The Mechanism Of HIF-2? Affecting The Proliferation And Metastasis Of HCC By Mediating The Expression Of DLST And PPP1R8

[Objective] Hypoxia is the most important microenvironmental feature of advanced malignant solid tumors.The Hypoxia-inducible factor-2 ?(HIF-2 ?)gene is involved in regulating a variety of molecular mechanisms of tumor progression in the hypoxic microenvironment.Hepatocellular carcinoma(HCC)is one of the most common malignant tumors in the tumor disease spectrum.HIF-2 ? and its downstream regulatory genes DLST and PPP1R8 are involved in regulating the proliferation,invasion,metastasis and apoptosis of HCC.The purpose of this study is to explore the expression of HIF-2 ? and its downstream genes DLST and PPP1R8 in clinical specimens and cell lines of liver cancer,to clarify their regulatory relationship,and to further explore the mechanism of DLST and PPP1R8 genes in HCC,so as to provide new scientific ideas for the prevention and treatment of HCC.[Methods] First of all,on the basis of the previous experimental demonstration of our group,we used gene chip technology to detect genome-wide differential expression profile of Hep3 B cell line after HIF-2 ? interference,and enriched the gene set and signal pathway.The differentially expressed genes with good correlation were screened in different cell lines for real-time fluorescence PCR verification.The downstream target genes DLST and PPP1R8 most likely to be related to HIF-2?-regulated tumor progression were screened and bioinformatics searched and compared.Secondly,we used immunohistochemistry and Western blotting to detect the expression of HIF-2 ?,DLST and PPP1R8 protein in clinical HCC tissues,and combined with clinicopathological data to explore the effect of DLST and PPP1R8 gene on the clinical prognosis of HCC.Finally,under the condition of simulated anoxic culture,we constructed Lvsh-HIF-2 ? Hep3 B and Hep G2 cell lines by lentivirus transfection RNAi interference technique,and overexpressed DLST and PPP1R8 genes in this cell line by transient liposome transfection.Cell functions such as cell proliferation,invasion,migration and apoptosis were verified by CCK8,Tanswell,Annexin V-FITC and flow cytometry.We use TCGA database and GSEA bioinformatics analysis methods to enrich the gene co-expression,gene function and signal pathway of DLST and PPP1R8 in hepatocellular carcinoma,and explore the most possible molecular biological mechanism.[Results] 1.Gene chip analysis showed that there were 1352 differentially expressed genes downstream of HIF-2 ?,including 674 up-regulation and 678 down-regulation.The differential genes were mainly enriched in metabolic and tumor-related pathways,and were closely related to cellular macromolecular metabolic function,MAPK signal pathway and Pathways in cancer signal pathway.The results of q RT-PCR verification of HIF-2 ? and its downstream genes DLST and PPP1R8 in Hep3 B and Hep G2 cell lines and bioinformatics mining analysis in TCGA and Oncomine database suggest that the high expression of DLST and PPP1R8 can be used as a kind of oncogene regulated by HIF-2 ? and may play an important role in the occurrence and development of hepatocellular carcinoma.2.In clinical trials,HIF-2 ?,DLST and PPP1R8 proteins were highly expressed in fresh frozen tissues and continuous pathological sections of the same liver cancer compared with adjacent tissues.Further analysis of the correlation with clinicopathological factors showed that the expression level of DLST was significantly correlated with vascular invasion,tumor Edmonson grade,tumor capsule and tumor TNM stage.The expression of PPP1R8 protein was significantly correlated with tumor diameter,tumor nodule,vascular invasion,Edmonson's stage,tumor capsule,serum alpha-fetoprotein(AFP)and tumor TNM stage.Survival analysis statistics showed that the overall survival time of patients with high expression of DLST and PPP1R8 protein was significantly lower than that of patients with low expression.3.Cell biological experiments showed that after interference with HIF-2 ? RNAi,the proliferation,invasion and migration ability of HCC stably transduced cell lines were significantly decreased,while the apoptosis rate was significantly increased.After overexpression of DLST and PPP1R8 in the same stable transfected cell line,the biological behavior of the cell line was reversed.Bioinformatics technology,using GO and KEGG analysis to enrich DLST and PPP1R8 coexpression genes and signal pathways,suggesting that DLST and its co-expression genes are mainly enriched in tricarboxylic acid cycle metabolic pathway,PPP1R8 and its co-expression genes are enriched in DNA damage repair metabolic pathway.[Conclusions] Clinical studies suggest that HIF-2 ?,DLST and PPP1R8 are involved in the proliferation,metastasis and other biological processes of HCC.Gene chip suggests that HIF-2 ? may affect the progression and prognosis of HCC by regulating multiple downstream genes.Cytological functional experiments suggest that HIF-2 ? promotes the proliferation and metastasis of HCC by regulating downstream target genes DLST and PPP1R8,and plays an important role in tricarboxylic acid cycle and DNA damage repair metabolic pathway.