Down-regulation of lncRNA UCA1 enhances radiosensitivity in prostate cancer by suppressing EIF4G1 expression via sponging miR-331-3p

Abstract

Background: We aimed to explore the role of long noncoding RNA urothelial carcinoma-associated 1 (lncRNA UCA1) and its underlying mechanism in the radioresistance of prostate cancer (PCa).

Methods: QRT-PCR was conducted to measure the expression of UCA1, microRNA-331-3p (miR-331-3p) and eukaryotic translation initiation factor 4 gamma 1 (EIF4G1) in PCa tissues and cells. The relative protein level was determined by western blot assay. Cell proliferation and apoptosis were detected by MTT, colony formation assay, and flow cytometry, respectively. The target interaction between miR-331-3p and UCA1 or EIF4G1 was predicted through bioinformatics analysis, and verified by dual-luciferase reporter gene assay system.

Results: The high levels of UCA1 and EIF4G1 as well as the low level of miR-331-3p were observed in PCa tissues and cell lines. UCA1 and EIF4G1 expression were significantly upregulated by Gy radiation treatement. UCA1 or EIF4G1 knockdown repressed cell growth and enhanced cell apoptosis in 22RV1 and DU145 cells under radiation. Moreover, overexpression of EIF4G1 abolished UCA1 knockdown-induced effect on 6 Gy irradiated PCa cells. UCA1 sponged miR-331-3p to regulate EIF4G1 expression.

Conclusions: LncRNA UCA1 deletion suppressed the radioresistance to PCa by suppressing EIF4G1 expression via miR-331-3p. UCA1 acted as a potential regulator of radioresistance of PCa, providing a promising therapeutic target for PCa.

Keywords: EIF4G1; Prostate cancer; Radioresistance; UCA1; miR-331-3p.

Fig. 1

UCA1 was positively related to EIF4G1 expression in PCa tissues. a, b The expression of UCA1 (a) and EIF4G1 (b) was measured by qRT-PCR analysis in PCa tissues (n = 40) or adjacent non-cancer tissues (n = 40). c The protein level of EIF4G1 was detected by western blot in PCa tissues (n = 40) or adjacent non-cancer tissues (n = 40). d Correlation between UCA1 and EIF4G1 expression in PCa tissues was analyzed. ^*P < 0.05

Fig. 2

Radiation treatment increased UCA1 expression and EIF4G1 protein level in PCa cells. a QRT-PCR analysis was conducted to detect UCA1 expression in PCa cell lines (22RV1 and DU145 cells) and human normal prostatic epithelial cells RWPE1. b, c The mRNA (b) or protein (c) level of EIF4G1 was determined by qRT-PCR or western blot in 22RV1 and DU145 cells, respectively. d, e UCA1 expression was detected in 22RV1 (d) and DU145 (e) cells every 3 h after 0 or 6 Gy radiation treatment. f, g UCA1 expression was measured in 22RV1 (f) and DU145 (g) cells after 0, 2, 4, 6 Gy radiation treatment for 24 h. h EIF4G1 protein level was upregulated after 22RV1 and DU145 cells treated with 6 Gy radiation for 24 h. ^*P < 0.05

Fig. 3

UCA1 knockdown enhanced the radiosensitivity of PCa cells. a, b The relative UCA1 expression was detected by qRT-PCR in 22RV1 (a) and DU145 (b) cells transfected with si-UCA1#1, si-UCA1#2, si-UCA1#3, or si-RNA. c–j 2RV1 and DU145 cells were transfected with si-NC or si-UCA1#3. c, d Cell viability was detected in transfected 22RV1 (c) and DU145 (d) cells at 0, 24, 48, and 72 h after Gy irradiation. e, f Transfected 22RV1 (e) and DU145 (f) cells were subjected to 0, 2, 4, 6 and 8 Gy irradiation. After 2 weeks, the colony survival fractions were measured. g, h Cell apoptosis was determined in transfected 22RV1 and DU145 cells at 24 h after 6 Gy radiotherapy. i, j The protein expression of Cyclin D1, Bcl-2, and Bax was examined in transfected 22RV1 (i) and DU145 (j) cells after 6 Gy irradiation. ^*P < 0.05

Fig. 4

EIF4G1 knockdown facilitated the radiosensitivity in PCa cells. a, b The relative mRNA and protein expression of EIF4G1 were determined in 22RV1 and DU145 cells transfected with si-EIF4G1#1, si-EIF4G1#2, si-EIF4G1#3 or si-NC by qRT-PCR and western blot, respectively. c–i Cells were transfected with si-NC or si-EIF4G1#3. c, d MTT assay was used to examine cell viability of transfected 22RV1 (c) and DU145 (d) cells after 6 Gy irradiation. e, f The colony survival fractions were measured in transfected 22RV1 (e) and DU145 (f) cells subjected to irradiation. g Cell apoptosis was determined in transfected 22RV1 and DU145 cells after 6 Gy radiotherapy. h, i The protein expression of Cyclin D1, Bcl-2, and Bax was examined in transfected 22RV1 (h) and DU145 (i) cells after 6 Gy irradiation. ^*P < 0.05

Fig. 5

Upregulation of EIF4G1 weakened the effect of UCA1 knockdown on the radiosensitivity of PCa cells. 22RV1 and DU145 cells were transfected with si-UCA1 or co-transfected with si-UCA1 and pcDNA-EIF4G1. a, b The mRNA (a) and protein (b) expression of EIF4G1 were measured in PCa cells. c, d Cell proliferation was detected using MTT assay at the indicated times after 6 Gy irradiation treatment. e, f The clonogenic survival curve was established at day 14 after transfected cells received indicated Gy irradiation. g Cell apoptosis was measured by flow cytometry after radiotherapy. h, i Protein expression of Cyclin D1, Bcl-2, and Bax was detected in 22RV1 (h) and DU145 (i) cells exposure to radiotherapy. ^*P < 0.05

Fig. 6

UCA1 regulated the expression of EIF4G1 via sponging miR-331-3p. a Bioinformatics (StarBase v3.0 software online) predicted the complementary binding sites between miR-331-3p and UCA1 or EIF4G1. b, c The direct combination between UCA1 and miR-331-3p in 22RV1 (b) and DU145 (c) was verified by dual-luciferase reporter assay. d, e The directive binding sequences between 3′UTR EIF4G1 and miR-331-3p in 22RV1 (d) and DU145 (e) were verified by dual-luciferase reporter assay. f–g QRT-PCR was used to evaluate the level of miR-331-3p in PCa tissues (f) and cells (g). h, i The correlation analysis between miR-331-3p and UCA1 or EIF4G1 was presented. j, k QRT-PCR or western blot assay was carried out to detect the mRNA (j) or protein (k) expression of EIF4G1 in PCa cells, respectively. ^*P < 0.05