Long non-coding RNA TRPM2-AS as a potential biomarker for hepatocellular carcinoma

One hundred eight fresh HCC tissue and matched normal adjacent tissue samples were selected from patients with HCC at our hospital (Yuhuangding Hospital of Qingdao University Medical College) between 2010 and 2012. None of the patients had received preoperative therapy. The diagnosis of each specimen was confirmed histopathologically. Letters of consent were obtained from all patients, and the experimental protocols were approved by the local ethics committee. Patient charts were reviewed to obtain clinical data including age, gender, tumor size, AFP, HBsAg, vascular invasion, TNM stage (AJCC), tumor differentiation, and death or time of last follow-up. Patient survival was calculated from the day of surgery until death in months.

The HCC cell lines HCCLM3, Huh7, SMMC-7721, SK-Hep1, and HepG2 and one normal liver cell line (QSG-7701) were maintained at our institute. HepG2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA), and SK-Hep1, SMMC-7721, Huh7, HCCLM3, and QSG-7701 cells were cultured in Medium 1640 (Gibco, Grand Island, NY, USA) supplemented with 10% heat-inactivated FBS (Sigma-Aldrich, St. Louis, MO, USA).

Si-NC and Si-TRPM2-AS were used to transfect the cells at a concentration of 20 nM. Oligonucleotide transfection was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Cells were collected 48 h after transfection. The siRNA sequences for TRPM2-AS1 were as follows: siRNA sense, 5′-GGGAAGAUGUCUCAGCAGACG-3′, and antisense, 5′-UCUGCUGAGACAUCUUCCCCU-3′.

Total RNA was extracted from tissues using TRIzol® Reagent (Life Technologies Corporation, Carlsbad, CA, USA) according to the manufacturer’s protocol, and cDNA was synthesized (Bio-Rad, Hercules, CA, USA). QPCR (7500Fast; ABI, Foster City, CA, USA) was performed to compare the mRNA expression levels of TRPM2-AS1 in 108 paired tumor and peritumor tissues from HCC patients. The primer sequences for TRPM2-AS1 were as follows: sense, 5′-CGTGACCAGGTTCAGACACA-3′, and antisense, 5′-TGGGCAGTTTGGTTCTGGTT-3′. The primer sequences for the house-keeping gene β-actin were as follows: sense, 5′-TGTCCACCTTCCAGCAGATG-3′, and antisense, 5′-TGTCACCTTCACCGTTCCAG-3′. Bax, Bcl-2, caspase-3, cyclin D1, cyclin E1, cyclin B1, and cyclin A2 expression was measured using the SYBR green qPCR assay (Takara, Dalian, China) and the primer sequences were shown in Table 1.

Cell viability assays were performed using the Cell Counting Kit-8 (CCK-8) (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instructions. Cells were cultured in 96-well plates at a cell density of 1000 cells per well for 24, 48, 72, and 96 h. The absorbance was read at 450 nm to determine the cell viability in each well. All experiments were independently repeated three times.

Cells were plated in 6-well plates, and 48 h after transfection, the cells were collected and resuspended in staining solution containing Annexin V-FITC and propidium iodide (PI; Tianjin Sungene Biotech Co. Ltd., Tianjin, China). The stained cells (1 × 10⁵) were analyzed using a flow cytometer.

Forty-eight hours after transfection, the cells were collected and fixed in 75% ethanol overnight at 4 °C and then incubated with 1 mg/mL RNase A for 30 min at 37 °C. Subsequently, cells were stained with propidium iodide (PI; Becton Dickinson, San Jose, CA, USA) in PBS according to the manufacturer’s instructions. The stained cells (1 × 10⁵) were analyzed by flow cytometry.

Data were analyzed using the Statistical Package for the Social Sciences (SPSS), Version 18 (SPSS, Chicago, IL, USA). Pearson’s chi-squared test was used to analyze the relationship between TRPM2-AS1 expression and clinicopathological characteristics. Kaplan-Meier plots were constructed to visualize survival outcomes, which were compared using a log-rank test. All experiments were performed in triplicate. The differences between the groups were analyzed using Student’s t test, and p < 0.05 was considered statistically significant in all cases.

We first detect the expression of TRPM2-AS in samples from 108 HCC patients using quantitative reverse transcription (qRT)-PCR. The expression level of TRPM2-AS in cancer tissues from patients with HCC was significantly higher than that in adjacent normal tissues (p < 0.001; Fig. 1a). Based on the relative expression levels in tumor and adjacent normal tissues, all 108 patients with HCC were divided into two subgroups. As shown in Fig. 1b, 32 patients (low-expression group) had a low expression level of TRPM2-AS in carcinoma tissues compared with that of adjacent normal liver tissues, and 76 patients (high-expression group) had a high expression level of TRPM2-AS.

We then determined whether the expression level of TRPM2-AS was associated with patient characteristics, such as gender, age, tumor size, tumor differentiation, and TNM stage. TRPM2-AS expression was significantly correlated with tumor size, AJCC stage, and tumor differentiation (p = 0.005, 0.014, and 0.021, respectively) (Table 2). However, there was no association between TRPM2-AS expression and age, sex, AFP, vascular invasion, or liver cirrhosis.

We then performed a survival analysis of the patients with HCC. Patients in the TRPM2-AS high-expression group had a worse overall survival than those in the low-expression group (Fig. 2). There was not much difference in the survival rate between the two groups after the first year. However, the 3-year and 5-year survival rates were worse for the TRPM2-AS high-expression group (32.9 and 28.7%, respectively) than those of the TRPM2-AS low-expression group (58.6 and 48.8%, respectively) (p = 0.021) (Table 3). Thus, high expression levels of TRPM2-AS in HCC predict a poor prognosis.

To further ascertain the function of lncRNA TRPM2-AS in HCC, we first examined the TRPM2-AS expression level in HCC cell lines using qRT-PCR. The expression of TRPM2-AS in five HCC cell lines, HCCLM3, Huh7, SMMC-7721, SK-Hep1, and HepG2, was significantly higher compared to that in the normal liver cell line QSG-7701 (Fig. 3a). We used SMMC-7721 and SK-Hep1 cells, which had the highest TRPM2-AS levels of these cell lines, to determine if Si-TRPM2-AS could effectively inhibit the expression of TRPM2-AS (Fig. 3b). The influence of TRPM2-AS on HCC cell proliferation was investigated using the CCK-8 assay. The inhibition of TRPM2-AS significantly inhibited SMMC-7721 and SK-Hep1 cell growth compared with that of the control, respectively (Fig. 3c, d)

(Table 3).

To evaluate the apoptotic effects of TRPM2-AS in HCC cells, the SMMC-7721 and SK-Hep1 cell lines were treated with Si-TRPM2-AS. The inhibition of TRPM2-AS significantly induces SMMC-7721 and SK-Hep1 cell apoptosis compared with that of the control. The downregulation of TRPM2-AS increased the apoptosis rate from 8.5 to 27.7% in SMMC-7721 cells and from 7.6 to 18.5% in SK-Hep1 cells (Fig. 4a, b).

We examined the effect of TRPM2-AS on apoptosis mediators, in order to elucidate the molecular mechanism of TRPM2-AS-induced apoptosis. As shown in Fig. 4c, Si-TRPM2-AS significantly increased the mRNA levels of caspase-3 and Bax, but not the expression of Bcl-2.

Then, we analyzed cell cycle alterations in HCC cells by flow cytometry. In the Si-TRPM2-AS group, cells in the G0/G1 phase increased significantly for both cell lines, accompanied by a significant decrease in cells at in G2/M phase (Fig. 5a, b). However, no statistically significant differences were observed for cells in the S phase. The percentage of SK-Hep-1cells in the G0/G1 phase was found to have increased from 33.69 to 47.11%, and those in G2/M phase decreased from 31.25 to 12.19%. This tendency was also observed in SMMC-7721 cells.

To understand these cell cycle alterations, we analyzed the expression of various cyclins, which are important regulators of the cell cycle. Inhibition of TRPM2-AS caused significant decrease in the mRNA level of cyclin D1, cyclin E1, and cyclin B1, but not cyclin A2 (Fig. 5c).