Human tissue specimens and cell lines
The study cohort comprised a consecutive series of 29 patients with thyroid cancer resected for cure at the First Hospital of Hunan University of Chinese Medicine from April 2015 to January 2016. With the formal consent form, we collected primary thyroid cancer specimens and adjacent noncancerous thyroid tissues from these patients by surgery before treatment and preserved them following the requirements of the experiments. Frozen specimens were used for MIR31HG, miR-761 and mitogen-activated protein kinase 1 (MAPK1) quantification as below, and formalin-fixed paraffin-embedded specimens were used for Ki67 staining by immunohistochemistry with a rabbit anti-Ki67 antibody (PA5-19462, Invitrogen, Basel, Switzerland) at a 1:200 dilution as described elsewhere . We collected the follow-up data of patients from the Registry of our hospital. Experimental protocol for human specimen collection and use was approved by the Ethics Committee of the First Hospital of Hunan University of Chinese Medicine.
We obtained human SW579 (thyroid squamous cell carcinoma) and TPC-1 (papillary thyroid cancer) cells from Procell (Wuhan, China) and HTH83 (anaplastic thyroid cancer) and Nthy-ori 3-1 (normal thyroid epithelial) cells from Bnbio (Beijing, China). We propagated the cells in the following media from Gibco (Paisley, UK): RPMI-1640 for TPC-1 and HTH83 cells, Leibovitz’s L-15 for SW579 cells, and DMEM for Nthy-ori 3-1 cells. All cells were grown at 5% CO2 at 37°C in media plus 10% FBS and 1% streptomycin/penicillin (all from Euroclone, Milano, Italy).
RNA preparation and quantitative real-time PCR (qRT-PCR)
To extract total RNA from cultured cells and tissue specimens, we applied a RiboPureTM RNA Kit (Invitrogen) based on the manufacturing recommendations. To prepare nuclear and cytoplasmic RNA of SW579 and TPC-1 cells, we employed a Cytoplasmic & Nuclear RNA Purification Kit from Norgen Biotek (Thorold, ON, Canada). For MIR31HG, MAPK1 mRNA and GAPDH mRNA analyses, cDNA was produced in a 10 µL of reaction volume containing 1 µg of RNA, 0.3 µg of oligo(dT)18 primer (TaKaRa, Dalian, China) and 100 U of M-MLV reverse transcriptase (Promega, Sydney, Australia); the cDNA in a 25 µL of reaction mixture was then amplified using VeriQuest SYBR Green (Affymeterix, Schwerte, Germany) and 10 pmol of each forward and reverse primers (Supplement Table 1). For miR-761 quantification, miScript RT Kit (Qiagen, Courtaboeuf, France), miScript SYBR Green PCR Kit (Qiagen) and specific primer for miR-761 (Supplement Table 1) were used in this study. A housekeeping gene β-actin or U6 was used to correct for differences in the amount of RNA in each sample. For relative quantification, we adopted the 2-ΔΔCt expression formula.
Plasmid, siRNA, miRNA mimic or inhibitor transfection
To silence MIR31HG in cells, we purchased Silencer® Select siRNA for MIR31HG (si-MIR31HG) from Thermo Fisher Scientific (Milan, Italy). To express miR-761 in cells, we transfected mirVana® miRNA mimic for miR-761 (Thermo Fisher Scientific) into cells. To knock down available miR-761 in cells, we obtained mirVana® miRNA Inhibitor for miR-761 (anti-miR-761) from Thermo Fisher Scientific and transfected it into cells. The siRNA-scramble (si-NC), miRNA-scramble (miR-NC), and inhibitor-scramble (anti-miR-NC) served as non-specific controls. To express MIR31HG or MAPK1 in cells, we cloned MIR31HG full-length sequence or MAPK1 coding sequence (lacking 3’UTR), synthesized by Abiocenter (Beijing, China), into the pcDNA3.1 vector (Thermo Fisher Scientific).
For transient transfection, we seeded SW579 and TPC-1 cells (100,000 cells/well) in 12-well culture dishes before transfection using Lipofectamine 2000 (Thermo Fisher Scientific) with plasmid (300 ng), miRNA mimic/inhibitor (50 nM) or siRNA (150 nM). We harvested the cells after 48 h for expression analysis and functional experiment.
MTS assay for cell viability
We plated SW579 and TPC-1 cells after the appropriate transfection into 96-well culture dishes at 5,000 cells per well and maintained them overnight at 37°C. Subsequently, each well received 10 µL of MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) solution as recommended by the manufacturers (BestBio, Shanghai, China). Following a 4-h incubation at 37°C, we measured the absorption by spectrophotometry (TECAN, Männedorf, Switzerland) at 490 nm.
5-Ethynyl-2’-Deoxyuridine (EdU) assay for cell proliferation
We incubated SW579 and TPC-1 cells after the appropriate transfection with EdU solution (50 µM, Yeasen, Shanghai, China) for 2 h before EdU staining with Apollo 488 (Ribobio, Guangzhou, China). Following the nuclei staining with 4’,6-diamidino-2-phenylindole (DAPI, Solarbio, Beijing, China), we analyzed the proliferation rate as the percentage of EdU positive cells (green) relative to total cells (blue) using a fluorescence microscope (Olympus, Hamburg, Germany).
Flow cytometry for cell apoptosis
We stained SW579 and TPC-1 cells (500,000 cells per sample) after the appropriate transfection with 25 µg/mL of Annexin V-FITC (Yeasen) and 50 µg/mL of propidium iodide (PI, MedChemExpress, Tokyo, Japan) in PBS. About 10,000 events were acquired in a FACS Aria III flow cytometer (BD Biosciences, North Ryde, Australia) and analyzed for percent cells undergoing apoptosis (Annexin V+/PI- and Annexin V+/PI+) with AccuriC6 software from BD Biosciences.
Transwell invasion assay
For evaluation of cell migration, we used 24-well, 6.5 mm internal diameter transwell plates (BD Biosciences) with Matrigel-coated membranes (8 µm pore size) separating the 2 chambers. SW579 and TPC-1 cells after the appropriate transfection were seeded in non-serum media in the upper chamber at 50,000 cells per well. 10% FBS medium served as chemoattractant in the lower chamber. 24 h post-seeding, non-invading cells were removed and invaded cells were fixed with methanol. After crystal violet (0.1%) staining, images were captured by Olympus CKX41 at 100× magnification and registered using the getIT software (Olympus).
Wound-healing assay for cell migration
We plated SW579 and TPC-1 cells after the appropriate transfection into 6-well dishes (500,000 cells/well) and cultured them until ~80% confluence. We created a scratch wound using a sterile plastic 200 µL pipette tip. Cell migration was photographed and measured by microscopy at 10× magnification.
For immunoblotting under standard methods , we isolated total protein using cold RIPA lysis buffer (Solarbio) from cultured cells and tissue specimens homogenized by a tissue grinder and separated it by SDS-PAGE, followed by blotting onto nitrocellulose membranes (Millipore, Oxen, UK). Antibodies against MAPK1 (sc-16472, Santa Cruz Biotechnology, Heidelberg, Germany), Cleaved-caspase-3 (ab32042, Abcam, Cambridge, UK), matrix metalloproteinase 9 (MMP9, ab137867, Abcam) and GAPDH (ab8245, Abcam) were used, which were visualized with IgG secondary antibody conjugated to HRP (ab97051 and ab97023, Abcam) and enhanced chemiluminescence (Millipore).
For prediction of miRNAs that potentially bind to MIR31HG, we used the computer algorithm LncBase Predicted v.2 (http://carolina.imis.athena-innovation.gr/diana_tools/web/index.php?r=lncbasev2/index-predicted). For prediction of miRNA-binding sites in human 3’UTRs, we employed the target prediction tool ENCORI (http://starbase.sysu.edu.cn/).
Dual-luciferase reporter assay
We obtained the fragments of MIR31HG and MAPK1 3’UTR encompassing the predicted miR-761 complementary sequence or mutated complementary seed region from Abiocenter and inserted them into a pmirGLO vector (Promega, Vienna, Austria). For luciferase assay, transfection experiments were done using Lipofectamine 2000 in SW579 and TPC-1 cells (100,000 cells/well) in 12-well dishes. The transfection mixture consisted of 200 ng of individual reporter construct and 30 nM of miRNA mimic. We harvested the cells at 48 h post-transfection for luciferase assay by Dual-luciferase Assay System (Promega).
Generation of stable MIR31HG depletion cell line
To generate TPC-1 cells stably expressing shRNA-MIR31HG (sh-MIR31HG), we obtained the lentivirus coding sh-MIR31HG from VectorBulider (Guangzhou, China). A shRNA-scramble (sh-NC, VectorBulider) served as a non-specific control. We transduced TPC-1 cells with the lentivirus and puromycin-selected to obtain stable cell lines.
All mouse studies adhered to protocols approved by the Animal Care and Use Committee of the First Hospital of Hunan University of Chinese Medicine. For formation of subcutaneous xenografts, we subcutaneously injected TPC-1 cells stably expressing sh-MIR31HG or sh-NC (5,000,000 cells/injection) in 150 µL PBS into the right flanks of male BALB/c nude mice aged 8-week (Beijing Vital River Laboratory Animal Technology Co., Ltd., Beijing, China). Measurement of xenograft growth was periodically conducted, and tumor volume was estimated using the formula 0.5 × (length × width2). All mice were sacrificed by anesthetic overdose on day 22, and the xenografts were harvested for weight and expression analysis. Sections of paraffin-embedded xenografts were processed by immunohistochemistry by staining with anti-Ki67 antibody (PA5-19462) as described elsewhere .
Unless otherwise noted, all experiments were repeated at least three times (in triplicate), with results presented as mean ± standard deviation. Two group means were analyzed using a Student’s t-test (two-tailed), and multiple group means were compared by two-way ANOVA, followed by the Tukey’s post hoc test. For analysis of overall survival of these patients, we used Kaplan-Meier method and log-rank test (for significance). For analysis of correlations of variables in tumor specimens, we employed Pearson’s rank correlation coefficient. Significance was defined as P < 0.05.