HCC patient samples
We obtained 135 HCC patient tissue specimens and their corresponding clinical data from the Department of Hepatobiliary and Pancreatic Surgery and Department of Pathology, Affiliated Hospital of Guilin Medical University, China from 2010 to 2016. The clinical data were reviewed and approved by the Ethics Committee of Guilin Medical University. We obtained written informed consent from all of the patients in accordance with the Declaration of Helsinki. The clinicopathological data included gender, age, tumour diameter, tumour differentiation, and clinical TNM staging of the HCC patients; all patients were classified and graded based on the pTNM classification advocated by the International Union against Cancer and were followed up until June 2017.
In situ hybridization (ISH)
The in situ hybridization assay to analyse miR-644a expression in HCC tissue samples was performed according to protocols described previously [31]. The miR-644a antisense oligonucleotide probes were obtained from Exiqon, Inc. (Woburn, MA, USA).
Immunohistochemistry
The 135 pairs of resected HCC and para-cancerous tissues were fixed overnight with 4% paraformaldehyde, embedded in paraffin and cut into 4 mm thick sections. Immunohistochemical staining was performed with an Envision IHC kit (Maxin Biotechnologies Inc., Fuzhou, Fujian, China). Briefly, the tissue sections were dewaxed and hydrated, followed by antigen retrieval with citrate buffer for 15 min at 100 °C in a microwave oven. The sections were incubated with the primary antibody against HSF1 (Santa Cruz Biotechnology, Inc., USA) at room temperature for 1 h at a dilution of 1:200 and were visualized using the UltraVision Quanto Detection System HRP DAB kit (Thermo Scientific) according to the manufacturer’s protocols. The stained sections were counterstained with haematoxylin and then developed according to the manufacturer’s instructions and scored using an Olympus X71 microscope. Based on the staining intensity, samples were divided into the following grades: 0: < 10% positive staining HCC cells; 1+: 11–25% positive staining HCC cells; 2+: 26–50% positive staining HCC cells; 3+: > 50% positive staining HCC cells. IHC and scoring analysis were performed independently by two investigators.
Western blotting
Total protein lysates were prepared from HCC cells and tissues in lysis buffer (50 mM Tris–HCl, 137 mM NaCl, 10% glycerol, 100 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mg/ml aprotinin, 10 mg/ml leupeptin, 1% Nonidet P-40, and 5 mM protease inhibitor cocktail; pH 7.4), and the protein concentration was measured by a BCA (bicinchoninic acid) protein assay (Beyotime, Inc., Shanghai, China). Equal amounts of protein lysates were separated on 10% SDS-PAGE at 100 mV for 2 h. Then, the separated proteins were transferred onto PVDF membranes at 80 mV for 1 h. The blots were first blocked with 5% nonfat milk, followed by incubation with primary antibodies overnight at 4 °C. The blots were then incubated with HRP-conjugated secondary antibody at room temperature for 1 h. Then, the blots were developed by an ECL chemiluminescence method and the specific protein bands were quantified with ImageJ software. β-Actin was used as an internal control. Antibodies against HSF1, caspase 3, caspase 9, BAD, Bcl-2, Bcl-xL, BIM and BID were purchased from Santa Cruz (Dallas, Texas, USA). Antibodies against β-actin, HSP90, HSP60, SMAC and Apaf-1 were purchased from Origene (Rockville, MD, USA).
Quantitative reverse transcription polymerase chain reaction (qRT-PCR)
Total RNA was extracted from HCC cells and tissues with TRIzol (Thermofisher Inc., Grand Island, NY, USA) and an RNAiso™ Plus kit (Takara, Japan) according to the manufacturer’s instructions. The RNA concentration was determined in a Beckman spectrophotometer (Beckman Coulter, USA). Reverse transcription was performed with the Fast Quant first strand cDNA synthesis Kit (TIANGEN, China). Then, we performed real-time PCR with Fast Start Universal SYBR Green Master Mix (Roche Diagnostics GmbH Mannheim, Germany) in an Applied Biosystems real-time PCR machine (Thermo Fisher Scientific, USA).
Cell culture and transfections
We obtained HCC cells (HepG2, SMMC-7721, sk-Hep1, MHCC-97 L, and QGY-7701) and normal liver cells (L-O2) from the Chinese Academy of Sciences cell bank (Shanghai, China). HepG2, sk-Hep1 and MHCC-97 L were grown in Dulbecco’s modified Eagle medium (Thermo Fisher Scientific, South America), and SMMC-7721, QGY-7701 and L-O2 cells were grown in RPMI-1640 medium (Thermo Fisher Scientific) supplemented with 10% foetal bovine serum (Thermo Fisher Scientific) at 37 °C and 5% CO2. The transfection of miR-644a mimic and HSF1-siRNA were prepared and used according to a previously described protocol [32].
CCK-8 cell proliferation assay
HCC cells were seeded in 96-well plates (3 × 103 cells in 100 μl per well). At different time points, 10 μl of cell counting kit-8 (CCK-8) solution (Dojindo, Shanghai, China) was added and incubated for 1–3 h. Absorbance was read at 450 nm in a microplate reader (Spectramax plus384, Molecular Devices, USA).
Flow cytometry analysis of apoptosis
After various treatments, HCC cells were washed twice with 1× PBS and centrifuged. Then, 100 μl of FITC-conjugated Annexin-V and PI (Sigma, USA) was added and the cells were incubated for 20 min at room temperature in the dark. Then, the cells were subjected to flow cytometric analysis (Merck Millipore, Germany). The percentage of apoptotic cells (Annexin V+ PI+) was determined by using FlowJo software 7.6 (Treestar, USA).
Colony formation assays
We seeded 600 HCC cells per well in a six-well plate for approximately two weeks in 10% DMEM or RPMI medium supplemented with 10% foetal bovine serum. Then, the colonies were fixed with 4% paraformaldehyde for 20 min and stained with 1% crystal violet (G1062, Solarbio, Japan) overnight. After washing the cells three times, the total number of colonies (> 10 cells per colony) per well was determined for all conditions. Images were also captured for documentation.
Dual luciferase reporter assay
We identified the miR-644a target site in the HSF1–3’UTR with TargetScan 6.2 (http://www.targetscan.org/vert_71/). We then designed primers to generate the mutant and wild type HSF1 based on the HSF1 mRNA sequence in NCBI GenBank. We PCR amplified the miR-644a target sequence in HSF1 from the total RNA from the HCC cells. The mutant version of the HSF1 3’UTR was generated by overlapping PCR with mutagenic primers. We cloned both wild-type and mutant HSF1–3’UTR sequences into the pMIR-REPORT-basic vector (Applied Biosystems, USA) and confirmed the products by DNA sequencing. For the luciferase reporter assay, we seeded 3 × 103 cells in 24-well plates and co-transfected 100 ng of the luciferase reporter vector with 20 nM miR-644a mimic or miRNA negative control into SMMC-7721 cells. The cells were lysed after 48 h, and the dual luciferase activities were determined using the luminescence reporter gene assay system (PerkinElmer, Norwalk, CT, USA) according to the manufacturer’s instructions.
Generation of stable miR-644a over-expressing HCC cell lines
We purchased lentivirus pEZX-MR04 plasmid clones with miR-644a mimics and negative control miRNA from GenePharma (Shanghai, China) and transfected them into HEK293T cells with EndoFectin Lenti transfection reagent (GeneCopoeia, Rockville, USA). The cells were centrifuged after 48 h at 10000 rpm for 10 min, and the lentiviral particles expressing miR-644a or negative control miRNAs in the supernatant were concentrated by ultrafiltration. Then, SMMC-7221 cells were infected with lentiviruses expressing miR-644a or negative control miRNAs in the presence of 2 μg/ml puromycin and cultured for 2 weeks to obtain stably transfected cells. The expression of miR-644a in the stably transfected control and miR-644a overexpressing SMMC-7221 cells was determined by qRT-PCR.
Xenograft nude mice model of in vivo tumourigenesis
We purchased forty 5–6-week-old male BALB/c nude mice from the Animal Experimental Center of Guilin Medical University. All animal experiments were approved by the Animal Care and Use Committee of Guilin Medical University. We randomly divided the nude mice into two groups and injected them subcutaneously into the right inguinal region with 200 μl (2 × 107) of SMMC-7221 cells that were stably transfected with either empty plasmid (control) or miR-644a mimic (experimental). Tumour growth was recorded at 7, 14, 21, 28 and 35 days. Then, the mice were sacrificed by cervical dislocation on day 35; the tumours were harvested by resection and weighed.
Protein chip analysis
We incubated the protein samples isolated from miR-644a overexpressing and control SMMC-7221 cells with the antibody chip AAH-APO-G1–8 (RayBiotech, Norcross, GA, USA). Then, the unbound proteins were removed by washing and the bound proteins were analysed by fluorescence scanning. We determined the differential expression of the proteins and performed clustering analysis with Cluster v3.0 software (Stanford University, USA). Then, we annotated the differentially expressed proteins and determined the molecular networks by gene ontology and KEGG enrichment analysis.
Statistical analysis
The statistical analysis was performed with GraphPad Prism 5 (GraphPad Software, Inc., San Diego, CA, USA) and SPSS v.18.0 (SPSS Inc., Chicago, IL, USA). The relationship between miR-644a expression and clinicopathological data was evaluated by the chi-square test (χ2). Quantitative data was analysed by the paired t test. Kaplan-Meier analysis was performed to estimate the survival rate and prognosis of patients with HCC expressing high or low miR-644a and HSF1 levels. The logarithmic rank test was used to compare the survival curves between different groups. All experiments were repeated three times, and the data are expressed as the mean ± SD. P < 0.05 was considered statistically significant.
