Hsa-microRNA-27b-3p inhibits hepatocellular carcinoma progression by inactivating transforming growth factor-activated kinase-binding protein 3/nuclear factor kappa B signalling

Patients and tissue specimens

A total of 71 pairs of HCC and ANTs from January 2015 to December 2016 were collected from patients with HCC who underwent hepatectomy at the Hepatic Surgery Center, Tongji Hospital of Huazhong University of Science and Technology (HUST) (Wuhan, China). HCC diagnosis was confirmed by histopathology. The median follow-up period was 27 months (range, 1–45 months). Forty-three pairs of snap-frozen HCC specimens were used to obtain total RNA and protein. All procedures were approved by the Ethics Committee of Tongji Hospital, HUST, and conducted according to the principles of the Declaration of Helsinki. Prior written and informed consent was obtained from each patient.

Cell lines and culture

The HCC cell lines HLF, MHCC97H and HCCLM3 were obtained from the Liver Cancer Institute, Zhongshan Hospital, Fudan University, as previously described [34]. The human normal liver cell line WRL68 (PWE-HU129) was purchased from Meilune Biological Technology Co. (Dalian, China). The HCC cell lines Hep3B (GDC0070) and Huh7 (GDC0134) were purchased from the China Center for Type Culture Collection (CCTCC, Wuhan, China). The HCC cell line PLC/PRF/5 (TCHu119) was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). For all cell lines, short tandem repeat (STR) analysis was performed to confirm cell identity. All cell lines were maintained in Dulbecco’s modified Eagle medium (DMEM) (HyClone, UT, USA) with 10% foetal bovine serum (FBS) (Gibco) at 37 °C in a 5% CO2 humid atmosphere.

ISH and IHC analysis

ISH was performed using the ISH Kit (Boster, Bioengineering Company, Wuhan, China). All procedures were performed following the manufacturer’s instructions. Samples were stained with haematoxylin, dehydrated with alcohol, washed with xylene and sealed with flavour sealing tablets. Oligo (5′ digoxin-ACAAAGTTCTGTAGTGC-ACTGA) was used as an ISH probe for hsa-miR-27b. IHC was performed as described previously [35]. The dilutions of antibodies for the IHC procedure are listed in Additional file 1: Table S1. Representative images of ISH and IHC were captured and processed using a DM2300 microscope and ScopeImage 9.0 software (Nanjing Jiangnan Novel Optics Co., Ltd., China). ISH and IHC staining scores were independently determined by three pathologists without prior knowledge of patient information. The overall scores were defined by multiplying the percentage of positive cells by the staining intensity score as described previously [35].

RNA isolation and qRT-PCR

Total RNA from human specimens and cancer cells was extracted by TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. We used a reverse-transcription system (Vazyme, Nanjing, China) and miRcute Plus miRNA First-Strand cDNA Synthesis (Tiangen, Beijing, China) kits to generate cDNA of mRNA and miRNA, respectively. Relative quantification of hsa-miR-27b and the mRNA expression levels were determined by a miRcute Plus miRNA qPCR Detection Kit (Tiangen, Beijing, China) and SYBR Green PCR kit (Vazyme, Nanjing, China) according to the manufacturers’ protocol. GAPDH and U6 small nuclear RNA were used as internal controls for mRNA and miRNA, respectively. We used the comparative CT (2−∆∆CT) method to perform relative quantification analysis. The primers used in this study are listed in Additional file 1: Table S2.

Cell transfection and infection

HCC cells were seeded in six-well plates at 70% density and cultured overnight. Hsa-miR-27b mimics and inhibitors and their respective NC (RiboBio, Guangzhou, China) were transfected with Lipofectamine 3000 (Invitrogen, USA). Small interference RNA (siRNA) targeting TAK1 was used to transiently knock down its expression in HCC cells. si-NC: 5′-UUGUACUACACAAAAGUACUG-3′; si1-TAK1: 5′-GGUAGUAAUUACAGUGAAA-3′; si2-TAK1: 5′-CCCGTGTGAACCATCCTAATA-3′.

Lentiviruses for stably overexpressing or knocking down TAB3 were generated by Shanghai GeneChem Co., Ltd. (Shanghai, China) and used to infect the indicated cells for 48 h. Transfected cells were then selected by culturing in medium containing 2.5 μg/ml puromycin for 2 weeks. The small hairpin RNA (shRNA) target sequences for knocking down the indicated genes were as follows: scramble: 5′-GCCTAAGGTTAAGTCGCCCTCG-3′; sh-TAB3: 5′-CCTCCTTCATACATGCACATA-3′.

CCK-8 assays

Cells were seeded into 96-well plates at 1500 cells per well and incubated at 37 °C. The culture medium was changed to 100 μl of 10% CCK-8 (Dojindo, Kumamoto, Japan) solution at the indicated time and incubated in a cell incubator for 2 h. Optical density (OD) was measured by a Universal Microplate Reader ELx 800 (Bio-Tek, USA) at a wavelength of 450 nm. For each group, the absorbance values were determined by five replicates. After extraction of the blank value, the average gross OD values were used for data analysis.

Transwell assays

Transwell chambers (8 μm pore size, Corning, NY, USA) were used for cell migration assays. In Transwell migration assays, HCC cells suspended in 200 μl of serum-free DMEM (HyClone, UT, USA) were added to the upper chambers, and the lower chambers were filled with 500 μl of DMEM with 10% FBS. In the Transwell invasion assay, before cells were seeded in the upper chamber, chambers were coated with Matrigel (BD Biosciences, NJ, USA) for 3 h. Following 24 h (migration assays) or 48 h (invasion assays) of incubation at 37 °C, the migrated or invaded cells were fixed with 4% paraformaldehyde and stained with 1% crystal violet solution. Stained cells were counted in three random microscopic fields per well by a Nikon Digital ECLIPSE C1 system (Nikon Corporation).

F-actin assay

The indicated cells were seeded in 96-well plates at a density of 1000 cells per well and cultured overnight. Cells were then fixed with 4% paraformaldehyde for 15 min at room temperature. After two washes with phosphate-buffered saline (PBS), 100 µl of Alexa Fluor 555-conjugated phalloidin (Life Technologies, USA) was added into each well for 1 h at room temperature. PBS washes were performed twice, and the nuclei were counterstained with DAPI. The cells were then observed using the EVOS FL auto-imaging system (Life Technologies, USA).

Dual-luciferase reporter assay

The DNA sequence containing the predicted binding site with hsa-miR-27b in the 3′-untranslated region (3′UTR) of TAB3 or the 3′UTR-TAB3 mutant was cloned into the psiCHECK-2-vector (Promega, Madison, WI, USA). Approximately 1 × 105 cells per well were seeded in 24-well plates. After 24 h, the recombinant plasmid psiCHECK-2-3′UTR-TAB3 or psiCHECK-2-3′UTR-TAB3 mutant was co-transfected into cells with hsa-miR-27b mimics, hsa-miR-27b inhibitors or their respective NC using Lipofectamine 3000 (Invitrogen, USA). Total cell protein was extracted with Passive Lysis Buffer (Promega, Madison, WI, USA), and luciferase activity was determined using the Dual-Luciferase Reporter 1000 Assay System (Promega, Madison, WI, USA) with a GloMax 20/20 luminometer (Promega, Madison, WI, USA). Renilla luciferase values were normalized against firefly luciferase activity, and the ratio of Renilla-to-firefly luciferase activity is presented. For NF-кB signalling activation analysis, NF-кB luciferase reporter plasmid containing the minimal promoter with multiple tandem NF-кB binding sites (pNF-кB-Luc, Clontech, Palo Alto, CA, USA) and its control vector (pTAL-Luc, Clontech) were transfected into HCC cells by Lipofectamine 3000 (Invitrogen, USA). The indicated cell lines were treated with TNF-α (10 ng/ml) (MCE, USA).

Immunofluorescence staining

HCC cells were cultured on sterile coverslips in 24-well plates and treated with 20 ng/ml TNF-α (MCE, USA) for 10 min. After treatment, the cells were fixed in 4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.5% Triton X-100 solution for 10 min and incubated with primary antibody at 4 °C overnight. Alexa Flour 488-conjugated anti-rabbit IgG and Alexa Flour 555-conjugated anti-mouse IgG (Beyotime Institute of Biotechnology, Shanghai, China) were used to incubate cells for 1 h at 37 °C. Nuclei were stained with DAPI (Wuhan Goodbio Biotechnology Co., Ltd., Wuhan, China). Images were captured by an EVOS FL auto imaging system (Life Technologies, USA).

Animal experiments

All BALB/c nude mice were purchased from Beijing HFK Bioscience Co., Ltd., and maintained under specific-pathogen-free conditions. For the xenograft tumour model, 1 × 106 indicated tumour cells were suspended in 100 μl of serum-free DMEM and inoculated subcutaneously into the flanks of 4-week-old nude mice. All experimental mice were monitored for 30 days and sacrificed to compare the volume and weight of subcutaneous tumours. For the in vivo metastasis assays, 1 × 106 cells in 100 μl of serum-free DMEM were injected into the tail vein of nude mice. After 2 months, the nude mice were sacrificed, and the lungs were dissected. All of the metastatic foci in the lung were calculated to evaluate the development of pulmonary metastasis.

For the OncoAd-mediated treatment of liver cancer, Hep3B tumour xenografts were established by subcutaneous inoculation of 5 × 105 cells into the right flank of 4-week-old nude mice. Twenty-one days after inoculation, the animals were randomly divided into three groups (n = 7 mice per group) and intra-tumourally injected with either PBS solution, OncoAd NC or OncoAdhsa-miR-27b (diluted in PBS) at multiplicities of infection (MOIs) of 5 × 108. The injections were performed once every other day for a total of four injections. Tumours were measured every 3 days, and volume was calculated by the following formula: volume = (length × width2)/2.

Recombinant adenovirus production

OncoAdhsa-miR-27b and OncoAd NC were constructed by Shanghai GeneChem Co., Ltd. (Shanghai, China). The u6-hsa-miR-27b cassette was cloned into the oncolytic adenoviral vector with the E1B 55-KD gene deletion. The recombinant adenovirus was transfected into 293T cells for amplification. The viral particles were collected, and the titre was detected according to the manufacturer’s operating instructions after purification.

Cytotoxicity assay

Hep3B and WRL68 cells were used for cytotoxicity assays in 96-well or 24-well plates by CCK-8 assays or crystal violet staining. Briefly, cells were pre-seeded in 96-well plates and cultured overnight. OncoAd at the indicated MOIs was used to treat cells for 24 h. Cell viability was measured by CCK-8 assays. Additionally, cells were prepared in the same manner in 24-well plates and cultured for 48 h. Then, the cells were fixed with 4% paraformaldehyde and stained with crystal violet staining solution.

Nuclear and cytoplasmic extraction assay

A total of 1 × 106 HCC cells were collected in a 1.5 ml microcentrifuge tube and then centrifuged at 500g for 5 min. The cells were washed by suspending the cell pellet with PBS. The supernatant was removed, leaving the cell pellet as dry as possible. Cytoplasmic and nuclear fraction protein extraction was performed using an NE-PER nuclear and cytoplasmic extraction reagent kit (Thermo Fisher Scientific, USA) according to the instructions.

In silico analysis

The miRNA expression profiles were retrieved from The Cancer Genome Atlas (TCGA) database (https://tcga-data.nci.nih.gov/tcga/) and the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/gds). Normalization and batch effect elimination were carried out for the two gene datasets using the Limma package in R. Statistical significance was calculated using the Kruskal–Wallis test. The correlation between the survival time and TAB3 expression in patients with HCC was assessed using the GEPIA website (http://gepia.cancer-pku.cn/). The miRDB (http://mirdb.org/miRDB), miRWalk (http://www.umm.uniheidelberg.de/apps-/zmf/mirwalk/) and TargetScan (http://www.targetscan.org/vert_71/) databases were used for miRNA target‐gene prediction.

EdU incorporation assays

HCC cells were seeded in 96-well plates at a density of 1000 cells per well and incubated overnight. Then, the cells were transfected with the indicated mimics or inhibitors for 72 h and subjected to an EdU incorporation assay using a Cell-Light EdU Apollo 567 In Vitro Imaging Kit (RiboBio, Guangzhou, China) according to the manufacturer’s instructions.

RIP assay

We performed RIP using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Darmstadt, Germany) following the manufacturer’s instructions. A detailed description was published previously [35].

Co-immunoprecipitation (co-IP)

The experiments were performed as described previously [36]. Briefly, a total of 1 × 107 cells were lysed with IP lysis buffer for 20 min at 4 °C (IP-lysis buffer: 50 mM Tris–HCl, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10% glycerol and protease inhibitor cocktail, pH 7.4) and then centrifuged at 13,000 rpm for 10 min. The supernatant was incubated with 30 μl of pre-cleared protein G-conjugated agarose (GE Healthcare Life Sciences) for 4 h at 4 °C. The indicated antibodies were added into cleared supernatants overnight at 4 °C. One per cent of the lysate fraction was reserved as an input. The next day, 25 μl of protein G-conjugated agarose was used to precipitate the antibody–protein mixture at 4 °C for 4 h. The beads were then washed five times in IP wash buffer (50 mM Tris–HCl, 300 mM NaCl, 1% Triton X-100 and 1 mM EDTA, pH 7.4) to remove unbound antibodies. The eluate was separated from the beads by heating at 95 °C for 10 min.

Statistical analyses

We used SPSS 19.0 (IBM, Chicago, IL, USA) or Prism 6.0 (GraphPad Software, La Jolla, CA, USA) software to analyse the data. Student’s t-test (two-tailed) or analysis of variance (ANOVA) test was used to compare tests under the assumptions of normality and the equality of variance. Otherwise, non-parametric tests (Mann–Whitney U-test or Kruskal–Wallis) were used. Categorical data were analysed by χ2 test or Fisher’s exact test. Kaplan–Meier analyses were used to assess the survival between subgroups (log-rank test). A Cox proportional hazards model was used to determine the independent factors of survival based on the variables selected in the univariate and multivariate analyses. The results are presented as mean ± standard deviation (SD). p-Values less than 0.05 were considered statistically significant.

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