MicroRNA-132 inhibits migration, invasion and epithelial-mesenchymal transition via TGFβ1/Smad2 signaling pathway in human bladder cancer

Abstract

Background and aim: Increasing evidence shows that microRNAs play an important regulatory role in the development of several types of cancers. However, the role of microRNA-132 (miR-132) in human bladder cancer (BC) metastasis remains unclear. In this research, we aimed to investigate the effect of miR-132 on the cell migration and relate potential mechanism in BC. Methods: miR-132 expression level was assessed by quantitative real-time PCR (qRT-PCR) in 32 BC tissues and BC cell lines (T24). The function of miR-132 was evaluated by Transwell assay. Gene expression was determined by using qRT-PCR or Western blot. Results: The results showed that miR-132 had a lower expression in BC tissues than in adjacent normal tissues. At the same time, compared to human normal urethral epithelium cells, the expression level of miR-132 was downregulated in T24 cell lines. miR-132 overexpression significantly inhibited migration and invasion capacities in T24 cells, while downregulation of miR-132 expression strengthened such capacities. Compared with those transfected with miR-132 mimic, EMT-related markers and TGFβ1/Smad2 expression levels were higher in T24 cells transfected with miR-132 inhibitor. Moreover, EMT-related markers and Smad2 expression levels was obviously increased in BC tissues compared to the adjacent normal tissues. The correlation result indicated that the expression of miR-132 and Smad2 was reversed. Conclusion: In short, our results suggest that miR-132 may play a suppressive role in the metastasis of BC cells via TGFβ1/Smad2 signaling pathway.

Keywords: epithelial-mesenchymal transition; human bladder cancer; metastasis; microRNA-132.

Figure 1

miR-132 expression in bladder cancer (BC) tissues and cells. (A) QRT-PCR analysis of miR-132 expression level in BC tissues is lower than in the matched normal tissues. (B) QRT-PCR analysis of miR-132 expression in normal human urethral epithelium cell is higher than in BC cells. Note: **p<0.01.

Figure 2

miR-132 inhibited the migration and invasion of BC cells. (A) QRT-PCR analysis of miR-132 expression in T24 cells transfected with miR-132 inhibitor was decreased compared to the mimic control cells. (B) miR-132 expression in cells transfected with miR-132 mimic is increased compared to the mimic control cells. (C) Wound-healing assays indicated that upregulation of miR-132 inhibits bladder cancer cell migration (×100). (D) Transwell experiments showed that the upregulation of miR-132 inhibits the invasion of bladder cancer cell (× 100). Notes: *p<0.05; **p<0.01.

Figure 3

The expression of EMT-related markers. (A and D) The mRNA level of mesenchymal markers N-Cadherin and Vimentin was elevated in miR-132 inhibited cells and was decreased in miR-132 overexpression cells. (B and C) The expression tendency of EMT-related transcription factors Zeb1 and Snail was the same as N-Cadherin and Vimentin in transfection cells. Notes: *p<0.05; **p<0.01. Abbreviation: NC, mimic control.

Figure 4

TGF-β1/Smad2 signaling was active in miR-132 inhibited cells. (A) The protein level of TGF-β1 was upregulated in miR-132 inhibition cells while it was downregulated in miR-132 mimic cells. (B) Expression of Smad2 and p-Smad2 was increased in miR-132 inhibition cells and was decreased in miR-132 overexpression cells, compared to miR-132 NC cells. Note: *p<0.05. Abbreviation: NC, mimic control.

Figure 5

Correlation between SMAD2 and miR-132 expression in bladder cancer. (A) The prediction of combine state between miR-132-3p and the 3ʹUTR of Smad2 on the website of www. targetscan. org. (B) Semi-quantitative results showed the expression of SMAD2 was increased in the tumor tissue compared to in the matched normal tissues in bladder cancer patients. (C) The mRNA expression of SMAD2 in bladder cancer tissues was significantly increased in bladder cancer tissues compared to in normal tissues. (D) Correlation between SMAD2 and miR-132 expression was negative by Pearson’s correlation analysis (r=−0.71, p<0.001). Note: **p<0.01. Abbreviations: N: normal control; T: bladder cancer patient.