| Objective: Prostate cancer(Pca)is the most common malignant tumor in the male urinary system and one of the leading causes of death in cancer patients.Current studies suggest that all stages of prostate cancer progression depend on androgen receptor signaling,and androgen receptor proteins promote the progression of prostate cancer by regulating the abnormal expression of genes and proteins in cells at the epigenetic,transcriptional and post-transcriptional levels.However,the specific molecular regulation mechanism and clinical application are still lack of in-depth understanding.In this study,LncRNA ARLNC1 was found to be abnormally expressed in androgen-dependent prostate cancer cells by verification in different cell lines.The relationship between androgen receptor and LncRNA ARLNC1 was further studied to explore the effect of LncRNA ARLNC1 on the biological function of prostate cancer cells and the potential mechanism of action.This study provides a strong reference for the exploration of targeted therapy and molecular diagnosis and treatment targets of prostate cancer.Methods: 1.Based on the LncRNA ARLNC1 that is significantly up-regulated in prostate cancer screened by TCGA and GEO databases,normal prostate epithelial cell WPMY-1 and prostate cancer cell lines LNCa P,VCa P,DU145,22RV1 and PC3 were cultured in vitro.RT-q PCR was used to verify the expression level of LncRNA ARLNC1 in different cell lines.2.LNCa P cells were stimulated with 100 n M DHT,and the expression level of LncRNA ARLNC1 was detected at different time points to observe the timedependent relationship.At the same time,LNCa P cells were stimulated with different concentrations of DHT(0n M,1n M,10 n M,100 n M)for 24 h to verify the expression of LncRNAARLNC1 and observe the concentration-dependent relationship.3.The JASPER database was used to predict the AR binding sites in the promoter region of LncRNA ARLNC1,and the localization of AR protein in cells was observed by cellular immunofluorescence.The promoter sequence of LncRNA ARLNC1 was constructed into a plasmid vector containing firefly luciferase reporter gene.After cotransfection with AR overexpression plasmid into LNCa P cells,the reporter gene activity was detected.4.Lentiviral interference of LncRNA ARLNC1 expression in prostate cancer LNCa P cells was used to investigate the effect of LncRNA ARLNC1 expression on the biological function of LNCa P cells in vitro using CCK-8,transwell,cloning and scratch experiments.Flow cytometry was used to detect the cell cycle changes of LNCa P cells after silencing LncRNA ARLNC1.In addition,the expression of epithelial-mesenchymal transition(EMT)markers in LNCa P cells after knocking down LncRNA ARLNC1 was detected by western blot(WB).LNCa P cells stably overexpressing LncRNAARLNC1 were implanted subcutaneously in nude mice in vitro to explore the effect of LncRNAARLNC1 on tumor formation in nude mice.5.To analyze the potential mechanism of LncRNA ARLNC1 regulating the biological function of prostate cancer cells by transcriptome sequencing and bioinformatics technology,and to verify it at the protein level.Results:1.RT-q PCR results showed that LncRNA ARLNC1 was almost not expressed in these three androgen receptor negative prostate cancer cell lines(22RV1,PC3,DU145);However,it was highly expressed in 2 androgen receptor-positive prostate cancer cell lines(LNCa P and VCa P).2.LNCa P cells were stimulated by DHT in a time-and concentration-dependent manner,and the expression of LncRNA ARLNC1 was increased at the transcriptional level(P<0.01).AR was mainly expressed in LNCa P nuclei by immunofluorescence.The results of dual luciferase reporter genes showed that the activity of promoter reporter genes in the experimental group was increased(P<0.05),while that in the Control group was almost unchanged(P>0.05)3.The results of CCK-8,transwell,cloning,and scratch experiments showed that the expression of LncRNAARLNC1 in LNCa P cells was decreased after lentivirus interference(P<0.01).Compared with the control group,the interference group could inhibit the cell cycle mainly in G1 and S phase(P<0.01).WB results also showed that the expression of cyclin D1 and CDK6 in the knockdown group was lower than that in the control group(P<0.05),while the expression of P27 protein was increased(P<0.01).Compared with the control group,the expression of epithelial cell marker E-cadherin protein in the ARLNC1 interference group was increased(P<0.01),while the expression of mesenchymal cell markers vimentin and N-cadherin protein was decreased(P<0.01).4.The prostate cancer cells with stable interference of LncRNAARLNC1 were implanted subcutaneously in nude mice.Compared with the control group,the growth of subcutaneous tumor in the interference group was slowed down(P<0.05).5.Compared with the control group,the expression of p-PI3 K,p-Akt and p-mtor proteins was inhibited in the knockdown group of LncRNA ARLNC1(P<0.05),but the levels of non-phosphorylated PI3 K,AKT and m TOR proteins did not change significantly in the cells(P>0.05).Conclusions:1.LncRNA ARLNC1 is a potential oncogene in prostate cancer.It is highly expressed in androgen-dependent prostate cancer cells,and androgen receptor protein can regulate the expression of LncRNAARLNC1 at the transcriptional level.2.LncRNA ARLNC1 can promote the proliferation,invasion,migration,colony formation ability and cell cycle progression of LNCa P cells in vitro,and play a cancer-promoting role through the process of epithelial mesenchymal transition.3.High expression of LncRNA ARLNC1 promotes subcutaneous tumor formation in nude mice in vivo.4.LncRNA ARLNC1 can regulate the malignant progression of prostate cancer through the PI3K/AKT/m TOR signaling pathway. |
