Study On NNMT Mediates The Invasion And Metastasis Of Hepatocellular Carcinoma Through CD44

Purpose1.To investigate the correlation between NNMT expression and prognosis and clinicopathological factors in patients with HCC.2.To explore the related mechanisms of NNMT affecting invasion and metastasis of HCC,and to provide a new perspective for the prevention and treatment of invasion and metastasis of HCC.Methods1.Paraffin samples containing carcinomas and adjacent tissues from 93 patients with hepatocellular carcinoma were collected and stained with IHC,and then divided into two groups according to IOD values: NNMT High and NNMT Low.2.We analyzed the NNMT and vascular invasion,liver cirrhosis status,serum HBV levels,and distant metastasiscorrelation by collecting follow-up data.3.In vitro,Transwell assay was used to detect the ability of HCC metastasis after knockdown and overexpression of NNMT.4.The expression of CD44 protein and mRNA was significantly decreased by NNMT knockdown in cells detected by WB and rt-pcr.Transwell assay was performed to analyze the invasion and metastasis abilities of Ctrl,PlvNNMT,PlvNNMT–shCD44,shCD44 in the four groups.Statistical analysis of the differences confirmed that NNMT up-regulated CD44 promoted invasion and metastasis of HCC.5.CD44 promoter-driven luciferase activity in 293 T cells transfected with NNMT knockdown or overexpression plasmid confirmed whether NNMT directly regulated CD44 promoter activity.6.WB examined the level of H3K27me3 in NNMT overexpression and NNMT-KD cells,and RT-PCR examined the level of CD44 mRNA after the treatment of NNMT-KD cells by the histone H3K27 me inhibitor gsk-126(5mM,72 h).NNMT was verified to promote the transcription of CD44 by regulating the methylation of histone H3K27.7.ChIP-qPCR analyses of H3K27me3 binding on CD44 promoter in NNMT-OE and NNMT-KD cells.8.We established anatopic xenograft model of HCC by subcutaneously transplanting nude mice.Metastatic tumors were analyzed by hematoxylin and eosin(H&E)staining.Results1.We examined NNMT expression levels in 93 pairs of HCC and corresponding adjacent tissue samples using immunohistochemistry(IHC).Based on the IOD values of IHC staining,the HCC patients were divided into the NNMT high(n=40)and NNMT low(n=40)groups.K-M analysis showed that patients with higher NNMT expression in their HCC tissues had a shorter overall survival rate(P=0.0168)and tumor-free survival(P=0.0183).2.Higher NNMT was significantly correlated with unfavorable prognosticfeatures such as vascular invasion(P=0.025),liver cirrhosis status(P=0.024),serum HBV levels(P=0.045),and distant metastasis(P=0.011).3.While shRNA-mediated knockdown of NNMT attenuated the expression of CD44 in the QGY-7701 and SMMC-7721 cells.To determine whether NNMT enhanced HCC cell invasion via CD44,we knocked down CD44 in SMMC-7721 cells overexpressing NNMT,and found that it significantly reversed the pro-metastatic effects of NNMT.4.Neither NNMT knockdown nor overexpression significantly affectedluciferase reporter activity.5.We performed a ChIP assay to analyze the H3K27 methylation status of theCD44 promoter,and found that NNMT overexpression diminished and NNMT knockdown increased H3K27me3 levels.6.We established anatopic xenograft model of HCC by subcutaneously transplanting nude mice.NNMT knockdown significantly inhibited the metastatic ability of the HCC cells.Conclusion1.Higher expression of NNMT was significantly correlated with unfavorable prognostic features such as vascular invasion,liver cirrhosis status,serum HBV levels,and distant metastasis.2.The down-regulation of NNMT significantly inhibits the metastasis of HCC cells,which is dependent on CD44.3.NNMT does not directly regulate the promoter activity of CD44.NNMT modulates intracellular methylation potential and thereby reduces the modification of CD44 promoter H3K27me3 to indirectly promote CD44 transcription.4.NNMT enhances tumor metastasis in vivo.