miR-106b promotes proliferation and invasion by targeting Capicua through MAPK signaling in renal carcinoma cancer

Abstract

Background: miR-106b has been reported to play a vital role in pathogenesis of some types of cancer, whilst the role of miR-106b in renal carcinoma cancer (RCC) remains unknown. Purpose: The objective of this study was to identify the mechanism of miR-106b regulating the progression of renal carcinoma. Method: The expression of miR-106b was analyzed in RCC cell lines, RCC and adjacent normal renal tissues through qRT-PCR assays. Target mRNA of miR-106b was predicted with databases and verified by luciferase reporter assays. And the effects of miR-106b or targeted mRNA on cell proliferation, invasion, the process of epithelial-mesenchymal transitions (EMTs) were assessed in vitrothrough CCK-8, transwell cell invasion assays, qRT-PCR and Western blotting assays respectively. In addition, the effects of miR-106b on the growth of xenografts mice were analyzedin vivo. Results: The results demonstrated that miR-106b was significantly increased both in RCC tissues and cell lines. Luciferase reporter assays revealed that miR-106b inhibited Capicua expression by targeting its 3'-UTR sequence. And miR-106b promoted cell proliferation, invasion, EMT progression in RCC cellin vitro, as well as promoted the tumor growth of 786-O cells derived xenografts mice. Additionally, loss of Capicua promoted the activation of MAPK signaling pathway. Conclusion: The study suggested that miR-106b regulated RCC progression through MAPK signaling pathway partly by targeting Capicua, which might provide valuable evidence for therapeutic target development of RCC.

Keywords: capicua; epithelial mesenchymal transitions; miR-106b; renal carcinoma.

Figure 1

Expression of miR-106b and CIC in RCC tissues and cell lines. Notes: (A) qRT-PCR analysis showed the expression of miR-106 in the RCC tissues (tumor) and the adjacent non-tumor tissues (normal) in patient samples (n=20). (B) qRT-PCR analysis of expression of miR-106b in RCC cancer cell lines (786–0, ACHN, Caki-1, Caki-2) and in normal renal tubules epithelium cell HK-2. (C) qRT-PCR analysis showed the expression of CIC in the RCC tissues (tumor) and the adjacent non-tumor tissues (normal) in patient samples (n=20). (D) qRT-PCR analysis of expression of CIC in RCC cancer cell lines (786–0, ACHN, Caki-1, Caki-2) and in normal renal tubules epithelium cell HK-2. **P<0.01, vs HK-2 cell; ^#P<0.05, vs 786-O cell.Abbreviations: CIC, capicua homology protein; RCC, renal call carvinoma cancer.

Figure 2

Ectopic expression of miR-106b promotes proliferation in RCC cells in vitro and in vivo. Notes: (A) miR-106b levels in 786-O cells transfected with scramble miRNA (miR-NC), miR-106b mimics, or miR-106b inhibitor. (B) CCK-8 assay of 786-O cells transfected with scramble miRNA, miR-106b mimics or miR-106b inhibitor. (C–D) Representative images and quantification of the BrdU cell proliferation assay (400×), *P<0.05, **P<0.01, vs mock group. (E) miR-106b silencing (anti-miR-106b) inhibits tumor growth in 786-O cells derived xenografts compared with negative control (NC). *P<0.05, vs NC.

Figure 3

miR-106b promotes invasion of RCC cells in vitro. Notes: Representative images and quantification of 786-O invasive cells transfected without (Mock) or within scramble miRNA, miR-106b mimics, or miR-106b inhibitor (100×). The cell number of invasive cells were counted in randomly selected fields and presented in the bar graph (mean ± SD; *P<0.05, **P<0.01, vs mock group).

Figure 4

miR-106b promotes EMT progression of RCC cells in vitro. Notes: (A–D) mRNA levels of EMT-related proteins and CIC examined by qRT-PCR in 786-O cells transfected with miR-106b mimics, miR-106b inhibitor, or scramble miRNA. (E–I) protein levels of EMT-related proteins and CIC examined by qRT-PCR in 786-O cells transfected with miR-106b mimics, miR-106b inhibitor or scramble miRNA. **P<0.01, vs mock group. Abbreviations: CIC, capicua hohology protein; EMT, epithelial-mesenchymal transition; NC, negative control.

Figure 5

miR-106b directly targets the 3ʹ-UTR of CIC in renal carcinoma cancer. Notes: Luciferase activity of 786-O cells transfected with plasmids carrying a wild-type or mutant 3ʹ-UTR of CIC, in response to miR-106b mimics or miR-NC. **P<0.01, vs miR-NC group. Abbreviations: CIC, capicua homology protein; NC, negative control.

Figure 6

CIC inhibits cell proliferation, invasion in RCC cells. Notes: (A) mRNA levels of CIC in 786-O cells transfected with CIC silencing plasmid (CIC-shRNA), CIC overexpressed plasmid (pcDNA3.1-CIC) or negative controls (pcDNA3.1). (B) CCK-8 assay of 786-O cells transfected with indicated molecular. (C) Representative images and quantification of the BrdU cell proliferation assay (400×). (D) Representative images and quantification of 786-O invasive cells transfected with indicated molecular. *P<0.05, vs mock group; **P<0.01, vs mock group. Abbreviations: CIC, capicua homology protein; RCC, renal cell carcinoma cancer.

Figure 7

CIC inhibits activation of MAPK signaling pathway in vitro. Notes: Changes of phosphorylation levels of MAPK in 786-O cells transfected with indicated molecular. **P<0.01, vs mock group. Representative images (A)  and quantification (B) of  levels of CIC, phosphorylated MAPK and total MAPK  in 786-O cells transfected with indicated molecular.  The  band density was qualified by Image J software, and β-actin was used as internal controls. Abbreviation: CIC, capicua homology protein.