Cancer tissue (LSCC group, n = 20) and paracancerous tissue (Normal group, n = 20) were extracted from LSCC1 patients2 admitted to our hospital.3 Besides, all patients offered the written informed consent4 forms, and the Ethics Committee of our hospital gave its approval to this study.

Human Bronchial Epithelial cells (16 HBE) and Human Epithelial cancer cells (HEp-2) were offered by American Type Culture Collection (USA). Next, DMEM medium (Gibco, USA) with 10% Fetal Bovine Serum (FBS) (Hyclone, USA) was adopted for 16 HBE cell culture, while 1640 medium (Gibco, USA) with 10% FBS (Hyclone, USA) for HEp-2 cell culture. All culture media were added with 1% 100 U/mL penicillin ‒ 0.1 mg/mL streptomycin (Beyotime, China) and cultured in a sterile incubator at 37 °C with 5% CO2.

HEp-2 cells were cultured to the logarithmic phase, followed by digestion and passage. Next, HEp-2 cells in the logarithmic phase were gathered, diluted to 2 × 106 cells/mL, and inoculated into 6-well plates for culture. Subsequent to the fusion degree of cells reached 80%–90%, the culture was discontinued, and the transfection was performed. Following the operating instructions of Lipofectamine™ 2000, miR-150-5p mimics, NC mimics, miR-150-5p inhibitor, NC inhibitor (GenePharma, China), vector, and PIN1 over-expression plasmid (ThermoFisher, USA) were transfected into HEp-2 cells using Lipo2000, respectively. Subsequently, the plates were developed for 24 h in a CO2 incubator at 37 °C. Finally, a light microscopy was applied to measure transfection efficiency.

Total RNA was obtained from the collected tissues and cells using Total RNA extraction kit (Takara, USA), and the miRNAs were extracted with miRNA extraction and isolation kit (Tiangen, China). Both extracted total RNA and miRNAs were stored at −80 °C. By reverse transcribing miRNA and mRAN with the directions on the reverse transcription PCR kit (Takara, USA) and the miRNA cDNA First Strand Synthesis Kit (Tiangen, China), the complementary DNA was synthesized. Checks were made on the complementary DNA that was created in terms of purity and concentration. Next, the Polymerase Chain Reaction (PCR) of mRAN was carried out based on the instruction of real-time PCR (Takara, USA). The reaction procedure went as follows: 95 °C for 30 s; 95 °C for 10 s; 57 °C for 20 s; 72 °C for 20 s, for 40 cycles. The PCR of miRNA was performed according to the miRNA fluorescence quantitative detection kit (Tiangen, China), and the reaction program went like this: 95 °C for 15 min; 94 °C for 20 s; 60 °C for 34 s, for 40 cycles. Taking GAPDH as an internal control, the 2−ΔΔCt method was utilized to perform data analysis. The sequences of primers adopted were shown in Table 1.

The transfected HEp-2 cells were put into a 96-well plate with 2000 cells/well, and 100 μL fresh complete medium was replaced at an interval of 24 h. Subsequently, 10 μL Cholecystokinin Octapeptide (CCK-8) solution (Abcam, USA) and 90 μL fresh complete culture were introduced to each tested wells. Next, the 96-well plate was positioned in an incubator and allowed to culture for a full hour. Lastly, molecular devices were employed to monitor the absorbance of 450 nm wavelength.

The transfected HEp-2 cells seeded in a 6-well plate were subjected to 2–3 weeks of static culture at 37 °C in an incubator containing 5% CO2. Upon macroscopic colony formation, the cell culture was discontinued. To count the number of colonies, the supernatant was aspirated, cells were fixed with methanol at ambient temperature for 30 min, and the staining was performed for 30 min with 0.5% crystalviolet (Sigma-Aldrich, USA). Later, the number of cell clones larger than 50 were counted under a microscope to determine the colony formation rate. The formula was displayed as follows: colony formation rate (%) = (number of colonies/number of the seeded cells) ×100%.

Matrigel (BD, Franklin Lakes, USA) was removed from −20 °C, left at 4 °C overnight to become liquid, and diluted in a ratio of Matrigel: 4 °C serum-free mediu = 6:1. Next, 100 μL diluted matrigel was supplemented to the upper transwell chamber at 37 °C for 3–5 h to turn it into solid. Then, the lower chamber received 500 μL of 10% FBS medium, whereas the upper chamber received 100 μL of cell suspension. The migrated cells were fixed with methanol for 30 min following a 24 h development. Upon removal of non-migrated cells, the staining was employed with 0.5% crystal violet for 30 min. Finally, cells were counted under a microscope.

Transfection was conducted once HEp-2 cells had fused to an extent of 80%–90%. Shortly speaking, miR-212-3pmimics were co-transfected into HEp-2 cells along with the constructed Mutant (PIN1-MUT) and Wild-Type (PIN1-WT) dual luciferase reporter vectors of PIN1 (ThermoFisher, USA). After transfection, the HEp-2 cell culture was performed for 48 h then collected for 20 min of lysis at ambient temperature. Next, the centrifugation was carried out and the supernatant was collected. Direct addition of the luciferase substrate was then made, followed by a measurement of the amount of luciferase activity by luminescence instrument. Later, relative firefly luciferase activity was calculated with Renilla luciferase activity serving as an internal control.

The tissues and cells of each group were lysed 30 min with RIPA lysis buffer, and then crushed by sonication in ice bath. Next, the protein concentration was measured once the proteins had been collected. The proteins were transferred to PVDF membrane after Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and sealed at ambient temperature for 1 h. Later, primary antibodies (Proteintech, USA): E-cadherin (Cat nº 20874-1-AP), N-cadherin (Cat nº 22018-1-AP), MMP-9(Cat nº 10375-2-AP), Vimentin (Cat nº 10366-1-AP), Matrix Metalloproteinase (MMP)-2 (Cat nº 10373-2-AP), PIN1 (Cat nº 10495-1-AP) were added respectively, then the incubation was conducted overnight at 4 °C. Washed twice with PBS, the membrane was supplemented with the diluted enzyme-labeled second antibody for 1 h incubation at indoor temperature. The protein level was determined with β-actin being an internal control.

Using the SPSS 26.0 program, statistical analysis was performed on the experimental data. When comparing two groups, the t-test was adopted, and when comparing multiple groups, one-way analysis of variance. The results were provided as the Mean ± Standard Deviation (Mean ± SD), and p < 0.05 represented a significant difference.

To clarify the role of miR-150-5p in LSCC, qRT-PCR was applied to observe its level in LSCC and normal tissues. We discovered that this RNA expression level was significantly reduced in LSCC tissue (Fig. 1A). Also, there was the same trend of expression at the cellular level. The expression of this RNA notably descended in HEp-2 cells relative to normal 16 HBE cells (Fig. 1B). In a word, reduced expression of this RNA may be related to the progression of LSCC.

Figure 1.

Down-regulated expression of miR-150-5p in laryngeal squamous cell carcinoma. (A) The expression of miR-150-5p in laryngeal squamous cell carcinoma and paracancerous tissues was detected by qRT-PCR; (B) The expression of miR-150-5p in normal Human Bronchial Epithelial cells (16 HBE) and Human Epithelial cancer cells (HEp-2) derived from a larynx carcinoma was tested by qRT-PCR; ***p < 0.001.

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Subsequently, for further observing the function of miR-150-5p in the development of LSCC, its expression level was under expressed (miR-150-5p inhibitor) or overexpressed (miR-150-5p mimics) in HEp-2 cells. The outcomes of qRT-PCR indicated that miR-150-5p mimics could remarkably enhance the expression level of this RNA in HEp-2 cells (p < 0.01), however, inhibitor of this RNA significantly declined it (p < 0.01) (Fig. 2A). Therefore, a successful transfection was obtained. Later, the cell proliferation of each group was observed with the help of cell colony formation assay and CCK-8 assay. The outcomes suggested that the miR-150-5p mimics group showed a markedly decreased cell proliferation rate (p < 0.01); however, the rate visibly climbed after inhibition of this RNA (p < 0.01) (Fig. 2B‒C). Furthermore, the invasion of cells in each group was observed by transwell assay. The findings exhibited that the invasion of cells was observably reduced after the overexpression of this RNA (p < 0.01) but enhanced after its inhibition (p < 0.01) (Fig. 2D). In brief, this RNA can prevent HEp-2 cells from proliferating and invasively spreading.

Figure 2.

MiR-150-5p inhibits the proliferation and viability of HEp-2 cells. (A) qRT-PCR to detect the expression level of miR-150-5p in each group of HEp-2 cells; (B) CCK-8 method to check the cell proliferation rate of cells; (C) Cell colony formation assay to observe the cell proliferation of each group; (D) Transwell assay to test the invasion ability of HEp-2 cells in each group. ***p < 0.001.

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Western blot analysis was performed to evaluate the expression of MMP and proteins associated with the Epithelial-Mesenchymal Transition (EMT) for further investigating the mechanism by which miR-150-5p prevents the invasion of HEp-2 cells. Shortly speaking, in the miR-150-5p mimics group, protein expression levels of MMP-2, MMP-9, Vimentin and N-cadherin in cells were notably down-regulated (p < 0.01) while the E- cadherin expression was markedly raised (p < 0.01). Nevertheless, miR-150-5p inhibitor group displayed a significant increase in the expression levels of MMP-2, MMP-9, Vimentin and N-cadherin (p < 0.01) while a notable reduction in the E-cadherin expression (p < 0.01) (Fig. 3A‒B). All in all, this RNA could not only suppress critical protein expression of MMPs but also inhibit the invasion of HEp-2 by inhibiting the EMT process.

Figure 3.

Effect of miR-150-5p on the expression of Matrix Metalloproteinase (MMP) and Epithelial-Mesenchymal Transition (EMT)-related proteins in Hep-2 cells. (A‒B) Western blot to assess the expression of Matrix Metalloproteinase (MMP)-related proteins MMP-2 and MMP-9 (A) and Epithelial-Mesenchymal Transition (EMT)-related proteins (N-cadherin, Vimentin and E- cadherin) (B) in Hep-2 cells; **p < 0.01; ***p < 0.001.

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TargetScan (http://www.targetscan.org/vert_72/), an online prediction network for miRNA target genes, was employed to predict target genes of miR-150-5p in order to reveal its mechanism. The prediction outcomes suggested an interaction between PIN1 and this RNA (Fig. 4A). Next, dual-luciferase reporter assays further uncovered that overexpression of this RNA noticeably inhibited luciferase activity of PIN1-WT (p < 0.01) but didn’t affect luciferase activity of PIN1-MUT (Fig. 4B). Additionally, qRT-PCR results exhibited that the overexpression of this RNA obviously lowered PIN1 expression, whereas its knockdown greatly increased PIN1 expression in cells (Fig. 4C). Moreover, the observation results of clinical tissues displayed that, relative to the Normal group, both protein and mRNA expression of PIN1 were significantly elevated in the LSCC group (Fig. 4D‒E). Similarly, PIN1 expression was notably increased in HEp-2 cells relative to that in 16 HBE cells (Fig. 4F). Furthermore, the Pearson correlation coefficient analysis disclosed the negative correlation between the expression of PIN1 and this RNA (Fig. 4G). Hence, this RNA could targetedly suppress PIN1 expression in LSCC.

Figure 4.

MiR-150-5p targeted inhibits PIN1 expression. (A) Potential target gene sequences of miR-150-5p were predicted by the online prediction network TargetScan; (B) Dual-luciferase reporter assay to verify the targeted relationship between miR-150-5p and PIN1; (C) QRT-PCR to detect PIN1 expression in cells with different miR-150-5p expression levels; (D) Expression of PIN1 detected by qRT-PCR in tissues from LSCC and Normal groups; (E) Western blot to meaure PIN1 protein expression in LSCC and Normal groups; (F) QRT-PCR for testing PIN1 expression in 16 HBE and Hep-2 cells; (G) Pearson correlation analysis for relation between miR-150-5p and PIN1 expression in laryngeal squamous cell carcinoma tissues (n = 8); **p < 0.01, ***p < 0.001.

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To further confirm the role of the association between miR-150-5p and PIN1 in HEp-2 cells, this RNA and PIN1 was simultaneously highly expressed in cells. According to the outcomes of transwell assay, colony formation assay and CCK-8 assay, miR-150-5p (miR-150-5p + NC vector) overexpression effectively weakened the ability of HEp-2 cells to proliferate and invasively spread; while on this basis, PIN1 (miR-150-5p + PIN1) overexpression could relieve the inhibition of this RNA on the proliferation and invasion of HEp-2 cells (Fig. 5A–C). Additionally, western blot results also presented that PIN1 (miR-150-5p + PIN1) overexpression could reverse the impact of this RNA on the MMP and EMT-related protein expression levels in HEp-2 cells (Fig. 5D‒E). It makes sense to assume that the inhibition of laryngeal cancer cell growth and invasion by miR-150-5p is likely reversed by PIN1 overexpression.

Figure 5.

Overexpression of PIN1 promotes proliferation and invasion of laryngeal cancer cells. (A) CCK-8 assay for detecting the cell proliferation rate of HEp-2 cells in each group; (B) Colony formation assay for observing the cell proliferation of Hep-2 cells in each group; (C) Transwell assay for checking the cell invasion of HEp-2 cells; (D‒E) Western blot for assessing the expression of Matrix Metalloproteinase (MMP)-related proteins MMP-2 and MMP-9 (D) and Epithelial-Mesenchymal Transition (EMT)-related proteins N-cadherin, Vimentin and E- cadherin (E) in HEp-2 cells. **p < 0.01, ***p < 0.001.

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