Cell lines
Human acute T-cell leukemia Jurkat cells., human embryonic kidney 293T and human oral cancer cells SCC-4, SCC-9, SCC-15, SCC-25, Cal-27, Cal-33, HSC-2, HSC-3, HSC-3-M3, and HSC-4, Ca9-22, OSC-19, OSC-20, and SAS were purchased from original resource and listed in Table S1. All cells were cultured in the standard culture condition by following instruction manual and maintained within 3 months. Additionally, all cells were regularly checked for mycoplasma infection and cell morphology to ensure cell health.
Activation of Jurkat T cell
The Jurkat cells were stimulated with 1.25 µg/ml of anti-CD3 (Clone: OKT3; BioLegend, San Diego, CA, USA) monoclonal antibody (mAb) and 1.25 µg/ml of anti-CD28 (Clone: CD28.2; BioLegend) mAb for 72 hours. Jurkat-CXCR3 cells were stimulated with the same amount of anti-CD3 and anti-CD28 mAb plus 10 ng/ml of IL-12 (R&D Systems, Inc, Minneapolis, MN, USA) for 72 hours.
T cell cytotoxicity assay against OSCC cells
2 × 104 Jurkat cells were seeded in the upper-transwell chamber and co-transfected with or without miR-762 mimics and CXCR3-CDS expression vector for 72 hours. For T cell cytotoxicity, 1 × 104 Cal-27 cells were seeded in the lower-well. After 48 hours, Cal-27 cells were fixed in 70% ice-cold ethanol for 60 minutes then stained with 0.1% crystal violet/20% methanol solution and recorded under a light microscope.
RNA extraction and real-time PCR (qRT‐PCR)
Total RNA was isolated from cell lines using TRIzol reagent, and cDNA was synthesized by Roche First Strand cDNA Synthesis Kit. All primers were listed in Table S2. qRT-PCR analysis was used ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) and QuantStudio 3 real-time PCR system. For mature miRNA detection, cDNA was prepared using the miScript II RT Kit (Cat: 218161, QIAGEN, Valencia, CA, USA). U6 was used as the internal control for human miRNAs. The expression level was defined based on the threshold cycle, and relative expression levels were calculated as ▵▵Ct after normalization with the reference control.
Protein extraction and western blot analysis
Cells were lysed in RIPA buffer with protease and phosphatase inhibitor cocktail. 10–30 µg of protein lysates were loaded onto 10% SDS-PAGE and transferred into PVDF membranes. All antibodies were listed in table S3. The membranes were exposed using ECL (Merck-Millipore) for the HRP-coupled secondary antibodies and analyzed using the e-BLOT Touch Imager.
Transwell migration assays
T cell migration assays were performed using a 3-µm Transwell chamber (SPL 36224, Korea). 2×104 Jurkat cells were seeded in the upper-chamber in serum-free medium, and 600 µL of medium containing 2% FBS was added to the lower well. The cells were incubated for 24 hours to allow them to attach. After 24 hours of incubation, migrated cells were fixed on the membrane with 70% ethanol for 60 minutes at 4°C, washed twice with PBS, and the interiors of the inserts were cleaned with wet cotton swabs. Cells were stained with a cell stain solution (0.1% crystal violet, 20% methanol) and visualized under an inverted microscope (200× magnification; Olympus Corporation, Hachioji, Tokyo, Japan). Images were then analyzed and counted using an analytical imaging station software package (Imaging Research, Ontario, Canada).
Plasmids and Transfection
The pLX304-CXCR3 plasmid was generated by inserting full-length cDNA (CXCR3: NM_001504) into the vector following previously established protocols. The miR-762 mimic (PM) (ID: 15805, Cat: AM17100, Thermo Fisher Scientific) transfection mix was prepared using TransIT-X2 Transfection Reagent (Mirus, Madison, WI, USA) according to the manufacturer's protocol and applied to Jurkat cells.
For lentivirus production, 293T cells were co-transfected with pMD2.G, pCMV▵R8.91, and lentiviral transfer plasmids using TransIT-LT1 Transfection Reagent (Mirus, Madison, WI, USA). The culture medium containing lentivirus was harvested 48 hours after transfection. Cells were removed from the culture medium by centrifugation at 500 g for 5 minutes and filtered through a 0.45-µm filter. The lentiviral stocks in the medium, containing polybrene (8 µg/mL), were used to infect the target cells for 6 hours.
CCK-8 Assay:
Equal numbers of Jurkat cells transfected with miR-762 mimics or non-targeting control (N.C.) for 72 hours were plated in a 96-well plate and allowed to grow for an additional 72 hours. Cell growth was determined using a Cell Counting Kit-8 (CCK-8) (Sigma-Aldrich, St Louis, USA). 10 µL of CCK-8 solution was added to each well of the plate, followed by incubation for 3 hours. The absorbance at 450 nm was measured using a 96-well plate SpectraMax 250 reader (Molecular Devices, CA, USA).
Luciferase Reporter Assays:
For NF-κB transcriptional activity and CDH1 promoter activity assay, the protocol was followed as per our previous work. For the 3’-UTR luciferase reporter assay, the CXCR3 3’-UTR region was generated by subcloning PCR-amplified full-length human CXCR3 3’-UTR (446 bp from the stop codon) into the modified 3’-UTR CpoI sites of the luciferase in the pGreenfire-CMV firefly luciferase-expressing vector (SBI, Palo Alto, CA, USA), using the primers: Forward: 5’-CGGACCGGGCCGGAATCCGGGCTCCCCTTTC-‘3 and Reverse: 5’-CGGTCCGTCCTGACGATCTTGTTTATT-3’. 293T cells were cultured in 24-well plates and co-transfected with 300 ng of CXCR3 3’-untranslated region (UTR) wild type or mutant type pGreenfire-CXCR3 3’-UTR reporter plasmid, along with 25 nM of miR-762 mimics (PM) or non-targeting control (N.C.), using TransIT-X2 Transfection Reagent (Mirus, Madison, WI, USA) according to the manufacturer’s instructions. The luciferase assay was performed 24 hours post-transfection with the ONE-Glo™ Luciferase Reporter Assay System (Promega, USA) following the manufacturer's protocol. 50 µL of ONE-Glo™ substrate was added into a white plate and the assay was conducted with a SpectraMax iD3 microplate reader.
Flow Cytometry:
The cells were harvested, washed twice with PBS, fixed in 1% paraformaldehyde (PFA) overnight at 4°C, washed again, and resuspended in flow cytometry buffer. To determine surface markers, these cells were labeled with the surface fluorochrome-conjugated antibodies CD25 and CD69 (BD Biosciences, Franklin Lakes, NJ) for 60 minutes on ice in the dark. The stained cells were analyzed using a Attune NxT Flow Cytometer and the quantitative analysis was performed using FlowJo v10 (Treestar, Ashland, OR).
Enzyme-linked immunosorbent assay (ELISA)
Cell supernatants were collected and processed using R&D Duoset ELISA kits (R&D Systems, Inc, Minneapolis, MN, USA) according to the manufacturer’s instructions for the detection of cell secretion of IL-12 (Cat# D1200).
Statistical analysis
All experiments were repeated at least 3 times. Data were presented as the mean ± standard deviation (SD) from repeated independent experiments. Differences between various treatment groups were assessed using the Student’s t test. Between-group differences were considered significant at P < 0.05. Data analyses were performed using GraphPad Prism Ver. 8 (San Diego, CA, USA).