Long non‑coding RNA CASC11 interacts with YBX1 to promote prostate cancer progression by suppressing the p53 pathway

Abstract

Prostate cancer (PCa) is one of the principal causes of cancer‑related death worldwide. The roles and mechanisms of long non‑coding RNA (lncRNA) involved in the development of PCa remain incompletely understood. The present study aimed to investigate the role and mechanism of lncRNA in PCa tumorigenesis. In the present study, lncRNA cancer susceptibility candidate 11 (CASC11) was revealed to be a crucial regulator of PCa progression. The expression profiles of CASC11 in PCa were identified through analysis of The Cancer Genome Atlas and Gene Expression Omnibus datasets, and validated in human PCa specimens and cell lines. Gain‑ and loss‑of‑function assays were utilized to explore the biological role of CASC11 in PCa initiation and progression. RNA‑sequencing, RNA pull‑down and RNA immunoprecipitation analyses were used to explore potential mechanisms with which CASC11 may be associated. Rescue experiments were further conducted to confirm this association. The present results revealed that CASC11 was dominantly distributed in the nuclei of PCa cells, and was highly expressed in PCa tissues and cells. Overexpression of CASC11 was markedly associated with increased tumor proliferation and migratory ability. Functionally, decreased proliferation and migration, as well as inhibited xenograft tumor growth, were observed in CASC11‑silenced PCa cells, whereas the opposite effects were detected in CASC11‑overexpressing cells. Mechanistically, CASC11 promoted progression of the cell cycle and competitively interacted with Y‑box binding protein 1 (YBX1) to block the p53 pathway. Given this, poly (β‑amino ester) (PBAE)/small interfering RNA‑CASC11 (si‑CASC11) nanoparticles were applied to inhibit CASC11 expression and enhance the antitumor effect in vivo. The results revealed that PBAE/si‑CASC11 nanoparticles augmented the antitumor efficacy of CASC11 knockdown in vivo. In conclusion, the present study suggested that CASC11 may regulate PCa progression and elucidated a novel CASC11/YBX1/p53 signaling axis, providing a potential lncRNA‑directed therapeutic strategy particularly for the treatment of patients with PCa.

Keywords: CASC11; Y‑box binding protein 1; p53 pathway; progression; prostate cancer.

Figure 1

Long non-coding RNA CASC11 expression is upregulated in PCa. (A) Differential expression profile of non-coding CASC gene family members in PCa samples and normal tissues. (B) Expression of CASC11 in prostate tumor and normal tissue samples with different Gleason scores in TCGA dataset. (C and D) Expression of CASC11 in prostate tumor and normal tissues in GSE46602 and GSE55945 datasets. (E) Expression of CASC11 in 66 paired prostate tumor and normal tissue samples. (F) Expression levels of CASC11 in PCa cell lines (LNCaP, PC-3, DU145 and 22Rv1) and a prostate epithelial cell line (RWPE-1). (G) Fluorescence in situ hybridization analysis of CASC11 in LNCaP and 22Rv1 cells. DAPI was used to stain the nuclei, and 18S was used as a positive control for cytoplasmic staining. Scale bar, 50 µm. ^*P<0.05, ^**P<0.01, ^***P<0.001, ^****P<0.0001 vs. normal or as indicated. CASC11, cancer susceptibility candidate 11; PCa, prostate cancer; TCGA, The Cancer Genome Atlas.

Figure 2

CASC11 promotes proliferation of PCa cells. Expression levels of CASC11 were (A) significantly downregulated in sh-CASC11-infected PCa cells and (B) significantly upregulated in OE-CASC11-transfected cells. (C and D) Cell Counting Kit-8 assay was used to investigate the proliferative effects of CASC11 knockdown or overexpression on PCa cells. (E-H) Colony formation assay was applied to investigate the proliferative ability of PCa cells. Scale bar, 500 µm. (I-N) EdU assay was applied to investigate the proliferative effects of CASC11 knockdown or overexpression in PCa cells. Scale bar, 100 µm. ^*P<0.05, ^**P<0.01 vs. sh-NC or vector, or as indicated. CASC11, cancer susceptibility candidate 11; EdU, 5-ethynyl-2′-deoxyuridine; NC, negative control; OE-CASC11, CASC11 overexpression plasmid; PCa, prostate cancer; sh, short hairpin.

Figure 3

CASC11 promotes migration of PCa cells. (A-C) CASC11 knockdown attenuated cell migration in LNCaP cells, whereas CASC11 overexpression exerted the opposite effect. (D-F) CASC11 knockdown attenuated cell migration in 22Rv1 cells, whereas CASC11 overexpression exerted the opposite effect. (G and H) Transwell assay demonstrated that CASC11 knockdown inhibited cell migration in LNCaP and 22Rv1 cells. (I and J) Transwell assay demonstrated that CASC11 overexpression promoted cell migration in LNCaP and 22Rv1 cells. Scale bar, 100 µm. ^*P<0.05, ^**P<0.01 vs. sh-NC or vector. CASC11, cancer susceptibility candidate 11; NC, negative control; OE-CASC11, CASC11 overexpression plasmid; PCa, prostate cancer; sh, short hairpin.

Figure 4

CASC11 promotes prostate cancer cell proliferation by stimulating G₁/S cell cycle transition. (A-C) CASC11 knockdown resulted in cell cycle arrest at G₁ phase in LNCaP and 22Rv1 cells. ^*P<0.05 vs. sh-NC (G1 and S stages). (D-F) CASC11 overexpression resulted in cell cycle arrest at S phase in LNCaP and 22Rv1 cells. ^*P<0.05 vs. sh-NC (S stage). Western blot analysis was used to detect the effects of CASC11 (G) knockdown and (H) overexpression on the expression levels of cell cycle-associated proteins in LNCaP and 22Rv1 cells. ^*P<0.05, ^**P<0.01 vs. sh-NC or vector, or as indicated. CASC11, cancer susceptibility candidate 11; NC, negative control; OE-CASC11, CASC11 overexpression plasmid; sh, short hairpin.

Figure 5

CASC11 knockdown inhibits prostate cancer cell tumorigenesis in vivo. (A) Following stable transduction of sh-NC, sh-CASC11-1 and sh-CASC11-2 into 22Rv1 cells, the cells were then injected into nude mice. (B) Tumor volumes were measured every 4 days following injection. (C) Tumor weight is presented as the mean ± SD. (D and E) Subcutaneous tumor proliferation was inhibited in the sh-CASC11 group, as determined by immunohistochemical analysis of Ki-67. Scale bars, 100 and 200 µm. ^**P<0.01 vs. sh-NC or as indicated. CASC11, cancer susceptibility candidate 11; H&E, hematoxylin and eosin; NC, negative control; sh, short hairpin.

Figure 6

CASC11 suppresses p53 signaling in prostate cancer cells by binding with YBX1. (A) Heat map displaying differentially expressed genes in sh-CASC11 groups and sh-NC groups by RNA sequencing. (B) Protein expression levels of p53 and p21 were measured by western blotting when CASC11 was knocked down in LNCaP and 22Rv1 cells. (C) SDS-PAGE gel stained with silver to show separated proteins. (D and E) Using the YBX1 or IgG antibody, reverse transcription-quantitative PCR was used to examine RNA enrichment in RIP assay. (F and G) Protein expression levels of YBX1 were measured by western blotting when CASC11 was knocked down or overexpressed in LNCaP and 22Rv1 cells. ^*P<0.05, ^**P<0.01, ^***P<0.001 vs. sh-NC or vector, or as indicated. CASC11, cancer susceptibility candidate 11; NC, negative control; ns, not significant; OE-CASC11, CASC11 overexpression plasmid; RIP, RNA immunoprecipitation; sh, short hairpin.

Figure 7

Knockdown of YBX1 in prostate cancer cells reverses the carcinogenic effects of CASC11. YBX1 knockdown reversed the OE-CASC11-induced (A and B) proliferation (scale bar, 500 µm) and (C and D) migration (scale bar, 100 µm) of LNCaP and 22Rv1 cells. ^*P<0.05, ^**P<0.01. (E) Western blot analysis demonstrated that YBX1 knockdown abolished the OE-CASC11-induced promotion of Cyclin A2, CDK2 and CDK4 proteins. (F) Western blot analysis demonstrated that YBX1 overexpression reversed the sh-CASC11-induced increase in the expression levels of p21 and p53 proteins. ^*P<0.05, ^**P<0.01 vs. the control group; ^#P<0.05, ^##P<0.01 vs. the OE-CASC11 or sh-CASC11 group. CASC11, cancer susceptibility candidate 11; NC, negative control; OE-CASC11, CASC11 overexpression plasmid; sh, short hairpin; YBX1, Y-box binding protein 1.

Figure 8

PBAE/si-CASC11 nanocomplexes may inhibit the progression of prostate cancer. (A-C) Weight and volume changes following 3 weeks of xenograft tumor treatment with saline, si-CASC11 or PBAE/si-CASC11. (D and E) Expression levels of Ki-67 were detected by immunohistochemistry. Scale bars, 100 and 200 µm. ^**P<0.01 vs. saline group or as indicated. CASC11, cancer susceptibility candidate 11; PBAE, poly (β-amino ester); si, small interfering.

Figure 9

Schematic diagram summarizing the present study results. CASC11, cancer susceptibility candidate 11; lncRNA, long non-coding RNA; PBAE, poly (β-amino ester); PCa, prostate cancer; si, small interfering.