miR-632 Promotes Laryngeal Carcinoma Cell Proliferation, Migration, and Invasion Through Negative Regulation of GSK3β

Abstract

Laryngeal cancer, one of the most common head and neck malignancies, is an aggressive neoplasm. Increasing evidence has demonstrated that microRNAs (miRNAs) exert important roles in oncogenesis and progression of diverse types of human cancers. miR-632, a tumor-related miRNA, has been reported to be dysregulated and implicated in human malignancies; however, its biological role in laryngeal carcinoma remains to be elucidated. The present study aimed at exploring the role of miR-632 in laryngeal cancer and clarifying the potential molecular mechanisms involved. In the current study, miR-632 was found to be significantly upregulated both in laryngeal cancer tissues and laryngeal cancer cell lines. Functional studies demonstrated that miR-632 accelerated cell proliferation and colony formation, facilitated cell migration and invasion, and enhanced the expression of cell proliferation-associated proteins, cyclin D1 and c-myc. Notably, miR-632 could directly bind to the 3'-untranslated region (3'-UTR) of glycogen synthase kinase 3β (GSK3β) to suppress its expression in laryngeal cancer cells. Mechanical studies revealed that miR-632 promoted laryngeal cancer cell proliferation, migration, and invasion through negative modulation of GSK3β. Pearson's correlation analysis revealed that miR-632 expression was inversely correlated with GSK3β mRNA expression in laryngeal cancer tissues. Taken together, our findings suggest that miR-632 functions as an oncogene in laryngeal cancer and may be used as a novel therapeutic target for laryngeal cancer.

Figure 1

miR-632 is significantly upregulated in laryngeal cancer tissues and cell lines. (A) Relative expression levels of miR-632 in 10 pairs of laryngeal cancer tissues and adjacent noncancerous tissues were measured using quantitative real-time PCR (qRT-PCR) analysis. (B) Relative expression levels of miR-632 in normal bronchial epithelial cell line BEAS-2B and three laryngeal cancer cell lines (SNU899, TU212, and Hep-2) were identified by qRT-PCR analysis. **p < 0.01.

Figure 2

miR-632 accelerates laryngeal cancer cell proliferation and colony formation. (A) miR-632 expression levels in Hep-2 cells were identified using qRT-PCR after transfection with miR-632 mimics or miR-632 inhibitor. (B) Hep-2 cell proliferation was assessed using MTT assays after transfection with miR-632 mimics or miR-632 inhibitor. (C) Clonogenic capability of Hep-2 cells was evaluated using colony formation assays after transfection with miR-632 mimics or miR-632 inhibitor. **p < 0.01.

Figure 3

miR-632 suppresses the expression of cell proliferation-associated proteins in Hep-2 cells. (A) Expression levels of cyclin D1 and c-myc in Hep-2 cells were determined using Western blot after transfection with NC-mimics or miR-632 mimics. (B) Expression levels of cyclin D1 and c-myc in Hep-2 cells were identified using Western blot after transfection with NC-inhibitor or miR-632 inhibitor. **p < 0.01.

Figure 4

miR-632 promotes laryngeal cancer cell migration and invasion. (A) Hep-2 cell migration was examined by wound healing assays after transfection with miR-632 mimics or miR-632 inhibitor. (B) Hep-2 cell invasion was identified using Transwell invasion assays after transfection with miR-632 mimics or miR-632 inhibitor. **p < 0.01.

Figure 5

Glycogen synthase kinase 3β (GSK3β) is a downstream target of miR-632. (A) A putative binding site of miR-632 in the 3′-untranslated region (3′-UTR) of GSK3β was predicted by TargetScan online software. (B) Luciferase activity of reporter vectors carrying wild-type or mutant GSK3β 3′-UTR in Hep-2 cells was detected in the presence of miR-632. (C) GSK3β mRNA expression in Hep-2 cells was examined using qRT-PCR after transfection with miR-632 mimics or miR-632 inhibitor. (D) Protein expression of GSK3β in Hep-2 cells was measured by Western blotting analysis after transfection with miR-632 mimics or miR-632 inhibitor. (E) Correlation between miR-632 expression and GSK3β mRNA expression in laryngeal cancer tissues was determined by Pearson’s correlation analysis. **p < 0.01.

Figure 6

Downregulation of GSK3β has similar effects with overexpression of miR-632 in laryngeal cancer cells. (A) Hep-2 cells were transfected with si-GSK3β to achieve the knockdown of GSK3β, as validated by Western blotting analysis. (B) Hep-2 cell viability was examined by MTT assays after transfection with si-NC or si-GSK3β. (C) Clonogenic ability of Hep-2 cells was evaluated by colony formation assays after transfection with si-NC or si-GSK3β. (D) Hep-2 cell migration was identified via wound healing assays after transfection with si-NC or si-GSK3β. (E) Hep-2 cell invasion was assessed using Transwell invasion assays after transfection with si-NC or si-GSK3β. **p < 0.01.

Figure 7

Downregulation of GSK3β has opposite effects to inhibition of miR-632 in laryngeal cancer cells. (A) Hep-2 cell viability was measured using MTT assays after cotransfection with miR-632 inhibitor and si-GSK3β. (B) Clonogenic capability of Hep-2 cells was identified by colony formation assays after cotransfection with miR-632 inhibitor and si-GSK3β. (C) Hep-2 cell migration was assessed by wound healing assays after cotransfection with miR-632 inhibitor and si-GSK3β after cotransfection with miR-632 inhibitor and si-GSK3β. (D) Hep-2 cell invasion was evaluated via Transwell invasion assays after cotransfection with miR-632 inhibitor and si-GSK3β. **p < 0.01 versus NC-inhibitor + si-NC group; ##p < 0.01 versus miR-632 inhibitor + si-NC group.