DPM1 Promotes The Invasion And Metastasis Of Hepatocellular Carcinoma By Upregulation Of VEGF And MMP2

Objective:Hepatocellular carcinoma(HCC)is one of the major cancer types in the world.The onset of HCC is insidious,and lack of definite clinical signs in the early stages of the patient.At the time of diagnosis,the patients are usually in the middle to advanced stages,with vascular invasion and metastasis of the tumour.Therefore,it is important to explore the biomolecular mechanisms associated with the invasive metastasis of HCC so as to explore new therapeutic targets.Dolichol phosphate mannose synthase(DPMS)is an essential mannosyltransferase that plays a crucial role in the posttranslational modification of proteins.DPMS promotes angiogenesis and its involvement in altered glycosylation has important implications for the development of cancer.DPM1 is a subunit of DPMS and is responsible for its catalytic function.However,the effect of DPM1 on the ability of hepatocellular carcinoma to migrate invasively and its specific regulatory mechanisms remain unclear.In order to further explore the molecular mechanism of DPM1 in HCC,we designed human histology experiments and in-vitro cytology experiments to verify that DPM1 can promote the invasion and metastasis of hepatocellular carcinoma through upregulating the expression of VEGF and MMP2.Methods:1.Hepatocellular carcinoma tissues and corresponding adjacent liver tissues of 90 patients with HCC who underwent hepatobiliary surgery in the First Affiliated Hospital of Gannan Medical University from January 2020 to October 2021.Real time quantitative q RT-PCR and Western blot were used to detect the expression of DPM1 m RNA and protein in hepatocellular carcinoma tissues and adjacent liver tissues;At the same time,the expression levels of DPM1,VEGF and MMP2 protein in HCC tissues and adjacent liver tissues were detected by immunohistochemistry,then analyze the correlation between DPM1 with VEGF and DPM1 with MMP2.2.The clinicopathological information of patients was counted,and the correlation between the expression level of DPM1 and the clinicopathological characteristics of patients was detected.3.q RT-PCR and Western blot were used to detect the expression of DPM1 m RNA and protein in hepatoma cell lines(Hep G2,Hep3 B,Huh7,MHCC-97H)and immortalized normal human liver cell lines(THLE-3),and to screen the hepatocellular carcinoma cell lines with high expression of DPM1.4.We used si RNA to interfere in DPM1 gene expression.Then used q RT-PCR and Western blot to detect the changes of the expression of DPM1 to verify the transfection results.Then we used Western blot to detect the changes of VEGF and MMP2 expression levels after interfering with DPM1 expression,so as to verify whether DPM1 can promote the expression of VEGF and MMP2.5.Transwell was used to detect the changes of invasion and migration of hepatoma cells before and after si RNA interference with DPM1 gene expression.To determine whether DPM1 is related to the invasion and migration of HCC,so as to verify that DPM1 promotes the invasion and migration of HCC through VEGF and MMP2.6.All statistical data shall be analyzed by SPSS 22.0 statistical software.The difference between groups shall be compared by t-test or chi square test,and the correlation analysis shall be conducted by Spearman correlation analysis.The difference is considered statistically significant with P<0.05.Results:1.The results of q RT-PCR and Western blot showed that the m RNA and protein expression level of DPM1 in HCC tissues was significantly higher than that in liver tissues adjacent to cancer.Immunohistochemical analysis showed that the positive rate of DPM1 protein expression in HCC was 70.0%(63/90),which was significantly higher than that in adjacent liver tissues(16.7%);The positive rate of VEGF protein expression was 72.2%(65/90),which was significantly higher than that of adjacent liver tissues(17.8%);The positive rate of MMP2 protein expression was 65.6%(59/90),which was significantly higher than that of adjacent liver tissues(13.3%),and the difference was statistically significant(P<0.05).The results of correlation analysis showed that there was a positive correlation between the expression of DPM1 protein and VEGF protein(r=0.575,P<0.001),and a positive correlation between the expression of DPM1 protein and MMP2 protein(r=0.743,P<0.001).2.The analysis of the correlation between DPM1 expression level and clinicopathologic features in hepatoma tissues showed that the expression of DPM1 was correlated with the number of tumors,vascular invasion,tumor envelope,tumor TNM stage and Edmondson grade(P<0.05),but not with sex,age,presence of hepatitis B,cirrhosis,AFP value and tumor size(P>0.05).3.The results of q RT-PCR and Western blot showed that compared with the immortalized normal human liver cell line THLE-3,the expression of DPM1 m RNA and DPM1 protein was significantly up-regulated in all hepatoma cell lines.In addition,the expression of DPM1 m RNA and DPM1 protein in MHCC-97 H was significantly higher than that in Hep G2,Hep3 B and Huh7 hepatoma cell lines(P<0.05).4.The MHCC-97 H cell was selected to construct a DPM1 interference cell.The results of Western blot showed that the expression of DPM1 protein in MHCC-97 H cells transfected with DPM1 si RNA decreased significantly,and the expression of VEGF protein and MMP2 protein decreased significantly(P<0.05).5.Transwell assay results showed that the invasion and migration ability of MHCC-97 H cells down-regulated DPM1 expression were significantly reduced(P<0.05).Conclusion:1.DPM1 was expressed at higher levels in hepatocellular carcinoma tissues than in paraneoplastic liver tissues,and DPM1 protein was positively correlated with the expression levels of both VEGF and MMP2 proteins respectively in HCC tissues.2.The DPM1 expression level was significantly correlated with the number of tumors,envelope invasion,vascular invasion,TNM stage and pathological grade of the tumors,while there was no significant correlation with gender,age,HBs Ag status,cirrhosis,AFP value and the size of the tumors.3.Compared with the cell line THLE-3,the expression of DPM1 was significantly up-regulated in all hepatoma cell lines.In addition,the expression of DPM1 in MHCC-97 H was significantly higher than that in Hep G2,Hep3 B and Huh7 hepatoma cell lines.4.Interfering with the expression of DPM1 in MHCC-97 H cells significantly could downregulated the expression of VEGF and MMP2.5.DPM1 could promote the invasion and metastasis of hepatocellular carcinoma by upregulating VEGF and MMP2.