This study included 64 patients with HCC (36 males, 28 females; age range, 33–67 years old; mean age, 51.8±6.0 years old) selected from the 136 patients with HCC admitted to the Union Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology (Wuhan, China) between April 2010 and April 2014. All patients were treated with surgical resection of the primary tumors and/or targeted therapies, radiotherapies and chemotherapies according to their conditions. The study was approved by the review board of the Ethics Committee of the aforementioned hospital. Inclusion criteria included: i) patients had not received radiofrequency ablation preoperatively; ii) newly diagnosed HCC case and iii) completed treatment and a 5-year follow-up in Union Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology. Exclusion criteria included: i) Recurrent patients with HCC, ii) previous history of cancer, iii) family history of cancer and iv) other clinical disorders. All 64 patients with HCC provided written informed consent.

Before initiation of therapies, liver biopsy was performed under the guidance of MRI to collect both HCC and non-tumor (within 3 cm of the tumor) tissues. All tissues were snap-frozen and then transferred into a −80°C freezer before use. HCC tissue samples were sectioned at 5-µm. The clinicopathological features of the patient specimens and the histological diagnosis of all HCC and normal tissues were independently reviewed and approved by two experienced pathologists. Pathologists performed a blinded assessment of the specimens. The TNM stage was evaluated based on the American Cancer Joint Commission Cancer Staging Manual (21). From the day of admission, all patients were followed-up for 5 years in a monthly manner through telephone to record their survival. The overall survival was defined as the day of diagnosis to the day of death or last follow-up. Patients who died of other causes or were lost during the follow-up were excluded. The 5-year overall survival rate was calculated as the proportion of the survivors among all patients with HCC at 5 years after hepatectomy.

Human HCC cell line SNU-398 (American Type Culture Collection) was used. A mixture of 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 90% RPMI 1640 medium was used to cultivate cells. Cells were cultivated under the conditions of 37°C, 95% humidity and 5% CO₂. Cells were harvested at the confluence of 70–80% to perform cell transfections. The overexpression pcDNA3.1-MT1JP (MT1JP), pcDNA3.1-PTEN (PTEN) and control pcDNA3.1 recombinant plasmids were constructed by Shanghai GenePharma Co., Ltd. miRNA-NC and miR-32 mimic were obtained from Guangzhou RiboBio Co., Ltd. Lipofectamine 2000^® (Sangon Biotech Co., Ltd.) was used to transfect 35 nM miRNA (miRNA-NC or miR-32) or 2 µg plasmid for 24 h in six-well plates. The control (C) cells for all transfections were untransfected cells. At 24 h post-transfection at the room temperature, cells were harvested to perform subsequent experiments. The sequences were as follows: miR-32 Mimic, 5′-AGUAUACGUCCUGCAGUCAGCU-3′) and miRNA-NC, 5′-UGCUCCGGACGUGUGAGGUGT-3′.

Total RNA from tissues and cells was extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. Total RNA was reverse transcribed into cDNA using oligo (dT)15, random primers (GenScript), M-MLV reverse transcriptase, dNTPs, 5X buffer and RNase inhibitor (all Tiangen Biotech Co., Ltd.) at 25°C for 10 min, 42°C for 50 min and 80°C for 10 min. QuantiTect SYBR^® Green PCR kit (Qiagen, Inc.) was used to prepare qPCR mixtures for measuring the expression levels of MT1JP and PTEN. GAPDH was used as the endogenous control of MT1JP and PTEN. To measure the expression levels of miR-32, all steps were performed using All-in-One™ miRNA qRT-PCR Detection kit (GeneCopoeia, Inc.). U6 was used as the endogenous control of miR-32. The following thermocycling conditions were used for the qPCR: Initial denaturation at 94°C for 5 min; followed by 40 cycles at 94°C for 10 sec, 60°C for 20 sec and 72°C for 30 sec; and final extension at 72°C for 150 sec. The following primer pairs were used for the qPCR: MT1JP forward: 5′-GCAAAGGGACGTCGGAGA-3′ and reverse: 5′-TCCAGGTTGCAGTTGTT-3′; PTEN forward: 5′-TGGCTTCGACTTAG-3′ and reverse: 5′-GGTTGGTTATGGTCTTTAAAAGG-3′; miR-32 forward: 5′-GCGGCGTATTGCACATTACT-3′ and reverse: 5′-TCGTATCCAGTGCAGGGTC-3′; GAPDH forward: 5′-AGATCATCAGCAATGCCTCCT-3′ and reverse: 5′-TGAGTCCTTCCACGATACCAA-3′ and U6 forward: 5′-CTCGCTTCGGCAGCACA-3′ and reverse: 5′-AACGCTTCACGAATTTGCGT-3′. The 2^−ΔΔCq method (22) was used for data analysis and all experiments were performed in three replicates.

SNU-398 cells were counted, and total proteins in 3×10⁵ cells were extracted using RIPA solution (Sangon Biotech Co., Ltd.). Protein samples were incubated in boiling water for 10 min to denature proteins, followed by protein lysates (30 µg/lane) of each group were separated using 10% SDS-PAGE. Proteins were then transferred to PVDF membranes and 5% non-fat milk was used to block membranes at room temperature for 80 min. Rabbit polyclonal PTEN (1:1,400; cat. no. ab31392; Abcam) and GAPDH (1:1,400; cat. no. ab37168; Abcam) primary antibodies were incubated with membranes at 4°C for 18 h, followed by incubation with goat anti-rabbit IgG H+L (1:1,000; cat. no. ab6721; Abcam) at room temperature for 2 h. After that, membranes were incubated with RapidStep™ ECL detection reagent (EMD Millipore) at room temperature for 10 min to develop signals. Data was processed using ImageJ v1.46 software (National Institutes of Health).

SNU-398 cells were counted, and 3×10⁴ cells per well were mixed with 1 ml mixture of 10% FBS and 90% RPMI 1640 medium to prepare single-cell suspensions. Cells were cultivated in a 96-well plate (0.1 ml per well) under conditions of 37°C, 95% humidity and 5% CO₂. Cell Counting Kit-8 solution (Sigma-Aldrich; Merck KGaA) was added into each well (10 µl per well) at 4 h before the end of cell culture. After cell culture, 10 µl DMSO was added and optical density values were measured at 450 nm wavelength using a microplate reader (Thermo Fisher Scientific, Inc.).

Statistical analyses were performed using SPSS version 23 (IBM Corp.). Data were expressed as mean values ± standard deviation of three biological replicates. Associations were analyzed by linear regression. Differences between two types of tissue and among different cell groups were analyzed using paired t-tests and ANOVA (one-way) in combination with the Tukey's test, respectively. Survival analyses were performed by dividing the 64 patients with HCC into high and low MT1JP level groups (n=32) according to the median of MT1JP expression (2.34) in HCC tissues. Kaplan-Meier plotter and log-rank tests were used to plot and compare survival curves. P<0.05 was considered to indicate a statistically significant difference.

To illustrate the role of MT1JP in the progression of HCC, the expression of MT1JP and the basic information, such as clinical pathological features of patients with HCC, are displayed in Table I. The expression levels of MT1JP in non-tumor and HCC tissues were measured by qPCR and compared using a paired t-test. Compared with non-tumor tissues, significantly lower expression levels of MT1JP were observed in HCC tissues (P<0.05; Fig. 1A). Compared with patients in the high MT1JP level group, a significantly lower overall 5-year survival rate was observed in the low MT1JP level group (P=0.0078; Fig. 1B). It is worth noting that the expression levels of MT1JP were not significantly affected by HBC and HCV infections as well as clinical stages (data not shown).

The expression levels of PTEN and miR-32 were also measured by qPCR and compared using paired t-tests between non-tumor and HCC tissues. Compared with non-tumor tissues, significantly lower expression levels of PTEN (Fig. 2A) and significantly higher expression levels of miR-32 (Fig. 2B) were observed in HCC tissues (both P<0.05). Regression analysis showed that the expression of MT1JP in HCC tissues was positively associated with the expression of PTEN (Fig. 2C) and negatively associated with the expression of miR-32 (Fig. 2D).

MT1JP and PTEN expression vectors, as well as miR-32 mimic, were transfected into SNU-398 cells and their expression levels were measured by qPCR at 24 h post-transfection. Compared with C and NC, the expression levels of MT1JP, miR-32 and PTEN were significantly increased (all P<0.05; Fig. 3A). Moreover, overexpression of MT1JP decreased the expression levels of miR-32 (P<0.05), whilst overexpression of miR-32 did not affect the expression of MT1JP (Fig. 3B). In addition, overexpression of miR-32 inhibited the mRNA and protein levels of PTEN (P<0.05), which could be reversed by simultaneous upregulation of MT1JP (Fig. 3C).

The effects of overexpression of MT1JP, miR-32 and PTEN on the proliferation of SNU-398 cells were analyzed using a cell proliferation assay. Compared with NC and C, overexpression of MT1JP and PTEN resulted in significantly inhibited proliferation of HCC cells (P<0.05). miR-32 promoted cell proliferation. Moreover, the suppressive effect of MT1JP on cell proliferation was reversed by miR-32 (P<0.05; Fig. 4).