The gene expression data of oral cancer patients were downloaded from the TCGA OSCC. We selected patients with cisplatin treatment and clear outcomes. Then, we filtered and standardized the data. Ultimately, a total of 72 patients with detailed clinical information and 724 genes were used for the subsequent analysis. All data were analyzed by R.4.0.2.
Identification of differentially expressed genes and weighted coexpression network constructionWe used the “limma” package to identify the differentially expressed genes between CR patients and CS patients. The genes with |logFoldChange| > 0 were considered significantly differentially expressed genes. We used “WGCNA” for the gene coexpression network construction, and the soft threshold for network construction was selected as 5. Then, we defined gene modules by gene branches using a dynamic tree-cutting algorithm. We displayed the relationship between gene modules and clinical signatures by a heatmap.
Survival analysis and functional enrichment analysisPatients were divided into two groups according to the expression level of MT3. The R packages “survival” and “ggplot” were applied to perform the survival analysis. We identified the differentially expressed genes between the high and low MT3 expression groups by using the “limma” package. Then, the functional enrichment analysis was performed via the “clusterProfiler” package.
Human samples, animals and cell linesParaffin-embedded tumor tissues were obtained from oral cancer patients at Zhongnan Hospital of Wuhan University. Ethical permission was granted by the Medical Ethics Committee, Zhongnan Hospital of Wuhan University. Six-week-old female NSG mice were purchased from HFK Inc. (Beijing, China) and raised in an SPF grade environment. In all animal experiments, we complied with the ethical norms of animal experiments. CAL27 and Fadu oral cancer cell lines were obtained from ATCC and cultured in DMEM. By continuously exposing CAL27 and Fadu oral cancer cell lines to stepwise increasing concentrations of cisplatin over 6 months, CIS-resistant CAL27-CISR and Fadu-CISR cells were generated. All media were supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) and 2 mM L-glutamine (Gibco, USA). All cells were grown at 37 °C in a 5% CO2 incubator.
Histological and immunohistochemical stainingTumor tissues were collected from patients with oral cancer at Zhongnan Hospital. Then, the samples were fixed in 10% formalin, embedded in paraffin and sectioned for H&E staining. The slides of tumor tissues were incubated with anti-MT3 or anti-Yap at 4 °C for 12 h. Afterward, the slides were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature, followed by development with DAB substrate and counterstaining with hematoxylin.
Knockdown of MT3 and YAP1Generation of MT3 or YAP1 knockdown using CRISPR‒Cas9. Fadu and CAL27 oral cancer cell lines were transfected with sgRNA against MT3 or YAP1 cloned in PX459 (Addgene, USA). Briefly, 2 × 105 Fadu and CAL27 oral cancer cells were seeded in 6-well plates for 24 h. Fadu and CAL27 cells were transfected with 5 µg of PX459 using Lipofectamine 8000 (Beyotime, China) in Gibco Opti-MEM reduced serum medium (Thermo Fisher Scientific, USA). After 48 h, puromycin (2 µg/ml) was used for selection for 14 days, and cells were maintained in puromycin (0.5 µg/ml).
The following primers were used:
SGGFP: 5’-CACCGGGGCGAGGAGCTGTTCACCG-3’;
MT3-SG1: 5’- GTGGCGTCGCCCTCTCTAGGTGG-3’;
MT3-SG2: 5’- GCGGACTCCTGCAAGTGCGAGGG-3’;
YAP1-SG1: 5’- GATGATGTACCTCTGCCAGC-3’; and
YAP1-SG2: 5’- GGGACAGCATGGCCTTCCTG-3’.
Western blottingCells were collected and lysed in RIPA lysis buffer at 4 °C for 30 min. Then, we centrifuged the pyrolysis solution at 12,000 × g for 15 min and collected the supernatant. After the determination of concentration, the protein was run on an SDS‒PAGE gel and transferred to a nitrocellulose membrane. The antibodies we used included anti-actin, anti-MT3, and anti-YAP1. The original gels and multiple exposure images are shown in Additional file 2.
Real-time PCRTotal RNA was extracted by using TRIzol (Beyotime, Shanghai) and reverse transcribed into cDNA by using a TAKARA reverse cDNA kit (TAKARA, Japan).
CCK-8 assayCell viability was assessed using the CCK-8 assay (Solarbio, China). Fadu and CAL27 oral cancer cells were seeded into 96-well plates in 100 µL medium and incubated for 48 h. The absorbance was measured by spectrophotometry following the manufacturer’s protocol.
Animal experiments and treatment protocolNude mice were purchased from HFK Animal BIOSCIENCE Co., LTD (Beijing) and kept in compliance with the Guidelines of Experimental Animal Welfare Ethics Committee, Zhongnan Hospital of Wuhan University. For the xenograft tumor experiment, the mice were randomly divided into 4 groups (6 mice/group) and subcutaneously inoculated with 1 × 106 Fadu-CISR or CAL27-CISR cells and intraperitoneally injected with DMSO (control group, 100 µl), verteporfin (100 µl), CIS (5 mg/kg) or CIS (5 mg/kg) combined with verteporfin (100 µl). The length and width of transplanted tumors were measured every 5 days, tumor volume was calculated as (length × width2) π/6, and a survival curve was drawn.
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