DDP, Radio Immunoprecipitation Assay (RIPA) lysis buffer, CCK-8 (Cell Counting Kit-8) kit were procured from Beyotime Biotechnology Co., Ltd. RPMI (Roswell Park Memorial Institute) 1640 medium was acquired from Wuhan Sewell Biotechnology Co., Ltd. miR-199a-5p mimics, inhibitors, and negative control reagents were obtained from Huzhou Hippo Biotechnology Co., Ltd (Table 1). The Lipofectamine™ 2000 Kit and Trizol Reagent were purchased from Invitrogen, Inc. miR-199a-5p primers and U6 were designed and synthesized by Sangon Bioengineering (Shanghai) Co., Ltd (Table 2). The MonAmp miRNA Universal Super Specificity qPCR Mix Reverse Transcription Kit and MonScript™ miRNA First Strand cDNA Synthesis Kit (Tailing Reaction) were acquired from Mona (Suzhou) Biotechnology Co., Ltd. Matrigel was sourced from BD in the United States. Transwell chamber was obtained from Corning, Inc., USA. The monoclonal antibody SLC1A5, E-cadherin, N-cadherin, vimentin, GAPDH, and goat anti-rabbit IgG Horseradish Peroxidase (HRP) antibodies were purchased from Jiangsu Affinity Biological Research Center Co., Ltd. The Annexin V-FITC/PI double-stained apoptosis detection kit was acquired from Bestbio.

Human HNE1 and HNE1/DDP cells were acquired from School of Pharmacy, Bengbu Medical University (Anhui, China), stored at −80 °C, thawed, and cultured with RPMI-1640 containing 10% FBS and penicillin/streptomycin in a CO2 incubator. Additionally, the culture medium of HNE1/DDP cells was supplemented with cisplatin at a concentration of 1 μg/mL.9

Lipofectamine™ 2000 (Invitrogen, USA) was used as directed for transfection with miR-199a-5p mimic, inhibitor, or blank control constructs. Experiments were performed after 48 h.

Proliferation was examined using CCK-8 assays. Cells plated in 96-well plates (5000 well) were cultured for 1–3 days, after which CCK-8 reagent (10 μL/well) was introduced for 2 h at 37 °C followed by the reading of absorbances (450 nm) with a microplate reader (Infinite F50; Tecan).

Colony formation assays were conducted by seeding cells in 6-well plates (1000 well) and culturing them for 14-days, after which they were fixed (4% paraformaldehyde, 20 min), stained for 20 min using crystal violet, rinsed using ddH2O, and imaged via microscopy. ImageJ (NIH, MD, USA) was used for image analysis.

For wound healing assays, cells were cultured in 6-well plates (2 × 105/well) until 90% confluent. Scratches were made in the monolayer with a 200 μL pipette tip. Imaging was then performed via microscopy after 0 and 24 h, and the resultant images were used to compute the rate of cellular migration.

Migration was measured using Transwell assay. 200 μL serum-free cell suspension was added to the upper part of the 24-well plate cell chamber (1 × 104/well), and 600 μL serum-containing medium was added to the lower chamber. After 24 h of culture, the suspension in upper chamber was discarded, while the cells were fixed with 4% paraformaldehyde, then stained with crystal violet, imaged and counted by microscope, and three randomly selected areas were counted.

Matrigel was added to the Transwell chambers before cell culture, and the other steps were the same as Transwell detection migration.

The total protein was obtained by lysing the cells with RIPA reagent, and its concentration was determined by the BCA method. The protein sample (30 μg) was subjected to SDS-PAGE and subsequently transferred to a PVDF membrane. Subsequently, the samples were incubated in TBST containing 5% skim milk for two hours. The primary antibody was then applied overnight at 4 °C, followed by a one-hour incubation at room temperature with the secondary antibody. The samples were then exposed to an ECL substrate, imaged, and documented.

Trizol reagent is employed for the extraction of total RNA from cells, with subsequent assessment of its purity and concentration conducted through the utilization of a micronucleic acid assay. The cDNA was synthesized in accordance with the instructions provided in the reverse transcription kit. The cDNA was then employed as a template to construct the reaction system for amplification, following the instructions set out in the fluorescence quantification kit. The expression was evaluated using the 2−ΔΔCt method with U6 as the internal reference standard.

The cells should be collected, subjected to centrifugation and then resuspended in PBS. Subsequently, the cells were stained using Annexin V-FITC and PI in accordance with the instructions provided. The extent of apoptosis was evaluated through flow cytometry following incubation.

Gating strategy: 1) Debris exclusion: Cells were gated on FSC-A vs. SSC-A to exclude debris (Gate P1). 2) Single-cell selection: FSC-H vs. FSC-A was used to eliminate doublets (Gate P2). 3) Viable cell gate: PI-negative cells were selected from Gate P2 to exclude dead cells (Gate P3). 4) Apoptosis analysis: Annexin V-FITC vs. PI fluorescence was analyzed on Gate P3. Quadrant gates were defined using unstained and single-stained controls: Annexin V-/PI-: Viable cells; Annexin V+/PI-: Early apoptotic cells; Annexin V+/PI+: Late apoptotic/necrotic cells; Annexin V-/PI+: Necrotic cells (if present).

Fluorescence compensation was performed with single-stained controls. Data analysis used FlowJo v10.8.

Nude mice, 5-weeks-old, were subcutaneously implanted in the right flank with 5 × 106 stably transfected cells. Tumor growth was monitored weekly and harvested 5-weeks after implantation, weighed, and used for H&E staining and immunohistochemical staining.

Luciferase reporter plasmids were transfected into appropriate cells, after which a dual-luciferase reporter assay kit (Beyotime, RG027) was utilized for determination of luciferase activity.

With “Naspharyngeal Carcinoma” as the keyword filtering in the GEO database (https://www.ncbi.nlm.nih.gov/geo/) to the relevant important data set of micrornas GSE32960 and GSE36682 data sets. The gene expression profiles of the above datasets were obtained using the “GEOquery” package and the differential expression maps were drawn using the “ggplot2” package. Wilcoxon test was used for statistical analysis. The rest of the database is used with ENCORI (https://rnasysu.com/encori/index.php), MiRWalk (http://mirwalk.umm.uni-heidelberg.de/) and miRmap (https://mirmap.ezlab.org/).

Results are reported as means ± SE and were compared in SPSS 26.0 (IBM, USA) using t-tests and ANOVAs; p < 0.05 was significant.

In GSE32960 and GSE36682 databases, miR-199a-5p was identified to be differentially expressed in NPC and downregulated in tumor tissues (Fig. 1A). Subsequently, we found that there was a certain correlation between it and prognosis, and patients with high expression had a significantly better prognosis (Fig. 1B). Therefore, we further speculated whether miR-199a-5p expression would be down-regulated in cisplatin resistant patients with poor prognosis. Based on this, we compared the non-resistant cell HNE1 with the cisplatin-resistant cell HNE1/DDP, and the drug resistance of HNE1/DDP cells has been verified. The IC50 values of drug-resistant strain (HNE1/DDP) and non-drug-resistant strain (HNE1) were 61.63 μM and 11.53 μM, respectively, and the drug resistance index was 5.35(61.63 μM/11.53 μM), as shown in Fig. 1 C. It was verified that the expression of miR-199a-5p in HNE1/DDP cells was significantly lower than that in HNE1 cells (Fig. 1D).

Fig. 1.

miR-199a-5p is differentially expressed in NPC and regulates cisplatin resistance. (A) The GSE32960 and GSE36682 datasets showed the expression of miR-199a-5p in NPC and normal tissues. (B) Overall survival of patients with different levels of miR-199a-5p was studied by Kaplan-Meier curve. (C) Study on the activity of two kinds of nasopharyngeal carcinoma cells under different DDP concentrations. (D) Expression of miR-199a-5p in two NPC cells. (E) The miR-199a-5p levels in the transfected cells were detected by RT-qPCR. (F) Regulation of HNE1/DDP cisplatin sensitivity by miR-199a-5p. Data are means ± SD, * p < 0.05.

After transfection (Fig. 1E), CCK-8 assay was used to investigate the effect of miR-199a-5p on cisplatin resistance (Fig. 1F). The results showed that the anti-tumor effect of DDP was more obvious in HNE1/DDP cells up-regulated by miR-199a-5p.

To verify the regulatory role of miR-199a-5p in NPC, its inhibition of proliferation was tested. The results of CCK-8 assay and colony formation assay showed that miR-199a-5p could significantly inhibit the proliferation of NPC cells (Fig. 2 A and B). In the flow cytometry assay, we found that miR-199a-5p could significantly promote the apoptosis of NPC cells (Fig. 2C). In the subsequent detection of apoptosis-related proteins, our study showed that miR-199a-5p significantly increased the expression of Caspase-3 and decreased the expression of Bcl-2 (Fig. 2D). These results suggested that miR-199a-5p could inhibit the proliferation of NPC cells.

Fig. 2.

Effect of miR-199a-5p on the proliferation of NPC cells. (A) CCK8, (B) Cell cloning was used to detect the effects of different miR-199a-5p levels on proliferation of HNE1 and HNE1/DDP cells. (C) Effects of different levels of miR-199a-5p on apoptosis of HNE1 and HNE1/DDP cells. (D) Western immunoblotting was used to study the effect of miR-199a-5p on apoptotic proteins. Data are means ± SD, * p < 0.05.

In the wound-healing assay, our study showed that miR-199a-5p significantly inhibited the migration ability of NPC cells, while its knockdown increased the migration ability (Fig. 3A). Transwell assay also showed that miR-199a-5p significantly inhibited the migration of NPC cells. Invasion assay showed that miR-199a-5p could significantly inhibit the invasion ability of NPC (Fig. 3B). When we examined the expression of EMT-related proteins, the results were equally surprising. The expression of interstitial markers N-cadherin and Viment could be inhibited by miR-199a-5p and the expression of epithelial marker E-cadherin was promoted (Fig. 3C). These results suggested that miR-199a-5p enhanced migration and invasion by promoting EMT.

Fig. 3.

Effect of miR-199a-5p on migration and invasion. (A) Effects of different levels of miR-199a-5p on the migration of nasopharyngeal carcinoma cells. (B) Transwell assay was used to analyze the effect of miR-199a-5p on the migration and invasion of NPC cells. (C) Effect of miR-199a-5p on EMT-related proteins. Data are means ± SD, * p < 0.05.

To further investigate the effect of miR-199a-5p on the malignant phenotype of NPC, animal experiments were performed. After lentivirus infection (Fig. 4A), the results of subcutaneous xenograft tumor experiments showed that miR-199a-5p could significantly inhibit tumor growth, on the contrary, knockdown of miR-199a-5p could promote tumor growth, and the tumor volume and weight were shown in Fig. 4 B and C. In the HE is staining of the tumors, we found that miR-199a-5p could significantly reduce the number of tumor cells, the cytoplasm and nucleus were lightly stained, and some of them showed karyolysis and cell disintegration (Fig. 4D). The expression of Ki-67 measured by IHC showed that miR-199a-5p reduced the expression of Ki-67, indicating that high miR-199a-5p expression is generally associated with lower malignancy (Fig. 4E).

Fig. 4.

In vivo experiments confirmed the effect of miR-199a-5p on NPC. (A) Detection of lentivirus transfection efficiency in NPC cells. (B and C) Effect of miR-199a-5p on subcutaneous xenograft tumor in nude mice. (D) The tumors in nude mice were stained with H&E staining. (E) The expression of Ki-67 was detected by IHC. Data are means ± SD, * p < 0.05.

In order to study the underlying mechanism of miR-199a-5p, the target of Mir-199a-5p was further explored. A total of 23 potential target genes were identified by searching in miRWalk, miRmap and ENCORI databases (Fig. 5 A and B). Among them, 6 genes were highly expressed in head and neck cancers, namely SLC1A5, ETS1, NDRG1, RHEB, HIF1A and CDH2 (Fig. 5C). Further analysis revealed that SLC1A5 has been shown to be a target gene of miR-199a-5p in thyroid cancer.10 Subsequently, using ENCORI and GEPIA2 online databases, we found that SLC1A5 showed significantly higher expression in HNSC (Fig. 5 D and E). Interestingly, higher SLC1A5 expression was associated with worse prognosis in female patients but not in male patients (Fig. 5F).

Fig. 5.

Targeting of SLC1A5 by miR-199a-5p. (A) Target analysis of miRWalk, miRmap, and ENCORI online databases. (B) Potential target genes were obtained by online database analysis. (C) Differentially expressed and up-regulated genes among the potential target genes. (D and E) Differential expression analysis of SLC1A5 in ENCORI and GEPIA2 online databases. (F) The effect of SLC1A5 expression level on the prognosis of patients. (G) Potential binding sites of miR-199a-5p and SLC1A5. (H) DLR analysis was used to verify the target between SLC1A5 and miR-199a-5p. (I‒J) Effect of miR-199a-5p on SLC1A5 levels by (I) Western immunoblotting and (J) RT-qPCR. Data are means ± SD, * p < 0.05.

The DLR assay was employed to substantiate the binding interaction between miR-199a-5p and SLC1A. The results demonstrated that the overexpression of miR-199a-5p markedly diminished the luciferase activity of SLC1A5-WT cells by 37% in comparison to the control cells. Following the SLC1A5 mutation, the luciferase activity in the SLC1A5-mut group exhibited a notable increase of 30.1% in comparison to the previous measurement (Fig. 5 G and H). The results of WB and RT-qPCR experiments demonstrated that the expression level of miR-199a-5p in NPC cells exerted a significant impact on the expression of SLC1A5 (Fig. 5 I and J). These findings suggest that miR-199a-5p directly targets SLC1A5 and regulates its expression (Fig. 6).

Fig. 6.

miR-199a-5p regulates the migration and invasion of nasopharyngeal carcinoma by inhibiting SLC1A5. CCK-8 assay (A) Clone formation assay (B) To assess the proliferative capacity of nasopharyngeal carcinoma cells; (C) Flow cytometry to detect the apoptotic ability of nasopharyngeal carcinoma cells; (D) Scratch test assesses the migration ability of nasopharyngeal carcinoma cells; (E) Transwell assay evaluates the migration and invasion capabilities of nasopharyngeal carcinoma cells, * p < 0.05.