Functional And Mechanistic Studies Of Histone Methyltransferase KMT2D Mediated Lipid Metabolism Via PPARγ In Prostate Cancer

Background and aimsProstate cancer(PCa)is one of the common malignancies in older men.Endocrine therapy is the first-line therapy for advanced prostate cancer.But as the disease progresses,patients tend to lose the sensitivity of the treatment.A large number of studies have shown that lipid metabolism plays an important role in the development of prostate cancer.Understanding the mechanism of lipid metabolism can provide new ideas for the treatment of advanced prostate cancer.Previous studies have found that histone methyltransferase KMT2D is highly expressed in prostate cancer cells and it is associated with a poor prognosis of the disease.Peroxisome proliferator-activated receptor gamma(PPARγ)is an important pathway molecule in lipid metabolism.PPARy agonists(such as rosiglitazone)can activate its transcription of downstream genes.Recent studies in the liver have found that KMT2D and PPARγ can form a protein complex,which prompts KMT2D to exert methylation to regulate the PPARγ signaling pathway,and PPARγ is also a target gene for its own regulation.This study will further explore the mechanism of the interaction between KMT2D and PPARy to regulate lipid metabolism in prostate cancer,and pro vide a new direction for the treatment of advanced prostate cancer.Methods1.Analysis of the correlation between KMT2D and PPARγ and their downstream regulatory genes(FASN,ACC and ACLY)in prostate cancer,based on the TCGA database.2.Construction of KMT2D silent cell model.After KMT2D expression was down-regulated in cells,we studied the changes in lipid droplet content in the cells.We also studied the changes in mRNA and protein levels of PPARyand downstream lipid metabolism-related genes(FASN,ACCand ACLY).And we further explored the effects of rosiglitazone on these changes.3.Using DNA pulldown technology to verify that KMT2D and PPARy were bound to the PPRE sequence of the FASN promoter..4.CCK-8 and cell scratch experiments to explore the effects of PPARy agonists on the proliferation and migration of prostate cancer cell lines before and after KMT2D silencing.Results1.Based on TCGA database analysis,it was found that KMT2D was positively correlated with the expression of FASN,ACC and ACLY(r>0.2,P<0.05),but had no significant correlation with the expression of PPARγ(r<0.2,P>0.05).2.We have successfully constructed three types of KMT2D silent cell models of prostate cancer cells.After KMT2D was down-regulated,oil red staining showed a decrease in lipid droplet content(p<0.005),the mRNA expression levels of lipid metabolism genes PPARy and downstream regulators(FASN,ACC and ACLY)were down-regulated(p<0.05).A corresponding decrease in protein levels.3.After addition of PPARy agonist,oil red O staining in both groups showed that lipid droplet content was up-regulated(p<0.05).The mRNA expression of PPARy was significantly up-regulated in the control group(p<0.001),and PPARy was also up-regulated in the cells of the KMT2D silencing group(p<0.05).The difference in expression between the two groups increased from 1.8 times without medication to 3.2 times after rosiglitazone.The downstream target genes also changed accordingly.4.DNA pulldown experiment was performed on the PPRE sequence in the FASN promoter:KMT2D and PPAR protein bands were detected only in the pull-down target group.5.Rosiglitazone can promote the proliferation(p<0.05)and migration(p<0.05)of prostate cancer cells,but when KMT2D silencing,rosiglitazone has no significant effect on cell proliferation and migration(p>0.05).Conclusions1.In prostate cancer cells,KMT2D can interact with PPAR gene,and bind to the PPRE sequence of the target gene promoter of PPAR gene,so that KMT2D plays an important role in the transcriptional regulation of PPAR gene and its target gene,and affects the lipid metabolism of cancer cells.2.In the absence of KMT2D,rosiglitazone,the agonist of PPAR,could not increase the ability of cell proliferation and migration,which indicat that KMT2D plays an important regulatory role in the biological role of PPAR.3.Advanced prostate cancer can be treated by intervention in the direction of lipid metabolism,and the specific mechanism of action still needs further study..