LncRNA MALAT1 promotes tumor growth and metastasis by targeting miR-124/foxq1 in bladder transitional cell carcinoma (BTCC)

Abstract

Mounting evidence shows that the long non-coding RNA MALAT1 plays a pivotal role in tumorigenesis and metastasis, but the functional significance of MALAT1 in bladder transitional cell carcinoma (BTCC) remains unclear. MALAT1 expression was measured in 56 BTCC patients and 2 BTCC cell lines by real-time PCR. The effects of MALAT1 on BTCC cells were investigated by over-expression approaches in vitro and in vivo. Insights of the mechanism of competitive endogenous RNAs (ceRNAs) were validated through bioinformatic analysis and luciferase assay. MALAT1 up-regulation positively correlated with advanced clinical pathological stage and shorter survival of BTCC patients. Furthermore, MALAT1 over-expression promoted proliferation, migration and invasion of BTCC cells in vitro and in vivo. Particularly, MALAT1 may function as a ceRNA to sponge miR-124, thus modulating the derepression of foxq1, miR-124 target gene, in post-transcriptional levels. The positive MALAT1/foxq1 interaction was confirmed by bivariate correlation analysis, and this positive correlation was of great significance in BTCC tumor growth and metastasis, also accompanied by EMT changes. Overall, this ceRNA regulatory network concerning MALAT1 and the positive MALAT1/foxq1 correlation benefit a better understanding of BTCC pathogenesis and promote the feasibility of lncRNA-directed therapy against this disease.

Keywords: Bladder transitional cell carcinoma (BTCC); MALAT1; competing endogenous RNA(ceRNA); foxq1; proliferation and invasion.

Figure 1

Expression of MALAT1, miR-124, foxq1 in BTCC. The relative expression was examined using real-time PCR in 56 patients with BTCC and 2 BTCC cell lines. Data are shown as mean ± SEM. A. MALAT1 was up-regulated in 56 tumor tissues compared with surrounding normal mucosa tissues; miR-124 was down-regulated in tumor tissues in comparison with matched normal mucosa tissues. B. MALAT1 and foxq1 were positively expressed in BTCC. C. MALAT1 and foxq1 expression positively correlated with BTCC clinical pathological stage; miR-124 expression inversely correlated with clinical pathological stage. D. MALAT1 and foxq1 were more abundant and miR-124 was weaker in T24 cells than BIU-87 cells. E. Kaplan-Meier survival curves revealed that patients with high levels of MALAT1 and foxq1, or weak levels of miR-124 exhibited an unfavorable clinical outcome. Patients were divided into MALAT1-high/low group; miR-124-high/low group; foxq1-high/low group based on the median of their expression in BTCC, respectively. *P < 0.05.

Figure 2

MALAT1 and foxq1 are direct targets of miR-124. Real-time PCR analysis of MALAT1, miR-124, and foxq1 mRNA expression in T24 cells (A) and BIU-87 cells (B) transfacted with Control, miR-124 mimics, respectively. Real-time PCR analysis of MALAT1, miR-124, and foxq1 mRNA expression in T24 cells (C) and BIU-87 cells (D) transfacted with Scramble, si-miR-124, respectively. (E) The putative miR-124-binding site in 3’UTR region of MALAT1. The relative luciferase activity was suppressed in cells transfected with the reporter vector MALAT1-WT, not in cells transfected with MALAT1-MUT. (F) The putative miR-124-binding site in 3’UTR region of foxq1. The relative luciferase activity was down-regulated in cells transfected with the reporter vector foxq1-WT, not in cells transfected with foxq1-MUT. Data are shown as mean ± SEM, *P < 0.05, ***P < 0.001.

Figure 3

MALAT1 regulates miR-124/foxq1 cascade expression in BTCC cells. The relative expression of MALAT1, miR-124, and foxq1 was examined using real-time PCR and western blot in T24 cells and BIU-87 cells. Data are shown as mean ± SEM. Real-time PCR analysis of MALAT1, miR-124, and foxq1 mRNA expression in T24 cells (A) and BIU-87 cells (B) transfacted with Control, MALAT1, respectively. Real-time PCR analysis of MALAT1, miR-124, and foxq1 mRNA expression in T24 cells (C) and BIU-87 cells (D) transfacted with Scramble, si-MALAT1, respectively. (E) Western blot analysis of foxq1 expression in T24 and BIU-87 cells both transfacted with Control, MALAT1+miR-124, respectively. (F) Western blot analysis of foxq1 expression in T24 and BIU-87 cells both transfacted with Scramble, si-miR-124+si-MALAT1, respectively. *P < 0.05.

Figure 4

Foxq1 deletion partly inverses the changes caused by MALAT1 in vitro. (A) T24 and BIU-87 cells were both transfected with MALAT1 or MALAT1+si-foxq1. Effects of down-regulated foxq1 in MALAT1 overexpressing cells on cell proliferation were measured. Effects of silencing foxq1 in MALAT1 overexpressing cells on cell migration (B) and invasion (C) were investigated. (D) Western blot analysis of foxq1, E-cadherin, and Vimentin expression in BIU-87 cells. Data are shown as mean ± SEM. *P < 0.05, Control vs. MALAT1; #P < 0.05, MALAT1 vs. MALAT1+si-foxq1.

Figure 5

MALAT1 promotes tumor growth and metastasis in vivo. MALAT1/T24 or Control/T24 cells were subcutaneously implanted into nude mice (n=12/group). A. Tumor volume was calculated every one week. 6 weeks later, tumors were removed and tumor weight was measured. B. Livers and lungs were dissected for histology analysis to evaluate distant metastasis. C. Real-time PCR analysis of miR-124 expression and western blot analysis of foxq1, E-cadherin, and Vimentin expression in tissues of resected tumors. Data are presented as mean ± SEM. *P < 0.05.