RNA-binding protein HuR promotes bladder cancer progression by competitively binding to the long noncoding HOTAIR with miR-1

Abstract

The elevated expressions of RNA-binding protein HuR and long noncoding HOX transcript antisense RNA (HOTAIR) are observed in numerous cancers. And HuR often exerts its promotive effects on tumorigenesis via binding to AU-rich elements in target transcripts and thus regulating the expression of target transcripts. However, the roles and related mechanisms of HuR/HOTAIR in bladder cancer progression have never been formally tested. Here, we found that the expression level of HuR was higher in clinical bladder cancer samples than in normal adjacent samples, mirroring that of HOTAIR, and their expression showed strong correlation. Knockdown of HuR/HOTAIR in bladder cancer inhibited cell proliferation, migration, invasion, and promoted cell apoptosis. Notably, HuR interacted and stabilized HOTAIR mRNA and knockdown of HuR decreased HOTAIR expression. Additionally, HOTAIR was identified as a potential target of miR-1 in bladder cancer cells. Interestingly, overexpression of HOTAIR enhanced HuR expression and increased cytoplasmic accumulation of HuR, thus enhancing HOTAIR expression in turn. But mutation of miR-1 binding site in HOTAIR canceled the effects of HOTAIR on HuR expression. Overall, we identified a regulatory loop between HOTAIR and HuR during the progression of bladder cancer, which could be exploited to curb bladder cancer progression.

Keywords: HOTAIR; HuR; bladder cancer; miR-1.

Figure 1

The association between HuR and HOTAIR expression levels in clinical samples. Notes: (A and B) The mRNA levels of HuR and HOTAIR were examined in 43 TCC tissues and 36 normal bladder tissues. (C and D) The protein level of HuR was examined in 43 TCC tissues and 36 normal bladder tissues. (E) Correlation between the levels of HuR and HOTAIR. (F) The mRNA levels of HuR and HOTAIR were examined in TCC cell lines and normal bladder epithelial cells via qRT-PCR analyses. (G) The protein level of HuR was determined in TCC cell lines and normal bladder epithelial cells via Western blot analysis. Data are presented as mean ± SD; **P<0.01 vs control. Abbreviations: HOTAIR, HOX transcript antisense RNA; qRT-PCR, quantitative real-time polymerase chain reaction; SD, standard deviation; TCC, transitional cell carcinoma.

Figure 2

Knockdown of HOTAIR and HuR inhibits TCC cell proliferation and promotes apoptosis. Notes: (A) The cell viability of J82 and T24 cells with or without HuR shRNA or HOTAIR shRNA stable expression was evaluated by MTT assays. (B) ki67, a proliferation marker, was detected in cells depicted in (A) via qRT-PCR assays. (C) Cell-cycle assays were carried out in cells described in (A). (D) The cell apoptosis was examined in the cells depicted in (A). (E and F) The antiapoptotic gene (Bcl-2) and proapoptotic gene (Bax) mRNA levels were evaluated in cells depicted in (A). (G) The anti-apoptotic gene (Bcl-2) and proapoptotic gene (Bax) protein levels were evaluated in cells depicted in (A). Data are presented as mean ± SD; **P<0.01 vs control. Abbreviations: HOTAIR, HOX transcript antisense RNA; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide; qRT-PCR, quantitative real-time polymerase chain reaction; SD, standard deviation; TCC, transitional cell carcinoma.

Figure 3

The effects of HOTAIR and HuR on TCC cell migration and invasion in vitro. Notes: (A and B) Knockdown of HuR and HOTAIR inhibited cell migration in J82 and T24 cells. (C and D) Knockdown of HOTAIR inhibited EMT in J82 and T24 cells. Data are presented as mean ± SD; **P<0.01 vs control. Abbreviations: EMT, epithelial–mesenchymal transition; HOTAIR, HOX transcript antisense RNA; SD, standard deviation; TCC, transitional cell carcinoma.

Figure 4

HuR could bind to HOTAIR and thus stabilizes HOTAIR mRNA stability in TCC cells. Notes: (A) The HOTAIR expression level was detected in J82 and T24 cells with or without HuR knockdown by qRT-PCR. (B and C) Cells depicted in (A) were treated with actinomycin D (2.5 µg/mL) for the indicated times. HOTAIR mRNA levels were measured by qRT-PCR and the percentage of mRNA that remained was plotted. (D) Ectopic HuR expression does not affect the promoter activity of HOTAIR. After treatment with HuR shRNA for 24 h, J82 and T24 cells were transiently transfected with the luciferase plasmids containing the HOTAIR promoter for 24 h. Luciferase activities in these cells were measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). (E) qRT-PCR was used to measure the abundance of HOTAIR mRNA present in the HuR-IP materials after the RIP assay. (F) HOTAIR level was examined in cells with HuR knockdown and Lenti-HOTAIR infection. (G) The apoptotic markers (Bcl-2 and Bax) expressions were determined as indicated. (H) The EMT markers (E-cadherin and vimentin) expressions were determined as indicated. (I and J) The apoptotic markers (Bcl-2 and Bax) expressions were determined as indicated. (K and L) The EMT markers (E-cadherin and vimentin) expressions were determined as indicated. Values are mean ± standard deviation. **P<0.01. Abbreviations: EMT, epithelial–mesenchymal transition; HOTAIR, HOX transcript antisense RNA; qRT-PCR, quantitative real-time polymerase chain reaction; RIP, RNA binding protein immunoprecipitation; TCC, transitional cell carcinoma.

Figure 5

miR-1 could target HOTAIR in TCC cells. Notes: (A) J82 and T24 cells were transfected with miR-1 mimics or inhibitor for 48 h. The level of HOTAIR mRNA was measured by qRT-PCR. (B) J82 and T24 cells were co-transfected with miR-1 mimics and Luc-HOTAIR-wt or Luc-HOTAIR-mut. The luciferase activity was detected. (C and D) HuR mRNA and protein levels were detected in Lenti-HOTAIR-wt-, Lenti-HOTAIR-mut-, or Lenti-HOTAIR-shRNA-infected J82 and T24 cells by qRT-PCR and Western blot. (E) The nucleo-cytoplasmic translocation of HuR was detected in J82 cells with or without HOTAIR knockdown. (F) The level of miR-1 was examined in 43 TCC tissues and 36 normal bladder tissues via qRT-PCR analyses. (G and H) Correlation between the levels of miR-1 and HuR or HOTAIR. Data are presented as mean ± SD; **P<0.01 vs control. Abbreviations: HOTAIR, HOX transcript antisense RNA; PCR, polymerase chain reaction; qRT-PCR, quantitative real-time polymerase chain reaction; SD, standard deviation; TCC, transitional cell carcinoma.