The Role Of MBD3 And Its Mechanism In The Growth And Metastasis Of Hepatocellular Carcinoma

Objective The methyl CpG binding domain protein 3(MBD3)is the core subunit of nucleosome remodelling and histone deacetylase(NuRD)complex,which is involved in the epigenetic regulation of the occurrence and development of tumors such as breast cancer and pancreatic cancer.However,the role of MBD3 in hepatocellular carcinoma(HCC)has not been reported yet.This study is aimed to analyze the different expression levels of MBD3 in liver cancer tissues and adjacent normal liver tissues,and the relationship between MBD3 expression and prognosis in HCC,and to investigate the role of MBD3 in the carcinogenesis,development and metastasis of HCC.Moreover,the specific molecular mechanisms that affect the proliferation and invasiveness of hepatoma cells will be elucidated thoroughly,providing a theoretical basis for the discovery of a new tumor marker for the prognosis and a new target for the treatment of HCC.Methods(1)HCC clinical samples were collected and prepared as tissue microarrays(TMA).The different expression levels of MBD3 in the liver cancer tissues and the adjacent normal liver tissues were detected by immunohistochemical staining(IHC).The relationship between the MBD3 expression in HCC with the overall survival(OS),the disease-free survival(DFS)and the metastasis-free survival(MFS)were statistically analyzed.(2)The effects of MBD3 on the proliferation and invasion of hepatoma cells were tested by the MTS,flat clone formation assay,soft agar clone spheres formation assay,wound healing,and Transwell assay in vitro.(3)The effects of MBD3 on the proliferation and invasion of hepatoma cells were tested by the subcutaneous tumor formation and lung metastasis model in vivo.(4)Analysis of whole genome transcriptome sequencing and The Cancer Genome Atlas(TCGA),rescue experiment of cell phenotype and animal phenotype,and the IHC staining in the consecutive clinical specimens of HCC will be implemented for exploring the molecular mechanism of MBD3 in the growth and metastasis of hepatocellular carcinoma comprehensively and thoroughly.Results(1)IHC staining of HCC samples showed that MBD3 was highly expressed in liver cancer tissues than that in the adjacent normal liver tissues.MBD3 expression in liver cancer tissues was negatively correlated with OS,DFS and MFS.(2)The results of MTS,flat clone formation and the soft agar clone spheres formation showed that MBD3 could promote the proliferation of hepatoma cells.The results of wound healing and transwell experiment showed that MBD3 can promote the invasion of hepatoma cells.(3)The results of subcutaneous tumorigenesis in nude mice showed that MBD3 can promote the growth of hepatoma cells.The model of lung metastasis by tail vein injection showed that MBD3 can promote the metastasis of hepatoma cells.(4)Sequencing analysis of whole gene transcriptome screened out the downstream target gene—tumor suppressor TFPI2.TCGA database analysis showed that the expression of MBD3 in liver cancer was significantly higher than that in normal liver tissues,and negatively correlated with prognosis.Further analysis showed that the expression of MBD3 was negatively correlated with TFPI2 protein level in HCC.Q-PCR and Western blot showed that the expression of MBD3 and TFPI2 were opposite in HCC cell lines.The results of rescue experiments suggested that MBD3 can promote the growth and metastasis of HCC by inhibiting TFPI2.The IHC results of MBD3,TFPI2 and CD34 staining in the consecutive clinical specimens of HCC showed that the MBD3 expression was negatively correlated with the TFPI2 expression,and positively correlated with the CD34 expression.Conclusion(1)MBD3 is highly expressed in the liver cancer tissues and closely related to the prognosis of HCC patients.It is an independent unfavourable prognostic factor for the patients with HCC.(2)MBD3 promotes the growth and metastasis of hepatoma cells by inhibiting the tumor suppressor TFPI2 expression.