Long non-coding RNA RNCR3 promotes prostate cancer progression through targeting miR-185-5p

Abstract

Long non-coding RNAs (lncRNAs) have been suggested to play important roles in the development of numerous kinds of human cancers. Increasing data has indicated that lncRNA RNCR3 has been involved in some human diseases. However, the exactly biological function and potential mechanisms of RNCR3 in the development of prostate cancer is still unclear. Here, our results confirmed that the RNCR3 expression was increased in prostate cancer compared to the corresponding adjacent normal prostate tissues. Moreover, our data showed that the increased expression of RNCR3 is significantly associated with tumor progression and poor survival of prostate cancer patients. Additionally, we found that RNCR3 knockdown could suppress the ability of proliferation, colony formation, and invasion of prostate cancer cells. Finally, we further confirmed that RNCR3 binds to miR-185-5p, which has been identified as a tumor suppressor in some human cancers, including prostate cancer. We also confirmed that the oncogenic function of RNCR3 in prostate cancer are partly mediated by negative regulation of miR-185-5p targeted BRD8 ISO2. Our data revealed that RNCR3 functions as an tumor-promoting lncRNA in prostate cancer and may serve as a novel important biomarker for the diagnosis and treatment of prostate cancer.

Keywords: RNCR3; miR-185-5p; prognosis; prostate cancer.

Figure 1

Clinical significance of relative RNCR3 expression levels in prostate cancer tissues. A. Relative expression of RNCR3 in 52 pairs of prostate cancer tissue and adjacent non-tumor tissues by qRT-PCR analysis. **P < 0.01. B. Upregulation of RNCR3 in prostate cancer cells compared with normal prostate epithelial cells RWPE-1. *P < 0.05. C. Kaplan-Meier survival curves for patients with prostate cancer tissues expressing low and high levels of RNCR3.

Figure 2

miR-185-5p expression was down-regulated in prostate cancer tissues and cell lines. A. Down-gulation of miR-185-5p in prostate cancer cells compared with normal prostate epithelial cells RWPE-1. *P < 0.05. B. Relative expression of miR-185-5p in 52 pairs of prostate cancer tissue and adjacent non-tumor tissues by qRT-PCR analysis. C. The expression of miR-185-5p was negatively related with the expression of RNCR3 in the prostate cancer tissues.

Figure 3

Knockdown of RNCR3 suppressed the miR-185-5p expression. A. The RNCR3 expression in the LNCaP and PC-3 cell was analyzed by qRT-PCR analysis. **P < 0.01. B. Knockdown of RNCR3 suppressed the expression of miR-185-5p. **P < 0.01. C. The mRNA expression of BRD8 ISO2 was analyzed by qRT-PCR analysis. **P < 0.01. D. The protein expression of BRD8 ISO2 was analyzed by western blot.

Figure 4

BRD8 ISO2 was a direct target gene of miR-185-5p. A. The potential target gene of miR-185-5p and the potential 3’UTR binding site of BRD8 ISO2 of miR-185-5p. B. The expression of miR-185-5p was measured in PC-3 cells. C. Overexpression of miR-185-5p decreased the luciferase activity of the WT BRD8 ISO2 3’UTR but not the mutant vector (Mut BRD8 ISO2 3’UTR). **P < 0.01. D. Overexpression of miR-185-5p suppressed the BRD8 ISO2 expression.

Figure 5

Knockdown of RNCR3 suppresses prostate cancer cell proliferation, colony formation, and invasion. A. The CCK-8 assay demonstrated that knockdown of RNCR3 suppressed LNCaP cell proliferation. P < 0.01. B. The CCK-8 assay demonstrated that knockdown of RNCR3 suppressed PC-3 cell proliferation. P < 0.01. C. Knockdown of RNCR3 inhibited colony formation of LNCaP and PC-3 cells. **P < 0.01. D. Knockdown of RNCR3 inhibited invasion of LNCaP and PC-3 cells. **P < 0.01.

Figure 6

Tumor suppression in prostate cancer cells induced by knockdown of RNCR3 was partially reversed by co-transfection with miR-185-5p inhibitor. A. qRT-PCR was used to measure miR-185-5p expression in PC-3 cells that were stably co-transfected with sh-RNCR3 and miR-185-5p inhibitor or sh-NC. **P < 0.01. B. CCK-8 assays were performed to measure proliferation of PC-3 cells that were stably transfected with sh-RNCR33 and miR-185-5p inhibitor or sh-NC. C. Colony formation of PC-3 cells that were stably transfected with sh-RNCR33 and miR-185-5p inhibitor or sh-NC. P > 0.05. D. Transwell assays were performed to measure invasion of PC-3 cells that were stably transfected with sh-RNCR33 and miR-185-5p inhibitor or sh-NC. P > 0.05.