Thirty pairs of NPC and para-cancer tissue samples were obtained from NPC patients in the Affiliated Hospital of North Sichuan Medical College. All patients signed informed consent of specimen retention before surgery, and all patients conforming to surgical conditions routinely collected pathological tissue samples. Professional doctors ensure that the NPC sample and its adjacent normal tissue are removed as accurately as possible during the procedure. Detailed pathological analysis was performed on the collected tissues, such as immunohistochemical staining to detect the expression of specific tumor markers in neighboring tissues, and cell types in neighboring tissues were further confirmed through experiments such as cell morphology observation. All specimens were confirmed by 2 or more pathologists in our hospital as NPC tissues and adjacent normal tissues, and no tumor cells and atypical hyperplasia cells were found in normal tissues. This study was approved by the Medical Ethics Committee of Affiliated Hospital of North Sichuan Medical College (No. 2022ER287-1).

Human nasal epithelial cell line HNEpC (nº BFN60806616) and human NPC cell line C666-1 (nº BFN608006727) and CNE-2 (nº BFN60700251) were purchased from BLUEFBIO (Shanghai, China). The three cells were cultured in DMEM high glucose medium (Hyclone, USA) containing 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin-streptomycin solution (Hyclone, USA) in a 5% CO2 incubator at 37°C.

The NC mimic (nº miR1N0000001-1-5) and the hsa-miR-186-5p mimic (nº miR10000456-1-5) were purchased from Ribobio (China). Interference sequences of si-RNA target Zinc Finger E-Box Binding Homeobox 1 (ZEB1) were also synthesized by Ribobio (China). Then, NC mimic, miR-186 mimic, si-NC and si-ZEB1 were used to transfect C666-1 and CNE-2 cells by Lipofectamine 2000 (nº 11668019; Invitrogen, USA). First, mimic, siRNA and lipofectamine 2000 reagents were diluted in Opti-MEM, incubated for 5min, and the diluted reagents and plasmids were mixed and placed at room temperature for 20min. When the cell density reached 70%, the transfection mixture was added and cultured at 37°C with 5% CO2. After transfection for 6h, fresh medium was exchanged, and the follow-up study was conducted 48h later.

Cell viability was analyzed by Cell Counting kit-8 (CCK8). After different transfections, CCK8 solution (nº C0037; Beyotime, China) was added and incubated with cells at 37°C for 1h. Absorbance was measured at 570nm using an enzyme marker, and the cell survival rate of each treatment group was calculated.

Cell invasion was carried out through transwell compartments coated with an artificial substrate gel Matrigel. The complete culture medium containing FBS was added to the lower chamber of the transwell, and 5×104 single-cell suspension suspended with the serum-free medium was added to the upper chamber. The cells were cultured in a cell incubator at 37°C for 24h, and the number of invaded cells was observed after fixing with 4% paraformaldehyde solution (nº P0099; Beyotime, China) for 20min, and staining with crystal violet staining solution (nº C0121; Beyotime, China) for 5–10min.

Cell migration was performed by a scratch assay. About 5×105 cells were evenly spread in the six-well plate, and two parallel lines were drawn after sticking to the wall and growing. The cells were continued to be cultured in a 37°C, 5% CO2 incubator for 24h. Observed and photographed under a microscope, the migration distance was calculated.

First, the Wild-Type (WT) and Mutant (MUT) sequences of ZEB-1 were constructed on luciferase vectors pGL3-basic. Luciferase vectors and miR-186 mimic or NC mimic were transfected into 293T cells according to experimental groups using transfection reagents Lipofectamine 2000. The dual luciferase reporter gene assay kit (nº RG027; Beyotime, China) was used to detect fluorescence intensity and analyze luciferase activity.

The total protein in NPC tissue or cells was obtained by RIPA lysis buffer (nº P0013B, Beyotime, China), the protein content was quantified by BCA protein assay kit (nº P0012S, Beyotime, China), and the total protein was separated by SDS-PAGE after protein denaturation. Then, the isolated proteins were transferred to the PVDF membrane, sealed with 5% skim milk, and incubated at 4°C overnight with the primary antibody β-actin (nº sc-8432; Santa Cruz Biotechnology, USA), ZEB1 (nº sc-515797; Santa Cruz Biotechnology, USA), E-cadherin (nº sc-8426; Santa Cruz Biotechnology, USA), N-cadherin (nº sc-8424; Santa Cruz Biotechnology, USA) or vimentin (no. sc-6260; Santa Cruz Biotechnology, USA). Then, the secondary antibody goat anti-mouse IgG-HRP (no. sc-2005; Santa Cruz Biotechnology, USA) was incubated at room temperature for 2h. ECL western blotting substrate (no. PE0010; Solarbio, China) and gel imaging analyzer (Bia-Rad, USA) was used to visualize the images. The relative protein expression was calculated using β-actin as an internal reference.

The gene expressions of miR-186 and ZEB1 in NPC tissue and cells were analyzed by RT-PCR. Total RNA was extracted by TRIzol reagent (nº 15596026; Invitrogen, USA). For the examination of miR-186, cDNA was obtained by miR-Quant TaqMan microRNA cDNA synthesis kit (no. MT0006; Biorab, China), and PCR reaction was performed by miR-Quant TaqMan microRNA qPCR kit (nº MTT02318; Biorab, Beijing, China). For the detection of ZEB1, cDNA was obtained by HiScript 1st strand cDNA synthesis kit (nº R111-01; Vazyme, Nanjing, China), and PCR reaction was performed by HiScript II one step RT-PCR kit (nº P611-01; Vazyme, Nanjing, China). The miR-186 and U6 sequence was provided by the kit, the ZEB1 sequence was F: 5′-ATGCAGCTGACTGTGAAGGT-3′, R: 5′-GCCCTTCCTTTCCTGTGTCA-3′, and the β-actin sequence was F: 5′-GAAGATCAAGATCATTGCTCC-3′, R: 5′-TACTCCTGCTTGCTGATCCA-3′. Using U6 as the internal reference of miR-186 and β-actin as the internal reference of ZEB1, the relative gene expression was calculated by the 2−△△CT method.

All data in this study were analyzed by SPSS software and expressed as mean±standard deviation. The difference analysis between the two groups was conducted by the Student t-test, and the difference analysis among the multiple groups was conducted by one-way analysis of variance; p<0.05 was considered statistically significant.

To analyze the effects of miR-186 on NPC, the expression of miR-186 was analyzed by RT-PCR, and the results showed that the expression of miR-186 in cancer tissues was significantly reduced (p<0.01, Fig. 1A). Similarly, miR-186 expression was significantly decreased in NPC cell lines C666-1 and CNE-2 compared to human nasal epithelial cell line HNEpC (p<0.01, Fig. 1B).

Figure 1.

Expression of miR-186 in NPC. The expression of miR-186 in NPC tissues (A) and cells (B) was detected by RT-PCR. *p<0 .05, **p<0.01, ***p<0.001.

(0.14MB).

Due to the low expression of miR-186 in C666-1 and CNE-2 cells, the miR-186 mimic was used to transfect C666-1 and CNE-2 cells, and RT-PCR was used to verify the expression of miR-186 after transfection. The results showed that the miR-186 mimic significantly increased the expression of miR-186 in two NPC cells compared with the NC mimic (p<0.01, Fig. 2A). The effects of miR-186 mimic on cell proliferation and metastasis were further analyzed, CCK8 experiment showed that the cell viability in miR-186 mimic transfected cells was significantly reduced compared with NC mimic in both two NPC cells (p<0.01, Fig. 2B). In addition, the invasion number (Fig. 2C and E) and migration distance (Fig. 2D and F) of C666-1 and CNE-2 cells in the miR-186 mimic group were significantly reduced compared with the NC mimic group (p<0.001), suggesting that miR-186 mimic inhibited NPC metastasis. The expression of EMT-related proteins was further analyzed, and results showed that miR-186 mimic significantly promoted the protein expression of E-cadherin, reduced the protein expression of N-cadherin and vimentin, and inhibited the EMT of NPC cells compared with the NC group (p<0.01, Fig. 2G and H).

Figure 2.

Effects of miR-186 on proliferation and metastasis of NPC cells. (A) The expression of miR-186 in cells transfected with miR-186 mimic was detected by RT-PCR. (B) Cell viability was detected by CCK8. (C) The number of cells invaded. (D) Cell migration distance. (E) Representative images of cell invasion measured by transwell. (F) Representative images of cell migration detected by scratch assay. (G) The protein expressions of E-cadherin, N-cadherin and vimentin were detected by WB. Full-length blots/gels are presented in Supplementary Fig. 1. (H) Gray scale analysis of protein bands. *p<0.05, **p<0.01, ***p<0.001, vs. NC mimic.

(1.12MB).

To further clarify the mechanism of miR-186 regulating NPC metastasis, the potential downstream genes of miR-186 were predicted through the RNAInter bioinformatics platform. The results showed that miR-186 and ZEB1 had a potential binding site (Fig. 3A). Dual luciferase assay showed that the fluorescence intensity in the miR-186 mimic+ZEB1-WT group was significantly reduced compared with the NC mimic+ZEB1-WT group (p<0.01, Fig. 3B), confirming the presence of a binding site between miR-186 and ZEB1. Then, the expression of ZEB1 in miR-186 mimic transfected C666-1 and CNE-2 cells was analyzed, and results showed that compared with the NC mimic group, the protein, and gene expressions of ZEB1 in the miR-186 mimic group were both significantly reduced (p<0.01, Fig. 3C and D), suggesting that miR-186 can target ZEB1 and negatively regulate its expression. In order to further clarify whether ZEB1 is involved in the regulation of NPC by miR-186, the protein and gene expressions of ZEB1 in NPC tissues and cells were also analyzed, and the results showed that the protein and gene expressions of ZEB1 in NPC tissues were significantly increased (p<0.01, Fig. 3E and F). Similarly, ZEB1 protein and gene expression in C666-1 and CNE-2 cells were also significantly increased compared with HNEpC cells (p<0.01, Fig. 3G and H).

Figure 3.

MiR-186 negatively regulated the expression of ZEB1 in NPC. (A) The predicted potential binding site between miR-186 and ZEB1. (B) Double luciferase reporter assay was used to detect luciferase activity. (C) The protein expression of ZEB1 in cells transfected with miR-186 mimic was detected by WB. Full-length blots/gels are presented in Supplementary Fig. 2. (D) The gene expression of ZEB1 in cells transfected with miR-186 mimic was detected by RT-PCR. (E) The protein expression of ZEB1 in NPC tissues was detected by WB. Full-length blots/gels are presented in Supplementary Fig. 3. (F) The gene expression of ZEB1 in NPC tissues was detected by RT-PCR. (G) The protein expression of ZEB1 in NPC cells was detected by WB. Full-length blots/gels are presented in Supplementary Fig. 4. (H) The gene expression of ZEB1 in NPC cells was detected by RT-PCR. *p<0.05, **p<0.01, ***p<0.001.

(0.44MB).

The si-RNA targeting ZEB1 was used to intervene the expression of ZEB1 in NPC cells, and the intervention effect was verified by WB and RT-PCR. The results showed that compared with the si-NC group, the protein and gene expressions of ZEB1 in the si-ZEB1 group were both significantly decreased (p<0.001, Fig. 4A and B). And compared with the si-NC group, the expression of miR-186 in the si-ZEB1 group was significantly increased, suggesting that ZEB1 could reverse regulate the expression of miR-186 in NPC (p<0.05, Fig. 4C). The role of the miR-186/ZEB1 regulatory network in NPC cell proliferation and metastasis was further analyzed. CCK8 results showed that miR-186 mimic and si-ZEB1 could significantly inhibit NPC cell viability (p<0.001, Fig. 4D). Transwell and scratch tests showed that miR-186 mimic and si-ZEB1 could significantly inhibit the number of invaded cells and the distance of migration (p<0.001, Fig. 4E‒H). Co-transfected with miR-186 mimic and si-ZEB1 showed more significant inhibitory effects on NPC cells than those of si-NC+miR-186 or si-ZEB1+NC mimic group (p<0.05, Fig. 4D‒H). In addition, the expressions of EMT-related proteins were analyzed, and results showed that miR-186 mimic and si-ZEB1 could significantly increase the protein expression of E-cadherin and reduce the protein expression of N-cadherin and vimentin in C666-1 and CNE-2 cells (p<0.001, Fig. 5). The regulation effect in the si-ZEB1+miR-186 mimic group on EMT protein expression was more obvious than that in the si-NC+miR-186 or si-ZEB1+NC mimic group (p<0.001, Fig. 5).

Figure 4.

Effects of miR-186/ZEB1 regulatory network on proliferation and metastasis of NPC cells. (A) The protein expression of ZEB1 in NPC cells transfected with si-ZEB1 was detected by WB. Full-length blots/gels are presented in Supplementary Fig. 5. (B) The gene expression of ZEB1 in NPC cells transfected with si-ZEB1 was detected by RT-PCR. (C) The gene expression of miR-186 in NPC cells transfected with si-ZEB1 was detected by RT-PCR. (D) Cell viability was detected by CCK8. (E) The number of cells invaded. (F) Cell migration distance. (G) Representative images of cell invasion measured by transwell. (H) Representative images of cell migration detected by scratch assay. *p<0.05, **p<0.01, ***p<0.001.

(1.06MB).

Figure 5.

Effects of miR-186/ZEB1 regulatory network on EMT-related protein expression in NPC cells. (A) The protein expressions of E-cadherin, N-cadherin and vimentin were detected by WB. Full-length blots/gels are presented in Supplementary.

(0.34MB).