Abstract：Objective To investigate the role of LINC00346 in the immune escape of prostate cancer cells and its related molecular mechanism.Methods Prostate cancer cells DU145, PC3 and LNCaP were cultured in vitro, and human normal prostate epithelial cell RWPE-1 was used as a control. PC3 cells were divided into NC-siRNA group, LINC00346-siRNA group, NC inhibitor group, miR-148a-3p inhibitor group, NC mimic group, miR-148a-3p mimic group, LINC00346-siRNA+NC inhibitor group, LINC00346-siRNA+miR-148a-3p inhibitor group and LINC00346-siRNA+pcDNA-PD-L1 group were transfected with Lipofectamine 2000 reagent, respectively. Real-time fluorescent quantitative PCR (qRT-PCR) was used to detect the expression of LINC00346 and miR-148a-3p in PC3 cells. CCK-8 assay and flow cytometry were used to detect the proliferation and apoptosis of PC3 cells in each group, respectively. Bioinformatics methods and dual luciferase reporter gene assay were used to predict and verify the targeting relationship between LINC00346 and miR-148a-3p and between miR-148a-3p and programmed death ligand 1 (PD-L1), respectively. qRT-PCR and Western blot were used to detect the expression of PD-L1 mRNA and protein in PC3 cells, respectively. The transfected PC3 cells were co-cultured with CD8+T cells, and the proliferation and apoptosis of CD8+T cells were detected by CCK-8 assay and flow cytometry, respectively. The levels of IFN-γ and TNF-α in CD8+T cells were measured by ELISA.Results Compared with human normal prostate epithelial cell RWPE-1, LINC00346 expression was significantly increased in prostate cancer cells DU145, PC3 and LNCaP (all P<0.05), and the expression level of LINC00346 was the highest in PC3 cells. Compared with the NC-siRNA group, the LINC00346 level and proliferation ability of PC3 cells were significantly decreased, and the apoptosis rate was significantly increased in the Linc00346siRNA group (all P<0.05). The results of dual luciferase reporter gene assay showed that compared with the NC mimic group, the luciferase activities of LINC00346-WT and PD-L1-WT in the cells transfected with miR-148a-3p mimic group were significantly decreased (all P<0.05). Compared with the NC-siRNA group, the expression of miR-148a-3p in the cells of the LINC00346-siRNA group was significantly increased (P<0.05). Compared with the NC-siRNA group, the proliferation ability of PC3 cells in the LINC00346-siRNA group was decreased, and the apoptosis rate was significantly increased (all P<0.05). Compared with the LINC00346-siRNA+NC inhibitor group, the level of miR-148a-3p and apoptosis rate of PC3 cells were significantly decreased, and the cell proliferation ability was significantly increased in the LINC00346-siRNA+miR-148a-3p inhibitor group (all P<0.05). Compared with NC inhibitor group, the expression of PD-L1 mRNA in miR-148a-3p inhibitor group was increased (P<0.05). Compared with the NC-siRNA group, the expression levels of PD-L1 mRNA and protein in PC3 cells were decreased in the LINC00346-siRNA group (all P<0.05). Compared with the LINC00346-siRNA+NC inhibitor group, the levels of PD-L1 mRNA and protein in the PC3 cells of the LINC00346-siRNA+miR-148a-3p inhibitor group were increased (all P<0.05). Compared with the LINC00346-siRNA group, the proliferation ability of CD8+T cells and the levels of IFN-γ and TNF-α were significantly decreased, and the apoptosis rate of CD8+T cells was significantly increased in the LINC00346-siRNA+miR-148a-3p inhibitor group and the LINC00346-siRNA+pcDNA-PD-L1 group (all P<0.05).Conclusions Knockdown of LINC00346 may inhibit the proliferation and immune escape of prostate cancer cells and promote cell apoptosis by regulating the miR-148a-3p/PD-L1 axis.
