miR‑490‑5p regulates the proliferation, migration, invasion and epithelial‑mesenchymal transition of pharyngolaryngeal cancer cells by targeting mitogen‑activated protein kinase kinasekinase 9

Abstract

MicroRNA (miRNA/miR) has been identified to be a promising tool in treating pharyngolaryngeal cancer. The present study aimed to investigate the role of miR‑490‑5p in the regulation of proliferation, migration, invasion and epithelial‑mesenchymal transition (EMT) of pharyngolaryngeal cancer cells. The data of miR‑490‑5p expression levels of 45 cases were obtained from the People's Hospital of Xinjiang Uygur Autonomous Region, and the prediction of the target of miR‑490‑5p was conducted by bioinformatics and verified using a luciferase assay. Cell viability was determined by cell counting kit‑8. Migration and invasion rates were measured by wound healing test and Transwell apparatus, respectively. Colony formation rate was measured by plate colony formation assay. mRNA and protein levels were determined by quantitative polymerase chain reaction and western blotting, respectively. miR‑490‑5p expression was significantly depressed in primary pharyngolaryngeal cancer tissues and cell lines, leading to an unfavorable prognosis. Evidently, miR‑490‑5p overexpression decreased the cell viabilities of BICR 18 and FaDu cells. Mechanically, miR‑490‑5p could target mitogen‑activated protein kinase kinasekinase 9 (MAP3K9). The overexpression of MAP3K9 could promote cell viability, migration and invasion rates, EMT process and ability of cloning, miR‑490‑5p could target MAP3K9 and further modulate the proliferation, migration, invasion and EMT of pharyngolaryngeal cancer cells. The results of the present study provide a novel entry point to the treatment of pharyngolaryngeal cancer.

Figure 1

Fold-change in expression of miR-490-5p relative to the internal control gene (U6) in laryngeal tumor, tumor-adjacent tissues and tumor cell lines as well as the overall survival curves of pharyngolaryngeal cancer with different expression levels of miR-490-5p and the alteration of cell viabilities in control, NC and miR-490-5p mimics groups of BICR 18 and FaDu cells. (A) Relative miR-490-5p expression in laryngeal tumor and tumor-adjacent tissues. (B) The overall survival curves of pharyngolaryngeal cancer with low or high expression level of miR-490-5p. (C) Relative miR-490-5p expression in Detroit 562, HNE-3, FaDu, BICR 18, HaCaT and NP 69 cell lines. (D) Relative miR-490-5p expression in each group of BICR 18 cells. (E) Cell viability of BICR 18 cells in each group following 24 and 48 h of culturing since the transfection. (F) Relative miR-490-5p expression in each group of FaDu cells. (G) Cell viability of FaDu cells in each group following 24 and 48 h of culturing since the transfection. Bars indicated the mean ± standard deviation. ^*P<0.05 vs. NP69 cells; ^#P<0.05 vs. HaCaT cells; ^P<0.05 vs. NC groups. NC, negative control; miR, microRNA.

Figure 2

Prediction of the target of miR-490-5p. (A) Venn diagram exhibiting the most possible targets of miR-490-5p (TNKS2, MAP3K9, NUFIP2, CLCC1 and ABCC2) predicting from targetscan, miRTarBase and miRDB databases. (B) Relative mRNA expressions of TNKS2, MAP3K9, NUFIP2, CLCC1 and ABCC2 in control, NC and miR-490-5p mimics groups of BICR 18 cells. (C) Relative mRNA expressions of TNKS2, MAP3K9, NUFIP2, CLCC1 and ABCC2 in control, NC and miR-490-5p mimics groups of FaDu cells. (D) The possible complementary sequences of MAP3K9 3′-UTR and miR-490-5p. (E) Relative luciferase activities in control, NC, mimics groups of BICR 18 cells when the sequences of MAP3K9-3′-UTR was wild or mutant type. (F) Relative luciferase activities in control, NC, mimics groups of FaDu cells when the sequences of MAP3K9-3′-UTR was wild or mutant type. (G) Relative miR-490-5p expression in laryngeal tumor and tumor-adjacent tissues. (H) Relative MAP3K9 expression in laryngeal tumor and tumor-adjacent tissues. Bars indicated the mean ± standard deviation. ^*P<0.05 vs. NC groups. miR, microRNA; NC, negative control; UTR, untranslated region; MAPK, mitogen activated protein kinase.

Figure 3

Overexpression of MAP3K9 in BICR 18 and FaDu cells as well as the alteration of cell viability in control, NC, miR-490-5p mimics, miR-490-5p mimics + MAP3K9 and MAP3K9 groups in BICR 18 and FaDu cells. (A) The protein expression of MAP3K9 in control, NC and MAP3K9 groups of BICR 18 cells. (B) Relative MAP3K9 mRNA expression in control, NC and MAP3K9 groups of BICR 18 cells. (C) The protein expression of MAP3K9 in control, NC and MAP3K9 groups of FaDu cells. (D) Relative MAP3K9 mRNA expression in control, NC and MAP3K9 groups of FaDu cells. (E) The cell viabilities in each group of BICR 18 cells following 24 and 48 h of culturing since the transfection. (F) The cell viabilities in each group of FaDu cells following 24 and 48 h of culturing since the transfection. Bars indicate the mean ± standard deviation. ^*P<0.05 vs. NC groups; ^#P<0.05 vs. miR-490-5p mimics groups. miR, microRNA; NC, negative control; MAPK, mitogen activated protein kinase.

Figure 4

Relative wound distances in the control, NC, miR-490-5p mimics, miR-490-5p mimics + MAP3K9 and MAP3K9 groups of BICR 18 and FaDu cells. (A) The images demonstrated the distances between the edges of gaps of BICR 18 cell monolayer following 0 and 24 h of culturing in each group. (B) The statistical results of relative wound distance in each group of BICR 18 cells. (C) The statistical results of relative wound distance in each group of FaDu cells. (D) The images demonstrated the distances between the edges of gaps of FaDu cell monolayer following 0 and 24 h of culturing in each group. Bars indicate the mean ± standard deviation. ^*P<0.05 vs. NC groups; ^#P<0.05 vs. miR-490-5p mimics groups. miR, microRNA; NC, negative control; MAPK, mitogen activated protein kinase.

Figure 5

Relative cell invasion rates in control, NC, miR-490-5p mimics, miR-490-5p mimics + MAP3K9 and MAP3K9 groups of BICR 18 and FaDu cells. (A) The images demonstrated the cells invaded into the bottom chamber of Transwell apparatus in each group of BICR 18 cells. (B) The statistical outcomes of relative cell invasion rates (of control) in each group of BICR 18 cells. (C) The statistical outcomes of relative cell invasion rates (of control) in each group of FaDu cells. (D) The images demonstrated the cells invaded into the bottom chamber of Transwell apparatus in each group of FaDu cells. Bars indicate the mean ± standard deviation. ^*P<0.05 vs. NC groups; ^#P<0.05 vs. miR-490-5p mimics groups. miR, microRNA; NC, negative control; MAPK, mitogen activated protein kinase.

Figure 6

Expression of MMP-9, TIMP-2, E-cadherin and vimentin in the control, NC, miR-490-5p mimics, miR-490-5p mimics + MAP3K9 and MAP3K9 groups of BICR 18 and FaDu cells. (A) Relative mRNA expressions of MMP-9, TIMP-2, E-cadherin and vimentin in each group of BICR 18 cells. (B) Relative mRNA expressions of MMP-9, TIMP-2, E-cadherin and vimentin in each group of FaDu cells. (C) The original outcomes of western blot of the expressions of MMP-9, TIMP-2, E-cadherin and vimentin in each group of BICR 18 cells. (D) Relative protein levels of MMP-9, TIMP-2, E-cadherin and vimentin (of β-actin) in each group of BICR 18 cells (E) The original outcomes of western blotting of the expression of MMP-9, TIMP-2, E-cadherin and vimentin in each group of FaDu cells. (F) Relative protein levels of MMP-9, TIMP-2, E-cadherin and vimentin (of β-actin) in each group of FaDu cells. Bars indicates the mean ± standard deviation. ^*P<0.05 vs. NC groups; ^#P<0.05 vs. miR-490-5p mimics groups. miR, microRNA; NC, negative control; MAPK, mitogen activated protein kinase; E, epithelial; TIMP-2, tissue inhibitor of metalloproteinase; MMP, matrix metalloproteinase.

Figure 7

Relative colony formation in the control, NC, miR-490-5p mimics, miR-490-5p mimics + MAP3K9 and MAP3K9 groups of BICR 18 and FaDu cells. (A) The images exhibited the stained colony in each group of BICR 18 cells. (B) Relative colony formation in each group of BICR 18 cells. (C) Relative colony formation in each group of FaDu cells. (D) The images demonstrated the stained colony in each group of FaDu cells. Bars indicates the mean ± standard deviation. ^*P<0.05 vs. NC groups; ^#P<0.05 vs. miR-490-5p mimics groups. miR, microRNA; NC, negative control; MAPK, mitogen activated protein kinase.