MicroRNA-644a promotes apoptosis of hepatocellular carcinoma cells by downregulating the expression of heat shock factor 1

Low miR-644a expression negatively correlates with HSF1 expression, tumour diameter and TNM stages in HCC

We performed in situ hybridization (ISH) and qRT-PCR analysis of miR-644a levels in surgically resected cancer and peri-cancerous liver tissues from 135 HCC patients. We observed lower expression of miR-644a in HCC tissues than in adjacent peri-cancerous liver tissues (P < 0.01, Fig. 1A–B). We also observed decreased miR-644a expression in HCC tissues (Fig. 1E).

Immunohistochemical analysis revealed higher HSF1 expression in HCC tissues than in adjacent peri-cancerous tissues (P < 0.01, Fig. 1C–D). Moreover, miR-644a expression was negatively correlated with HSF1 levels in HCC (Fig. 1F). Therefore, we postulated that miR-644a negatively regulates HSF1 expression.

Prognostic significance of miR-644a expression in HCC patients

Correlation between miR-644a and HSF1 expression in HCC cell lines

We verified the correlation between miR-644a and HSF1 by analysing their expression in multiple HCC cell lines and normal hepatocytes by qRT-PCR and western blotting, respectively. QRT-PCR analysis showed that miR-644a expression was lower in HepG2 and SMMC-7721 cells than in normal hepatocytes, L-O2, and the HCC cell lines sk-Hep1, MHCC-97 L and QGY-7701 (P < 0.05; Fig. 2A). Conversely, the mRNA and protein levels of HSF1 were higher in HepG2 and SMMC-7721 cells than in L-O2, sk-Hep1, MHCC-97 L and QGY-7701 cells (P < 0.05, Fig. 2B–C). Spearman’s correlation analysis showed that miR-644a levels were negatively correlated with HSF1 mRNA levels (P < 0.05, Fig. 2D).

High miR-644a levels inhibit proliferation and promote apoptosis of HCC cells

Next, we investigated the role of miR-644a in proliferation and survival of HCC cells by transfecting miR-644a mimics into HepG2 and SMMC-7721 cells (Fig. 3A). The CCK-8 assay showed that miR-644a over-expression decreased the proliferation of HepG2 and SMMC-7721 cells more than in the corresponding controls (Fig. 3B). We further analysed the effects of miR-644a over-expression on apoptosis of HepG2 and SMMC-7721 cells by Annexin V/PI double staining. We observed that HepG2 and SMMC-7721 cells transfected with miR-644a mimics showed increased apoptosis compared with the corresponding controls (Fig. 3C). These results suggest that miR-644a negatively regulates proliferation and apoptosis of HCC cells.

MiR-644a down-regulates HSF1 expression by binding to the HSF1 3’UTR

TargetScan and miRanda analysis showed a putative miR-644a binding site in the 3’-UTR of HSF1 (Fig. 4A). Therefore, to prove that HSF1 was the target gene of miR-644a, we analysed HSF1 mRNA and protein levels in HepG2 and SMMC-7721 cells transfected with miR-644a mimics or miR-644a inhibitors. We observed that the miR-644a inhibitor increased HSF1 mRNA and protein expression, whereas the miR-644a mimic decreased HSF1 mRNA and protein levels in HepG2 and SMMC-7721 cells (Fig. 4B). Then, we investigated the correlation between miR-644a and HSF1 by using a dual luciferase reporter assay after co-transfecting HepG2 and SMMC-7721 cells with the luciferase reporter vector containing wild-type (wt) or mutant (mut) HSF1–3’UTR segments and miR-644a mimic or control miRNA. We observed a decreased relative luciferase activity in HCC cells co-transfected with miR-644a mimic and wild-type HSF1 3’UTR, whereas relative luciferase activity was normal in cells co-transfected with mutant HSF1 3’UTR and miR-644a mimics (Fig. 4D–E). These results showed that miR-644a down-regulates HSF1 expression.

HSF1 promotes proliferation and survival of HCC cells

Next, we analysed the proliferation, colony formation and apoptosis of HCC cells transfected with HSF1-siRNA. We observed that HSF1 silencing decreased proliferation and colony formation (Fig. 5A–B), but increased cellular apoptosis (Fig. 5C). Since miR-644a negatively regulates HSF1 expression, we postulated that up-regulation of HSF1 would increase proliferation and survival of HCC cells. Therefore, we over-expressed HCC cells with miR-644a mimic and wild type HSF1 or mutant HSF1. We observed that over-expression of wild type HSF1 increased HCC cell proliferation and colony formation (Fig. 6A–B) and decreased apoptosis (Fig. 6C). These data suggest that HSF1 up-regulation reverses the inhibitory effects of miR-644a in HCC cells.

The miR-644a/HSF1 axis modulates expression of BH3 only apoptotic proteins

To determine the role of the miR-644a/HSF1 axis in HCC, we performed protein chip analysis of apoptosis-related proteins in control and HSF1-siRNA transfected SMMC-7721 cells. HSF1 silencing resulted in changes in the expression of apoptosis-related proteins (Fig. 7A). Furthermore, HSF1 silencing up-regulated many apoptosis-related signalling pathways such as BH3-only apoptotic proteins, DNA damage signalling and HDAC class III regulated apoptotic proteins (Fig. 7B). The BH3-only apoptotic proteins were the most enriched apoptotic pathway (Fig. 7B). These results suggest that the miR-644a/HSF1 axis regulates apoptosis by modulating the expression of BH3-only proteins.

Next, we transfected SMMC-7721 cells with miR-644a mimics to down-regulate HSF1 expression and analysed the expression of BH3-only proteins by western blotting. We observed decreased expression of HSF1, HSP90, HSP60, Bcl-2, and Bcl-xL proteins and increased expression of BID, BAD, BIM, SMAC, Apaf-1 and cleaved caspases-3 and -9 (Fig. 7C). We further analysed the expression of Bcl-xl and BID in 135 HCC and adjacent normal liver patient tissues. We observed higher Bcl-xL and lower BID expression in HCC tissues than in adjacent normal liver tissues (Additional file 1: Figure S1D & Additional file 1: Figure S1E). BID expression was inversely correlated with Bcl-xL levels in HCC tissues (Additional file 1: Figure S1C). In addition, we confirmed that the BID and Bcl-xL expression levels in the xenograft model were consistent with that of the cell models (Additional file 2: Figure S2A & Additional file 2: Figure S2B). These results suggest that the miR-644a/HSF1 axis regulates BH3-only proteins in the apoptotic pathway.

MiR-644a inhibits in vivo xenograft tumour growth by down-regulating HSF1

To verify the effects of miR-644a on HCC cell proliferation in vivo, we generated stably transfected SMMC-7721 and HepG2 cell lines with miR-644a mimics and control miRNA. Then, we injected these cells into the right groin region of BALB/c nude mice and followed the tumour growth. We sacrificed the nude mice on day 35 and harvested the tumours and weighed them. We observed decreased tumour growth from HCC cells transfected with the miR-644a mimic than in the control groups (Fig. 8A–B). Subsequently, we observed low expression of HSF1 in the miR-644a mimic group tumours by IHC and western blotting (Fig. 8C–D). These results demonstrate that miR-644a inhibits HCC growth and survival by down-regulating HSF1. Thus, our results show that miR-644a inhibits HCC tumourigenesis by inhibiting HSF1 expression and promoting expression of BH3-only proteins (Fig. 9).

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.