| Objective:Prostate cancer is the most common malignancy of the male reproductive system,and prostate cancer is one of the leading causes of cancer death in men worldwide.The incidence and mortality of prostate cancer in China has been increasing in the last decade,and the proportion of progressive prostate cancer is high.Progressive prostate cancer is a therapeutic challenge,and the study of its progression mechanism is expected to break the current therapeutic bottleneck.Non-coding RNAs have been the focus and hot spot in the field of oncology research in recent years,and numerous studies have confirmed that they are closely related to the occurrence and progression of tumors.Previous studies in our group found that miR-141-3p expression is upregulated in prostate cancer tissues and cells,and can play a pro-cancer role by targeting downregulation of KLF9.The N~6-methyladenosine(m~6A)methylation of RNA is a major form of RNA modification and is involved in the genesis and metastasis of various malignancies.No study has analyzed the regulatory role of miR-141-3p on m~6A.Therefore,the aim of this study was to investigate whether miR-141-3p has a regulatory role on m~6A modification in prostate cancer and whether this regulatory role is one of the mechanisms promoting prostate cancer progression,and to further explore the underlying molecular mechanisms.Research is designed to provide a basis for exploring new targets of personalized targeted therapy.Methods:1)Thirty cases of prostate cancer tissues and benign prostatic hyperplasia tissues were collected.The expression of miR-141-3p in the tissues was detected by q RT-PCR,and the expression of ALKBH5 in the tissues was detected by Western blot.The differences in the expression of miR-141-3p and ALKBH5 in prostate cancer tissues and benign prostatic hyperplasia tissues were analyzed.The correlation between the expression ofmiR-141-3p and ALKBH5 in prostate cancer tissueswere was analyzed.The correlation between the expression of the two in prostate cancer tissues and the clinicopathological data was also analyzed;2)The protein expression of ALKBH5 in prostate cancer cell lines PC-3,LNCa P,DU145,22RV1,and prostate immortalized normal epithelial cells RWPE-1 were detected by Western blot,and suitable prostate cancer cell lines were selected for subsequent experiments;3)The si-ALKBH5 and OE-ALKBH plasmids were constructed and transfected into prostate cancer cells,and the effect of ALKBH5 on the proliferation ability of prostate cancer cells was detected by CCK-8 assay.The effect of ALKBH5 on the migration and invasion ability of prostate cancer cells was detected by wound healing assay and transwell assay.The effect of ALKBH5 on stem cell characteristics of prostate cancer cells was detected by cell sphere formation and colony formation assays;4)The targeted relationship between miR-141-3p and ALKBH5 was verified by dual-luciferase reporter assay,and the effect of miR-141-3p on ALKBH5 expression in prostate cancer cells was detected by Western blot;5)Me RIP-PCR experiments were used to verify the m~6A modification effect of ALKBH5 on PRMT6,and the effect of ALKBH5 on the m~6A modification of PRMT6 was detected by Western blot;6)Western blot was used to detecte the effects of miR-141-3p and ALKBH5 on PRMT6 expression in prostate cancer cells,and thus analyzed the regulatory effect of the miR-141-3p/ALKBH5signaling axis on PRMT6 expression;7)Western blot and q RT-PCR were detected the PRMT6’s m RNA and protein expression of PRMT6 in prostate cancer tissues,and the correlation between its expression and the expression of miR-141-3p and ALKBH5 was analyzed;8)CCK-8 assay,wound healing assay,transwell assay and cell sphere-forming assay were put into use to verify the effect of miR-141-3p/ALKBH5signaling axis regulating PRMT6 expression on the biological function of PC3 cells;9)A model of subcutaneous prostate cancer tumorigenesis in nude mice was constructed to detect the effect of miR-141-3p on the tumorigenic ability of prostate cancer cells,and to assess the effect of miR-141-3p on ALKBH5 and PRMT6expression from in vivo experimental levels using q RT-PCR and immunohistochemical staining.Results:1)The expression of miR-141-3p was significantly higher in prostate cancer tissues than in prostate hyperplasia tissues(P<0.001),and its expression showed a significant positive correlation with Gleason score and T-stage of prostate cancer(P<0.001);2)The expression of ALKBH5 was significantly lower in prostate cancer tissues than in normal prostate tissues(P<0.001),and its expression was negatively correlated with the T-stage of prostate cancer(P=0.012);3)Mi R-141-3p was negatively correlated with the expression of ALKBH5 in prostate cancer tissues(r=-0.517,P=0.003);4)The expression of ALKBH5 in prostate cancer cell lines was significantly lower than that in normal prostate epithelial cells,and the expression was relatively lowest in PC3.Therefore,PC3 cell line were selected for subsequent experiments;5)ALKBH5 inhibited the proliferation,migration and invasive ability of PC3 cells and was able to suppress the stem cell properties of PC3 cells;6)ALKBH5 was a downstream target gene of miR-141-3p;7)ALKBH5 reduces the protein expression of PRMT6 by performing m~6A demethylase modifications of the m RNA of PRMT6;8)PRMT6 expression was significantly higher in prostate cancer tissues than in prostate hyperplasia tissues(P<0.001),and PRMT6 expression in prostate cancer tissues was positively correlated with miR-141-3p expression(r=0.440,P=0.015).PRMT6 expression was negatively correlated with ALKBH5 expression(r=-0.539,P=0.002);9)Mi R-141-3p/ALKBH5signaling axis was able to promote the proliferation,invasion and sphere-forming ability of prostate cancer cells by regulating the expression of PRMT6;10)Animal experiments further confirmed that miR-141-3p was able to promote the tumorigenic ability of prostate cancer cells,and it could inhibit the expression of ALKBH5 protein in transplanted tumors.While miR-141-3p can promote the expression of PRMT6.Conclusion:1)High expression of miR-141-3p in human prostate cancer tissues correlates with the degree of malignancy of prostate cancer,and low expression of ALKBH5 also correlates with the malignancy of prostate cancer;2)ALKBH5 can inhibit the proliferation,invasive ability and stem cell properties of prostate cancer cells;3)The miR-141-3p regulates the biological function of prostate cancer cells by targeting inhibition of the ALKBH5expression;4)ALKBH5reduces the protein expression of PRMT6 by m~6A demethylase modification of PRMT6m RNA;5)Mi R-141-3p/ALKBH5signaling axis regulates biological functions of prostate cancer cells by m~6A modification of PRMT6 m RNA. |
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