Human LSCC cell lines (LSC-1, FaDu, FD-LSC-1, and TU177) and a Normal Laryngeal Epithelial Cell Line (NPEC) purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China) were cultured in RPMI-1640 medium (GIBCO, Thermo Fisher Scientific) added with 10% fetal bovine serum at 37°C in an atmosphere of 5% CO2. When cell confluence reached 90%, cell passage was carried out, and the cell lines were screened by qRT-PCR.

miR-375 mimic (miR-375-mimic, 50nM), miR-375 inhibitor (miR-375-inhibitor, 100nM), CST1 overexpression plasmid (oe-CST1, 2μg), and their corresponding negative controls (mimic-NC, inhibitor-NC, and oe-NC) were all purchased from GeneChem (Shanghai, China). Cell transfection was performed based on the instruction of the Lipofectamine 2000 kit (Invitrogen, Carlsbad, CA, USA). The following experiments were performed 48h after the transfection.

The extraction of Total RNA (Invitrogen, Car, USA) and the reverse transcription into cDNA were executed using Trizol and a PrimeScript RT kit (RR037A, Takara, Japan), respectively. Fluorescent quantitative PCR was performed per the instructions of SYBR®Premix ExTaqTM Ⅱ Kit (RR820A, TaKaRa). The cDNA templates were subjected to real time fluorescence quantitative PCR (ABI 7500, ABI, Foster City, CA, USA). The relative expression of target genes was internalized to that of GAPDH or U6 and calculated by the 2−ΔΔCt method: ΔΔCt=ΔCt experimental group−ΔCt control group, ΔCt=Ct target gene−Ct internal control. Each experiment was run in triplicate. Relevant primers were designed by Shanghai Sangon Biotech (Table 1).

The LSCC cells were inoculated onto 96-well plates with 100μl diluted cell suspension (1×105cells/mL) per well. Each sample had triplicate wells. Then 10μL of CCK-8 reagent (Tokyo, Dojindo, Japan) was appended into each well and incubated for 2h after cells cultures for 0, 24, 48, and 72h. The absorbance was measured at a wavelength of 450nm.

Cells (5×104) were suspended in serum-free DMEM and inoculated onto transwell chambers coated with matrixgel (BD Biosciences, Bedford, MA, USA), and 600μL medium containing 10% FBS was added into lower chambers. After 48-h incubation, the cells invaded to the lower surface of the membrane were fixed in 100% methanol and stained with 0.1% crystal violet. The non-invaded cells on the upper surface were removed with cotton swabs. Cells on the lower surface were photographed using a microscope (Olympus, Tokyo, Japan) in five random fields.

The cells were inoculated onto 6-well plates at a density of 5×105 per well. When grew to 90% confluence, the cells were scratched with a 200μL pipette tip. The cells were washed three times with DPBS (14190250, Gibco, New York, USA), and the culture medium was refreshed with DMEM containing 2% FBS. The gap between the cells was observed under a low-power contrast microscope (Olympus MK, Tokyo, Japan). Cells in the same field were photographed after continuous culture for 24h, and the change of the scratch width was observed. Migration rate=(0h scratch distance−24h scratch distance)/0h scratch distance. The experiment was run in triplicate.

After transfection for 48h, cells were digested with 0.25% trypsin (without EDTA) (YB15050057, Yubo Biotechnology Co., Ltd., Shanghai, China), and then collected for centrifugation with the supernatant discarded. After that, the cells were washed three times with cold PBS before centrifugation again and the supernatant was removed. Annexin-V-FITC apoptosis assay kit (K201-100, Biovision, USA) was used for measurement. The cells (1×106) were suspended with 100μL of PI solution, and mixed. After 15min of incubation, the cells were mixed with 1mL HEPES buffer (PB180325, Procell). Cell apoptosis was measured by flow cytometry. The experiment was repeated three times.

Enhanced RIPA cell lysis (Wuhan Boster Biological Technology., Ltd., Wuhan, China) containing protease inhibitors was used for cell lysis, and the protein concentration was determined using a BCA protein quantitative kit (Boster). The extracted protein was separated by 10% SDS-PAGE and transferred onto PVDF membranes before being blocked with 5% BSA for 2h to block non-specific binding. Diluted primary antibodies of MMP-2 (ab92536, 1:1000, Abcam, Cambridge, UK), MMP-9 (ab76003, 1:1000, Abcam), mouse anti-human CST1 (ab68329, 1:1000, Abcam), and GAPDH (ab9485, 1:2500, Abcam) were added for incubation at 4°C overnight. Then the protein was washed and incubated with HRP labeled goat anti-rabbit secondary antibody (ab6721, 1:2000, Abcam) or rabbit anti-mouse secondary antibody (ab6728, 1:2000, Abcam) for 1h, followed by treatment with ECL solution (EMD Millipore company, USA) at room temperature for 1min. The membrane was exposed to X-Ray films for 5–10min for color development. Image J analysis software (National Institutes of Health, NIH) was used for gray quantification of western blot images, with GAPDH as the internal reference. Each group had three parallel experiments.

Jefferson database (https://cm.jefferson.edu/) was implied to predict the binding site of miR-375 with CST1, according to which the wild-type fragments and mutant fragments were designed and inserted into pMIR-report plasmids. The Wild-Type luciferase Reporter Plasmid (WT-CST1) and mutant luciferase reporter plasmid (MUT-CST1) were co-transfected into HEK-293T cells with miR-375-mimic or mimic-NC (Shanghai North Connaught Biotechnology Co., Ltd., Shanghai, China). After transfection (48h), the cells were collected and lysed before centrifugation for 3–5min. The supernatant was collected to determine the luciferase activity using the luciferase assay kit (Dual-Luciferase Reporter Assay System, Promega, USA). The ratio of target luciferase activity and reference luciferase activity was used as the relative luciferase activity, and the luciferase activity was measured by a fluorescence detector (Promega). Three independent experiments were carried out.

Data were analyzed by GraphPad prism 6 software and expressed as mean±standard deviation (Σ±s). When two groups were analyzed, statistical comparisons were made by using t-test. One-way analysis of variance was adopted followed by Tukey’s multiple comparisons test for comparisons among more than two groups. p<0.05 was thought to have statistical significance.

TCGA database showed that miR-375 was decreasingly expressed in HNSC (Fig. 1A). As demonstrated in Fig. 1B, qRT-PCR results revealed that the expression of miR-375 in LSC-1, FaDu, FD-LSC-1, and TU177 cells was clearly lower than in NPEC cells, especially in LSC-1 and TU177 cells (p 0.05). Therefore, LSC-1 and TU177 cells were selected for subsequent experiments.

Figure 1.

miR-375 expression was downregulated in LSCC cell lines. (A) lower expression of miR-375 in HNSC tissues was shown in TCGA database when compared with normal tissues. (B) qRT-PCR was used to measure the expression of miR-375 in human LSCC cell lines. * Compared with NPEC, p<0.05. HNSC, Head and Neck Squamous Cell Carcinoma; LSCC, Laryngeal Squamous Cell Carcinoma.

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miR-375-mimic or miR-375-inhibitor was transfected into LSC-1 and TU177 cells. The transfection efficiency was measured, and the results displayed that miR-375 expression was clearly increased in miR-375-mimic group (vs. mimic-NC group) and decreased in miR-375-inhibitor group (vs. inhibitor-NC group) (Fig. 2A, p<0.05).

Figure 2.

miR-375 overexpression in LSCC cells resulted in suppressed migration and elevated apoptosis. miR-375-mimic, miR-375-inhibitor or their controls were transfected into human LSCC cell lines LSC-1 and TU177. (A) the expression of miR-375 was tested by qRT-PCR. (B) cell viability in each group was measured by CCK-8 assay. (CD) cell invasive and migration in each group were evaluated by scratch and transwell tests. (E) cell apoptosis in each group was measured by flow cytometry. (FG) the protein expression of MMP2 and MMP9 in each group was assessed by western blot. * Compared with mimic-NC group or inhibitor-NC group, p<0.05. LSCC, Laryngeal Squamous Cell Carcinoma.

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The viability of LSCC cells in each group was measured by CCK-8 assay. The results showed that cell viability was signally inhibited in miR-375-mimic group compared with mimic-NC group, but markedly enhanced in miR-375-inhibitor group compared with inhibitor -NC group (Fig. 2B, p<0.05). Results from scratch and transwell tests revealed that the invasive and migration abilities of cells were clearly decreased in miR-375-mimic group compared with mimic-NC group, but clearly increased in miR-375-inhibitor group compared with inhibitor-NC group (Fig. 2C‒D, p<0.05). Moreover, flow cytometry results displayed that the apoptosis rate in miR-375-mimic group was clearly increased compared with mimic-NC group, while decreased cell apoptosis was found in miR-375-inhibitor group compared with inhibitor-NC group (Fig. 2E, p<0.05).

Extracellular matrix degradation-related proteins, MMP-2 and MMP-9 were highly expressed in laryngeal carcinoma, overexpression of which was related to tumor migration and invasion.18 The expression of MMP-2 and MMP-9 in lSC-1 and TU177 cells was further measured by western blot. The results showed that the protein expression of MMP-2 and MMP-9 was obviously dropped in miR-375-mimic group compared with mimic-NC group, but elevated in miR-375-inhibitor group compared with inhibitor-NC group (Fig. 2F, p<0.05). Above results indicated that upregulated expression of miR-375 inhibited the malignancy of LSCC cells and promoted cell apoptosis, while downregulation of miR-375 expression resulted in reverse patterns.

Differentially expressed genes in LSCC were analyzed by GEO database GSE84957 dataset and TCGA database, and it was found that CST1 was highly expressed in HNSC (Fig. 3A‒C). However, there was no study exploring the mechanism of CST1 regulation in LSCC. As reflected by qRT-PCR and western blot, the expression of CST1 in LSC-1 and TU177 cells was markedly higher than in NPEC cells (Fig. 3D‒E, p<0.05).

Figure 3.

miR-375 targeted CST1. (AC) GEO and TCGA database analysis showed that CST1 was highly expressed in HNSC. (D) the expression level of CST1 was examined by qRT-PCR and western blot. (E) Jefferson database showed that CST1 had direct binding sites with miR-375. GH, qRT-PCR and western blot were used to detect the expression level of CST1. * Compared with NPEC or mimic-NC or inhibitor-NC group, p<0.05. HNSC, Head and Neck Squamous Cell Carcinoma.

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Jefferson database showed that CST1 had a direct binding site with miR-375 (Fig. 3F). The binding relationship between miR-375 and CST1 was evaluated by dual luciferase reporter gene assay. The results showed that the luciferase activity of cells transfected with WT-CST1 was clearly decreased in miR-375-mimic group compared with mimic-NC group (Fig. 3G, p<0.05). The luciferase activity of cells transfected with MUT-CST1 showed significant difference neither in miR-375-mimic group nor in mimic-NC group. qRT-PCR and western blot were used to assess the expression level of CST1 in cells after miR-375-mimic or miR-375-inhibitor was transfected. The experiment result indicated that the expression of CST1 in miR-375-mimic group was signally decreased compared with mimic-NC group, while that in miR-375-inhibitor group was dramatically increased compared with inhibitor-NC group (Fig. 3H‒I, p<0.05). From these results, it was clear that miR-375 targeted CST1 in LSCC cells.

LSC-1 and TU177 cells were introduced with miR-375-mimic+oe-CST1 and miR-375-mimic+oe-NC, and the impact of miR-375-mediated CST1 inhibition on the viability of LSCC cells was further explored. First, results from qRT-PCR and western blot showed that the expression of CST1 in miR-375-mimic+oe-CST1 group was memorably increased compared with miR-375-mimic+oe-NC group (Fig. 4A‒B, p<0.05).

Figure 4.

miR-375 suppressed CST1 to inhibit LSCC cell proliferation. (AB) the expression level of CST1 was measured by qRT-PCR and western blot. (C) cell viability was tested by CCK-8 assay. (DE) cell invasive and migration were measured by scratch test and transwell assay. (F) the apoptosis of cell in each group was evaluated by flow cytometry. (GH) the protein expression of MMP2 and MMP9 in each group was assessed by western blot, * Compared with miR-375-mimic+oe-NC group, p<0.05. LSCC, Laryngeal Squamous Cell Carcinoma.

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Moreover, CCK-8, scratch, and transwell tests revealed that the malignancy of LSCC cells in miR-375-mimic+oe-CST1 group was dramatically enhanced compared with miR-375-mimic+oe-NC group (Fig. 4C‒E, p<0.05). To the contrary, LSCC cell apoptosis was significantly decreased in miR-375-mimic+oe-CST1 group compared with miR-375-mimic+oe-NC group (Fig. 4F, p<0.05). Western blot results indicated that the expression of MMP-2 and MMP-9 in miR-375-mimic+oe-CST1 group was clearly increased compared with miR-375-mimic+oe-NC group (Fig. 4G‒H, p<0.05). Taken together, miR-375 inhibited the growth and invasiveness and facilitated the apoptosis of LSCC cells by targeting CST1.