Silencing Of MBD2 And EZH2 Inhibit The Proliferation Of Colorectal Carcinoma Cells By Rescuing The Transcription Of SFRP

Objective Secreted frizzled related proteins(SFRPs),as extracellular inhibitors of Wnt pathway signaling,was found to be underexpressed in the early stage of colorectal tumorigenesis due to hypermethylation of the promoter.Our previous study found that restoring the expression of SFRP can inhibit the activation of Wnt pathway.MBD2 and EZH2 are core members of MBD and PCG protein families respectively,which have been known as crucial proteins of epigenetic regulation.The aim of this study is to figure out the potential role of MBD2 and EZH2 proteins in colorectal cancer(CRC)and its effects on the expression of SFRP.Methods The m RNA expression of SFRPs in CRC and adjacent noncancerous tissues are analyzed by bioinformatics.Real-time quantitative polymerase chain reaction(q RTPCR)and western blot were used to detect the expression of MBD2,EZH2 and SFRP in CRC cell lines and human normal intestinal mucosa cell NCM460.We knockdown of MBD2 and EZH2 using small interfering RNA(si RNA)to clear its role in the regulation of SFRP gene expression.The function of MBD2 and EZH2 in cell proliferation,cycle,apoptosis and invasion was examined in CRC cell lines.Results The m RNA expression level of SFRPs was significantly decreased in CRC tissues and cell lines compared to adjacent tissues and NCM460(P<0.05).However,the m RNA level of EZH2 and MBD2 were highly expressed in CRC cell lines(P<0.05).In SW480 cells,interference with MBD2 alone could restore the expression of SFRP1,but could not restore the expression of SFRP2,SFRP4 and SFRP5.Interference with EZH2 alone could restore the expression of SFRP2,SFRP4 and SFRP5,but could not restore the expression of SFRP1.In HCT116 cells,the expression of SFRP1,SFRP2,SFRP4 and SFRP5 could not be restored after interfering with EZH2 alone,but the expression of SFRP1 gene could be restored after interfering with MBD2 alone,but there was no significant effect on the expression of SFRP2,SFRP4 and SFRP5.However,simultaneous interference with MBD2 and EZH2 in SW480 and HCT116 cells could restore the expression of SFRP1,SFRP2,SFRP4 and SFRP5,and there was significant difference compared with MBD2 and EZH2,respectively.Western blotting showed that interference with MBD2 and EZH2 had no significant difference in the expression of SFRP1,SFRP2,SFRP4 and SFRP5 in SW480 and HCT116 cells compared with NC in the control group,while interference with MBD2 and EZH2 could significantly restore the protein levels of SFRP1 and SFRP4.There was no significant difference between SFRP2 and SFRP5.Compared with interfering with MBD2 or EZH2 alone,simultaneous interference with MBD2 and EZH2 could significantly inhibit the proliferation,migration and invasion of CRC,could block the cell cycle and increase apoptosis more effectively(P < 0 05).Conclusion Compared with silencing MBD2 or EZH2 alone,silencing MBD2 and EZH2 together can inhibit the growth of CRC cells by restoring the expression of SFRP more effectively.MBD2 and EZH2 may be potential therapeutic targets for colorectal tumors.However,the regulatory mechanism of SFRP remains to be further studied in the future.