The Mechanism Of LncRNA GSEC Regulating The Progression Of Hepatocellular Carcinoma Through The MiR-101-3p/PSPH/VEGF Axis

Objective: Long non-coding RNAs(LncRNAs)regulate downstream gene expression through sponge adsorption of micrornas(miRNAs),thus constructing endogenous competitiveRNAs(Competing endogenous RNAs,CeRNA)network.Long non-coding RNA GSEC(G-quadruplex forming sequence containing lncRNA(LncRNA GSEC)is overexpressed in many cancers and diseases,and is quadruplex forming sequence containing lncRNA(LncRNA GSEC)quadruplex forming sequence containing LncRNA(LncRNA GSEC)is upregulated in liver cancer.This study aims to investigate the mechanism of LncRNA GSEC regulating the progression of hepatocellular carcinoma(HCC)through the miR-101-3p/PSPH/VEGF axis.To elucidate the oncogenic mechanism and interaction network of LncRNA GSEC and its downstream gene PSPH(Phosphoserine Phosphatase)in hepatocellular carcinoma,providing a new theoretical basis for early clinical diagnosis and targeted therapy of HCC.Methods: The ceRNA regulatory network was constructed through bioinformatics website,and the expression levels of PSPH and LncRNA GSEC in liver cancer cells and normal cells were verified by RT-qPCR and Western blot,and the plasmids knockdown and overexpression of PSPH and LncRNA GSEC were constructed.Stable cell lines with knockdown and overexpression of PSPH and LncRNA GSEC were constructed in SMMC-7721 and Hep G2 cell lines,respectively,and the expression levels of marker genes related to angiogenesis were verified by Western blot assay.The effects of PSPH and LncRNA GSEC on angiogenesis,migration,invasion and proliferation of hepatocellular carcinoma cells in vitro were verified by matrix gel angiogenesis,wound healing,Transwell matrix gel and colony formation experiments.The endogenous expression of miR-101-3p was verified by RT-qPCR assay.The mimics and inhibitors of miR-101-3p and its control group were transitively transfected in hepatoma cell lines Hep G2 and SMMC-7721.The expression levels of marker genes related to angiogenesis were verified by Western blot assay,and the effects of miR-101-3p on cell migration and invasion ability of hepatoma cells in vitro were verified by wound healing assay and Transwell matrix gel assay.The wild-type and mutant plasmids of LncRNA GSEC and miR-101-3p and PSPH 3’utr and miR-101-3p were constructed,and the binding of the two plasmids was verified by double Luciferase reporter gene assay.The SMMC-7721 cell line with stable knockout of PSPH and LncRNA GSEC was constructed,and the SMMC-7721 cell line with overexpression of PSPH on the basis of stable knockout of LncRNA GSEC was constructed,and the transplanted tumor model of nude mice was established.The effect of LncRNA GSEC and PSPH on HCC cell tumorigenesis was verified by measuring tumor size and volume and immunohistochemical staining in nude mice.A response experiment was set up to overexpress PSPH after LncRNA GSEC knockdown,transfect miR-101-3p inhibitor after LncRNA GSEC knockdown,and overexpress PSPH after miR-101-3p overexpression.Western blot assay was used to verify the changes in the expression of marker genes associated with angiogenesis after the relevant response experiment,and the influence of signal axis on the progression of hepatocellular carcinoma was further verified by matrix gel angiogenesis,wound healing,Transwell matrix gel and colony formation in vitro experiments.Results: The results of RT-qPCR and Western blot showed that the expression of PSPH was up-regulated in liver cancer cells,and the results of Western blot showed that PSPH promoted the expression of the marker gene of VEGF signaling pathway.The results of matrix glue angiogenesis experiment,wound healing experiment,Transwell matrix glue experiment and colony formation experiment showed that PSPH can promote the angiogenesis,migration,invasion and proliferation of hepatocellular carcinoma cells in vitro.The results of tumor transplantation in nude mice showed that PSPH knockdown could inhibit the tumorigenesis of hepatocellular carcinoma cells in vivo.The results of Luciferase experiment showed that there was a binding site between PSPH 3’utr and miR-101-3p.The results of RT-qPCR showed that the expression of miR-101-3p was downregulated in liver cancer cells.Western blot showed that miR-101-3p inhibited the expression of PSPH and VEGF signaling pathway marker genes,and the gene expression level was restored to a certain extent after overexpression of PSPH.In vitro experiments of hepatoma cells showed that overexpression of miR-101-3p inhibited the ability of angiogenesis,migration,invasion and proliferation of hepatoma cells in vitro,while overexpression of PSPH restored the ability of angiogenesis,migration,invasion and proliferation.Luciferase assay showed that there was a binding site between LncRNA GSEC and miR-101-3p.The results of RT-qPCR assay showed that LncRNA GSEC was up-regulated in liver cancer cells,and down-regulated after overexpression of miR-101-3p.The expression of LncRNA GSEC was up-regulated after the inhibition of miR-101-3p expression.Bioinformatics analysis of LncRNA GSEC was mainly localized in the cytoplasm.Western blot results showed that LncRNA GSEC inhibited the expression of PSPH and VEGF signaling pathway marker genes,while overexpression of PSPH or inhibition of miR-101-3p could partially restore the expression of related genes.In vitro experiments of hepatocellular carcinoma cells showed that knockdown of LncRNA GSEC inhibited the ability of angiogenesis,migration,invasion and proliferation of hepatocellular carcinoma cells in vitro,while overexpression of PSPH or inhibition of miR-101-3p restored the ability of angiogenesis,migration,invasion and proliferation of hepatocellular carcinoma cells.The results of tumor transplantation experiments in nude mice showed that the knockdown of LncRNA GSEC inhibited the tumorigenesis of hepatocellular carcinoma cells in vivo,while the overexpression of PSPH weakened the tumorigenesis effect.Conclusions: In conclusion,LncRNA GSEC removes the inhibition of miR-101-3p on the downstream gene PSPH through sponge adsorption of miR-101-3p,and PSPH promotes the development of hepatocellular carcinoma through VEGF signaling pathway.In this study,the regulatory mechanism of LncRNA GSEC/miR-101-3p/PSPH/VEGF axis on the occurrence and development of liver cancer was initially elucidate at the molecular,cellular and animal levels.The results of this study may provide a new theoretical basis for the occurrence and development of hepatocellular carcinoma,and may provide a new marker for clinical validation of HCC.