A total of 64 patients with HCC (38 men and 26 women; mean age, 52.1±6.9 years; age range, 40–64 years) at the First Affiliated Hospital of Shenzhen University were enrolled in the present study between January 2013 and January 2015. The inclusion criteria were as follows: i) Patients with pathologically confirmed HCC; ii) patients who underwent curative surgical resection and iii) patients >18 years. The exclusion criteria were as follows: i) Patients receiving preoperative chemotherapy or radiotherapy and ii) patients who have two or more primary tumors simultaneously or asynchronously. Prior to therapy, HCC tissues and paired adjacent normal tissues (>5 cm away from cancer tissues) were collected from all patients via biopsy. All the histological diagnoses for HCC and normal tissues were confirmed by two pathologists from the First Affiliated Hospital of Shenzhen University and immediately subjected to total RNA extraction following collection. Based on the medical records of all patients, HBV infection was observed in 26 cases, HCV infection was observed in 16 cases, both HBV and HCV infections were observed in 16 cases, and no infections were observed in 6 cases. The present study was approved by the Ethics Committee of the First Affiliated Hospital of Shenzhen University (Shenzhen, China; approval no. 2013006) and written informed consent was provided by all patients prior to the study start.

Based on the tumor-node-metastasis (TNM) staging system (16), of the 64 patients included, 28 cases were in stages I and II, while 36 cases were in stages III and IV. Treatment approaches were determined based on the TNM stage and health conditions of the patients. From June 30, 2015, all patients were followed-up for 5 years in a monthly manner through telephone to record their survival. The survival status of patients was recorded during follow-up until June 30, 2020. Patients who died of causes unrelated to HCC were excluded.

Survival analysis was performed using the Kaplan-Meier method and log-rank test. Patients were divided into high and low TONSL-AS1 expression groups (n=32/group and median TONSL-AS1 expression in HCC tissues was set as the cut-off value).

The interaction between TONSL-AS1 and miR-135a was predicted using IntaRNA2.0 (http://rna.informatik.uni-freiburg.de/IntaRNA).

The HCC cell lines, SNU-398 and SNU-475, were purchased from the American Type Culture Collection. Cells were maintained in RPMI-1640 medium (Sigma, India; 90%) supplemented with 10% fetal bovine serum (MP Biomedicals), at 37°C with 5% CO₂ and 95% humidity. Cells were collected once they reached ~85% confluence and used in subsequent experiments.

The TONSL-AS1 expressing vector was constructed with pcDNA3.1 vector as the backbone (Invitrogen; Thermo Fisher Scientific, Inc.). miR-135a mimic and negative control (NC) miRNA were synthesized by Invitrogen; Thermo Fisher Scientific, Inc. Vector (1 µg) or miRNA (50 nM) were transfected into SNU-398 and SNU-475 cells using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. Transfection with empty vector or NC miRNA served as the control groups, and control cells were untransfected cells. Cells were cultured for 48 h at 37°C with 5% CO₂ and harvested for subsequent experimentation. Sequences of transfection vectors were as follows: miR-135a mimics forward, 5′-UAUGGCUUUUUAUUCCUAUGUGA-3′ and reverse, 5′-GUGCCGAGGAUUAGGGAUAUACU-3′; and NC mimics forward, 5′-UUCUCCGAACGUGUCACGUTT−3′ and reverse, 5′-ACGUGACACGUUCGGAGAATT−3′.

Total RNA was extracted from tissues and cells using RiboZol™ reagent (Amresco, LLC). RNA samples were digested to remove genomic DNA using DNase I (Invitrogen; Thermo Fisher Scientific, Inc.), at 37°C for 100 min. RNA integrity was assessed using 5% urine-PAGE gel.

Total RNA was reverse transcribed into cDNA using the PrimeScript RT reagent kit (Takara Bio, Inc.) at 37°C for 15 min. qPCR was subsequently performed using LightCycler^® 480 SYBR Green I Master (Roche Diagnostics GmbH). The following thermocycling conditions were used for qPCR: Initial denaturation at 95°C for 30 sec, followed by 40 cycles at 95°C for 5 sec and at 60°C for 45 sec (17). The following primer sequences were used for qPCR: TONSL-AS1 forward, 5′-AGCCCTCTCTCCCTGCACCAC−3′ and reverse, 5′-AGCTGAGCACCTTCCTGAAACCC-3′; miR-135a forward, 5′-AGCATAATACAGCAGGCACAGAC-3′ and reverse, 5′-AAAGGTTGTTCTCCACTCTCTCAC−3′; and U6 forward, 5′-GCUUCGGCAGCACAUAUACUAAAAU-3′ and reverse, 5′-CGCUUCACGAAUUUGCGUGUCAU-3′. TONSL-AS1 expression was measured with 18S rRNA as the internal control.

miR-135a expression levels were measured as follows: i) Addition of poly (A); ii) RT of miRNA and iii) miRNA qPCR. U6 was used as the internal control. Relative expression levels were calculated using the 2^−ΔΔcq method (18). All experiments were performed in triplicate.

The Cell Counting Kit-8 (CCK-8) assay (cat. no. ab228554; Abcam) was performed to assess the proliferative ability of SNU-398 and SNU-475 cells. Cells were seeded into 96-well plates at a density of 3,000 cells/well (in 0.1 ml medium) and cultured at 37°C, and optical density (OD) values were measured at a wavelength of 450 nm every 24 h for a total of 4 days. CCK-8 solution was added to each well for 2 h to reach 10% concentration, prior to analyzing cell proliferation.

All experiments were performed in triplicate and data are presented as the mean ± standard deviation. Differences between two groups were compared using an unpaired two-tailed Student's t-test, while one-way ANOVA and Tukey's post hoc test were used to compare differences between multiple groups. Unpaired Student's t-test was performed to assess the significance of cell proliferation assay. Pearson's correlation coefficient was used to determine the correlation between TONSL-AS1 and miR-135a expression levels. Survival analysis was performed using the Kaplan-Meier method and log-rank test. P<0.05 was considered to indicate a statistically significant difference.

RT-qPCR analysis was performed to detect TONSL-AS1 expression in HCC tissues and paired adjacent normal tissues from 64 patients with HCC. The results demonstrated that TONSL-AS1 expression was significantly downregulated in HCC tissues compared with adjacent normal tissues (P<0.001; Fig. 1A). In addition, survival analysis demonstrated that patients with low TONSL-AS1 expression had significantly lower overall survival rates than those with high TONSL-AS1 expression (Fig. 1B). Notably, no significant differences in TONSL-AS1 expression were observed between patients in different clinical stages or patients with (mean value, 3.23±0.97) or without HBV/HCV (mean value, 3.04±0.72) infections (P>0.05) (data not shown). Taken together, these results suggest that TONSL-AS1 may be an independent prognostic factor for HCC.

The interaction between miR-135a and TONSL-AS1 was predicted using IntaRNA2.0. The results indicated that miR-135a targets TONSL-AS1 (Fig. 2A). To further investigation their interaction, SNU-398 and SNU-475 cells were transfected with miR-135a mimic or TONSL-AS1 expression vector, and transfection efficiency was assessed via RT-qPCR analysis 48 h post-transfection (P<0.05; Fig. 2B). The results demonstrated that overexpression of miR-135a downregulated TONSL-AS1 expression (Fig. 2C), while overexpression of TONSL-AS1 had no effect on miR-135a expression (Fig. 2D).

RT-qPCR analysis was performed to detect miR-135a expression in HCC tissues and paired adjacent normal tissues from 64 patients with HCC. The results demonstrated that miR-135a expression was significantly upregulated in HCC tissues compared with adjacent normal tissues (P<0.001; Fig. 3A). In addition, Pearson's correlation coefficient analysis demonstrated that TONSL-AS1 and miR-135a expression levels were inversely correlated with each other in both HCC tissues (Fig. 3B) and adjacent normal tissues (Fig. 3C). Collectively, these results suggest that TONSL-AS1 and miR-135a interact with each other.

The CCK-8 assay was performed to assess the effects of miR-135a and TONSL-AS1 on the proliferative ability of HCC cells. The results demonstrated that overexpression of miR-135a increased the proliferation of SNU-398 and SNU-475 cells that compared with the control group. Conversely, overexpression of TONSL-AS1 decreased cell proliferation. In addition, overexpression of miR-135a attenuated the effect of overexpression of TONSL-AS1 on cell proliferation (P<0.05; Fig. 4A and B).