Expression And Function Of N~6-methyladenosine (m6A) Methylase METTL3 In Prostate Cancer

Objective:Our aim of this study is to explore the expression and clinical significance of Methyltransferase-like 3(METTL3):in prostate cancer,to observe the effect of METTL3 silenced by small interfering RNA(siRNA)on proliferation,invasion and cloning of prostate cancer cells,to explore the possible molecular mechanism of METTL3 in prostate cancer,and to provide a new theoretical basis for the diagnosis and treatment of prostate cancer.Methods:1.The TCGA datasets were used to analyze the differential expression of METTL3 mRNA in prostate cancer and adjacent tissues.Western blot(WB)and immunohistochemistry(IHC)were used to detect the differential expression of METTL3 protein in prostate cancer and adjacent tissues.2.Spearman statistical methods were used to analyze the correlation between the expression of METTL3 mRNA and protein and the clinicopathological data of prostate cancer.3.The siRNA silencing METTL3 gene was screened.The prostate cancer DU 145 and PC-3 cells were randomly divided into blank control group,Mock group,negative control group and experimental group.Quantitative real-time PCR(RT-PCR)and western blot assays were used to detect the expression levels of METTL3 mRNA and protein to verify interference efficiency.4.Cell proliferation,invasion and cloning ability were measured by using CCK-8 assay,transwell chamber assay and plate colony formation assay,respectively.5.After cell transfected,dot blot was used to detect the expression of total m6A in cellular RNA,and western blot was used to detect the expression of prostate tumor-associated proteins Bcl-2,c-Myc and EGFR.Results:1.TCGA datasets showed that METTL3 mRNA expression in prostate cancer tissue was significantly higher than that in adjacent tissues(p<0.01).METTL3 mRNA expression level was positively correlated with Gleason score(p<0.01).However,METTL3 mRNA expression was not significantly correlated with age,T stage and N stage(p>0.05).2.IHC assay showed that METTL3 protein was detected in the nucleus and cytoplasm of prostate cancer and adjacent tissues,mainly expressing in the nucleus.IHC and Western Blot assay showed that METTL3 protein expression was significantly up-regulated in prostate cancer tissues compared with adjacent tissues(p<0.01).The expression of METTL3 protein was positively correlated with PSA value and Gleason score(pPSA<0.01,pGleason<0.05).The expression of METTL3 protein was not significantly correlated with age,T stage and N stage(p>0.05).3.After cell transfected for 48 hours,RT-PCR results showed that the interference efficiencies of siRNA-1400 fragments on METTL3 mRNA in DU 145 and PC-3 cells were 90%,91%and 72%,73%,respectively,compared with the blank group and the negative control group(p<0.01).Western blot results showed that the interference efficiency of siRNA-1400 fragment on METTL3 protein in DU 145 and PC-3 cells was 87.2%,85.7%and 97.8%,97.7%,respectively(p<0.01).Western blot results were consistent with RT-PCR's.The siRNA-1400 interference fragment successfully down-regulated METTL3 expression.4.CCK-8 assay showed that the OD values of the experimental group were significantly lower than those of the blank and negative control groups in DU 145 and PC-3 cells(p<0.01).Transwell chamber assay showed that the number of cells passing through Matrigel basement membrane was significantly lower than that of blank control and negative control group in DU145 and PC-3 cells(p<0.01).Plate cloning assay showed that compared with blank control group and negative control group,the number of clones in DU 145 and PC-3 cells in experimental group decreased significantly(PDU145<0.01,pPC-3<0.05).These results showed that down-regulation of METTL3 gene significantly inhibited the proliferation,invasion and cloning ability of DU 145 and PC-3 cells.5.Dot blot assay showed that the total m6A level of RNA in the experimental group was significantly lower than that in the blank control group and the negative control group in DU145 and PC-3 cells(P<0.01),which indicated that inhibiting the expression of METTL3 gene would down-regulate the total m6A level of RNA.Western blot assay showed that the expressions of Bcl-2,c-Myc and EGFR were significantly lower than those in the blank control group and the negative control group in DU145 and PC-3 cells(p<0.01),which indicated that down-regulation of METTL3 would significantly inhibit the expression of Bcl-2,c-Myc and EGFR.Conclusions:1.METTL3 protein is expressed in the cytoplasm and nucleus of prostate cancer and adjacent tissues,mainly expressing in the nucleus.The expression of METTL3 mRNA and protein was significantly up-regulated in prostate cancer tissues.2.The expression of METTL3 was positively correlated with Gleason score and PSA value.High expression of METTL3 may inhibit the tissue differentiation of prostate cancer,and METTL3 may have the value of being a diagnostic marker combined with PSA for prostate cancer.3.METTL3,which is highly expressed in prostate cancer,may promote the expression of Bcl-2,c-Myc,EGFR through m6A modification pathway or independent of methyltransferase activity,may enhance the proliferation,invasion and cloning ability of cancer cells,and may be a potential therapeutic target for prostate cancer.