MiR-5195-3p functions as a tumor suppressor in prostate cancer via targeting CCNL1

Abstract

Background: Accumulating evidence indicates that miR-5195-3p exerts tumor-suppressive roles in several tumors. However, the clinical significance and biological function of miR-5195-3p in prostate cancer (PCa) have not been reported yet.

Methods: The expression levels of miR-5195-3p and Cyclin L1 (CCNL1) were determined using quantitative real-time PCR in clinical specimens and cell lines. The clinical significance of miR-5195-3p in patients with PCa was evaluated using Kaplan-Meier survival analysis and Cox regression models. Cell proliferation and cell cycle distribution were measured by CCK-8 assay and flow cytometry, respectively. The association between miR-5195-3p and CCNL1 was analyzed by luciferase reporter assay.

Results: MiR-5195-3p expression levels were significantly downregulated in 69 paired PCa tissues compared with matched adjacent normal tissues. The decreased miR-5195-3p expression was associated with Gleason score and TNM stage, as well as worse survival prognosis. The in vitro experiments showed that miR-5195-3p overexpression suppressed the proliferation and cell cycle G1/S transition in PC-3 and DU145 cells. Elevated miR-5195-3p abundance obviously impaired tumor formation in vivo using PC-3 xenografts. Mechanistically, CCNL1 was a direct target of miR-5195-3p in PCa cells, which was inversely correlated with miR-5195-3p in PCa tissues. Importantly, CCNL1 knockdown imitated, while overexpression reversed, the effects of miR-5195-3p overexpression on PCa cell proliferation and cell cycle G1/S transition.

Conclusions: Our data suggest that miR-5195-3p functions as a tumor suppressor by targeting CCNL1 in PCa.

Keywords: CCNL1; Proliferation; Prostate cancer; miR-5195-3p.

Fig. 1

MiR-5195-3p was downregulated in PCa tissues and correlated with overall survival. A MiR-5195-3p expression in 69 paired HCC and the matched adjacent normal tissue samples was measured by quantitative real-time PCR. B The correlation between miR-5195-3p expression and overall survival was analyzed with the Kaplan–Meier method. The p-value was obtained using the log-rank test

Fig. 2

Effects of miR-5195-3p overexpression on PCa cell proliferation and cell cycle progression. A MiR-5195-3p expression in PCa cell lines (PC-3, 22RV1, DU145, and LNCaP) and a nontransformed but immortalized prostate cell line RWPE-1 was measured by quantitative real-time PCR. B PC-3 and DU145 cells were transfected with the miR-5195-3p mimics or miR-NC. MiR-5195-3p expression in PC-3 and DU145 cells was detected by quantitative real-time PCR. C–D Cell proliferation was tested with CCK-8. MiR-5195-3p overexpression significantly inhibited the proliferation of PC-3 and DU145 cells. E–F Cell cycle distribution was determined by PI staining and flow cytometry analysis in PC-3 and DU145 cells. Data are presented as mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001 compared with miR-NC group. PI propidium iodide; NC negative control

Fig. 3

CCNL1 was a direct target of miR-5195-3p in PCa. A The sequences of the putative miR-5195-3p binding sites in the wild-type and mutant CCNL1 3′-UTR. B–C Luciferase reporter plasmids carrying the CCNL1 wild-type 3′-UTR (CCNL1-WT) or CCNL1 mutant 3′-UTR (CCNL1-MUT) were transfected into PC-3 and DU145 cells with miR-5195-3p mimics or miR-NC. MiR-5195-3p upregulation suppressed luciferase activity of the wild-type but not the mutant 3′-UTR of CCNL1. Renilla luciferase activity was used as a control. D mRNA and E protein expression levels of CCNL1 following miR-5195-3p mimics or miR-NC transfection. F CCNL1 mRNA expression levels in 69 pairs of human PCa and matched adjacent normal tissues were measured by quantitative real-time PCR. Data are presented as mean ± standard deviation. **p < 0.01, ***p < 0.001 compared with miR-NC group. G MiR-5195-3p expression was inversely correlated with CCNL1 miRNA expression in PCa tissues, as demonstrated by the Spearman’s correlation coefficient

Fig. 4

MiR-5195-3p suppressed cell proliferation and cell cycle G1/S transition through CCNL1 mediation. PC-3 cells were transfected with si-CCNL1 or si-NC, as well as co-transfection with miR-5195-3p mimics and pcDNA3.1-CCNL1. A Western blot was used to determine the Cyclin L1 protein expression levels in the above transfected PC-3 cells. B Cell proliferation was tested with CCK-8 assay in the above transfected PC-3 cells. C–D Cell cycle distribution was determined by PI staining and flow cytometry analysis in the above transfected PC-3 cells. Data are presented as mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001 compared with si-NC or miR-5195-3p mimics

Fig. 5

MiR-5195-3p induced the regression of prostate tumorigenesis in vivo. A MiR-5195-3p expression in stable miR-5195-3p mimics or miR-NC overexpression PC-3 cells determined by quantitative real-time PCR. B Image of tumor xenografts in nude mice injected subcutaneously with miR-5195-3p-overexpressing PC-3 cells. C Tumor volume was measured every 5 days. D Changes in the tumor weight in mice after miR-5195-3p overexpression. E MiR-5195-3p expression in tumor tissue isolated from miR-5195-3p mimics and miR-NC groups of nude BALB/c mice. F Protein expression levels of Cyclin L1, CDK4, and Cyclin D1 expression were determined by western blot analysis. Data are presented as mean ± standard deviation. **p < 0.01, ***p < 0.001 compared with miR-NC