Effect Of DNA Methylation On The Expression Of CLDN4 In Laryngeal Carcinoma Hep-2 Cells By Inhibiting Sp1 Binding

Research Background:Several studies have shown that the tight junction protein CLDN4 is abnormally expressed in a variety of epithelial-derived tumors.CLDN4 is usually silenced by hypermethylation produced by the promoter region of the gene.DNA methylation is a means of epigenetic regulation of tumors,and there is evidence that this regulation plays an important role in laryngeal squamous cell carcinoma.DNA methylation can be regulated by inhibiting the binding of transcription factors to their corresponding sites,and this process may be associated with certain binding proteins,such as the MBPs protein family.The previous work of the research team found that the expression of CLDN4 was down-regulated in human laryngeal squamous cell carcinoma,and the expression of methylation-specific binding protein Me CP2 was up-regulated,and the two were significantly correlated.Therefore,we hypothesized that there is a correlation between the down-regulation of CLDN4 expression in human laryngeal carcinoma and the occurrence of DNA methylation.Subsequent studies at the cellular level revealed that CLDN4 expression was down-regulated in laryngeal squamous cell carcinoma Hep-2 cells,and its expression was regulated by DNA methylation.At the same time,it was found that the change of CLDN4 expression was related to the migration and invasion of Hep-2 cells.However,the mechanism by which DNA methylation regulates CLDN4 expression remains unclear.In order to further investigate the mechanism of DNA methylation down-regulating CLDN4 expression,Immunohistochemistry was used to detect the expression of transcription factor Sp1 and tight junction protein CLDN4 in laryngeal carcinoma tissues,and to analyze the correlation between them;bisulfite sequencing method(bisulfite)Sequencing PCR,BSP)detection of DNA methylation sites in the promoter region of CLDN4 in Hep-2 cells;detection of the regulation of CLDN4 expression by Sp1 inhibitor MTM by PCR and Western blot;Binding of the transcription factors Sp1 and Me CP2 to the CLDN4 gene was detected by Ch IP technology.Objective:To investigate the role or the mechanisms of transcription factors Sp1 and Me CP2 in down-regulating CLDN4 expression in laryngeal carcinoma Hep-2 cells by DNA methylation.Method:1.The human laryngeal carcinoma tissues and adjacent tissues were selected as the research objects.The expression of Sp1 and CLDN4 in laryngeal carcinoma tissues was detected by immunohistochemistry,and the correlation between Sp1 and CLDN4 expression in laryngeal carcinoma tissues was analyzed;2.Detection of methylation status of Cp G locus in the promoter region of CLDN4 gene before and after treatment of demethylated drug 5-aza-d C by bisulfite sequencing technology(BSP);3.The expression of CLDN4 was detected by RT-PCR and Western blot after treatment of Hep-2 cells with 5-aza-d C and Sp1 inhibitor MTM.4.The effect of DNA methylation on the binding of Sp1 and Me CP2 to CLDN4 gene was detected by immunoprecipitation(Ch IP)technique in the treatment of demethylated drug 5-aza-d C before and after treatment of laryngeal carcinoma Hep-2cells.Result:1.The results of immunohistochemistry showed that Sp1 was highly expressed in laryngeal carcinoma tissues compared with paracancerous tissues,CLDN4 was down-regulated in laryngeal carcinoma tissues,and Sp1 expression was positively correlated with CLDN4 expression;2.BSP results showed that in Hep-2 cells,compared with the original CLDN4 gene sequence,the CLDN4 promoter region of the control group was hypermethylated,and the specific site contained the Sp1 binding region;compared with the control group,the 5-aza-d C group demethylation occurs and demethylation occurs in the Sp1 binding region;3.The results of RT-PCR showed that the expression of CLDN4 was down-regulated in a concentration-and time-dependent manner when Hep-2 cells were treated with 20?M 5-aza-d C and administered at a concentration of 300,400,500 n M MTM for 36 h.Western Blot showed that compared with the control group,the expression of CLDN4 was significantly down-regulated at 36 h and 48 h after MTM treatment.4.The results of Ch IP showed that there was a small amount of binding between Sp1 and CLDN4 promoter region in Hep-2 cells,and 5-aza-d C demethylation treatment promoted the binding of Sp1 to CLDN4 gene,and the binding of Me CP2 to CLDN4 promoter region gene,5-aza-d C demethylation inhibits the binding of Me CP2 to the CLDN4 gene.Conclusion:1.DNA methylation inhibits the binding of Sp1 and CLDN4 gene to down-regulate the expression of CLDN4 in Hep-2 cells;2.Me CP2 plays a role in DNA methylation down-regulation of CLDN4 expression in Hep-2 cells.