Silencing of LINC01963 enhances the chemosensitivity of prostate cancer cells to docetaxel by targeting the miR-216b-5p/TrkB axis

Abstract

Docetaxel (DTX) treatment effectively prolongs the overall survival of patients with prostate cancer. However, most patients eventually develop resistance to chemotherapy and experience tumor progression or even death. Long noncoding RNAs (lncRNAs) affect docetaxel chemosensitivity. However, the biological role and regulatory mechanisms of lncRNAs in docetaxel-resistant prostate cancer remain unclear. Differences in lncRNAs were evaluated by lncRNA sequencing and evaluated using quantitative real-time polymerase chain reaction, and TrkB expression was measured through western blot analysis. Proliferation was measured using the MTS, while apoptosis and cell cycle were measured using flow cytometry. In addition, migration and invasion were measured using transwell assays. Forty-eight female BALB/c nude mice were used for subcutaneous tumorigenicity and lung metastasis assays. We found that LINC01963 was overexpressed in the PC3-DR cells. LINC01963 silencing enhanced the chemosensitivity of PC3-DR to docetaxel and inhibited tumorigenicity and lung metastasis, while LINC01963 overexpression enhanced the chemoresistance of PC3 cells to docetaxel. It was found that LINC01963 bind to miR-216b-5p. The miR-216b-5p inhibitor reversed the suppressive effect of sh-LINC01963 on PC3-DR cell proliferation, migration, and invasion. Furthermore, miR-216b-5p can bind to the 3'-UTR of NTRK2 and inhibit TrkB protein levels. TrkB enhances docetaxel resistance in prostate cancer and reverses the effects of LINC01963 silencing and miR-216b-5p overexpression. In conclusion, silencing LINC01963 inhibited TrkB protein level to enhance the chemosensitivity of PC3-DR to docetaxel by means of competitively binding to miR-216b-5p. This study illustrates that LINC01963 is a novel therapeutic target for treating prostate cancer patients with DTX resistance.

Fig. 1. The expression of four lncRNAs in PC3 and PC3-DR cells.

PC3 and PC3-DR cells were not treated with DTX in those studies. a The expression of four lncRNAs in PC3 and PC3-DR cells is shown according to the results of lncRNA sequencing. b The expression of four lncRNAs in PC3 and PC3-DR cells was analyzed by qRT-PCR. Data are shown as the mean ± standard deviation of three technical replicates. *P < 0.05.

Fig. 2. LINC01963 expression was analyzed in PC3 cells and in the PC3-DR stable cell line.

PC3 and PC3-DR cells were not treated with DTX in those studies. LINC01963 expression in the PC3-DR and PC3 stable cell line was analyzed by qRT-PCR. Data are shown as the mean ± standard deviation of three technical replicates. shRNA: short hairpin RNA, ov: ovexpression, NC: empty control lentivirus. ***P < 0.001 vs. NC group.

Fig. 3. LINC01963 silencing reduced PC3-DR cell survival while LINC01963 overexpression increased PC3-DR cell survival.

PC3-DR cells were treated with 40 nmol/L DTX in those studies. a Proliferation of PC3-DR cells was analyzed by MTS. b, d PC3-DR cell apoptosis was analyzed by flow cytometry. Apoptosis is shown as the mean ± SD (b), and a representative image of apoptosis is shown (d). c, e The cell cycle of PC3-DR was analyzed by flow cytometry. G1 phase distribution is shown as the mean ± standard deviation of three technical replicates (c) and a representative image of the cell cycle is shown (e). f Tumor tissues in nude mice were excised and photographed 30 days after xenograft study. g, h Tumor volumes and weights were detected. i The degree of lung metastasis in nude mice was evaluated by an in vivo imaging system. ***P < 0.001 vs. NC group.

Fig. 4. LINC01963 overexpression increased PC3 cell survival while LINC01963 silencing reduced PC3 cell survival.

PC3 cells were treated with 4 nmol/L DTX in those studies. a Proliferation of PC3 cells was analyzed by MTS. b, d PC3 cell apoptosis was analyzed by flow cytometry. Apoptosis is shown as the mean ± standard deviation of three technical replicates (b), and a representative image of apoptosis is shown (d). c, e The cell cycle of PC3 was analyzed by flow cytometry. G1 phase distribution is shown as the mean  ± SD (c) and a representative image of the cell cycle is shown (e). f Tumor tissues in nude mice were excised and photographed 30 days after xenograft study. g, h Tumor volumes and weights were detected. i The degree of lung metastasis in nude mice was evaluated by an in vivo imaging system. ***P < 0.001 vs. NC group.

Fig. 5. miR-216b-5p was bound by LINC01963.

a StarBase 3.0 and LncBase 2.0 analysis found that 11 common miRNAs had a promising binding site with LINC01963. b miRNA expression was measured by qRT-PCR in PC3 and PC3-DR cells. PC3 and PC3-DR cells were not treated with DTX in those studies. c miR-216b-5p expression in the Blank, NC, ov-LINC01963, sh-LINC01963 groups was measured by qRT-PCR in PC3 and PC3-DR cells. PC3 cells were treated with 4 nmol/L DTX and PC3-DR cells were treated with 40 nmol/L DTX. d The binding sites between LINC01963 and miR-216b-5p are shown. e The binding sites between LINC01963 and miR-216b-5p were determined using a dual luciferase assay. f The binding sites between LINC01963 and miR-216b-5p were determined using an AGO-RIPA assay. g The binding sites between LINC01963 and miR-216b-5p were determined using a pull-down assay. Data are shown as the mean ± standard deviation of three technical replicates. ***P < 0.001.

Fig. 6. miR-216b-5p reduced DTX resistance of prostate cancer and reversed the effect of LINC01963 on cell proliferation.

a miR-216b-5p expression in PC3-DR cells was analyzed by qRT-PCR. PC3-DR cells were not treated DTX. b Transfected-PC3-DR cell proliferation was analyzed by MTS after treatment with 40 nmol/L DTX. c, d The apoptosis (c) and G1 phase distribution (d) of transfected-PC3-DR cells after treatment with 40 nmol/L DTX is shown. Results are shown as the mean ± SD. e The apoptosis and cell cycle were analyzed by flow cytometry, and a representative image of apoptosis and the cell cycle are shown. Data are shown as the mean ± standard deviation of three technical replicates. ***P < 0.001 vs. Blank group. ^###P < 0.001 vs. sh-LINC01963-2+NC inhibitor group.

Fig. 7. NTRK2 is the target gene of miR-216b-5p.

PC3-DR cells were treated with 40 nmol/L DTX and PC3 cells were treated with 4 nmol/L DTX in those studies. a The binding sites between the 3′-UTR of NTRK2 and miR-216b-5p were analyzed by Targetscan 7.2 and miRWalk. b The binding sites between the 3′-UTR of NTRK2 and miR-216b-5p were determined using a dual luciferase assay. c TrkB protein level was analyzed by western blot assay in PC3 cells treated with 4 nmol/L DTX and PC3-DR cells treated with 40 nmol/L DTX. ***P < 0.001 vs. Blank group. d TrkB protein level was analyzed by western blot assay in PC3-DR cells treated with 40 nmol/L DTX. ***P < 0.001 vs. Blank group. e NTRK2 mRNA expression was measured by qRT-PCR in PC3 cells treated with 4 nmol/L DTX and PC3-DR cells treated with 40 nmol/L DTX. Data are shown as the mean ± standard deviation of three technical replicates.