miR-34a通过靶向ATG7调节结直肠癌细胞自噬与增殖

miR-34a regulates autophagy and proliferation of colorectal cancer cells by targeting ATG7

目的探讨miR-34a对结直肠癌细胞自噬和增殖的调控机制。方法采用实时定量反转录PCR检测2019年5月至2020年5月期间中国医科大学附属第四医院结直肠癌手术切除的30例结直肠癌组织以及相应癌旁组织中miR-34a的表达水平,同时检测结直肠癌细胞(RKO、LoVo、SW480和SW620)和人正常结肠上皮细胞(FHC)中miR-34a的表达水平。通过pcDNA3.1-miR-34a转染结直肠癌细胞株SW480以过表达miR-34a。CCK-8法检测细胞活性。Western blot检测自噬相关蛋白微管相关蛋白1轻链3(microtubular associated protein 1 light chain 3,LC3)-Ⅱ/LC3-Ⅰ和自噬相关基因7(autophagy associated gene7,ATG7)的表达。免疫荧光检测LC3荧光斑点表达。Starbase数据库分析miR-34a与ATG7的相关性,并采用荧光素酶报告基因实验验证miR-34a与ATG7的结合情况。SW480细胞共转染pcDNA3.1-miR-34a和pcDNA3.1-ATG7后,CCK-8法检测细胞活性,克隆形成实验检测细胞增殖水平。结果miR-34a在结直肠癌组织的表达低于相应癌旁组织,在结直肠癌细胞(RKO、LoVo、SW480和SW620)中的表达低于FHC细胞(均P<0.05)。过表达miR-34a72h后SW480细胞活性降低(P<0.01),自噬相关蛋白LC3-Ⅱ/LC3-Ⅰ降低(P<0.01),免疫荧光显示LC3荧光斑点减少(P<0.01)。过表达miR-34a 24 h后SW480细胞ATG7mRNA表达降低(P<0.01)。Starbase分析结果表明,miR-34a可以靶向结合ATG7。荧光素酶报告基因实验显示,miR-34a抑制野生型ATG7-3’UTR质粒转染的荧光素酶活性(P<0.01),而对突变型ATG7-3’UTR质粒转染的荧光素酶活性无影响(P>0.05)。共转染pcDNA3.1-miR-34a和pcDNA3.1-ATG772h后,SW480细胞活性相对增加(P<0.01),细胞增殖能力增强(P<0.01)。结论miR-34a通过靶向ATG7抑制结直肠癌细胞自噬和增殖。

Objective To explore the mechanism of miR-34 a on the autophagy and proliferation of colorectal cancer cells.Methods Quantitative reverse transcription PCR was used to detect the expression level of miR-34 a in colorectal cancer tissues and corresponding adjacent tissues of 30 patients who underwent colorectal cancer surgery in the Fourth Affiliated Hospital of China Medical University from May 2019 to May 2020.The expression levels of miR-34 a in colorectal cancer cells(RKO,Lo Vo,SW480 and SW620)and human normal colon epithelial cells(FHC)were also detected.Colorectal cancer cell line SW480 was transfected with pc DNA3.1-miR-34 a to overexpress miR-34 a.CCK-8 kit was used to detect cell viability.Western blot was used to detect the expression of autophagy related proteins microtubular associated protein 1 light chain 3(LC3)-Ⅱ/LC3-Ⅰand autophagy associated gene 7(ATG7),and immunofluorescence was used to detect the number of LC3 fluorescent spots.Starbase database was used to analyze the correlation between miR-34 a and ATG7,and luciferase reporter gene experiment was used to verify the binding of miR-34 a and ATG7.After the SW480 cells were co-transfected with pc DNA3.1-miR-34 a and pc DNA3.1-ATG7,the cell viability was detected by CCK-8 kit,and the cell proliferation level was detected by clone formation assay.Results The expression level of miR-34 a in colorectal cancer tissues was lower than that in adjacent tissues(P<0.05),and the expression level of miR-34 a in colorectal cancer cells(RKO,Lo Vo,SW480 and SW620)was lower than that in human normal colon epithelial cells(all P<0.05).After overexpression of miR-34 a for 72 h,the cell viability of SW480 cells decreased(P<0.01),the level of autophagy related proteins LC3-Ⅱ/LC3-Ⅰdecreased(P<0.01),and the immunofluorescence showed that the fluorescence spots of LC3 decreased(P<0.01).The expression of ATG7 m RNA in SW480 cells decreased 24 h after overexpression of miR-34 a(P<0.01).Starbase analysis showed that miR-34 a could target ATG7.Luciferase reporter gene assay showed that miR-34 a significantly inhibited the luciferase activity of wild-type ATG7-3’UTR plasmid transfected cells(P<0.01),but had no effect on luciferase activity of mutant ATG7-3’UTR plasmid transfected cells(P>0.05).After co-transfection of pc DNA3.1-miR-34 a and pc DNA3.1-ATG7 for 72 h,the cell viability of SW480 cells was relatively increased(P<0.01),and the proliferation ability was relatively enhanced(P<0.01).Conclusions miR-34 a inhibits the autophagy and proliferation of colorectal cancer cells by targeting ATG7.