miR-195 inhibits cell proliferation and angiogenesis in human prostate cancer by downregulating PRR11 expression

Abstract

hsa-miR-195-5p (miR-195) has been proven to be a critical regulator in the progression of prostate cancer (PCa). To identify additional targets and molecular functions of miR-195, we overexpressed miR-195 by transient oligonucleotide transfection in DU145 and LNCaP cells and examined the effects. RNA-based microarray and dual-luciferase assays were carried out to identify novel targets of miR-195, while in vitro functional assays, a subcutaneous xenograft model, tissue microarray (TMA) analysis and a cohort of publicly available data (Taylor cohort) were used to investigate the biological function and clinical value of miR-195 targeting. The results shown that miR-195 overexpression could markedly suppress cellular proliferation and tube formation compared with miR-negative control. The RNA-based microarray identified a total of 153 differentially regulated genes with fold changes of ≤|1.5|, including 138 (90.2%) downregulated and 15 (9.8%) upregulated genes. Among the downregulated genes, we found that proline-rich protein 11 (PRR11) combined with miR-195 expression (miR-195/PRR11) could be used as an independent predictor of the risk of biochemical recurrence in the Taylor cohort. Additionally, the dual-luciferase assay identified PRR11 as a novel target of miR-195, and the in vitro assays indicated that PRR11 abrogated the suppressive effects of miR-195 on cell proliferation, tube formation and cell cycling. Furthermore, the subcutaneous tumor xenograft model indicated that knockdown of PRR11 inhibited xenograft growth and angiogenesis, while the results of the TMA and Taylor cohort analyses collectively demonstrated that PRR11 expression was upregulated in aggressive tumors and is associated with poor clinical outcome. Taken together, these findings further illustrate the suppressive role of miR-195 in PCa, and indicate a novel role of PRR11 in PCa. Importantly, the newly identified miR-195/PRR11 axis may aid with identifying potential therapeutic targets in PCa.

Figure 1.

Overexpression of miR-195 suppresses the proliferation of LNCaP and DU145 cells and inhibits human umbilical vein endothelial cell (HUVEC) tube formation in vitro. (A) miR-195 levels were determined by RT-qPCR at 48 h after transient transfection of DU145 and LNCaP cells with miR-195 mimics. miR-195 expression (miR-195/U6) was calculated as fold change relative to the negative control (NC). (B) HUVEC tube formation was inhibited by the addition of conditioned media from DU145 and LNCaP cells transfected with miR-195 mimics. (C) The number of complete tubes per field (×40) was calculated. (D) Enforced expression of miR-195 inhibited the proliferation of DU145 and LNCaP cells. Statistical analysis was performed on the results from three independent experiments. Data are presented as means ± SD. **P<0.01 compared with the negative control.

Figure 2.

Analysis of RNA-based microarray following miR-195 overexpression. (A) Supervised hierarchical clustering of the genes significantly differentially expressed after miR-195 overexpression in LNCaP cells (fold changes ≤-1.5 or ≥1.5). (B) Top 10 enriched KEGG pathways associated with the differentially expressed genes induced by miR-195.

Figure 3.

PRR11 is a direct target of miR-195. (A) Six genes were shared between the results of the TargetScan miRNA target prediction and RNA microarray following miR-195 overexpression. (B) Spearman correlation analysis clearly indicated an inverse relationship between PRR11 and miR-195 expression in the Taylor cohort (r=−0.355, P<0.001). (C) miR-195 expression level combined with PRR11 expression level was able to stratify patients into different groups according to BCR-free survival. (D) RNA sequence alignment of the 3′ UTR of PRR11 mRNA indicating a complementary site for the seed region of miR-195 at 4827–4833 bp. PRR11-mut was a mutant sequence with substitutions in the complementary region (negative control). (E) Luciferase activity was decreased after transfection of psi-PRR11-3′ UTR-2-WT into miR-195-transfected LNCaP cells. (F) RNA sequence alignment of the 3′ UTR of PRR11 mRNA indicating a complementary site for the seed region of miR-195 at 3490–3496 bp. (G) Luciferase activity was unchanged after transfection of psi-PRR11-3′ UTR-1-WT into miR-195-transfected LNCaP cells. Data are presented as means ± SD.

Figure 4.

miR-195 exerts its tumor suppressive role by downregulating PRR11 expression. (A and B) Endogenous PRR11 expression levels were detected by RT-qPCR and western blotting in DU145 and LNCaP cells transfected with miR-195 mimic in the presence of PRR11, with miR-195 mimic in the presence of vector control, with PRR11 siRNA or with vector control for 48 h. (C-I) Expression of PRR11 using a construct lacking the 3′ UTR of PRR11 abrogated the biological effects associated with miR-195 overexpression, and knockdown of endogenous PRR11 expression with siRNA generated similar effects as those induced by miR-195 on HUVEC tube formation, cell cycling and cell proliferation. Data were obtained from three independent experiments and are presented as means ± SD. *P<0.05, **P<0.01 compared with control.

Figure 5.

Knockdown of PRR11 suppresses tumor growth and angiogenesis in vivo. (A and B) Western blotting showed that PRR11 expression in PCa cells was inhibited by three lentivirus expression plasmids containing small interfering RNAs against PRR11. The cells transfected with the third lentivirus expression plasmid were used in subsequent experiments. (C and D) Knockdown of PRR11 by the lentivirus-shPRR11 vector in DU145 and LNCaP cells inhibited subcutaneous tumor growth over the 42-day monitoring period after tumor cell injection. (E-H) The tumor growth curves are shown. Knockdown of PRR11 suppressed the growth of tumor nodules and reduced the weight of tumors in the PRR11-shRNA groups on day 42 compared with the control groups (scramble). (I and J) Immunochemistry analysis of the tumor xenografts. CD31 stained the cytomembrane or cytoplasm of pan-endothelial cells undergoing angiogenesis. Ki67 stained the nucleus of proliferative PCa cells (shown in the fields at a magnification of ×400). The CD31 staining results indicated that PRR11 downregulation reduced angiogenesis in tumor xenografts compared with control tumor xenografts. The Ki67 staining results indicated that the tumor xenografts established by cells with low expression of PRR11 expressed less Ki67 protein. The results are presented as means ± SD. *P<0.05, **P<0.01 compared with the negative control.

Figure 6.

Upregulation of PRR11 expression occurs in aggressive tumors and is associated with poor clinical outcome. (A) A full view of the immunohistochemistry staining for PRR11 in our TMA cohort. (B-D) The immunohistochemistry staining indicated that PRR11 was mainly expressed in the cytoplasm and cellular membrane of PCa and benign glandular epithelium cells. The intensities of the PRR11 immunostaining were weak (B), intermediate (C) and strong (D) (left panel: magnification ×40; right panel: magnification ×200). (E and F) In the Taylor cohort, PRR11 expression served a prognostic role regarding BCR-free survival but not overall survival, as indicated by Kaplan-Meier analysis. PRR11 overexpression was frequently observed in patients with shorter BCR-free survival time.