Micro-ribonucleic acid 29b inhibits cell proliferation and invasion and enhances cell apoptosis and chemotherapy effects of cisplatin via targeting of DNMT3b and AKT3 in prostate cancer

Abstract

Background: Micro-ribonucleic acids (miRNAs) are crucial regulators in malignant tumors. miRNA-29b (miR-29b) has been identified as a tumor suppressor in prostate cancer (PCa). However, very few studies have investigated the effects of miR-29b in PCa, especially the mechanism and its association with chemotherapy. Our study aimed to explore the role and mechanism of miR-29b in PCa.

Materials and methods: The expression levels of miR-29b were detected in ten clinical PCa specimens and four different PCa cell lines through quantitative real-time polymerase chain reaction. After miR-29b mimics and inhibitors were successfully transfected into LNCaP, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was then used to investigate cell proliferation and cisplatin sensitivity of PCa cells. Cell cycle, cell apoptosis, and cell invasion were detected via flow cytometry, annexin V-fluorescein isothiocyanate labeling, and transwell assay, respectively. Based on bioinformatic methods, Western blot analysis, and dual-luciferase reporter assay, novel target genes of miR-29b were identified.

Results: miR-29b was downregulated in PCa tissues compared with matched adjacent nontumor tissues. In the androgen-independent PCa cell line (LNCaP-AI), the expression of miR-29b was much lower than the androgen-dependent PCa cell line (LNCaP). Subsequent studies showed that forced expression of miR-29b inhibited cell proliferation and cell invasion and induced cell apoptosis in PCa. Upregulation of miR-29b also enhanced the chemosensitivity of PCa cells to cisplatin. Moreover, we identified DNMT3b and AKT3 as novel target genes of miR-29b in PCa.

Conclusion: Taken together, the results showed that miR-29b plays a tumor-suppressive role in PCa. It inhibits cell biological behavior and enhances the chemotherapy effects of cisplatin through its involvement in epigenetic regulation and PI3K/AKT pathway.

Keywords: AKT3; DNMT3b; cell biological behavior; chemosensitivity; miRNA-29b; prostate cancer.

Figure 1

miR-29b is downregulated in prostate cancer. Notes: (A) Transfection efficiency of miR-29b mimics was evaluated, and more than 80% of cells were found to be transfected in light microscopy and fluoroscopy, suggesting the transfection was successful. (B) Relative miR-29b expression in the miR-29b mimic group (miR-29b group) was significantly higher than the negative-control miR (Cont-miR) group (*P<0.05). (C) Comparison of miR-29b expression in prostate cancer tissues (Cancer) and compared with patient-matched control noncancerous prostate tissues (Control). miR-29b expression was significantly lower in the cancer group (*P<0.05). (D) Comparison of miR-29b expression in different prostate cancer cell lines. miR-29b expression was significantly lower in LNCaP-AI cell lines (*P<0.05). Abbreviation: miR, micro-ribonucleic acid.

Figure 2

Upregulation of miR-29b expression inhibited growth and invasion and induced apoptosis of prostate cancer cells. Notes: (A) In the miR-29b mimic-transfected group (miR-29b), the proportion of sub-G₀/G₁-phase cells increased significantly, and the proportion of S-phase and G₂/M-phase cells decreased significantly compared with the negative-control group (Cont-miR). (B) The percentage of apoptotic cells in the miR-29b group was significantly higher than in the Cont-miR group (*P<0.05). (C) The invasion cells were significantly reduced in the miR-29b-transfected group compared to the negative-control group (*P<0.05). Abbreviations: miR, micro-ribonucleic acid; FITC, fluorescein isothiocyanate.

Figure 3

Overexpression of miR-29b enhances chemosensitivity of cisplatin on prostate cancer cells. miR-29b targets DNMT3b and AKT3. Notes: (A) Compared with the blank-control (blank) and negative-control miR (Cont-miR) groups, the relative cell-growth rate in the miR-29b group was significantly lower along with action time, indicating that miR-29b inhibited cell proliferation of prostate cancer. Cell viability in the miR-29b group plus cisplatin was significantly lower than the miR plus cisplatin-control group and the miR-29b group, suggesting that overexpression of miR-29b enhanced the chemotherapy of cisplatin for prostate cancer. (B) Western blotting of eight potential genes’ expression at the protein level in the blank, negative-control miRNA, miR-29b inhibitor-transfection, and miR-29b mimic-transfection groups. (C) The quantitative protein expression of DNMT3b, AKT3, and Mcl-1 were significantly increased in the miR-29b mimic-transfection group (Mimics) and decreased in the miR-29b inhibitor-transfection group (Inhibitors). *P<0.05. Abbreviation: miR, micro-ribonucleic acid.

Figure 4

miR-29b directly targets DNMT3b and AKT3. Notes: (A) Computational analysis of 3′UTR of DNMT3b and AKT3 revealed the 3′-UTR-binding sites. The miR-29b target regions of DNMT3b and AKT3 are indicated. (B) Luciferase assay showing increased reporter activity after cotransfection of DNMT3b with miR-29b inhibitors and decreased reporter activity after cotransfection of DNMT3b with miR-29b mimics (*P<0.05). (C) Luciferase assay showing increased reporter activity after cotransfection of AKT3 with miR-29b inhibitors and decreased reporter activity after cotransfection of AKT3 with miR-29b mimics (*P<0.05). Abbreviations: UTR, untranslated region; miR, micro-ribonucleic acid.