Downregulation of RBM17 enhances cisplatin sensitivity and inhibits cell invasion in human hypopharyngeal cancer cells

2.1 Patient samples and cell line

HSCC tissues and adjacent normal tissues were collected from six patients, who were hospitalized in The First Affiliated Hospital of Bengbu Medical College from 2021 to 2022. FaDu cells were purchased from BeNa Culture Collection Incorporation Co., Ltd., and maintained at 37°C in a humidified 5% CO₂ atmosphere in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin, and 100 µg/ml streptomycin (Hyclone, GE).

1. Ethics approval and consent to participate: The present human and animal studies were approved by the Ethics Committee of The First Affiliated Hospital of Bengbu Medical College, and patients provided written informed consent.

2. Informed consent: Informed consent has been obtained from all individuals included in this study.

2.2 Lentiviral construction for shRNA treatment

RBM17-targeted shRNA (5′-ACTTAAGTGTCCTACTAAA-3′, GenBank NM_032905) and the scramble negative control sequence (5′-AATTCTCCGAACGTGTCACGT-3′) were cloned into the pGV115-green fluorescent protein lentiviral vector (Shanghai Genechem Co., Ltd., Shanghai, China).

2.3 Cell proliferation assays

Cell proliferation was evaluated by cell counting kit-8 (CCK-8) assay. In brief, FaDu cells were seeded in a 96-well plate at 5,000 cells per well and cultured in 37°C incubator for 24 h. After treatment with different concentrations of cisplatin for 24 h, 100 μl of CCK-8 solution was added to each well. Two hours later, absorbance was measured at a wavelength of 450 nm with a plate reader.

2.4 Wound healing assays

Twelve-well plate was seeded with LV-scramble or LV-shRBM17-infected FaDu cells at a density of 100,000 cells/well. The monolayer cells were scratched by a sterile pipette tip and washed three times by phosphate-buffered saline (PBS). Photographic images were taken at 0, 24, and 48 h.

2.5 Cell migration and invasion assays

The migration and invasion abilities of FaDu cells were investigated by using a 24-well Transwell chamber (diameter 6.5 mm, 8 μm pore size; Corning, Corning, NY, USA). Briefly, the infected FaDu cells were resuspended in serum-free medium and seeded in the upper chamber at a density of 3 × 10⁴ cells/well. The medium with 10% FBS was added to the bottom chamber. For invasion assays, the upper chamber was pre-treated with Matrigel (50 μl; BD Biosciences). After incubation in a 5% CO₂ humidified incubator at 37°C for 24 h, the non-migrating cells were scraped with cotton swabs and the migrated and invaded cells were fixed with 4% paraformaldehyde and then stained with 5% crystal violet. Cells were quantified in three random fields using an optical inverted microscope at a magnification of 200×.

2.6 Liquid chromatography–mass spectrometry (LC–MS)

The tryptic peptides were dissolved in 0.1% formic acid (solvent A), and then separated using the EASY-nLC 1000 ultra-high performance liquid system. The peptides are separated by the ultra-high-performance liquid system and injected into the NSI ion source for ionization and then analyzed by a Q Exactive mass spectrometer (Thermo Fisher Scientific). The mass spectrometer was operated in a positive ion mode. Mass spectrometry (MS) data were acquired using a data-dependent top 10 method dynamically choosing the most abundant precursor ions from the survey scan (350–1,800 m/z) for HCD fragmentation. Survey scans were acquired at a resolution of 70,000 at m/z 200 with an AGC target of 3e6 and a maxIT of 50 ms. MS2 scans were acquired at a resolution of 17,500 for HCD spectra at m/z 200 with an AGC target of 2e5 and a maxIT of 45 ms, and isolation width was 2 m/z. Only ions with a charge state between 2 and 6 and a minimum intensity of 2e3 were selected for fragmentation. Dynamic exclusion for selected ions was 30 s. Normalized collision energy was 30 eV. MS/MS raw files were processed using MASCOT engine (Matrix Science, London, UK; version 2.6) embedded into Proteome Discoverer 2.2 and searched against the Uniprot_HomoSapiens_20367_20200226 database. Except for TMT labels, carbamidomethyl (C) was set as a fixed modification. Variable modifications were oxidation (M) and acetyl (protein N-term). A peptide and protein false discovery rate of 1% were enforced using a reverse database search strategy. Proteins with fold change >1.2 and P value (Student’s t test) <0.05 were considered to be differentially expressed proteins [13,14].

2.7 qRT-PCR

Total RNA was extracted from tissue and FaDu cells using the TRIzol reagent (Invitrogen). Complementary DNA (cDNA) was reverse transcribed by the PrimeScriptÔ 1st Strand cDNA Synthesis Kit (Takara, Kusatsu, Japan) according to the manufacturer’s instructions. The SYBRR Premix Ex TaqÔ (TaKaRa) was used for qRT-PCR. The2^−ΔΔCt method was adopted and applied to calculate the relative expression. mRNA was normalized using GADPH, which were reported in our previous study [15]. The list of primers is shown in Table 1.

2.8 Western blot analysis

FaDu cells and tissue were collected and resuspended in cell lysis buffer for western blot on ice. BCA protein quantification kit was used to detect the protein concentration. Protein from each sample was loaded onto an SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore, Temecula, CA, USA). Membranes were blocked with 5% skim milk in phosphate-buffered saline containing 0.1% Tween-20 (PBS-T). To detect the target proteins, membranes were incubated with primary antibodies Vimentin (1:2000, Proteintech Group), E-cadherin (1:5000, Proteintech Group), GAPDH (1:3000, Proteintech Group), ZEB1 (1:500, Proteintech Group), ARHGDIB (1:5,000; Abcam), UHRF1 (1:500; Abcam), CCN1 (1:1,000; Abcam) and β-actin (1:2,000; Abcam) overnight at 4°C. Membranes were then washed for 10 min with PBS-T for three times and incubated with secondary antibody (1:2,000; Abcam) for 2 h at room temperature. The membranes were washed and developed using chemiluminescence reagents (Millipore) and visualized with gel imaging equipment (Bio-Rad, USA).

2.9 In vivo tumorigenesis

The experiments were performed with the approval of Animal Care and Committee of Bengbu Medical College. Six-week-old BALB/c female nude mice were purchased from Beijing Vital River Animal Company. A number of 1 × 10⁷ logarithmically growing FaDu cells (LV-scramble and LV-shRBM17) were subcutaneously inoculated into the left flank of mice. The diameters of the tumors were measured every 2 days, and the tumor volumes were calculated.

2.10 Bioinformatic analysis

The “Expression analysis-Box Plots” module was used to get box plots of the expression difference between these tumor tissues and the corresponding normal tissues of the GTEx (Genotype-Tissue Expression) database and “Survival Analysis” module was applied to obtain OS and disease-free survival (DFS) data with 50% cutoff by online tool of Gene Expression Profiling Interactive Analysis (GEPIA) (http://gepia.cancer-pku.cn) database [16]. We used the UALCAN portal to analyze cancer omics data and conduct protein expression analysis of the clinical proteomic tumor analysis consortium (CPTAC) dataset [17].

2.11 Statistical analysis

Data were analyzed using GraphPad Prism 8.0 software (GraphPad Software Inc., CA) and were expressed as mean ± standard error of mean. Difference among the groups was determined using analysis of variance or unpaired t test. All experiments were repeated at least three times. P < 0.05 was considered to be statistically significant.

3.1 Gene expression profiling of RBM17 in normal human versus tumor tissues

First, we applied the TCGA and GTEx datasets to evaluate the expression of RBM17 in tumor tissues and normal tissues by the GEPIA. As shown in Figure 1a, compared to normal tissues, RBM17 transcript levels were significantly increased in tumor tissues. In addition, the CPTAC dataset displayed increasing level of RBM17 protein in the primary tumor tissues (Figure 1b). In addition, qRT-PCR and western blot results confirmed that RBM17 expression was upregulated in HSCC tissues compared with adjacent normal tissues (Figure 1c and d). Kaplan–Meier survival curves from the GEPIA database demonstrated that high expression of RBM17 was significantly associated with shorter OS time and DFS time (P = 0.017; P = 0.012) (Figure 1e and f).

Figure 1

RBM17 is upregulated and associated with a poorer prognosis in HNSCC. (a) The expression of RBM17 in the TCGA database was compared with the expression of the corresponding normal tissues in the GTEx database, which was analyzed by GEPIA. (b) The protein level of RBM17 in primary tumor tissues and normal tissues from CPTAC database. (c and d) Expression level of RBM17 was confirmed by qRT-PCR and western blot in HSCC tissues and normal tissues. (e and f) Kaplan–Meier survival curve analysis of RBM17 for the OS and DFS in HNSCC from GEPIA database. **P < 0.01, ***P < 0.001.

3.2 Downregulation of RBM17 inhibits HSCC proliferation and sensitizes HSCC to cisplatin

To investigate the effect of cisplatin on the growth and proliferation of HSCC, FaDu cells were treated with gradient concentration of cisplatin. Cisplatin significantly decreases the proliferation of FaDu cells; the IC₅₀ value is about 3.26 μg/ml (Figure 2a). Furthermore, we examined whether cisplatin can regulate the expression of RBM17. Interestingly, real-time PCR result indicated that cisplatin can increase the RBM17 mRNA level (Figure 2b). The effect of RBM17 knockdown on the sensitivity of FaDu cells to cisplatin was then evaluated by CCK-8 assays. The results showed that downregulation of RBM17 by lentiviral vector significantly decreases cell viability of cisplatin-treated FaDu cells (Figure 2c).

Figure 2

Downregulation of RBM17 enhances cisplatin sensitivity in vitro. (a) Inhibition of cisplatin on FaDu cells was in a concentration-dependent manner (b) FaDu cells were treated with 3.26 μg/ml cisplatin for 24 h, and the mRNA level of RBM17 was detected by real-time PCR. (c) LV-shRBM17- or LV-scramble-infected cells were incubated with different concentrations of cisplatin for 24 h, and the cell viability was determined by CCK-8 assays. **P < 0.01.

3.3 Downregulation of RBM17 inhibits the invasion, migration, and epithelial–mesenchymal transition (EMT) processes of FaDu cells

Our previous study demonstrated that the acquisition of cisplatin resistance is related with a more aggressive and invasive phenotype in nasopharyngeal carcinoma [15]. To assess whether knockdown RBM17 can affect the migration and invasion ability of FaDu cells, we performed wound-healing and Transwell assays. We found that cell migration and invasion were remarkably decreased in LV-shRBM17 cells compared with LV-scramble cells (Figure 3a and b). In these results, we deduced that the RBM17 pathway may induce EMT in HSCC. We detected mRNA and protein expression of EMT-related genes. Protein and mRNA levels of EMT markers Vimentin and transcript factor ZEB1 were significantly decreased, but the expression level of epithelial marker E-cadherin was remarkably increased in LV-shRBM17 cells, compared with LV-scramble cells (Figure 3c and d).

Figure 3

Knockdown of RBM17 affects FaDu cell invasion, migration, and EMT. (a) Cell migration ability was examined by the wound-healing assay. Images were taken using a 4× objective lens. (b) The cell invasion ability was investigated using a Transwell assay. (c and d) ZEB1, Vimentin, and E-cadherin expressions in LV-shRBM17 and LV-scramble cells were detected by qRT-PCR and western blot. *P < 0.05, **P < 0.01.

3.4 Downregulation of RBM17 decreases tumor growth in an animal model

We evaluated the effect of RMB17 downregulation on the HSCC growth in a xenograft mice model. Flank tumors were established in nude mice inoculated with FaDu cells stably expressing shRNA directed against the RBM17. Downregulation of RBM17 significantly inhibited tumor growth (Figure 4a) and reduced tumor burden (Figure 4b and c), relative to the control mice.

Figure 4

Knockdown RBM17 inhibits FaDu HSCC tumor growth in vivo. (a) Tumor volume was measured every 2 days from day 7 to day 21. (b) Tumor weight was calculated at day 21. (c) Representative images of FaDu HSCC xenograft tissues in different groups. *P < 0.05, ***P < 0.001.

3.5 Analysis of differentially expressed proteins of RBM17-downregulated cells vs control cells

LC–MS analysis was performed to explore RBM17-regulated proteins. Expression alterations in the proteomes were analyzed on RBM17 downregulation in FaDu cells (Figure 5a). Comparative proteome analysis was administrated on three samples per group, and these differentially expressed proteins were identified with ＞1.2-fold changes and P-values ＜0.05. There were 62 differentially expressed proteins, 21 upregulated and 41 downregulated. To understand the significant biological function and pathways associated with RBM17, GO enrichment analysis was carried out for biological process, molecular function, and cellular components (Figure 5b). Biological process category analysis showed that these proteins with enhanced localization in the cell surface were associated with cell cycle regulation. Molecular function category analysis demonstrated that these differential proteins of cell membrane were involved in kinase activity and signaling receptor binding. The cellular component category indicated that these differential proteins of cell surface were enriched in the negative regulation of apoptotic process.

Figure 5

Comparative profiling of the proteomes after downregulation of RBM17. (a) Volcano plots of the proteomes in LV-shRBM17 vs LV-scramble. (b) Bubble chart displaying GO terms for biological process and cellular components. GO terms were functionally grouped depending on Kappa scores and only the most significant GO terms were displayed. The significance of each GO term was displayed as −log10 (P-value).

To further verify the abundance of proteins of interest, parallel reaction monitoring (PRM) was utilized for label-free quantification by an MS. Due to the limitation of proteins’ characteristics and their expression abundance, 28 potential proteins were further independently validated by PRM analysis. More than two unique peptides were used for quantifying most protein; however, some proteins were identified only by one peptide because of sensitivity and other reasons. As shown in Figure 6a, 12 proteins (GABARAPL2, HMGCS1, ARHGDIB, CCN1, SCD, SQLE, CNN3, CKS2, RAB5A, MCM2, MORF4L1, and UHRF1) were enriched in RBM17-dowregutated cells, and 9 proteins (HMBS, FERMT2, VCL, SDC4, ATP1B1, ARCN1, SOD2, IL1A, ICAM1) were reduced in RBM17-downregulated cells.

Figure 6

PRM analysis of the differentially expressed proteins. (a) PRM analysis of the differentially expressed proteins after downregulation of RBM17 compared to LV-scramble groups. (b) Selected differentially expressed proteins were confirmed by western blot.

We further confirmed the expression results of three representative proteins by western blot (Figure 6b). Compared with lv-scramble cells, the expressions of ARHGDIB, UHRF1 and CCN1 in LV-shRBM17 cells were increased, which was consistent with the results of PRM.