Functional And Mechanistic Studies Of Histone Methyltransferase KMT2D Promotes Prostate Cancer Progression Through IL-6 Signaling

Background and objectiveGenomics and tumor biology are still hot spots and difficulties in prostate cancer research.In my research group,previous studies have found that KMT2D in the histone methylated transferase KMTs family has mutations and high expression in prostate cancer,and the two are significantly correlated.Further studies confirmed that KMT2D up-regulates the expressions of LIFR and KLF4 genes through histone modification enzyme activities,thus activating PI3K/Akt and EMT signaling pathway,to promote the proliferation,invasion and metastasis of prostate cancer cells.Meanwhile,KMT2D can enhance enhancer activity,prevent the binding of.FOXO3,a transcription factor mediating oxidative stress,to downstream target genes,enhance antioxidant activity,resist ROS mediated DNA damage,and inhibit the apoptosis of prostate cancer cells.As a common epigenetic modification enzyme,KMT2D may be involved in the occurrence and progression of prostate cancer through multiple mechanisms at the same time.In order to further explore the significance of KMT2D in prostate cancer,a more comprehensive study of its mechanism of action in prostate cancer is still needed.MethodsKyoto Encyclopedia of Genes and Genomes(KEGG)and Gene Set Enrichment Analysis(GSEA)were conducted by RNA-seq.GSEA enrichment analysis was performed in the TCGA prostate cancer cohort.Prostate cancer cells were cultured with KMT2D knockout cell culture supernatant,cell proliferation toxicity was detected by cell viability assay kit(CCK-8),cell proliferation was detected by EdU cell proliferation,migration ability was detected by scratch assay,and cell apoptosis was detected by flow cytometry.The phenomenon of epithelial mesenchymal transformation was investigated by Western blot.Cytokine screening and identification of KMT2D knockout cell culture supernatant were performed by semiquantitative sandwich membrane chip detection,and enzyme linked immunosorbent assay,ELISA),quantitative reverse transcription polymerase chain reaction(qRTPCR),Western blot were used to detect the cytokine levels in the cell supernatant,mRNA and cell protein knockout of KMT2D.Clinical prostate cancer tissue specimens were obtained and assessed by immunohistochemical staining(IHC)while correlation analysis was conducted through TCGA database.After knockout of IL6R,CCK-8 was used to detect cell proliferation toxicity,EdU cell proliferation,scratch test to detect migration ability,and flow cytometry to detect cell apoptosis in prostate cancer.The mechanism was explored by chromatin immunoprecipitation technology-polymerase chain reaction(Chip-PCR)and Western blot.ResultsThe results of this study can be divided into five parts.First.After KMT2D silencing,signal pathways such as cytokines are enriched;Second,KMT2D silencing cell supernatant inhibits the proliferation and migration of prostate cancer cells and promotes cell apoptosis,and Epithelial mesenchymal transformation was observed.Third,IL-6 decreased most obviously after KMT2D silencing,and IHC suggested KMT2D was positively correlated with IL-6.TCGA also suggested that KMT2D was correlated with IL-6 and STAT3.Forth,IL-6R silencing also inhibited the proliferation,migration and apoptosis of prostate cancer cells and acted on KMT2D silenced cells supernatant treatment group was equivalent.Fifth,The mechanism was explored and discovered by Chip-PCR and Western blot:KMT2D enhances IL-6 transcriptional activity by methylating H3K4,KMT2D silenced cells supernatant and blocking of IL-6 signaling pathway inhibited STAT3 phosphorylation.ConclusionOur study showed that the loss of KMT2D inhibited prostate cancer progression through the downregulation of IL-6 signaling.These results suggest that KMT2D could be a potential therapeutic target for PCa treatment.