Patients with liver cancer (40–50 years old, male) were recruited from Henan Provincial People's Hospital (Zhengzhou, China) from January to May 2017 and divided into a TACE-untreated group (n=22) and a TACE-treated group (n=20). Necrotic parts of liver tumors were excluded from our study. The expression levels of MIAT, miR-203a and HIF-1α were assessed by reverse transcription-quantitative PCR (RT-qPCR) assay in liver tumors without necrosis.

Patients in the TACE-treated group received ≥1 session of TACE alone without other treatments prior to resection. Patients with liver cancer in the TACE-untreated group did not receive any treatment before surgery. The present experiments were approved by the Research Ethics Committee of Henan Provincial People's Hospital. Written informed consent was obtained from all participants prior to the experiments.

The liver cancer cell line HepG2 (Cell Bank of the Chinese Academy of Sciences) was cultured in Dulbecco's modified Eagle's medium (cat. no. 11965092; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (cat. no. 16140071; Thermo Fisher Scientific, Inc.) under normoxia (20% O₂) or hypoxia (1% O_2, 5% CO₂) conditions at 37°C. This cell line was derived from a 15 year-old Caucasian male with liver cancer. The cell line was authenticated by STR profiling. The results were as follows: D5S818: 11,12; D13S317: 9,13; D7S820: 10,10; D16S539: 12,12; vWA: 17,17; THO1: 9,9; Amelogenin: XY; TPOX: 8,9; and CSF1PO: 10,11. Normoxia or hypoxia was created using the Anoxomat Mark II Anaerobic system (Mart Microbiology B.V.).

Small interference (si)RNAs targeting MIAT (si-MIAT#1, si-MIAT#2) and a scramble control (si-NC), as well as siRNA targeting HIF-1α (si-HIF-1α) and its negative control (NC) (si-nc) were obtained from Shanghai GenePharma Co., Ltd. miR-203a mimic (cat. no. miR10000264-1-5) and its NC (miR-NC, cat. no. miR1N0000001-1-5), and miR-203a inhibitor (anti-miR-203a; cat. no. miR20000264-1-5) and its NC (anti-miR-NC; cat. no. miR2N0000001-1-5) were ordered from Guangzhou RiboBio Co., Ltd. The HIF-1α coding region was cloned into the pcDNA3.1 vector by Shanghai Genomeditech Co., Ltd. to obtain a pcDNA-HIF-1α overexpression plasmid. The target sequences of si-MIAT#1 and si-MIAT#2 were 5′-CCAGGCTCCTTTAAACCAA-3′ and 5′-GCAGTTCTTAGCTCATATA-3′, respectively. The target sequences of si-HIF-1α were 5′-GGCCGCTCAATTTATGAAT-3′ and 5′-GCTGGAGACACAATCATAT-3′.

HepG2 cells were transfected with corresponding biotin-labeled miRNAs, biotin-labeled probes, miRNA mimics, miRNA inhibitors, siRNAs or plasmids, and cultured for 24 h under normoxia or hypoxia conditions. Cell transfection was performed using DharmaFECT 4 reagent (cat. no. T-2004-0X) or DharmaFECT Duo transfection reagent (cat. no. T-2010-03) (both from Thermo Fisher Scientific, Inc.) according to the instructions of the manufacturer.

Total RNA was isolated from HepG2 cells and liver tumors using TRIzol reagent (cat. no. 15596018; Thermo Fisher Scientific, Inc.) following the protocols of the manufacturer. The miR-203a level was measured using Bulge-Loop miRNA qRT-PCR Starter kit (cat. no. C10211-1; Guangzhou RiboBio Co., Ltd.) and Bulge-Loop miRNA primer sets (cat. no. MQPS0000787-1-100; Guangzhou RiboBio Co., Ltd.) with small nuclear RNA U6 (cat. no. MQPS0000002-1-100; Guangzhou RiboBio Co., Ltd.) as the internal control. cDNA first strand synthesis reaction programs included reverse transcription at 42°C for 60 min and enzyme inactivation at 70°C for 10 min. Quantitative PCR reaction programs consisted of pre-denaturation at 95°C for 10 min and 40 cycles of denaturation at 95°C for 2 sec, annealing at 60°C for 20 sec and extension at 70°C for 10 sec.

For detection of MIAT, HIF-1α and GAPDH levels, cDNA was synthesized from an RNA template using M-MLV reverse transcriptase (cat. no. 28025021; Thermo Fisher Scientific, Inc.) and quantified using Fast SYBR™ Green Master mix (cat. no. 4385617; Thermo Fisher Scientific, Inc.). GAPDH functioned as the house-keeping gene to normalize the expression of MIAT and HIF-1α. The primer sequences for MIAT, HIF-1α and GAPDH were as follows: HIF-1α forward, 5′-CCACCTATGACCTGCTTGGT-3′ and reverse, 5′-TGTCCTGTGGTGACTTGTC-3′; MIAT forward, 5′-CAAAGAGCCCTCTGCACTAG-3′ and reverse, 5′-ACCTTGGTTACCCCTGTGATG-3′; and GAPDH forward, 5′-GTCTCCTCTGACTTCAACAGCG-3′ and reverse, 5′-ACCACCCTGTTGCTGTAGCCAA-3′. Reverse transcription reaction samples without M-MLV reverse transcriptase and ribonuclease inhibitor were incubated at 65°C for 5 min and then immediately chilled on ice. Next, the reverse transcription reaction was performed at 37°C for 50 min and the reaction was subsequently inactivated at 70°C for 15 min. Quantitative PCR reaction programs included pre-denaturation at 95°C for 5 min and 40 cycles of denaturation at 95°C for 5 sec and annealing/extension at 60°C for 30 sec. Quantitative PCR reactions were run on Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). Relative expression patterns of genes were analyzed using the 2^−ΔΔCT method (30).

Proteins were extracted from HepG2 cells and liver tumors by centrifugation using RIPA Lysis Buffer (cat. no. P0013E; Beyotime Institute of Biotechnology) containing a protease inhibitor cocktail (cat. no. 5892970001; Roche Diagnostics) and then quantified using Bio-Rad Bradford Protein Assay kit (cat. no. 5000002; Bio-Rad Laboratories, Inc.). Next, 30 µg protein samples were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (cat. no. IPVH00010; EMD Millipore). Then, the membranes were blocked with 5% non-fat milk for 1 h at room temperature and incubated overnight at 4°C with primary antibodies against HIF-1α (product code ab16066; 1:2,000 dilution), Ki-67 (product code ab16667; 1:50 dilution), vascular endothelial growth factor (VEGF; product code ab106580; 1:1,000 dilution) and GAPDH (product code ab181602; 1:5,000 dilution; all from Abcam). Subsequently, the membranes were incubated for 1 h at room temperature with a horseradish peroxidase-conjugated secondary antibody (product codes ab205718 or ab205719; 1:5,000 dilution; Abcam). Finally, the protein signals were detected using SignalFire™ ECL Reagent (product no. 6883; Cell Signaling Technology, Inc.) on a Bio-Rad ChemiDoc XRS imaging system (Bio-Rad Laboratories, Inc.) and quantified using Quantity One 1-D Analysis Software (version 4.6.6; Bio-Rad Laboratories, Inc.).

The cell proliferative capacity was assessed by CCK-8 assay kit (cat. no. CK04-01; Dojindo Molecular Technologies, Inc.) referring to the protocols of the manufacturer. Briefly, cells (50,000 cells/well) transfected with corresponding oligonucleotides or plasmids, alone or in combination, were plated into 96-well plates and maintained under the hypoxia condition for 24 h. Next, 10 µl CCK-8 solution was added into each well and incubated for 3 h. Finally, the absorbance was determined at 450 nm using a SpectraMax M3 Multi-Mode microplate reader (Molecular Devices, LLC).

Cell migratory and invasive potential was estimated using 24-well Transwell chambers (cat. no. 3422; Corning Inc.) containing 8.0 µm pore filter membrane inserts. For cell invasion assay, the membranes on the chambers were precoated with Matrigel (cat. no. 356234; BD Biosciences). At 24 h after transfection, the transfected cells (3×10⁴) were resuspended into serum-free medium and then added into the upper chambers. Complete medium supplemented with 20% FBS was added into the lower chambers. After 24 h of incubation under hypoxia conditions, cells on the upper chambers were removed using a cotton swab. The cells attached on the bottom surface of the membranes were fixed with pre-cold methanol for 20 min at 4°C, stained with crystal violet solution (0.1%; cat. no. V5265; Sigma-Aldrich; Merck KGaA) for 30 min at room temperature and counted in 5 random fields under a light microscope (magnification ×200). The Transwell migration assay was performed following the same procedures as described in the cell invasion assay, i.e., in Transwell chambers, but without Matrigel.

Potential interaction between miR-203a and MIAT or HIF-1α was predicted through miRcode (http://www.mircode.org/) or TargetScan Human7.2 (http://www.targetscan.org/vert_72/), respectively. Partial sequences of MIAT and HIF-1α 3′ untranslated region (UTR) containing the corresponding miR-203a binding sites were cloned into the psiCHECK-2 vector by Hanbio Biotechnology Co., Ltd. to generate MIAT-wild-type (Wt) reporter and HIF-1α-Wt reporter, respectively. MIAT-mutant (Mut) reporter and HIF-1α-Mut reporter were also produced by mutating matching miR-203a complementary sites by Hanbio Biotechnology Co., Ltd. HepG2 cells were co-transfected with MIAT-Wt reporter, MIAT-Mut reporter, HIF-1α-Wt reporter, or HIF-1α-Mut reporter and miR-NC or miR-203a mimic. At 48 h after transfection, luciferase activities were measured using the Dual-Luciferase Reporter Assay system (cat. no. E1910; Promega Corporation) according to the instructions of the manufacturer.

An RNA pull-down assay was carried out as previously described (31) to explore whether MIAT could bind to miR-203a through putative binding sites. Biotin-labeled wt miR-203a (Bio-miR-203a), biotin-labeled mutant miR-203a containing mutated MIAT binding sites (Bio-miR-203a-Mut) and their NC (Bio-miR-NC), as well as biotin-labeled wt MIAT probe (Bio-MIAT), biotin-labeled mutant MIAT probe (Bio-MIAT-Mut) and a scramble probe (Bio-NC) were obtained from Sangon Biotech Co., Ltd. HepG2 cells were transfected with matching biotin-labeled miRNAs or probes. After 48 h of culture, cells were collected and lysed with lysis buffer (20 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl₂, 0.3% IGEPAL CA-630 (cat. no. I8896; Sigma-Aldrich) supplemented with protease inhibitor (cat. no. 5892970001; Roche Diagnostics) and RNase inhibitor (cat. no. EO0381; Thermo Fisher Scientific, Inc.). Next, cell lysates were co-incubated overnight at 4°C with pre-treated Dynabeads^® M-270 Streptavidin (cat. no. 11205D; Thermo Fisher Scientific, Inc.). Subsequently, RNA was isolated and purified from the beads. Finally, MIAT and miR-203a enrichment levels were determined by RT-qPCR assay.

A RIP assay was carried out in HepG2 cells using the EZ-Magna RIP kit (cat. no. 17-701; EMD Millipore) and anti-argonaute 2 (Ago2; cat. no. 03-110; Part #CS204386; EMD Millipore; 1:200 dilution) or IgG (cat. no. 03-110; Part #CS200621; EMD Millipore; 1:200 dilution) antibodies following the instructions of the manufacturer. RNA was isolated and purified from IgG or Ago2 immunoprecipitation complexes. MIAT and miR-203a levels were assessed by RT-qPCR assay.

Adult male Wistar rats (200–220 g) were obtained from the Laboratory Animal Center of Zhengzhou University and housed in a pathogen-free environment with a temperature of 22±2°C and a relative humidity of 50±5% under light-dark cycles (12 h light/dark). In addition, food and water were provided ad libitum. Animal experiments were performed with the approval of the Animal Ethics Committee of Henan Provincial People's Hospital and the procedures for the Care and Use of Laboratory Animals in cancer research. Sustained short hairpin RNA against MIAT (sh-MIAT) and a scramble control (sh-NC) were obtained from Hanbio Biotechnology Co., Ltd. In this study, a total of 60 rats were used in the animal experiments. Rat liver cancer cells (10⁶ cells/0.1 ml of normal saline) infected with or without sh-NC or sh-MIAT lentiviruses were injected into the fascia under the neck skin of rats (10 rats in each group). All rats were euthanized using intraperitoneal phenobarbital injection (100 mg/kg; Guangdong Bangmin Pharmaceutical Co., Ltd.) at 14 days after injection (experimental animals are euthanized when they are dying or obsolete due to various factors or they have fulfilled the tasks of experiments). After breathing arrest, tumors with a size of ~1 cm³ were harvested. Then, fresh tumors (~1 mm in diameter) without tumor capsules and necrotic tissues were implanted into the livers of rats as previously described (32). On day 14 after tumor transplantation (~1 cm³ in tumor volume), TAE was performed following previously described procedures (32,33). The NC, TAE+sh-NC, and TAE+sh-MIAT groups contained 10 rats prior to tumor transplantation. However, 2 rats after sham or TAE treatment in NC, 1 rat after TAE+sh-NC treatment, and 1 rat after TAE+sh-MIAT treatment, were revealed to be dead. These deaths may have been caused by anaesthetization, surgery or liver cancer. Five rats in the NC, TAE+sh-NC, or TAE+sh-MIAT groups were euthanized and tumors were resected 2 weeks later, and the tumor volumes were measured using the formula: V = 0.5 × L (longest tumor diameter) × S (shortest tumor diameter) × S (shortest tumor diameter). The expression levels of MIAT, miR-203a, HIF-1α and VEGF were measured by RT-qPCR and western blot assay in non-necrotic liver tumors. Animal health and behavior were monitored every 3 days post-surgery.

H&E staining of liver tumor tissues was performed 2 weeks after TAE. In brief, formaldehyde-fixed tumor tissues were embedded in paraffin. Thereafter, the sections (5 µm) were dewaxed with xylene, hydrated with gradient ethanol solutions, rinsed with water and stained with hematoxylin staining solution for 10 min and eosin staining solution for 2 min at room temperature. Images of the tissue sections were obtained using an optical microscope (magnification ×200).

Data were obtained from >3 independent experiments and analyzed with GraphPad Prism 7 software (GraphPad Software, Inc.). Analysis of differences between groups was conducted using Student's t-test (2-groups data) or one-way ANOVA with Tukey's post hoc test. (>2-groups data). P<0.05 was considered to indicate a statistically significant difference.

Clinical data revealed that the MIAT level was notably upregulated and miR-203a expression was markedly downregulated in the liver tumors of 20 patients with liver cancer who underwent TACE treatment compared with 22 patients with liver cancer without TACE treatment (Fig. 1A and B). Moreover, the miR-203a level was negatively associated with the MIAT level in the liver tumors of 20 patients with liver cancer who underwent TACE treatment (Fig. 1C). Consistently, higher MIAT expression and lower miR-203a expression were observed in HepG2 cells under hypoxia conditions compared with cells under normoxia conditions (Fig. 1D). These data indicated that MIAT and miR-203a may play vital roles in TACE treatment for liver cancer.

Next, si-MIAT#1, si-MIAT#2 and a scramble control (si-NC) were synthesized to evaluate the effect of MIAT loss on the proliferative, migratory and invasive capacities of HepG2 cells under hypoxic conditions. Firstly, transfection efficiency analysis revealed that the transfection of si-MIAT#1 or si-MIAT#2 could significantly reduce the expression level of MIAT in hypoxia-treated HepG2 cells compared with cells transfected with si-NC (Fig. 2A). A CCK-8 assay revealed that MIAT depletion inhibited proliferation in HepG2 cells under hypoxic conditions (Fig. 2B). The present results also demonstrated that the expression of proliferative markers such as Ki-67 was markedly decreased in hypoxia-exposed HepG2 cells following MIAT knockdown (Fig. 2C). Moreover, MIAT loss led to a noticeable reduction in the migratory and invasive abilities of HepG2 cells exposed to hypoxic conditions (Fig. 2D and E).

Subsequently, bioinformatics analysis indicated that MIAT could interact with miR-203a (Fig. 3A). To further demonstrate this prediction, MIAT-Wt reporter containing wt miR-203a binding sites and MIAT-Mut reporter containing mutant miR-203a binding sites were constructed. Following luciferase reporter assay, the results revealed that miR-203a overexpression led to a significant downregulation of luciferase activity of the MIAT-Wt reporter, but did not have a marked effect on the luciferase activity of the MIAT-Mut reporter (Fig. 3B). Ago2, a core component of the RNA-induced silencing complex, plays vital roles in miRNA-mediated gene silencing. Hence, a RIP assay was performed using an anti-Ago2 antibody in HepG2 cells to explore whether MIAT could bind miR-203a. The results revealed that MIAT and miR-203a were significantly enriched in the anti-Ago2 group (Fig. 3C), indicating a possible interaction between MIAT and miR-203a in HepG2 cells. Moreover, RNA pull-down assay revealed that MIAT was significantly enriched by Bio-miR-203a but not by Bio-miR-203a-Mut (Fig. 3D). Similarly, Bio-MIAT could pull down abundant miR-203a in HepG2 cells, while Bio-MIAT-Mut had little enrichment effect on miR-203a (Fig. 3E). In summary, these outcomes revealed that MIAT could directly bind to miR-203a in HepG2 cells. Next, it was further demonstrated that MIAT loss mediated by si-MIAT#1 or si-MIAT#2 led to a notable increase in the expression level of miR-203a in HepG2 cells exposed to hypoxic conditions (Fig. 3F).

Next, an RT-qPCR assay validated that the transfection of miR-203a mimic led to a marked increase in the expression level of miR-203a, and the addition of anti-miR-203a inhibited the increase in the expression level of miR-203a induced by MIAT knockdown in HepG2 cells under hypoxic conditions (Fig. 4A). Furthermore, miR-203a overexpression decreased cell proliferation (Fig. 4B), inhibited the expression of proliferative markers such as Ki-67 (Fig. 4C), and decreased cell migratory and invasive abilities in HepG2 cells under hypoxic conditions (Fig. 4D and E). Moreover, it was further demonstrated that miR-203a loss weakened the detrimental effects of MIAT knockdown on the proliferation, migration and invasion of HepG2 cells under hypoxic conditions (Fig. 4B-E).

Prediction results revealed the existence of some complementary bases between HIF-1α 3′UTR and miR-203a (Fig. 5A), indicating that HIF-1α is a potential target of miR-203a. The results of luciferase reporter assay revealed that miR-203a overexpression led to a significant decrease in the luciferase activity of the HIF-1α-Wt reporter, while miR-203a upregulation did not have a marked effect on the luciferase activity of the HIF-1α-Mut reporter, which contains mutant miR-203a binding sites (Fig. 5B), suggesting that miR-203a could directly bind to HIF-1α 3′UTR via putative binding sites. In addition, the present study demonstrated that the expression level of HIF-1α was significantly increased in the liver tumors of 20 patients with liver cancer who underwent TACE treatment compared with the 22 patients with liver cancer without TACE treatment (Fig. 5C). Consistently, higher HIF-1α expression was observed in HepG2 cells under hypoxic conditions than in cells under normoxic conditions (Fig. 5D). Moreover, RT-qPCR and western blot assays revealed that miR-203a overexpression or MIAT knockdown led to a significant reduction in HIF-1α mRNA and protein levels in hypoxia-treated HepG2 cells (Fig. 5E and F). miR-203 loss weakened the inhibitory effect of MIAT knockdown on HIF-1α mRNA and protein expression in hypoxia-stimulated HepG2 cells (Fig. 5E and F).

si-HIF-1α and pcDNA-HIF-1α overexpression plasmid were synthesized to further explore HIF-1α function in liver cancer. A western blot assay demonstrated that the addition of si-HIF-1α effectively triggered a reduction in the expression level of HIF-1α in hypoxia-treated HepG2 cells (Fig. 6A). Conversely, transfection of pcDNA-HIF-1α led to a significant increase in the protein expression level of HIF-1α in hypoxia-stimulated HepG2 cells (Fig. 6A). Subsequent loss-of-function analysis revealed that depletion of HIF-1α decreased cell proliferation (Fig. 6B), migration (Fig. 6D) and invasion (Fig. 6E), and suppressed Ki-67 expression (Fig. 6C) in hypoxia-treated HepG2 cells. Conversely, enforced expression of HIF-1α induced cell proliferation (Fig. 6B), migration (Fig. 6D) and invasion (Fig. 6E), and increased Ki-67 expression levels (Fig. 6C) in hypoxia-stimulated HepG2 cells.

The present study demonstrated that TAE inhibited the growth of rat liver tumors and MIAT knockdown enhanced the TAE-mediated antitumor effect in rat liver tumors (Fig. 7A). Lack of MIAT led to markedly fewer blood vessels (Fig. 7B). Moreover, TAE treatment led to a significant increase in MIAT and HIF-1α mRNA levels, and a significant reduction in the expression level of miR-203a in rat liver tumors (Fig. 7C). Furthermore, the MIAT and HIF-1α mRNA levels were significantly reduced, while the expression level of miR-203a was significantly increased, in TAE-treated rat liver tumors following MIAT knockdown (Fig. 7C). In addition, the protein levels of HIF-1α and VEGF were significantly increased in TAE-treated rat liver tumors compared with untreated tumors (Fig. 7C). Moreover, MIAT knockdown inhibited the increase in HIF-1α and VEGF protein levels induced by TAE in rat liver tumors (Fig. 7D).