miR-105-5p靶向SIRT1调控脂肪酸代谢影响胃癌细胞生物学行为的研究

MiR-105-5p regulates fatty acid metabolism and biological behaviors of gastric cancer cells by targeting SIRT1

目的探讨miR-105-5p对胃癌细胞增殖、侵袭、迁移及脂肪酸代谢的影响,阐明miR-105-5p与沉默信息调节因子1(SIRT1)基因的靶向关系。方法选取人胃黏膜上皮细胞GES-1、人胃癌HepG2细胞,将不同质粒转染到HepG2细胞,分为miR-105-5p组(转染miR-105-5p mimic)、si-miR-105-5p组(转染miR-105-5p inhibitor)、miR-NC组(转染mimic NC)、NC组(转染inhibitor NC)、Scramble组(转染Scramble)、miR-105-5p+Scramble组(转染miR-105-5p mimic、Scramble)及miR-105-5p+sh-SIRT1组(转染miR-105-5p mimic、sh-SIRT1)。MTT检测细胞增殖能力,Transwell小室实验检测细胞迁移、侵袭能力,ELISA检测细胞磷脂、三酰甘油含量,实时荧光定量PCR检测细胞中miR-105-5p、SIRT1 mRNA表达水平;Western blot检测细胞SIRT1、FASN、ACC1及FABP5表达水平。生物信息预测miR-105-5p与SIRT1的靶向作用关系,并使用荧光素酶报告实验验证。结果HepG2细胞miR-105-5p表达水平高于GES-1细胞,SIRT1蛋白、基因mRNA表达水平低于GES-1细胞(P<0.01)。miR-105-5p组细胞48 h、72 h OD值,侵袭及迁移细胞数量,磷脂含量、三酰甘油含量及FASN、ACC1、FABP5蛋白水平高于miR-NC组,si-miR-105-5p组细胞48 h、72 h OD值,侵袭及迁移细胞数量,磷脂含量、三酰甘油含量及FASN、ACC1、FABP5蛋白水平低于NC组(P<0.01)。miR-105-5p+Scramble组细胞48 h、72 h OD值,侵袭及迁移细胞数量,磷脂含量、三酰甘油含量及FASN、ACC1、FABP5蛋白水平高于Scramble组,miR-105-5p+sh-SIRT1组细胞48 h、72 h OD值,侵袭及迁移细胞数量,磷脂含量、三酰甘油含量及FASN、ACC1、FABP5蛋白水平低于miR-105-5p+Scramble组(P<0.01)。miR-105-5p组细胞SIRT1蛋白水平低于miR-NC组,si-miR-105-5p组细胞SIRT1蛋白水平高于NC组(P<0.01)。miR-105-5p与SIRT13’UTR存在碱基互补配对位点。miR-105-5p+WT-SIRT1组细胞荧光素酶活性低于miR-NC+WT-SIRT1组(P<0.01)。结论miR-105-5p可通过SIRT1调节脂肪酸代谢来抑制胃癌细胞增殖、侵袭及迁移。

Objective To investigate the effects of miR-105-5p on the proliferation,invasion,migration,and fatty acid metabolism of gastric cancer cells,and to elucidate the targeted relationship between miR-105-5p and silent information regulatory factor 1(SIRT1)gene.Methods Human gastric mucosa epithelial cells GES-1 and human gastric cancer HepG2 cells were selected.Different plasmids were transfected into HepG2 cells and they were divided into miR-105-5p group(transfected with miR-105-5p mimic),si-miR-105-5p group(transfected with miR-105-5p inhibitor),miR-NC group(transfected with mimic NC),NC group(transfected with inhibitor NC),Scramble group(transfected with Scramble),miR-105-5p+Scramble group(transfected with miR-105-5p mimic and Scramble)and miR-105-5p+sh-SIRT1 group(transfected with miR-105-5p mimic and sh-SIRT1).MTT assay was used to detect cell proliferation.Transwell assay was used to detect cell migration and invasion.The enzyme-linked immunosorbent assay(ELISA)was used to detect the content of phospholipid and triacylglycerol.The real-time quantitative PCR(RT-qPCR)was used to detect the mRNA levels of miR-105-5p and SIRT1 in cells.Western blot was used to detect the protein levels of SIRT1,fatty acid synthase(FASN),Acetyl-CoA carboxylase 1(ACC1),fatty acid binding protein 5(FABP5)in cells.The targeting relationship between miR-105-5p and SIRT1 was predicted by bioinformatics,and verified by dual-luciferase reporter assay.Results The level of miR-105-5p in HepG2 cells was higher than that in GES-1 cells,while the mRNA and protein levels of SIRT1 were significantly lower than those in GES-1 cells(P<0.01).The optical density(OD)values at 48h and 72h,invasive and migratory cell numbers,phospholipid content,triacylglycerol content,and protein levels of FASN,ACC1 and FABP5 in the miR-105-5p group were significantly higher than those of the miR-NC group,which,in the si-miR-105-5p group were significantly lower than those of the NC group(P<0.01).OD values at 48h and 72h,invasive and migratory cell numbers,phospholipid content,triacylglycerol content,and protein levels of FASN,ACC1 and FABP5 in the miR-105-5p+Scramble group were significantly higher than those of the Scramble group,which,in the miR-105-5p+sh-SIRT1 group were significantly lower than those of the miR-105-5p+Scramble group(P<0.01).The protein level of SIRT1 in the miR-105-5p group was significantly lower than that of the miR-NC group,which,in the si-miR-105-5p group was significantly higher than that of the NC group(P<0.01).MiR-105-5p had a complementary base pairing site with SIRT13’UTR.The luciferase activity in miR-105-5p+WT-SIRT1 group was significantly lower than that of miR-NC+WT-SIRT1 group(P<0.01).Conclusion MiR-105-5p may inhibit the proliferation,invasion,and migration of gastric cancer cells by regulating fatty acid metabolism through SIRT1.