Upregulation of kazrin F by miR-186 suppresses apoptosis but promotes epithelial-mesenchymal transition to contribute to malignancy in human cervical cancer cells

Abstract

Objective: Previous studies have identified that kazrin is a constituent of desmosome and influences intercellular adhesion, growing development and morphology. We previously cloned another new isoform, kazrin F and found that it has anti-apoptotic effects on human glioma cell line. To further explore whether kazrin F is involved in tumorigenesis, we investigated its expression and role in cervical cancer (CC) cells.

Methods: The role of kazrin F and miR-186 in CC was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony formation, transwell, and apoptosis assays. Using enhanced green fluorescent protein (EGFP) reporter assays, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis, we identified kazrin F post-transcriptional regulation by miR-186.

Results: We demonstrate that kazrin F is highly expressed in CC tissues compared with the adjacent noncancerous tissues and promotes cell proliferation, colony formation, migration and invasion in HeLa and C33A cells by suppressing apoptosis and facilitating epithelial-to-mesenchymal transition (EMT). Furthermore, miR-186 was confirmed as a regulator of kazrin F dysregulation. An EGFP reporter assay proved that miR-186 directly targets the 3'-untranslated region (3'UTR) of kazrin F and downregulates its expression, and miR-186 expression showed an inverse correlation with kazrin F levels in CC tissues. In addition, overexpression of miR-186 suppressed the malignant behaviors of CC cells. The ectopic expression of kazrin F rescued the inhibitory effects of miR-186.

Conclusions: Our findings indicate that the upregulation of kazrin F due to downregulated miR-186 levels contributes to malignancy, and highlight the significance of kazrin F in CC tumorigenesis.

Keywords: EMT; Kazrin F; apoptosis; cervical cancer; miR-186.

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Kazrin F is upregulated and promotes the proliferation of cervical cancer (CC) cells. (A) The relative mRNA expression level of kazrin F in 18 pairs of CC tissues and adjacent non-tumor tissues was detected with RT-qPCR. β-actin was used for normalization; (B) The mRNA and protein levels of kazrin F were determined using RT-qPCR and western blot analysis in HeLa and C33A cells, respectively; (C) MTT assay was used to measure cell viability 48 h, 72 h and 96 h after transfection in HeLa and C33A cells; (D) The proliferative activity of HeLa and C33A cells was tested with the colony formation assays 14 d after transfection; (E) After transfection with kazrin F for 24 h, the relative apoptosis rate of HeLa cells was monitored with Annexin V staining and flow cytometry analysis; (F) The protein level of caspase 3 in HeLa cells was assessed by western blot. GAPDH served as a loading control. Quantification of the bands is shown on the left. All data represent ±s of three independent experiments. *, P<0.05; **, P<0.01; ***, P<0.001.

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Kazrin F promotes migration and invasion of cervical cancer cells. (A) Transwell assays were performed to detect the migration (left panel) and invasion ability (right panel) of HeLa and C33A cells; (B) External cell morphology of HeLa and C33A cells was evaluated with a phase contrast microscope. All photomicrographs were taken at 200× magnification; (C) The protein levels of epithelial-to-mesenchymal transition (EMT)-associated molecules (E-cadherin and vimentin) were determined using a western blot. GAPDH served as a loading control. All data represent the ±s of three independent experiments. *, P<0.05; **, P<0.01; ***, P<0.001.

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Kazrin F rescues the suppression of malignancy induced by miR-186 in cervical cancer cells. (A) MTT assay tested the viability of cells co-transfected with pcDNA3/pri-miR-186 and pcDNA3/kazrin F; (B) Colony formation assays were used to test the proliferation of the transfected cells; (C) Apoptosis assays were performed to assess the cell apoptotic ability in the rescue experiment; (D) Transwell migration and invasion assays were used to test the migration and invasion abilities of the co-transfected cells; (E) Western blot analysis of E-cadherin and vimentin protein expression levels in the transfected cells. All data represent the ±s of three independent experiments. *, P<0.05; **, P<0.01; ***, P<0.001.