The Expression Of HMMR In Hepatocellular Carcinoma And Its Influence On Tumorigenesis And Metastasis

Objective:Analyze the expression of Hyaluronan Mediated Motility Receptor(HMMR)in hepatocellular carcinoma(HCC)and its relationship with patient prognosis,and explore the role and mechanism of HMMR in the tumorigenesis and metastasis of hepatocellular carcinoma.Methods:1.Bioinformatics methods was used to analyze the expression and regulation of HMMR in HCC.1)By using the data from the TCGA and Oncomine database,we analyzed the m RNA expression level of HMMR in HCC and did a survival analysis combined with the patient’s clinical information.2)GSEA enrichment was used to find the pathways related to HMMR expression in HCC.3)Using RNA-seq technology,we analyzed differentially expressed genes in Hep G2 cell line after knocking down the expression of HMMR.Kegg enrichment analysis was used to find pathways that are significantly enriched in differential expressed genes.4)We used the m6A2 Target database to analyze the N6-methyladenosine(m6A)modification changes in HMMR in liver cancer cells.Combined with the RNA-seq and Me RIP-seq data of liver cancer cells which knocked out the expression of METTL3,and HCC tissue RNA-seq data,we did Venn analysis to find overlapping candidate genes.5)Using the STRING database and Cytoscape software,we analyzed the interactions between the proteins encoded by the overlapping candidate genes and calculated the key genes.6)TCGA database was used to analyze the correlation between the expression of METTL3 and HMMR in HCC.2.Immunohistochemistry was used to detect the expression level of HMMR protein in HCC tissue microarray chip.3.Small interfering RNA(si RNA)was used to knock down HMMR expression in liver cancer cells.1)The CCK-8 experiment was used to detect the changes in the number of cell proliferation.The Ed U experiment was used to detect the cell DNA synthesis ability.The clone formation experiment was used to detect the cell colony growth ability.And the Transwell experiment was used to detect the cell migration ability.2)Flow cytometry was used to detect cell cycle progress.Western blotting was used to detect the expression level of HMMR,cell-cycle-related proteins(Cyclin E1,p-CDK2,CDK-2),and epithelial-mesenchymal transition(EMT)related proteins(CDH1,ZO-1,FSP-1).4.si RNA was used to knock down the expression of METL3 in Hep G2 cells.Real-time quantitative PCR was uesd to detect the expression of HMMR in transcription level.Western blotting was used to detect expression of METTL3 and HMMR in protein level.5.After treating with m6 A inhibitor 3-Deazaadenosine(DAA),we detected HMMR expression in liver cancer cells.6.Using tumor xenotransplantation and in vivo metastasis models,we explored the effect of HMMR on the growth and metastasis of liver cancer.Results:HMMR was significantly up-regulated in HCC.Highly expressed HMMR was closely related to the poor prognosis of patients.Using si RNA to knock down the expression of HMMR could effectively inhibit the proliferation,migration and clonal formation of liver cancer cells.GSEA enrichment results showed that high expression of HMMR was significantly related to cell cycle and cell adhesion.The results of flow cytometry showed that knocking down HMMR can significantly block the progression of liver cancer cells in G1 phase.Western blot results showed that the expression of intracellular cycle-related proteins Cyclin E1 and p-CDK2/CDK2 was significantly reduced after HMMR was knocked down,the expression of epithelial cell markers CDH1 and ZO-1 was significantly up-regulated,while the mesenchymal cell marker FSP-1 was significantly down-regulated.Transcriptome sequencing analysis revealed that 117 genes were differentially expressed in Hep G2 cells after knocking down HMMR.Kegg enrichment results showed that differentially expressed genes were significantly enriched in cell cycle,cell adhesion and other related pathways.The m6A2 Target database analysis revealed that the m6 A modification mediated by METTL3 may be involved in regulating the expression of HMMR in Hep G2 cells.Venn analysis found 110 overlapping candidate genes.Through STRING database and Cytoscape software analysis,it was found that HMMR was among the top ten key genes.The results of expression correlation analysis showed that the expression of METTL3 and HMMR in HCC were highly positively correlated.The results of RT-PCR and Western blot experiments showed that knockdown of METTL3 or treatment with m6 A inhibitor DAA both significantly down-regulated the expression of HMMR in liver cancer cells.The results of subcutaneous tumor implantation experiments in nude mice showed that the size and quality of tumors in the knockdown HMMR group showed a consistent decrease.The results of lung metastasis of liver cancer model showed that knockdown of HMMR can significantly reduce the tumor lung metastasis process.HE staining showed that the metastatic tumor nodules in the lung tissue of nude mice in the knockdown HMMR group were significantly reduced.Conclusions:The expression level of HMMR in HCC is significantly up-regulated.Highly expressed HMMR is associated with poor prognosis of patients.HMMR can promote cell proliferation and migration by regulating cell cycle and EMT of liver cancer cells.Knockdown of HMMR can effectively inhibit tumor growth and metastasis.HMMR may be an effective prognostic marker and potential therapeutic target for HCC.