Molecular Mechanism Of CFLAR_L Regulation By PRMT5 And PRMT1 In Tumor Cells

Cancer is the leading cause of morbidity and mortality around the world.Current clinical treatment is mainly chemoradiation and surgery.Induction of tumor cell apoptosis has become the usual way of treating cancer.L-type variants of CFLAR is an important regulatory protein in extrinsic apoptotic signaling pathway.It inhibits the apoptosis-related signal transduction by combination with Caspase8 to formate heterodimers,affecting negative CASP cascades activation,acting as an anti-apoptotic protein.According to Phosphosite,CFLARL has a mono-methylation modification at the R122 site,and the methylation modification of this site plays an important regulatory role on the stability of the protein itself.However,there are not many reports on methyltransferase of CFLARL.Therefore,looking for CFLARL methyltransferase and exploring the effect of CFLARL will provide more ideas for us to explore the regulation of CFLARL protein and apoptosis,and help us better explore CFLARL specific genes,and in accordance with these design more effective drug targets.The protein arginine methyltransferases PRMT5 and PRMT1 are members of the PRMT family.PRMT5 catalyzes the production of monomethylated or symmetric dimethylated arginine for the substrate protein.PRMT1 catalyzes the monomethylation or asymmetric dimethylation of arginine in substrate proteins.Both are important transferases for methylation modification of histones and non-histones,and can be investigated as candidate methyltransferases for CFLARL.In the course of our experiments,we found that there were three methylation modifications in CFLARL and were affected by the chemotherapeutic drug doxorubicin.The existence of methylation modification in CFLARL has been confirmed by our results.To investigate whether PRMT5 and PRMT1 affected CFLARL,we knocked down PRMT5 by RNA interference technology and CFLARL expression was significantly decreased.Knockdowning PRMT1,CFLARL expression levels were significantly increased.As a result of our co-immunoprecipitation,both PRMT5 and PRMT1 interacted with CFLARL.Considering the methyltransferase activities ofPRMT5 and PRMT1,we found that overexpression of PRMT5 decreased the monomethylation level of CFLARL and increased the symmetric bimethylation level of CFLARL;overexpression of PRMT1 also reduced CFLARL monomethylation levels but increased the asymmetric bimethylation levels of CFLARL.Next,we examined the effects of PRMT5 and PRMT1 on CFLARL stability and ubiquitination.By interfering with PRMT5 siRNA or overexpressing PRMT1 and treating cells with E64D and MG132,respectively,Western blotting experiments showed that PRMT5 and PRMT1 affected the ubiquitin-proteasomal degradation pathway of CFLARL.ITCH is an E3 ligase of CFLARL,and XRCC6 regulates the stability of CFLARL.Overexpression of PRMT5 causing acceleration of its binding to XRCC6 increased the stability of CFLARL,lowered the binding of CFLARL to the E3 ubiquitin ligase ITCH,thereby suppressing polyubiquitination of CFLARL;Overexpression of PRMT1 inbibited the ability of CFLARL binding to XRCC6,which loweded the stability of CFLARL and caused CFLARL binding to E3 ubiquitin ligase thereby lifting polyubiquitination of CFLARL.Our previous results showed that chemotherapy drugs counld promote apoptosis by down-regulating CFLARL expression.Therefore,we speculated that PRMT5 and PRMT1 regulate apoptosis.Using human non-small cell lung cancer cells as a model,we used chemotherapeutic drugs doxorubicin to promote the degradation of CFLARL and induce apoptosis.We used cellular and molecular biological techniques to further study the role of PRMT5 and PRMT1 to regulate apoptosis by CFLARL in this process.Finally,we explored the relationship between PRMT5 and PRMT1,co-immunoprecipitation results showed that there was competitively bound to CFLARL between PRMT5 and PRMT1.In summary,this study explored the mechanism that arginine methyltransferases PRMT5 and PRMT1 regulate CFLARL and their role in apoptosis.From this hypothesis:PRMT5 and PRMT1 competitively bound CFLARL,which caused different regulation of CFLARL relying on different enzyme catalytic function and different methylation patterns.There were influence in CFLARL binding with XRCC6 and E3 ubiquitin ligase,and thus the stability of CFLARL and ubiquitination.It was showed that changes in protein levels ultimately played a positive or negative regulatory role in apoptosis of non-small cell lung cancer cells.