The miR-136-5p/ROCK1 axis suppresses invasion and migration, and enhances cisplatin sensitivity in head and neck cancer cells

Abstract

Laryngeal squamous cell carcinoma (LSCC) and hypopharyngeal squamous cell carcinoma (HPSCC) are two types of head and neck cancers with high incidence rates and relatively poor prognoses. The aim of the present study was to determine the effects of microRNA (miR/miRNA)-136-5p and its downstream target, Rho-associated coiled-coil containing protein kinase 1 (ROCK1), on LSCC and HPSCC progression and cisplatin sensitivity. The miRNA and protein expression levels in head and neck cancer cell lines were evaluated using reverse transcription-quantitative PCR and western blotting, respectively. MTT, wound healing assays, transwell assays and flow cytometry analysis were performed to measure cell properties. The binding between miR-136-5p and ROCK1 was detected using a dual-luciferase reporter assay. Autophagy double-labeled adenoviral infection assays were used to assess cell autophagy. The results showed that miR-136-5p was expressed in LSCC and HPSCC cells. Functional experiments showed that the expression of miR-136-5p in LSCC and HPSCC cells was negatively correlated with cell viability, invasion and migration. Additionally, miR-136-5p overexpression inhibited epithelial-mesenchymal transition, whereas miR-136-5p knockdown had the opposite effect. Dual-luciferase reporter assays confirmed the targeting relationship between miR-136-5p and ROCK1. miR-136-5p overexpression increased the cisplatin sensitivity of LSCC and HPSCC cells by reducing cell viability, as well as promoting cell apoptosis and autophagy. miR-136-5p overexpression decreased the expression levels of its downstream target ROCK1 and attenuated activity of the Akt/mTOR signaling pathway in cisplatin-treated LSCC and HPSCC cells. Conversely, miR-136-5p knockdown increased ROCK1 levels and decreased cisplatin sensitivity of the LSCC and HPSCC cells by increasing cell viability and inhibiting cell apoptosis, which was reversed by ROCK1 inhibition using the ROCK1 inhibitor, Y27632. Taken together, the results showed that the miR-136-5p/ROCK1 axis inhibits cell invasion and migration, and increases the sensitivity of LSCC and HPSCC cells to cisplatin.

Keywords: Rho-associated coiled-coil containing protein kinase 1; cell invasion; cell migration; cisplatin sensitivity; hypopharyngeal squamous cell carcinoma; laryngeal squamous cell carcinoma; microRNA-136-5p.

Figure 1

The expression levels of miR-136-5p in LSCC and HPSCC cells. (A) The expression level of miR-136-5p in LSCC and HPSCC cells. Following transfection of the cells with (B) miR-136-5p mimics, mimics-NC; or (C) miR-136-5p inhibitor or inhibitor-NC for 24 h, the expression levels of miR-136-5p were measured using reverse transcription-quantitative PCR. Data are presented as the mean ± SD, n=3. ^*P<0.05. HPSCC, head and neck squamous cell carcinoma; LSCC, laryngeal squamous cell carcinoma; miR, microRNA; NC, negative control.

Figure 2

miR-136-5p suppresses viability, invasion and migration of LSCC and HPSCC cells. (A) After LSCC and HPSCC cells transfected with miR-136-5p mimics, mimics-NC, miR-136-5p inhibitor or inhibitor-NC for 24 h, cell viability was detected using MTT assays, n=6. Cell migratory abilities were measured using wound healing assays in (B) FD-LSC-1 cells and (C) FaDu cells, scale bar=200 µm. (D) Migratory rates were then quantified. (E) Cell invasion was observed and (F) measured using transwell assays, scale bar =100 µm. (G) The protein expression levels of E-cadherin, N-cadherin and vimentin were measured using western blot analysis and normalized to the levels of β-actin. Data are presented as the mean ± SD, n=3. ^*P<0.05. HPSCC, head and neck squamous cell carcinoma; LSCC, laryngeal squamous cell carcinoma; miR, microRNA; NC, negative control.

Figure 3

miRNA-136-5p directly targets ROCK1 in LSCC and HPSCC cells. (A) Binding sites between ROCK1 wt and the mut-type miR-136-5p are shown. (B) miR-136-5p mimics or mimic-NC and luciferase plasmid containing ROCK1-wt or ROCK1-mut transcript were co-transfected into LSCC and HPSCC cells, luciferase activity was measured using dual-luciferase reporter assays. Data are presented as the mean ± SD, n=3. ^*P<0.05. HPSCC, head and neck squamous cell carcinoma; LSCC, laryngeal squamous cell carcinoma; miR, microRNA; NC, negative control; UTR, untranslated region; wt, wild-type.

Figure 4

miR-136-5p overexpression increases the cisplatin sensitivity of laryngeal squamous cell carcinoma and head and neck squamous cell carcinoma cells. (A) After cells were transfected with miR-136-5p mimics or mimics-NC and treated with cisplatin (2.6 µM) for 24 h, cell viability was detected using MTT assays, n=6. (B) Flow cytometric detection of apoptosis using annexin V-FITC/PI staining and (C) quantification of apoptosis. Autophagy was detected using an autophagy double-labeled adenovirus infection assay in (D) FD-LSC-1 and (E) FaDu cells, scale bar=50 µm. Free yellow puncta indicates the autophagosome, free red puncta indicates the autophagolysosome. (F) The protein expression levels of ROCK1, p-Akt/Akt (Ser473), GSK-3β, p-GSK-3β (Ser9), p-mTOR (S2448)/mTOR, LC3II/I and P62 were measured using western blot analysis and normalized to the levels of β-actin. Data are presented as the mean ± SD, n=3. ^*P<0.05. GSK-3β, glycogen synthase kinase-3β; LC3, microtubule-associated protein 1 light chain 3; miR, microRNA; mTOR, mammalian target of rapamycin; NC, negative control; p-, phosphorylated.

Figure 5

miR-136-5p downregulation decreased the cisplatin sensitivity of laryngeal squamous cell carcinoma and head and neck squamous cell carcinoma cells by targeting ROCK1. (A) After cells were transfected with miR-136-5p inhibitor or inhibitor-NC for 24 h, as well as with cisplatin (2.6 µM) and/or Y27632 (25 µM, ROCK inhibitor) for 24 h, cell viability was detected using MTT assays, n=6. (B) Flow cytometric detection of apoptosis using annexin V-FITC/PI staining and (C) quantification of apoptosis. (D) The protein expression levels of ROCK1, p-GSK-3β (Ser9)/GSK-3β, cleaved caspase-3, Bax and Bcl-2 were measured using western blot analysis and normalized to the levels of β-actin (D). Data are presented as the mean ± SD, n=3. ^*P<0.05. GSK-3β, glycogen synthase kinase-3β; miR, microRNA; NC, negative control; p-, phosphorylated; ROCK, Rho-associated coiled-coilcontaining protein kinase.