In silico analysis of miRNAs targeting TSC1
Five miRNA target prediction programs (DIANA-microTv3.0, microRNA, miRDB, Targetscan and PicTar) were used to identify miRNAs targeting the 3’UTR of TSC1 (GenBankTM accession number NM_7428) (Supplementary Table S1).
A total of 36 paired normal oral tissue and OSCC samples were collected from the HCG-Bangalore Institute of Oncology, Bangalore, India (Supplementary Table S2). Tissue samples were stored in RNAlater® (Sigma-Aldrich, St. Louis, MO) and frozen in a -80°C freezer until use. Oral cancer patients enrolled in the study were not under any treatment at the time of the surgery. OSCC samples were classified according to the TNM (Tumor, Node and Metastasis) classification based on the UICC (Union for International Cancer Control, Switzerland; http://www.uicc.org/resources/how-use-tnm-classification). All the samples were obtained with informed consent from the patients and approval from the ethics committee of the Indian Institute of Science, Bangalore. All experiments were performed in accordance with relevant guidelines and regulations. Details of patients are given in Supplementary Table S2.
RNA extraction and cDNA preparation
Total RNA including miRNA was isolated using TRI-Reagent® (Sigma-Aldrich, St. Louis, MO), and quantitated using NanoDropTM 1000 Spectrophotometer (Thermo Fischer Scientific, Waltham, MA). First-strand cDNA was synthesized using 2 µg of total RNA and a Verso cDNA Synthesis Kit (Thermo Fischer Scientific, Waltham, MA).
The expression level of miR-130a was determined by RT-qPCR as suggested by Sharbati-Tehrani et al.48. Details of the primers are given in Supplementary Table S3. The RT-qPCR analysis was carried out using the DyNAmo ColorFlash SYBR qPCR kit in a StepOnePlus™ Real-Time PCR System (Thermo Fischer Scientific, Waltham, MA). GAPDH and 5S rRNA were used as normalizing controls. The following equation ΔCtgene= Ctgene - Ctnormalizing control, was used to calculate the fold change. Ct represents cycle threshold value, and ΔCt represents the gene expression normalized to GAPDH or 5S rRNA. A two-tailed unpaired t-test was performed using the GraphPad PRISM5 software (GraphPad Software Inc., San Diego) to analyze the statistical significance of the difference in mRNA expression.
MiR-130a (pmiR-130a) and TSC1-ORF (pTSC1) constructs were generated in pcDNA3-EGFP and pcDNA3.1 (+) vectors, respectively, using human genomic DNA or cDNA as templates as required and gene specific primers (Supplementary Table S4) by a standard laboratory method. A 1,384 bp long 3’UTR of TSC1 was cloned at the 3’end of the luciferase ORF in the pMIR-REPORT® miRNA Expression Reporter Vector System (Thermo Fischer Scientific, Waltham, MA) in both sense and antisense orientations using human genomic DNA as a template. Primers were designed (Supplementary Table S4) using specific DNA sequences retrieved from the UCSC Genome Browser (https://genome.ucsc.edu), and PCR was performed using a standard laboratory procedure to facilitate directional cloning. The site-directed mutagenesis was carried out to generate constructs with the 3’UTR of TSC1 harboring mutations in the TS1 (target site 1), TS2 (target site 2) or TS3 (target site 3) or all the TSs, according to Rather et al.49. The pTSC1-3’UTR-S, pTSC1-3’UTR-AS or pTSC1-3’UTR-S-M constructs were generated by cloning the relevant 3’UTR sequences downstream to the TSC1-ORF at EcoR V and Not I sites in the pTSC1 construct. All the constructs were sequenced on an ABI PRISM®R A310-automated sequencer (Thermo Fisher Scientific, Waltham, MA) to confirm the directionality and error-free sequence of the inserts.
Human oral squamous cell carcinoma cell lines, UPCI: SCC084 and UPCI: SCC131, were a gift from Prof. Susanne Gollin, University of Pittsburgh, Pittsburgh, PA. Cells were maintained in DMEM supplemented with 10% FBS and 1X antibiotic/antimycotic solution (Sigma-Aldrich, St. Louis, MO) at 37°C in 5% CO249.
Transfection and reporter assays
SCC131 or SCC084 cells were seeded at a density of 2 x 106 cells/well in a 6-well plate and transiently transfected with an appropriate construct or a combination of constructs using the LipofectamineTM 2000 Transfection Reagent (Thermo Fisher Scientific, Waltham, MA). After 48 h, cells were harvested for either total RNA isolation using TRI-ReagentTM or total protein lysate preparation using the CelLyticTM Cell Lysis Reagent (Sigma-Aldrich, St. Louis, MO). For the dual-luciferase reporter assay, 5 x 104 cells/well were transfected with different constructs as mentioned above. The assay was carried out after 48 h of transfection in SCC084 cells, using the Dual-Luciferase®Reporter Assay System (Promega, Madison, WI)49. The pRL-TK control vector, coding for Renilla luciferase, was co-transfected for normalizing the transfection efficiency 49.
Protein lysates from cells and tissue samples were prepared using the CelLyticTM M Cell Lysis Reagent (Sigma-Aldrich, St. Louis, MO). The proteins were resolved on an SDS-PAGE and then transferred to a PVDF membrane (Pall Corp., Port Washington, NY). The signal was visualized using an appropriate antibody and the ImmobilonTM Western chemiluminescent HRP substrate (Milipore, Billerica, MA). The anti-β-actin antibody (Cat# A5441) purchased from Sigma-Aldrich (St. Louis, MO) was used as a loading control. Antibodies such as anti-TSC1 (Cat# 6935S), anti-TSC2 (Cat# 3612), anti-phospho-p70S6 Kinase (Thr389) (Cat# 9205), anti-p70S6 Kinase (49D7) (Cat# 2708), anti-phospho-ULK1 (Ser757) (Cat# 6888) and anti-SQSTM1/p62 (Cat# 5114) were purchased from Cell Signalling Technologies (Danvers, MA). The anti-mouse HRP-conjugated secondary antibody (Cat# HP06) and anti-rabbit HRP-conjugated secondary antibody (Cat# HP03) were purchased from Bangalore GeneiTM (Bangalore, India). The anti-phospho-TSC2 (S939) (Cat# ab52962) was purchased from Abcam (Cambridge, MA).
Cell proliferation assay
CHEMICON BrdU Cell Proliferation Assay Kit (Milipore Corporation, Billerica, MA) was used to determine cell proliferation50. To this end, 2,000 cells were seeded in 96-well plates and transiently transfected with different constructs. The BrdU label was added at 2, 4, or 6 days and incubated for 20 h in a humidified CO2 incubator. The remaining protocol was followed as per the manufacturer’s instructions, and absorbance was measured at 450 nm using Infinite® 200 PRO Plate reader (Tecan Group Ltd, Mannedorf, Switzerland).
The CaspGLOWTM fluorescein active caspase-3 staining kit (Biovision, Mountain View, CA) was used to quantify the apoptosis of cells transfected with the appropriate constructs49. After transfection, FITC-DEVD label was added to cells, and rest of the steps were followed as per the protocol. The fluorescence intensity was measured, using Infinite® 200 PRO Plate reader (Tecan Group Ltd, Mannedorf, Switzerland).
Soft agar colony forming assay
The ability of cells to grow independently of solid surface, also known as anchorage independent growth, was assessed by the number of colonies formed in soft agar 49,50. After transfecting cells with appropriate constructs, they were harvested and 5,000 cells were plated in 1 ml of 0.35% noble agar (Difco, Mumbai, India) diluted with culture media in a 35 mm dish. After 21 days, colonies were counted and imaged using Leica Inverted Microscope DMi1 (Leica Microsystems, Wetzlar, Germany).
Cell invasion assay
The Corning® BioCoat™ Matrigel® Invasion Chamber was used for analyzing the invasion of cells (Corning Inc., Corning, NY) after transfection with different constructs49. Next, 50,000 cells were added to each transwell coated with Matrigel membrane (upper chamber) and placed in a chamber of 24-well plate containing 0.75 ml of culture media supplemented with 10% fetal bovine serum. Cells were allowed to invade by incubating them for 36 h in a humidified CO2 incubator. The non-invading cells were removed from the inside of transwell inserts using a cotton swab, and the invading cells were fixed using methanol and stained with 0.01% crystal violet (Sigma-Aldrich, St. Louis, MO)49. Cells were then imaged using Leica Inverted Microscope DMi1 and the number of cells invaded was estimated by counting in three random microscopic fields under a 10X objective50.
Nude mouse xenograft model
To analyze the effect of miR-130a-mediated targeting of TSC1 on tumor growth, 2 x 106 SCC131 cells were transfected with 1,200 nM of antagomiR-130a (miR-130a inhibitor) or 1200 nM of mock (scrambled oligos) separately. After 24 h of transfection, cells from both groups were suspended separately in 150 μl of incomplete DMEM and then injected into the left flank of a female BALB/c athymic 5-week-old nude mouse subcutaneously49. Tumor growth was monitored by measuring its volume using a digital caliper every 3 days until 26 days49. The following equation was used to measure the tumor volume (V): V = (W2 X L)/2, where L and W represent length and width, respectively49,50. Excised tumors were weighed at the end of 26 days. Data is an average of three nude mice. miRIDIAN microRNA hsa-miR-130a Hairpin Inhibitor/AntagomiR-130a (Cat# IH-300598-05-0005) and miRIDIAN microRNA Hairpin Inhibitor Negative Control #1/Mock (Cat# IN-001005-01-05) were purchased from Dharmacon (Lafayette, CO). The experiment was performed with approval from the animal ethics committee of the Indian Institute of Science, Bangalore. All experiments were carried out in accordance with relevant guidelines and regulations. The study was carried out in compliance with the ARRIVE guidelines.