the C5orf66-AS1,miR-637 expression levels,apoptosis rate and Bax protein expression in AGS cells were significantly increased,and cell viability(48 h,72 h,96 h),CyclinD1 and Bcl-2 proteins were reduced(P<0.05).There was an interaction between C5orf66-AS1 and miR-637.Moreover,the overexpression of miR-637 could make the overexpression of C5orf66-AS1 more effective in inhibiting AGS cell proliferation and promoting apoptosis(P<0.05).Conclusions:C5orf66-AS1 and miR-637 are lowly expressed in GC tissues and cells.Overexpression of C5orf66-AS1 and up-regulation of miR-637 can synergistically inhibit the proliferation of GC cells and promote apoptosis.