microRNA-211 promotes proliferation, migration, and invasion ability of oral squamous cell carcinoma cells via targeting the bridging integrator 1 protein

Abstract

Oral squamous cell carcinoma (OSCC), the most common pathological type of oral cancer, is still a frequent malignancy with unsatisfactory prognosis. Accumulating studies have proven some microRNAs (miRNAs) can function as oncogenes in OSCC by targeting tumor suppressors. In this study, we first investigated the expression and role of tumor suppressor bridging integrator-1 (BIN1) in OSCC tissues and cells. Our results indicated that BIN1 was low expressed in the OSCC tissues and cell lines (SCC6, SCC9, SCC25, HN4, and HN6) along with miR-211 was highly expressed in OSCC tissues and cell lines, and BIN1 overexpression could evidently inhibit their proliferation, migration, and invasion abilities. Next, we used bioinformation algorithms to predict the potential miRNA targeting BIN1 and chose miR-211 for further study. miR-211, a highly expressed miRNA in OSCC cells, could specifically bind with the 3'-untranslated region (3'-UTR) of BIN1 to trigger its degradation. Addition of miR-211 inhibitor could evidently suppress the malignant behaviors of OSCC cells by upregulating BIN1 expression and inhibit the activation of the EGFR/MAPK pathway. Taken together the findings of the study indicated that miR-211 mediated BIN1 downregulation had crucial significances in OSCC, suggesting the miR-211 might be a novel potential therapeutic target for the OSCC treatment.

Keywords: BIN1; OSCC; invasion; miR-211; proliferation.

Figure 1

Expression of BIN1 and miR‐211 in OSCC tissues and cell lines. A, Protein expression of BIN1 was low in OSCC tissues used by immunohistochemistry (SP × 100). I: Representative positive staining of BIN1 in OSS tissue; II: Representative negative staining of BIN1 in OSCC tissue. B, The gene expression of BIN1 and miR‐211 in OSCC tissues and matched normal tissues. C, The correlation between BIN1 and miR‐211 in OSCC tissues. D, BIN1 was attenuated in OSCC cell lines (SCC6, SCC9, SCC25, HN4, and HN6) compared with normal human oral keratinocytes (HOKs) used by Western blot analysis assay. and F, mRNA expression status of BIN1 and miR‐211 in OSCC cell lines and HOK cells examined by qRT‐PCR. Significant P values marked by the asterisk: * P < 0.05 and ** P < 0.001. BIN1, bridging integrator‐1; miR, microRNA; mRNA, messenger RNA; qRT‐PCR, quantitative real‐time polymerase chain reaction; OSCC, oral squamous cell carcinoma

Figure 2

Effect of BIN1 overexpression on malignant behaviors of HN4 cells. A, Effect of BIN1 overexpression on the proliferation ability inhibition of HN4 cells, analyzed with CCK‐8 assay. B, Effect of BIN1 overexpression on the migration ability suppression of HN4 cells, assayed with wound healing experiment. C, Effect of BIN1 overexpression on the invasion ability of HN4 cells, assayed with transwell experiment. D,Effect of BIN1 overexpression on the apoptosis ability of HN4 cells. Significant P values marked by asterisk: *P < 0.05 and **P < 0.001. BIN1, bridging integrator‐1

Figure 3

Effect of BIN1 overexpression on malignant behaviors of SCC6 cells. A, Effect of BIN1 overexpression on the proliferation ability inhibition of SCC6 cells analyzed with the CCK‐8 assay. B, Effect of BIN1 overexpression on the migration ability suppression of SCC6 cells assayed with wound healing experiment. C, Effect of BIN1 overexpression on the invasion ability of SCC6 cells assayed with transwell experiment. D, Effect of BIN1 overexpression on the apoptosis ability of SCC6 cells. Significant P values marked by the asterisk: *P < 0.05 and **P < 0.001. BIN1, bridging integrator‐1; NC, negative control

Figure 4

Interaction between miR‐211 and BIN1 in OSCC HN4 and SCC6 cells. A, The predicted site in BIN1 3′‐UTR for binding miR‐211. B‐F, Luciferase reporter assay of HN4 and SCC6 cells were transfected with the BIN1 3′‐UTR‐WT or BIN1 3′‐UTR‐M in the binding sites. Differences were observed when miR‐211 inhibitor was added, and miR‐NC inhibitor was used as the control. G and H, miR‐211 inhibitor could reverse the BIN1 protein expression assayed by Western blot analysis experiment in HN4 and SCC6 cells. Significant P values marked by the asterisk: *P < 0.05 and **P < 0.001. BIN1, bridging integrator‐1; miR, microRNA; OSCC, oral squamous cell carcinoma; 3′‐UTR, 3′‐untranslated region; WT, wild type

Figure 5

The effects of miR‐211 inhibitor on OSCC cells. A, Effect of miR‐211 inhibitor on the inhibition of migration ability of HN4 cells assayed with the wound healing experiment. B, Effect of miR‐211 inhibitor on the suppression of invasion ability of HN4 cells assayed with transwell experiment. C, Effects of miR‐211 inhibitor on the activation of EGFR/MAPK pathway analyzed with Western blotting experiment. D, Effect of BIN1 overexpression on EGFR/MAPK pathway OSCC cells. Significant P values marked by the asterisk: *P < 0.05 and **P < 0.001. BIN1, bridging integrator‐1; miR, microRNA; NC, negative control; OSCC, oral squamous cell carcinoma