miR-125a-5p inhibits colorectal cancer cell epithelial-mesenchymal transition, invasion and migration by targeting TAZ

Abstract

Background: miR-125a-5p regulated biological processes in various types of cancer, including colorectal cancer (CRC). TAZ, a vital transcriptional coactivators of the Hippo pathway, was found to be overexpressed in various cancers. Objectives: This study aims to study the effect of miR-125a-5p on the progression of CRC by regulating TAZ expression. Methods: In this study, miR-125a-5p and TAZ expression in CRC tissue and cell lines were detected by real-time polymerase chain reaction (RT-PCR). Luciferase reporter assay was applied to detect whether TAZ was a target of miR-125a-5p. Cell migration and invasion were detected in vitro by wound-healing assay and cell invasion assay. Western blot was used to detect the expression of epithelial-mesenchymal transition (EMT)-related proteins. Findings: The results revealed downregulation of miR-125a-5p, as well as upregulation of TAZ in CRC tissue and cell lines. TAZ was identified as a direct target of miR-125a-5p, and its expression was negatively regulated by miR-125a-5p in CRC cell lines. The functional studies revealed that overexpression of miR-125a-5p inhibited the migration, invasion and EMT of CRC cells, while upregulation of TAZ reversed the inhibitory effect caused by miR-125a-5p. Conclusion: Our data suggest that miR-125a-5p inhibits CRC cell migration, invasion and EMT by targeting TAZ. These results suggest that miR-125a-5p serves as a potential therapeutic biomarker for CRC patients.

Keywords: TAZ; colorectal cancer; epithelial–mesenchymal transition; invasion; miR-125a-5p; migration.

Figure 1

miR-125a-5p was downregulated in CRC tissues and cell lines. (A) The expression of miR-125a-5p in colorectal cancer and adjacent tissues was determined by qRT-PCR. (B) The expression of miR-125a-5pin five CRC cell lines LoVo, SW480, SW620, HCT116, HT29 and NCM460 was detected by qRT-PCR. Dates are expressed by mean ± standard deviation. (A) ***P<0.001 vs colorectal cancer adjacent tissues; (B) **P<0.01, ***P<0.001 vs NCM460. Abbreviations: CRC, colorectal cancer; NCM460, normal colonic mucosa epithelial cells; miR-125a-5p, microRNA-125a-5p.

Figure 2

TAZ was upregulated in CRC cells and is a direct target gene of miR-125a-5p. (A) The mRNA expression of TAZ in CRC tissues was determined by qRT-PCR. The mRNA and protein expression of TAZ in CRC cell lines LoVo, SW480, SW620, HCT116, HT29 was determined by qRT-PCR (B) and Western blot (C). (D) Luciferase activity was detected in cells co-transfected with TAZ 3ʹUTR WT/MUT and miR-125a-5p mimic/mimic control by dual-luciferase reporter assay. Protein expression levels of TAZ in LoVo (E) and SW480 (F) cells transfected with miR-125a-5p mimic or mimic control was detected by Western blot. mRNA expression levels of TAZ in LoVo (G) and SW480 (H) cells transfected with miR-125a-5p mimic or mimic control was detected by qRT-PCR. Dates are expressed by mean ± standard deviation. (A) ***P<0.001 vs colorectal cancer adjacent tissues. (B and C) *P<0.05, **P<0.01, ***P<0.001 vs NCM460. (D–H) **P<0.01, ***P<0.001 vs control. Abbreviations: CRC, colorectal cancer; NCM460, normal colonic mucosa epithelial cells; miR-NC, microRNA mimic control; miR-125a-5p, microRNA-125a-5p.

Figure 3

miR-125a-5p inhibits migration and invasion of CRC cells via directly targeting TAZ. Protein expression levels of TAZ in LoVo (A) and SW480 (B) cells transfected with miR-125a-5p mimic or mimic control were detected by Western blot. (C) Wound-healing assays were used to examine cell migration of LoVo and SW480 cells transfected with miR-NC, miR-125a-5p mimic or co-transfected with miR-125a-5p mimic and TAZ overexpression plasmids. (D) Transwell assays were used to examine cell invasion of LoVo and SW480 cells transfected with miR-NC, miR-125a-5p mimic or co-transfected with miR-125a-5p mimic and TAZ overexpression plasmids. Dates are expressed by mean ± standard deviation. **P<0.01, vs control. ^##P<0.01, ^###P<0.001 vs miR-125a-5p mimic. Abbreviations: miR-NC, microRNA mimic control; PCDNA3-TAZ, TAZ overexpression plasmids.

Figure 4

miR-125a-5p inhibits epithelial–mesenchymal transition of CRC cells via directly targeting TAZ. Western blot was used to examine the protein expression of EMT markers E-cadherin, N-cadherin, vimentin and β-catenin in LoVo (A) and SW480 (B) cells transfected with miR-NC, miR-125a-5p mimic or co-transfected with miR-125a-5p mimic and TAZ overexpression plasmids. Dates are expressed by mean ± standard deviation. **P<0.01, ***P<0.01 vs miR-NC. ^##P<0.01, ^###P<0.01 vs miR-125a-5p mimic. Abbreviations: miR-NC, microRNA mimic control; PCDNA3-TAZ, TAZ overexpression plasmids.