Long non-coding RNA TMCC1-AS1 predicts poor prognosis and accelerates epithelial-mesenchymal transition in liver cancer

Long non-coding RNA transmembrane and coiled-coil domain family 1 antisense RNA 1 (TMCC1-AS1) has been frequently reported to be associated with prognosis in patients with liver cancer (LC). However, the biological role of TMCC1-AS1 in LC in vitro remains unclear. The expression levels of TMCC1-AS1 in primary tumor tissues and LC cell lines were determined using reverse transcription-quantitative PCR. The associations between TMCC1-AS1 expression and the clinicopathological factors of patients with LC were statistically analyzed using the χ2 test. The role of TMCC1-AS1 in LC prognosis was assessed using Kaplan-Meier curves and proportional hazards model (Cox) analysis. Cell proliferation was determined by Cell Counting Kit-8 and colony formation assays. Transwell assays were performed to determine migration and invasion. TMCC1-AS1 expression was found to be significantly upregulated in LC tissues and cell lines compared with the corresponding controls. High TMCC1-AS1 expression was associated with advanced TNM stage and lymph node metastasis. Furthermore, high TMCC1-AS1 expression predicted poor survival in patients with LC. Knockdown of TMCC1-AS1 significantly inhibited the proliferation, migration and invasion of HepG2 and SNU-182 cells, while overexpression of TMCC1-AS1 had the opposite effect in HepG2 and SNU-182 cells. At the molecular level, downregulation of TMCC1-AS1 expression resulted in increased E-cadherin expression and decreased proliferating cell nuclear antigen, Ki67, N-cadherin and Vimentin expression in HepG2 cells. Overexpression of TMCC1-AS1 had the opposite effects on these factors in SNU-182 cells. In conclusion, the present findings indicated that TMCC1-AS1 might be considered as a novel oncogene, which promotes cell proliferation and migration, and may be a potential therapeutic target for LC.


Introduction
It has been estimated that about 841,000 newly diagnosed liver cancer cases and 782,000 liver cancer-associated mortality cases appeared worldwide according to the 2018 Global Cancer Statistics, of which hepatocellular carcinoma (HCC) accounts for 75-80% of all liver cancer cases (1,2). Numerous risk factors, including diabetes, alcohol consumption, and especially chronic hepatitis B virus (HBV) infection, have been reported to be associated with the pathogenesis of HCC (3,4). Although great progress has been achieved in innovative therapeutic strategies for HCC, the five-year survival rate remains low (~46.8%) due to high rates of recurrence and metastasis (5,6). Therefore, having an improved understanding of the molecular mechanism underlying HCC pathogenesis is helpful for developing clinical applications for its treatment.
Long non-coding RNAs (lncRNAs) are a class of mRNA-like transcripts with a length of >200 nucleotides, which participate in cellular physiological and pathological processes, including but not limited to cell proliferation, cell cycle, apoptosis and motility (7,8). In recent years, a variety of lncRNAs have been demonstrated to be dysregulated in HCC and to be associated with the initiation and development of HCC. For example, Zhang et al (9) reported that ROR1-AS1 is upregulated in HCC, which is associated with clinical stage and poor prognosis and serves as an independent risk factor for HCC. Upregulated GMAN expression is associated with TNM stage, short overall survival and disease-free survival in patients with HCC (10). Similarly, Luo et al (11) observed that PCAT6 is upregulated in HCC and associated with poor prognosis of patients with HCC. In functional experiments, lncRNAs, including MYCNOS (12), NEAT1 (13) and SNHG11 (14), exert oncogenic effects on HCC cells by promoting cell proliferation, migration and invasion, whereas MAGI2-AS3 (15), ID2-AS1 (16) and HHIP-AS1 (17) have the opposite effects on HCC cell functions. Although the oncogenic or tumor-suppressive role of these lncRNAs has been well characterized in LC, investigations on the function and mechanisms of novel lncRNAs are still necessary to identify functional biomarkers in LC progression.
In the present study, the expression levels of TMCC1-AS1 in LC tissues and cell lines were determined. By performing Cell Counting Kit-8 (CCK-8), colony formation and transwell assays, the present study investigated the effects of TMCC1-AS1 on LC cell proliferation, migration and invasion. The present study also evaluated whether TMCC1-AS1 served as an independent predictor for overall survival in LC.

Materials and methods
Clinical tissues and cell culture. Tumor tissues and matched adjacent normal tissues (at least 5 cm away from the edge of the tumor) were collected from 68 patients (age range, 28-68 years; mean age, 45.7 years) diagnosed with LC who underwent routine curative surgery at Affiliated Hospital of Hebei Engineering University (Handan, China) between December 2016 and November 2019. All tissue samples were immediately frozen in liquid nitrogen and kept at -80˚C for further experiments. Before surgery, all patients who received any anticancer therapies were excluded. Some major clinical and pathological information is summarized in Table I. Clinical staging was performed according to American Joint Committee on Cancer/International Union Against Cancer TNM staging system (21). The survival information was obtained through monthly follow-up telephone calls. The present study has been approved by the Ethics Committee of Affiliated Hospital of Hebei Engineering University (Handan, China) and the participants provided written informed consent.
A total of two LC cell lines (HepG2 and SNU-182) and normal liver THLE-3 cells were purchased from American Type Culture Collection, which were identified using short tandem repeat DNA profiling analysis. These cell lines were cultured in DMEM (Thermo Fisher Scientific, Inc.) with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and antibiotics (100 µg/ml streptomycin and 100 U/ml penicillin; Sigma-Aldrich; Merck KGaA) at 37˚C in a humidified incubator containing 5% CO 2 .
Reverse transcription-quantitative PCR (RT-qPCR). Total RNA was extracted from tissue samples or cell lines using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) and reverse transcribed into cDNA using the PrimeScript™ RT reagent kit (Takara Biotechnology Co., Ltd.) according to the manufacturer's instructions. The primer sequences used were as follows: TMCC1-AS1 forward, 5'-AGC GAG GGA TCG AGT TGA GA-3' and reverse, 5'-TAG TCA TGT CCC CGT TGG TG-3'; and GAPDH forward, 5'-CGA CTT ATA CAT GGC CTT A-3' and reverse, 5'-TTC CGA TCA CTG TTG GAA T-3'. RT-qPCR was performed on an Applied Biosystems 7900 Fast Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) using SYBR ® Premix Ex Taq™ II (Takara Biotechnology Co., Ltd.). The PCR reactions conditions were as follows: 95˚C for 1 min, followed by 40 cycles of 95˚C for 10 sec and 57˚C for 40 sec. Relative expression levels of TMCC1-AS1 were calculated using the 2 -ΔΔCq method (22) with GAPDH as the endogenous control.

CCK-8 assay.
After 48 h of transfection, cells at a density of 3x10 3 cells per well were seeded into 96-well plates. Subsequently, 10 µl CCK-8 solution (Dojindo Molecular Technologies, Inc.) was added to the cells in each well at 0, 24, 48 and 72 h after seeding. At each time point, cells were incubated for another 2 h and the absorbance was measured at 450 nm using a microplate reader.
Colony formation assay. For the colony formation assay, ~500 transfected cells were plated in six-well plates and cultured for 2 weeks. The naturally formed colonies were fixed with 4% paraformaldehyde for 15 min at room temperature, stained with 0.5% crystal violet (Wuhan Servicebio Technology Co., Ltd.) for 15 min at 37˚C and manually counted by visual inspection using light microscopy.
Transwell assay. After 48 h of transfection, a total of 5x10 4 cells suspended in 200 µl serum-free medium were seeded into the upper Transwell chamber (8-µm pore size; Merck KGaA) coated with Matrigel (final concentration, 250 µg/ml/well; BD Biosciences) at 37˚C for the invasion assay and without Matrigel for the migration assay. DMEM supplemented with 10% FBS was added to the lower chamber. After incubation for 24 h at 37˚C, cells that migrated into the lower chamber were fixed with methanol for 20 min, then stained with 0.5% crystal violet for 15 min at 37˚C and counted in five randomly selected fields under a light microscope (magnification, x100).
Western blot analysis. Extraction of total protein samples from cell lines was performed using RIPA lysis buffer (Beyotime Institute of Biotechnology) and the protein concentration was determined using a bicinchoninic acid kit (Beyotime Institute of Biotechnology). Equal amounts of protein sample (30 µg per lane) were separated by 10% SDS-PAGE and then transferred onto PVDF membranes (Millipore, Sigma). After blocking with 5% skim milk for 2 h at room temperature, the membranes were incubated with primary antibodies against proliferating cell nuclear antigen (PCNA; 1:1,000; ab18197), Ki67 (1:500; ab254123), E-cadherin (1:1,000; ab238099), N-cadherin (1:1,000; ab76059), Vimentin (1:1,000; ab137321) and GAPDH (all from Abcam) at 4˚C overnight. Subsequently, the membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies at room temperature for 2 h. The protein bands were detected using an enhanced chemiluminescence kit (Pierce; Thermo Fisher Scientific, Inc.).
Statistical analysis. All statistical analyses were performed using SPSS software version 20.0 (IBM Corp.) or GraphPad Prism 6.0 (GraphPad Software, Inc.). The differences of expression levels of TMCC1-AS1 between tumor and normal group were compared using a paired sample t-test. All LC patients were divided into high and low expression groups according to the median value of TMCC1-A1 expression. The association between TMCC1-AS1 expression and clinicopathological features of patients with LC was evaluated using the χ 2 test. Kaplan-Meier and log-rank analyses were used to evaluate the prognosis of patients with LC, and proportional hazards model (Cox) regression was utilized for univariate and multivariate analyses. Quantitative in vitro data are presented as the mean ± SD of three independent experiments. One-way ANOVA followed by Dunnett's test was applied for analyzing the differences in quantitative data. P<0.05 was considered to indicate a statistically significant difference.

TMCC1-AS1 is upregulated in LC and increased TMCC1-AS1
expression is associated with worse prognosis. To explore the functional role of TMCC1-AS1 in LC, the present study examined the expression levels of TMCC1-AS1 in LC tissues and cell lines using RT-qPCR. As shown in Fig. 1A, the expression levels of TMCC1-AS1 were significantly increased in tumor tissues compared with matched adjacent normal tissues derived from 68 patients with LC. Consistently, TMCC1-AS1 expression was higher in the LC cell lines (HepG2 and SNU-182) compared with normal liver THLE-3 cells (Fig. 1B). To understand the clinical significance of TMCC1-AS1 upregulation in LC, the present study investigated the potential associations between TMCC1-AS1 expression and clinicopathological features of patients. The results of the χ 2 test demonstrated that high TMCC1-AS1 expression was significantly associated with advanced TNM stage and lymph node metastasis, but not associated with age, sex, HBV infection and tumor size (Table I). Furthermore, Kaplan-Meier analysis with log-rank test was performed to evaluate the association between TMCC1-AS1 expression and overall survival of patients with Table I. Association between TMCC1-AS1 expression and clinicopathological characteristics of patients with hepatocellular carcinoma (n=68). LC. As shown in Fig. 2, patients with high TMCC1-AS1 expression had a shorter overall survival than patients with low TMCC1-AS1 expression (log-rank P=0.0022). Furthermore, univariate analyses suggested that TMCC1-AS1 expression, as well as TNM stage and lymph node metastasis, were significantly associated with overall survival of patients with LC (Table II). Notably, TMCC1-AS1 expression and lymph node metastasis served as independent prognostic factors for poor overall survival (P=0.021; hazard ratio, 2.013; 95% confidence interval, 1.485-2.696).

TMCC1-AS1 upregulates PCNA expression and activates the epithelial-mesenchymal transition (EMT) process in LC cells.
Furthermore, the present study detected the effects of TMCC1-AS1 on the expression levels of proteins associated with proliferation and migration in LC cells. In HepG2 cells, knockdown of TMCC1-AS1 markedly downregulated the expression levels of a proliferation indicator (PCNA and Ki67) and suppressed the EMT process, as reflected by increased E-cadherin, and decreased N-cadherin and Vimentin protein expression (Fig. 5A). By contrast, overexpression of TMCC1-AS1 upregulated the expression levels of PCNA, Ki67, N-cadherin and Vimentin, but downregulated E-cadherin expression in SNU-182 cells (Fig. 5B). These data further demonstrated the positive regulation of TMCC1-AS1 on LC cell proliferation and migration.

Discussion
The present study revealed that TMCC1-AS1 expression levels were significantly upregulated in LC tissues and LC cell lines, which indicated that TMCC1-AS1 may contribute to the progression of LC. Furthermore, the present study demonstrated that high TMCC1-AS1 expression was associated with advanced TNM stage and lymph node metastasis. Notably, it was demonstrated that TMCC1-AS1 expression was significantly associated with overall survival and could serve as an independent potential prognostic biomarker for patients with LC. In line with our clinical data analysis, Cui et al (18) and Zhao et al (19) reported that patients with HCC with higher levels of lncRNA TMCC1-AS1 had a shorter overall survival time based on the lncRNA expression profiles of 370 patients with HCC from TCGA. Furthermore, Deng et al (20) demonstrated that TMCC1-AS1 was one member of the constructed nine-lncRNA prognosis model as a reliable tool for the prediction of the prognosis of HCC. The present study also demonstrated that TMCC1-AS1 promoted the proliferation, migration and invasion of HepG2 and SNU-182 cells, which was consistent with the clinical observation that high TMCC1-AS1 expression was closely associated with lymph node metastasis. These data indicated that TMCC1-AS1 was important in controlling hepatocellular carcinogenesis, even though, to the best of our knowledge, there are no studies reporting the oncogenic role of TMCC1-AS1 in tumor cells at present.
Next, the present study focused on the EMT signaling pathway to explore the potential mechanism by which TMCC1-AS1 promoted the metastasis of LC cells. EMT is a process in which cells change their epithelial phenotype and lose cell polarity, causing enhancement of migratory and wandering ability (23). The hallmarks of EMT include decreased epithelial marker E-cadherin expression and increased expression of mesenchymal markers, such as N-cadherin and Vimentin, which is a key element in tumor metastasis (24,25). The present study modulated the expression levels of TMCC1-AS1 and western blotting was performed to observe the effect of TMCC1-AS1 on EMT-related factors. Downregulation of TMCC1-AS1 expression resulted in increased E-cadherin expression and decreased N-cadherin and Vimentin expression. Overexpression of TMCC1-AS1 had the opposite effects on EMT-related factors. These data suggested that TMCC1-AS1 exerted its oncogenic effect on LC cell migration and invasion via modulation of the EMT pathway. Similarly, numerous lncRNAs, such as LOC105372579 (26), DANCR (27), CASC2 (28) and CRNDE (29), have been demonstrated to modulate the EMT pathway in HCC. In addition, PCNA, which is associated with cell proliferation, was downregulated in HepG2 cells after TMCC1-AS1 knockdown and upregulated in SNU-182 cells after TMCC1-AS1 overexpression. These regulatory effects of TMCC1-AS1 on PCNA and EMT-related factors further demonstrated the accelerative effects of TMCC1-AS1 on LC cell proliferation, migration and invasion. In addition, there were certain limitations of the present study, including a lack of in vitro experiments using additional cell lines and a lack of in vivo experiments.
In conclusion, the present data not only demonstrated that increased TMCC1-AS1 expression was associated with poor prognosis in patients with LC, but also demonstrated that TMCC1-AS1 promoted the proliferation, migration, invasion and EMT process of LC cells. The present findings may contribute to the understanding of the mechanisms underlying LC progression and promote the development of novel therapeutic strategies for LC. drew the figures. YS was involved in drafting the manuscript and revising it critically for important intellectual content. All authors read and approved the final manuscript.

Ethics approval and consent to participate
The present study has been approved by the Ethics Committee of Affiliated Hospital of Hebei Engineering University (Handan, China) and the participants provided written informed consent.

Patient consent for publication
Not applicable.