miR-148b-3p通过调节脂代谢基因对肝癌细胞恶性生物学行为的影响

Effects of miR-148b-3p on malignant biological behavior of hepatocellular carcinoma cells through regulation of lipid metabolism genes

目的探讨miR-148b-3p通过调节脂代谢基因对肝癌细胞增殖、转移及侵袭能力等恶性生物学行为的影响。方法通过癌症和肿瘤基因图谱(TCGA)数据库寻找miR-148b-3p、肝癌及脂代谢三者的交集基因,筛选miR-148b-3p治疗肝癌的潜在靶点基因;采用荧光定量逆转录-聚合酶链反应(RT-PCR)检测miR-148b-3p在正常肝脏细胞LO2和肝癌细胞Huh7、Hep1中的表达水平,选取miR-148b-3p表达量最低的细胞Huh7作为研究对象;Huh7肝癌细胞分为对照组(转染miR-148b-3p NC)和实验组(转染miR-148b-3p mimics),CCK8实验和克隆形成实验、划痕实验、Transwell实验、以及油红O染色分别检测两组肝癌细胞的增值、迁移、侵袭能力及脂滴形成情况,采用RT-PCR和Western blot检测交集基因及脂肪生成指标基因FASN、PPAR-γ及SCD1的mRNA和蛋白表达水平。结果miR-148b-3p在Huh7细胞中mRNA相对表达量显著低于Hep1细胞(P<0.01)和LO2细胞(P<0.001);与对照组相比,实验组Huh7细胞的增殖受到抑制(P<0.05)、转移能力减弱(P<0.05)、侵袭能力降低(P<0.05),细胞脂滴形成明显减少(P<0.01);交集基因PRKAG2、INSIG2的mRNA水平的表达量明显升高(P<0.01、P<0.01)、CHD9表达量明显降低(P<0.001),脂代谢FASN、PPAR-γ、SCD1基因的mRNA水平明显降低(P<0.001、P<0.001、P<0.01);交集基因PRKAG2、INSIG2蛋白水平的表达量升高(P<0.01、P<0.05)、CHD9表达量降低(P<0.05),脂代谢FASN、PPAR-γ、SCD1基因蛋白水平的表达量均明显降低(P<0.01)。结论过表达miR-148b-3p可抑制肝癌细胞的增殖、转移和侵袭能力,其机制可能与miR-148b-3p抑制了肝癌细胞的脂代谢有关。

Objective To investigate the effect of miR-148b-3p on the malignant biological behavior of hepatocellular carcinoma cells through the regulation of lipid metabolism genes,such as proliferation,metastasis and invasive ability.Methods The cancer genome atlas(TCGA)database was used to find the intersection of miR-148b-3p,hepatocellular carcinoma and lipid metabolism to obtain the potential target genes(intersection genes)for the treatment of hepatocellular carcinoma with miR-148b-3p;fluorescence quantitative reverse transcription-polymerase chain reaction(RT-PCR)was used to detect miR-148b-3p in normal liver cells LO2 and hepatocellular carcinoma cells Huh7 and Hep1 expression;the hepatocellular carcinoma cell line with the lowest expression was selected for the experiment.The cells were divided into control group(miR-148b-3p NC)and experimental group(miR-148b-3p mimics)for transfection.The transfected cells were subjected to CCK8 assay and clone formation assay,scratch assay,Transwell assay,and oil red O staining to detect the value-added,migration,invasion and lipid droplet formation of hepatocellular carcinoma cells respectively.The mRNA levels and protein expression levels of target genes and adipogenic index genes(FASN,PPAR-γ,SCD1)were examined by RT-PCR and Western blot respectively.Results The relative mRNA expression of miR-148b-3p was significantly lower in Huh7 cells than in Hep1 cells(P<0.01)and LO2 cells(P<0.001);Compared with the control group,the proliferation of Huh7 cells in experimental group was inhibited(P<0.05),metastatic ability was diminished(P<0.05),the invasion ability was reduced(P<0.05),lipid droplet formation was significantly reduced(P<0.01);the expression of PRKAG2 and INSIG2 gene mRNA levels were significantly increased(P<0.01,P<0.01),and CHD9 expression was significantly reduced(P<0.001);Lipid metabolism FASN,PPAR-γ,and SCD1 genes mRNA levels were significantly decreased(P<0.001,P<0.001,P<0.01).The protein expression of intersection genes PRKAG2 and INSIG2 increased(P<0.01,P<0.05),the expression of CHD 9 decreased(P<0.05),and the expression of lipid metabolism FASN,PPAR-γand SCD 1 genes all significantly decreased(P<0.01).Conclusion Overexpression of miR-148b-3p could inhibit the proliferation,metastasis and invasion ability of hepatocellular carcinoma cells,which might be related to the inhibition of lipid metabolism of hepatocellular carcinoma cells by miR-148b-3p.