TSCC patient cohort

32 pairs of tumor tissues and adjacent paracancerous tissues were obtained from the TSCC patients. We employed the qRT-PCR method for comparing the mRNA expression of ITGB1-7 in TSCC patients’ samples. RNA was extracted from TSCC patient’s fresh dissected tissues using the RNA Quick Purification kit (ES science, Cat#RN001). qRT-PCR was performed using SYBR Green Real-time PCR Master Mix (ReverTra Ace, Toyobo) and a LightCycler 480 (Roche, Basel, Switzerland). Based on the obtained Ct values, we compared the expression of ITGB1-7 in TSCC tumor tissue as the relative expression levels in paracancerous tissues were set to 1. A total of 103 cases of TSCC and its adjacent tissues were collected from the Department of Oral and Maxillofacial Surgery of Sun Yat-sen University’s Sun Yat-sen Memorial Hospital (83 cases) and the Department of Stomatology of Guangzhou First People Hospital (20 cases) from June 2008 to October 2015 to perform a retrospective analysis.

Inclusion criteria is as following: All patients were primary TSCC patients. Tissues were diagnosed as tongue squamous cell carcinoma by pathological biopsy according to 8th edition of AJCC Staging System. No distant organ metastasis was found in all patients. All enrolled patients did not receive any other anti-tumor treatment before surgery. Exclusion criteria is as following: Patients with recurrent tongue squamous cell carcinoma. Patient received chemotherapy, radiotherapy or other non-surgical treatment before surgery.

The removed tissue specimens were fixed in 4% paraformaldehyde, embedded in paraffin, and 4 μm -thick tissue sections were prepared for following study. All patients have been informed and signed an accurate informed consent form approved by the Ethics Committee of the two hospitals, and all the experiments were performed in accordance with approved guidelines and regulations.

GEPIA database analysis

By analyzing the expression level of ITGB5 gene in head and neck squamous cell carcinoma through the GEPIA database (http://gepia.cancer-pku.cn/) (Tang et al. 2017). Gene Expression Profile was used to analyze the expression of ITGB5 in the cancer and adjacent tissues of head and neck squamous cell carcinoma.

Immunohistochemistry

The 4 μm tissue sections of tongue squamous cell carcinoma were dewaxed with xylene, hydrated with gradient ethanol (95%, 90%, 85%, 75%), and then repaired with antigen by microwave oven. After that, tissue sections were incubated at room temperature with 3% hydrogen peroxide for 10 min to remove endogenous peroxidase. Next, tissue sections were permeated with PBS solution containing 0.1% Triton X-100 for 5 min, washed with PBS, added 5% goat serum, and sealed at room temperature for 1 h. Excess liquid was tossed off from the tissue sections, ITGB5 antibody (abcepta, Cat#AP14000a) were added and incubated in a refrigerator at 4 ℃ overnight. On the second day, the first antibody solution of the tissue sections was removed, washed with PBS, and then the pika universal second antibody was added dropwise to incubate at room temperature for 30 min. DAB was colored under microscope for 3–5 min, and hematoxylin was re-stained for 1–2 min. Dehydrated, transparent, neutral resin seal, observed under a microscope. Read 1000 cells per tissue section, and if the number of positive cells is greater than 350, it indicates high expression.

Cell culture

Cal27 and Scc9 were purchased from the American Type Culture Collection. cisplatin (Sigma, St. Louis, MO, USA) at concentrations of 10− 7 M to 10− 5 M were used to treat Cal27 or Scc9 to establish the stable cisplatin-resistant lines Cal27-re and Scc9-re. Dulbecco’s modified Eagle’s medium (Gibco, Rockville, MD, USA) supplemented with 10% fetal bovine serum (ABW) were supplied to cultivate Cal27 and Cal27-re cells. Dulbecco’s modified Eagle’s medium-F12 (Gibco) supplemented with 10% fetal bovine serum (ABW) were supplied to cultivate Scc9 and Scc9-re cells.

Transfections

The shRNA was purchased from TranSheep Bio. The various targeting sequences were as follows: sh-ITGB5, 5-CCCGCTATGAAATGGCTTCAA-3. ITGB5 cDNA was purchased from TranSheep Bio. To generate lentivirus expressing sh-ITGB5 or ITGB5, HEK 293T cells grown on a T75 dish were transfected with 15 µg pCDH-sh-ITGB5 or pCDH-ITGB5 or control vector (pCDH), 10 µg psPAX2, and 5 µg pMD2G. 6 h after the transfection, cells were cultured with DMEM containing 10% FBS. Virus supernatant was collected twice at 48 and 72 h after transfection with a 0.45 μm Filter and centrifuged in a 40 mL ultracentrifugation tube at 4 ℃ for 120 min at 72,000 g/min. Resuspended lentivirus using PBS. Then lentivirus along with polybrene were used to transfect TSCC cells. Puromycin (1 ug/mL) was used as a selection marker for the infected cells. The expression efficiency was evaluated by qRT-PCR and Western blot.

RNA and protein extraction

For RNA extraction, total RNA was extracted by the RNA Quick Purification kit (ES science, Cat#RN001), according to the instructions of the manufacturer. For protein extraction, the cells were rinsed three times with PBS for 5 min each, followed by the addition of radioimmunoprecipitation assay (RIPA) Buffer (Kangweishiji, Cat# CW2333S) containing a protease inhibitor cocktail (APExBIO, Cat# K1007). The cells were lysed for 0.5 h at 4 °C. Then, the samples were scraped down and centrifuged at a speed of 12,000 rpm for 20 min at 4 °C. The supernatants were collected as the total protein.

qRT-PCR

qRT-PCR was performed using SYBR Green Real-time PCR Master Mix (ReverTra Ace, Toyobo) and a LightCycler 480 (Roche, Basel, Switzerland) according to the manufacturer’s instructions. Reactions were performed in triplicate in three independent experiments. The sequences of all primers were listed in sup Table 1. The relative expression levels in the control were set to 1.

Western blot

Protein extracts were resolved via 8% SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes (BioRad, Berkeley, CA, USA), probed with antibodies against human ITGB5 (abcepta, Cat#AP14000a), GAPDH (HUABIO, Cat#HA1031), E-cadherin (Immunoway, Cat#YT1454), Vimentin (Immunoway, Cat#YT4880) and then with a peroxidase-conjugated secondary antibody (Beijing Ruikang); the bands were visualized via chemiluminescence (BIO-RAD, ChemiDocTMImaging System).

Modified boyden chamber assay

In all, 1 × 105 TSCC cells were plated into the upper chamber of a polycarbonate transwell filter chamber (Corning, NewYork, NY, USA) and incubated for 22 h. For invasion assay, the upper chamber was coated with Basement Membrane (R&D, Minneapolis, MN, USA). Cells on the lower membrane surface were fixed in 4% paraformaldehyde, stained with crystal violet and counted (5 random 100× fields per well). Three independent experiments were performed and the data are presented as the average ± s.d.

ROS production assay

The intracellular ROS levels were measured by detecting the conversion of cell-permeable 2,7-dichlorofluorescein diacetate (DCFH-DA) to fluorescent dichlorofluorescein (DCF) (Beyotime Biotechnology, S0033). The cells were seeded in 6-well plates and treated under different conditions. Then, all wells were washed with PBS three times, and the cells were incubated with DCFH-DA at 37 °C for 25 min. The cells were washed with serum-free culture medium. Then the cells were fixed with 4% paraformaldehyde and DAPI staining. Finally, the DCF fluorescence distribution was detected by a confocal microscopy (ZEISS, LSM900).

mRNA array and KEGG

mRNA array analyses were compared between Cal27 with or without ITGB overexpression. The differentially expressed mRNAs and KEGG enrichment were calculated, identified, and visualized (KangchengBio Corporation, Shanghai, China).

Xenografts

The animal experiments were approved by the Sun Yat-sen University Laboratory Animal Care and Use Committee. Male BALB/c-nu mice (4–6 weeks old) were randomly divided into 4 groups (n = 6) and applied to evaluate the effects of ITGB5 in TSCC cells in vivo. TSCC cells with ITGB5 knock down or overexpressed were transfected with luciferase reporter plasmid (OBiO Technology, Shanghai). 1 × 106 cells

in 200 mL of PBS were injected into the mice via the tail vein. The mice were imaged 1.5 months later with luciferase-based IVIS Lumina Imaging System (Xenogen, Alameda, CA, USA) after injection of 100 mL of D-luciferin (15 mg/mL) (Yeasen, Shanghai) 10 min before imaging. After imaging, lungs were harvested from euthanized mice and fixed in paraformaldehyde for immunohistochemistry.

Statistical analysis

SPSS 25.0 statistical software was used to analyze and process the data. Chi-square test was used to compare the inter group rates. Student’s t test was used to compare the mean between the two groups to analyze the correlation between the expression of ITGB5 and the clinical status of patients. The Kaplan Meier was used to analyze the survival of patients. P < 0.05 indicates statistical significance.