SETD4 Inhibits The Proliferation Of Prostate Cancer By Catalyzing H3K27 Methylation And Regulating Cell Cycle

Background and purposeProstate cancer,as a common malignancy,poses a serious threat to men’s health in China.SETD4 is a histone methyltransferase that is reported involved in many disease processes including tumorigenesis and inflammation,but its role in prostate cancer is still elusive.Hence,in this research we intend to clarify the biological function of SETD4 in prostate cancer in vitro and in vivo,and elucidate the underlying mechanism,thereby providing a potential target for clinical prognosis prediction and intervention treatment of prostate cancer.MethodsThe expression of SETD4 in tumors was identified by bioinformatics analysis;the expression of SETD4 in prostate cancer clinical samples and its correlation with clinicopathological parameters were detected by immunohistochemistry;the expression of SETD4 in prostate cancer cells was detected by qRT-PCR and Western blotting.Prostate cancer cell lines with stable SETD4 silencing or overexpression were constructed by lentiviral vectors,and the effect of SETD4 on the proliferation ability of prostate cancer cells in vitro was clarified by CCK8,plate cloning and EdU assays,the cycle distribution of prostate cancer cells were detected by flow cytometry,the expression of key proteins in cell cycle that regulated by SETD4 was detected by Western blotting;The effects of SETD4 on the migration,invasion and apoptosis of prostate cancer cells were determined by Transwell migration assay,scratch healing assay,stromal gel invasion assay and flow cytometry apoptosis assay respectively;the role of SETD4 in the tumorigenic of prostate cancer cells in vivo were determined by subcutaneous xenograft tumor model in nude mice.The histone methylation sites catalyzed by SETD4 were screened verified by histone methylation sites sequencing,ELISA and Western Blotting;the downstream targets of SETD4 were screened by RNA-seq and the regulatory relationship was verified by qRT-PCR;the locating of H3K27me3 to the promoter region of NUPR1 was analyzed by ChIP.The effect of NUPR1 on the altered biological function of prostate cancer cells caused by SETD4 was verified by CCK8 assay,plate cloning assay,EdU incorporation assay and flow cytometric cycle assay,and the signaling pathways mediating SETD4-induced functional alterations were explored by Western Blotting.ResultsThe expression level of SETD4 in prostate cancer tissues was down-regulated compared to normal prostate tissue,and negatively correlated with the pathological grade,clinical stage,Gleason score and T-stage of prostate cancer;SETD4 expression was down-regulated in prostate cancer cell lines compared with normal prostate epithelial cells.Cell function assays demonstrated that SETD4 significantly delayed the proliferation and cycle of prostate cancer cells in vivo,but had no effect on migration,invasion and apoptosis of prostate cancer cells.Subcutaneous xenograft tumor assay in nude mice demonstrated that SETD4 inhibited the tumorigenic capacity of prostate cancer cells in vivo.Mechanistic studies demonstrated that SETD4 inhibits NUPR1 expression by catalyzing H3K27me3.Functional reversion experiments and pathway experiments confirmed that NUPR1 mediates SETD4-induced alterations in prostate cancer cell cycle progression through regulation of the AKT pathway,thereby affecting the proliferative capacity of prostate cancer.ConclusionSETD4 expressed a reduced level in prostate cancer and correlates closely with the malignancy of prostate cancer.Mechanically,SETD4 inhibits the proliferation of prostate cancer by inducing the inhibition of NUPR1 expression through catalyzing H3K27me3,which further inhibits the activity of AKT pathway and induces cycle arrest in prostate cancer cells.