| Objectives To explore the biological activity of let-7b in human HCC,and try to find a potential signal transduction pathway.Methods In this experiment,HepG2 human liver cancer cells purchased from American Type Culture Collection(ATCC)were selected as the research object.To explore the important regulatory role of let-7b expression level in the development of hepatocellular carcinoma,RNA extraction and quantitative real-time PCR(q RT-PCR)were used to detect let-7b expression levels in HCC cells and non-cancerous cells.In order to determine whether let-7b can inhibit the proliferation of Hep G2 cells in vitro,lentivirus transfection technology was used,the multiplicity of infection MOI was set to 50,and let-7b was transfected into Hep G2 cells.The experiment was divided into three groups,namely the Hep G2 group(Hep G2-let-7b)infected with lentiviral let-7b(LV-let-7b)as the experimental group,transfected with a blank lentiviral vector(LV-VC)Hep G2 group(Hep G2-VC)as a negative control group,virus-free Hep G2 cells(Hep G2-BC)as a blank control group,first detect the expression level of let-7b in each group,and apply Cell Counting Kit-8(CCK-8)kit and clony formation technology to detect cell proliferation ability.In order to explore the effect of let-7b on the Wnt/β-catenin signaling pathway.The experiment was divided into three groups: Hep G2-let-7b as the experimental group,Hep G2 cells infected with control mi RNA(Hep G2-con-mi R)as the negative control group,and Hep G2-BC as the blank control group,the dual luciferase reporter gene detection system and Western blot analysis were used to determine the activity of Wnt/β-catenin signaling pathway and the expression levels of β-catenin,c-Myc and E-cadherin in the three groups.In order to explore the effect of Wnt/β-catenin signaling pathway on the proliferation of HCC cells,the Hep G2-group of LV-let-7b with let-7b inhibitor(Hep G2-let-7b inhibitor)and Wnt/β-catenin pathway inhibitory factor 1 blocked Wnt/β-catenin activity in Hep G2 group(Hep G2-WIF1)as experimental group,and Hep G2 without virus infection as blank control group,using CCK-8 kit and clony formation test cell proliferation ability.Results The study samples included 19 HCC tissue samples and 15 non-cancerous tissue samples.The experimental results showed that the expression level of let-7b in HCC tissues was reduced by about 2.1 times compared with non-cancerous tissues(P<0.05).Cell proliferation is the process that results in an increase of the number of cells over time.Hep G2-let-7b group’s Hep G2 cell proliferation ability was reduced from day 2 to day 5 than LV-VC and BC groups(P<0.05).In addition,compared to the Hep G2-BC group,the number of Hep G2 colonies in the Hep G2-let-7b group was reduced,while the number of Hep G2 colonies in the Hep G2-anti-let-7b group was increased.We monitored the effect of let-7b on the activity of Wnt/β-catenin signaling pathway and the components contained in the pathway.Compared with Hep G2-con-mi R,let-7b overexpression significantly downregulated the activity of Wnt/β-catenin signaling pathway and protein expression levels of β-catenin and c-Myc,but increased the protein expression level of E-cadherin(P<0.05).Compared with the Hep G2-BC group,cell proliferation was significantly inhibited in the Hep G2-WIF1 group from day 2 to day 5,and WIF1 also significantly blocked the increase in Hep G2 proliferation caused by let-7b inhibitor(P<0.05).In addition,after inhibiting Wnt/β-catenin with WIF1,the number of colonies of Hep G2 cells decreased.Conclusions The expression level of let-7b plays an important regulatory role in the development of hepatocellular carcinoma.Let-7b can inhibit the proliferation of Hep G2 cells in vitro.Wnt/β-catenin signaling pathway plays a crucial role in the proliferation of Hep G2 cells.Let-7b targets the Wnt/β-catenin signaling pathway and has an effect on hepatocellular carcinoma cells.Figure 11;Table 3;Reference 134... |
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