Long non-coding RNA CASC2 regulates cell biological behaviour through the MAPK signalling pathway in hepatocellular carcinoma

Abstract

Long non-coding RNAs have previously been demonstrated to play important roles in regulating human diseases, especially cancer. However, the biological functions and molecular mechanisms of long non-coding RNAs in hepatocellular carcinoma have not been extensively studied. The long non-coding RNA CASC2 (cancer susceptibility candidate 2) has been characterised as a tumour suppressor in endometrial cancer and gliomas. However, the role and function of CASC2 in hepatocellular carcinoma remain unknown. In this study, using quantitative real-time polymerase chain reaction, we confirmed that CASC2 expression was downregulated in 50 hepatocellular carcinoma cases (62%) and in hepatocellular carcinoma cell lines compared with the paired adjacent tissues and normal liver cells. In vitro experiments further demonstrated that overexpressed CASC2 decreased hepatocellular carcinoma cell proliferation, migration and invasion as well as promoted apoptosis via inactivating the mitogen-activated protein kinase signalling pathway. Our findings demonstrate that CASC2 could be a useful tumour suppressor factor and a promising therapeutic target for hepatocellular carcinoma.

Keywords

Long non-coding RNA cancer susceptibility candidate 2 hepatocellular carcinoma mitogen-activated protein kinase

Introduction

Hepatocellular carcinoma (HCC) is the sixth most common malignancy worldwide, and it is second only to lung cancer in terms of mortality in human cancer.¹ Because of chronic hepatitis B infection and liver cirrhosis, China accounts for 55% cases of the world each year.² Despite the therapeutic advances that have been made in HCC, such as surgical resection, liver transplantation and adjuvant therapy, the efficacy and prognosis of HCC patients remain unsatisfactory.³ Patients with this malignancy have a low 5-year-survival rate largely due to the lack of effective tools for the early diagnosis and high incidence of tumour recurrence or metastasis after treatment.⁴ Earlier studies have indicated that hepatocarcinogenesis is a multistep process characterised by many abnormal molecules that are not all known.⁵ To develop better preventive and diagnostic methods, as well as more effective treatment approaches, a deep understanding is required for the molecular mechanisms implicated in the complex process of liver carcinogenesis.

In recent years, studies have shown that only 1% of the genome can be transcribed into RNA that is further translated into protein, the other RNAs are collectively known as non-coding RNAs (ncRNAs).⁶ Long non-coding RNAs (lncRNAs) are a group of non-protein-coding RNAs with a length of >200 nucleotides that can regulate gene expression at the transcriptional or posttranscriptional level.⁷ In addition, studies have shown that lncRNA regulates various biological processes, including cell growth, cell cycle, differentiation and apoptosis.⁸ Benefiting from the advance of high-throughput transcriptome analyses, increasingly new lncRNAs have been found and identified as oncogenes or anti-oncogenes in HCC, such as H19, MALAT-1 (metastasis-associated lung adenocarcinoma transcript 1), HOTAIR (HOX transcript anti-sense RNA) and HULC (highly upregulated in liver cancer).^9–12 However, most of the roles of lncRNAs in HCC remain unknown yet.

The lncRNA CASC2 (cancer susceptibility candidate 2), which is located on chromosome 10q26, was initially found to be downregulated in endometrial cancer and was considered to be a tumour suppressor.^13,14 Furthermore, studies have reported that CASC2 is abnormally expressed in certain cancers and affects the development of tumours, including glioma, lung cancer, colon cancer and renal carcinoma.^15–18 However, the expression and functional role of lncRNA CASC2 in HCC are still unknown. Thus, the potential relationship between CASC2 expression levels and the clinicopathological factors of patients with HCC was investigated. Moreover, we demonstrated that the upregulation of CASC2 expression inhibited hepatoma cell proliferation, migration and invasion and induced apoptosis in vitro. Furthermore, we found that the overexpression of CASC2 can result in the inactivation of mitogen-activated protein kinase (MAPK) pathway. Thus, these results may provide new insight into the role of lncRNAs in the development of HCC and indicate the potential application of CASC2 in the treatment of HCC.

Materials and methods

Cell lines and samples

The HepG2, HuH7, Hep3B, SMMC7221 and Bel7402 human HCC cell lines and LO2 human immortalised normal hepatocyte cells were maintained in our laboratory. All the cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) high glucose medium (HyClone, Logan, UT, USA) containing 10% foetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA) and were incubated at 37°C in a humidified environment containing 5% CO₂. Samples of HCC tissues and corresponding normal liver tissues were collected from patients who had undergone surgery for HCC at Renmin Hospital of Wuhan University and Hubei Provincial Tumor Hospital between 2013 and 2015. None of the patients underwent radiation therapy, chemotherapy, biological therapy, or other treatment preoperatively. All the HCC and normal liver tissue samples were pathologically confirmed. The tissue samples were snap-frozen with liquid nitrogen after surgery and were stored at −80°C prior to RNA extraction. The study was approved by the Ethics Committee of Renmin Hospital of Wuhan University, and written informed consent for the biological studies was obtained from each patient involved in the study.

RNA extraction

Total RNA was extracted from the cells or tissue samples using TRIzol reagent (Invitrogen) according to the manufacturer’s recommended instructions. The RNA purity and integrity were analysed using an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). Quantified total RNA was further purified using an RNeasy Mini Kit (Qiagen, Hilden, Germany) and an RNase-Free DNase Set (Qiagen).

Quantitative real-time polymerase chain reaction

Complementary DNA (cDNA) was synthesized using the Revert Aid First Strand cDNA Synthesis Kit instructions (Fermentas, Vilnius, Lithuania). The RNA expression levels were detected using quantitative real-time polymerase chain reaction (qRT-PCR), which was performed using an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and SYBR Green Mix (TaKaRa Bio Inc., Shiga, Japan). CASC2 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primers were synthesized by Wuhan Biofavor Biotech company. The expression of GAPDH was used as a reference to normalise the amount of lncRNA in each sample. All the qRT-PCR reactions were performed in triplicate. The relative RNA expression was calculated using the 2^−ΔΔCt method. The primer sequences that were tested in this study were as follows—CASC2: 5′-GCACATTGGACGGTGTTTCC-3′ (forward) and 5′-CCCAGTCCTTCACAGGTCAC-3′ (reverse); GAPDH: 5′-AGAAGGCTGGGGCTCATTTG-3′ (forward) and 5′-AGGGGCCATCCACAGTCTTC-3′ (reverse).

RNA transfection

A specific plasmid targeting CASC2 RNA (pcDNA3.1-CASC2) and a negative plasmid were synthesised by Shanghai OE biotech (Shanghai, China). HCC cells were transfected with 4.0 µg of either plasmid targeting CASC2 using Lipofectamine 2000 Transfection Reagent (Invitrogen) according to the manufacturer’s recommended instructions. To monitor the transfection efficiency, the negative plasmid was used as a control. The cells were harvested 48 h after transfection.

Cell Counting Kit-8 assay

Exponentially growing HepG2 and HuH7 cells were digested and centrifuged and were then collected and inoculated into 96-well culture plates at a density of 4 × 10³ cells/well. After culturing for 24 h in DMEM containing 10% FBS, the cells were transfected with the pcDNA3.1-CASC2 or negative plasmid. Short-term proliferation activity was analysed using the Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Kumamoto, Japan) assay at the indicated time points. After transfection for 0, 24, 48, 72 and 96 h, the CCK-8 reagent was added to the culture wells, which were then incubated at 37°C for an additional 2 h. To plot the growth curve, a microplate reader (Bio-Rad Laboratories, Los Angeles, CA, USA) was used to measure the absorbance at 450 nm. Experiments were performed three times.

Tablet cloning experiments

HepG2 and HuH7 cells were inoculated into a six-well plate at a density of 1 × 10⁵/well and were then cultured in a 37°C incubator with 5% CO₂. On the following day, the cells were transfected with the pcDNA3.1-CASC2 or negative plasmid. After culturing for 24 h, the transfected cells were seeded on six-well plates at 500 cells per well for a 2-week incubation at 37°C and 5% CO₂. The colonies were fixed in paraformaldehyde solution for 10 min and were then stained with 0.2% crystal violet (KaryoMAX; Gibco, Grand Island, NY, USA) for 10 min. The colonies with a diameter greater than 1 mm were counted under a microscope. Experiments were performed three times.

Flow cytometry assay

After 48 h of transfection, the cells were harvested and washed with ice-cold phosphate-buffered saline (PBS). Next, the HCC cells were resuspended in 500 µL of 1× binding buffer, 10 µL of propidium iodide and 5 µL of Annexin V–fluorescein isothiocyanate (FITC; MultiSciences Biotech, Hangzhou, China) to detect cell apoptosis in a FACSCalibur flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Data analysis was performed using FCS express version 3 software (De Novo Software, Los Angeles, CA, USA). Experiments were performed three times.

Cell migration and invasion assays

Transwell Chambers (Becton, Dickinson and Company) were used to detect the activities of cell migration and invasion after CASC2 overexpression. For the migration assay, cells with a density of 5 × 10⁴ in 200 µL of serum-free DMEM medium were inoculated in the upper compartment of the chamber. A total of 600 µL of DMEM medium containing 10% FBS was added to the lower chamber. After the cells were cultivated at 37°C in a 5% CO₂ incubator for 24 h, the top layer of the insert was wiped off with a sterile cotton swab to remove any remaining cells. Next, the cells that had invaded the microporous membrane were washed with PBS and fixed with paraformaldehyde solution at 4% for 15 min; then, they were dyed with crystal violet for 10 min. Finally, a 400× inverted microscope (Olympus BX51 Olympus, Tokyo, Japan) was selected to calculate the invading cells in 10 randomly microscopic fields in every group. Experiments were repeated three independent times. For the invasion assay, the experimental operation steps were similar to those of the migration assay, except that the transwell membranes were pre-coated with 50 µL of Matrigel (Becton, Dickinson and Company). Every procedure was repeated three times in each group.

Western blot analysis

After transfecting with plasmid for 48 h, the cells were lysed in radioimmunoprecipitation assay (RIPA) buffer containing fresh protease and phosphatase inhibitor incubated at 4°C for 20 min. Identical quantities of proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred onto polyvinylidene difluoride (PVDF) membranes. After incubation with specific antibodies overnight at 4°C, the blots were incubated with sheep anti-mouse or anti-rabbit secondary antibodies for 1-2 h. GAPDH expression was used as an internal control. Finally, the bands were scanned using the Odyssey Infrared Imaging System (LI-COR, Houston, TX, USA), which can analyse the optical density value of particular strip. Details of the antibodies are shown in Table 1.

Table 1.

The details of antibody solutions.

Name	Producers	Article number	Dilution ratio	Species	Molecular weight (kDa)
c-Jun	Santa Cruz Biotechnology, CA, USA	sc-44	1:500	Rabbit	43
c-Myc	Abcam, Cambridge, England	ab32072	1:2000	Rabbit	57
Bax	Abcam, Cambridge, England	ab32503	1:1000	Rabbit	21
Caspase-3	TDY Biotech, Beijing, China	TDY411	1:1000	Rabbit	35
Bcl2	TDY Biotech, Beijing, China	TDY061	1:1500	Mouse	26
MMP9	Abcam, Cambridge, England	ab52496	1:500	Rabbit	92
MMP2	Abcam, Cambridge, England	ab86607	1:1000	Mouse	72
p-P38	Abcam, Cambridge, England	ab178867	1:1000	Rabbit	42
P38	Abcam, Cambridge, England	ab32142	1:1000	Rabbit	42
p-JNK	Abcam, Cambridge, England	ab76572	1:1000	Rabbit	48
JNK	Abcam, Cambridge, England	ab179461	1:1000	Rabbit	46, 54
p-ERK	CST, Hochschule Darmstadt, Germany	#4370	1:1000	Rabbit	42, 44
ERK	Abcam, Cambridge, England	ab17942	1:1000	Rabbit	42, 44
GAPDH	Abcam, Cambridge, England	ab37168	1:10,000	Rabbit	37

MMP: matrix metalloproteinase; p-JNK: phospho c-Jun N-terminal kinase; p-ERK: phospho extracellular signal–regulated kinase; CST: Cell Signaling Technology; GAPDH: glyceraldehyde 3-phosphate dehydrogenase.

Statistical analyses

All statistical analyses were performed using SPSS 20.0 (IBM, NY, USA) software. The differences between independent groups were analysed using analysis of variance (ANOVA) followed by Student’s t test. The correlations between CASC2 and clinicopathological characteristics were analysed using Pearson’s chi-square test or Fisher’s exact test. GraphPad Prism 5.0 (GraphPad Software, La Jolla, CA, USA) and Adobe Photoshop CS6 (Adobe, San Jose, CA, USA) were adopted for image analysis. p < 0.05 was considered to be statistically significant.

Results

The lncRNA CASC2 is downregulated in HCC tissues and cell lines

We determined the expression levels of CASC2 in liver cancer tissues and cell lines. qRT-PCR analysis showed that the CASC2 expression level was obviously decreased in liver cancer samples compared with that in paracarcinoma liver tissue samples (Figure 1(a); *p < 0.05). Furthermore, the expression of CASC2 was decreased in liver cancer cell lines (HepG2, HuH7, Hep3B, SMMC7221 and Bel7402) compared with that in the normal liver cell line (LO2; Figure 1(b); *p < 0.05). As CASC2 expression was lowest in HepG2 and HuH7 cells, we chose them for the following cytology experiment.

Figure 1.

LncRNA CASC2 expression is downregulated in HCC tissues and cells. (a) Quantitative real-time PCR was used to detect the expression level of CASC2 in cancerous tissues and paracancer tissues, indicating that CASC2 expression was significantly downregulated in cancerous tissue compared with that in adjacent tissues (*p < 0.05). (b) qRT-PCR was performed to assess CASC2 expression in LO2, HepG2, HuH7, Hep3B, SMMC7721 and Bel7402 cells, indicating that CASC2 expression in HCC cells was lower than that in normal hepatocyte LO2 cells, especially HepG2 and HuH7 cells (*p < 0.05). Therefore, HepG2 and HuH7 cells were used in the subsequent experiments.

According to the association between CASC2 expression and clinicopathological characteristics of liver cancer, the downregulated expression of CASC2 was not correlated with tumour size, metastasis and tumor–node–metastasis (TNM) stage (p > 0.05; Table 2). This result may be due to an insufficient sample size. In future experiments, the sample size of liver cancer must be expanded and additional medical records must be collected to verify the correlation.

Table 2.

The relationship between CASC2 expression and clinicopathological characteristics in 50 cases of hepatocellular carcinoma patients.

characteristics	Number of patients	CASC2		p value
characteristics	Number of patients	low	high	p value
All cases	50	31 (62%)	19 (38%)
Age (years)				0.833
≥50	28	17	11
<50	22	14	8
Gender				0.498
Male	39	23	16
Female	11	8	3
Tumour number				0.715
Single	41	26	15
Multiple	9	5	4
Tumour size (cm)				0.382
<5	25	14	11
≥5	25	17	8
Lymph node metastasis				1.000
Yes	12	8	4
No	38	23	15
Distant metastasis				0.629
Yes	4	2	2
No	46	29	17
TNM stage				0.640
I + II	19	11	8
III + IV	31	20	11
Histological differentiation				0.960
High + moderate	34	21	13
Low	16	10	6
PVTT				0.332
Absent	38	22	16
Present	12	9	3
AFP level (ng/mL)				0.13
≤400	36	20	16
>400	14	11	3
HBV				0.742
Yes	37	22	15
No	13	9	4

CASC2: cancer susceptibility candidate 2; TNM: tumor–node–metastasis; PVTT: portal vein tumor thrombosis; AFP: alpha-fetoprotein; HBV: hepatitis B virus.

Expression of CASC2 in HepG2 and HuH7 cells after plasmid interference

After HepG2 and HuH7 cells were transfected with the CASC2 plasmid for 48 h, the expression of CASC2 in liver cancer cells was examined by qRT-PCR. The results showed that the expression levels of CASC2 was significantly higher in the CASC2 plasmid-transfected group than in the other two groups (Figure 2; *p < 0.05).

Figure 2.

LncRNA CASC2 expression is detected in HepG2 and HuH7 cells by qRT-PCR after transfection of the lncRNA CASC2 plasmid, indicating that CASC2 expression was significantly higher in the pcDNA3.1-CASC2 group than in the blank and negative groups (*p < 0.05).

Inhibitory role of CASC2 in cell proliferation and colony formation

The obvious downregulation of CASC2 in HCC tissues and cells prompted us to study the potential biological functions of CASC2 in carcinogenesis. First, the CCK-8 assay showed that the proliferation of HepG2 and HuH7 HCC cells was remarkably inhibited after CASC2 expression was enhanced for 24, 48, 72 and 96 h compared with the blank and negative groups (Figure 3(a) and (b); *p < 0.05 in the experimental groups). Because colony formation is another important indicator of the viability of cells, we further detected the effect of CASC2 on cell colony formation. According to the proliferation assay, the colony formation assay also indicated that the clonogenic survival of HepG2 and HuH7 cells was evidently suppressed following CASC2 overexpression (Figure 3(c)–(e); *p < 0.05). Furthermore, western blotting indicated that the protein expression levels of c-Myc and c-Jun were downregulated in the pcDNA3.1-CASC2 group compared with those in the blank and negative groups (Figure 3(f)–(i); *p < 0.05).

Figure 3.

LncRNA CASC2 inhibits HCC cell proliferation and tumour growth. (a and b) The CCK-8 assay was performed to determine the cell proliferative activities after transfection for 0, 24, 48, 72 and 96 h, showing that CASC2 overexpression significantly decreased cell proliferation in a time-dependent manner compared with that in the blank and negative groups (*p < 0.05). (c–e) The clone-formation assay revealed that the quantity and size of the colony in the pcDNA3.1-CASC2 group were significantly decreased compared with those in the blank and negative groups (*p < 0.05). (f–i) Western blotting indicated that protein expression levels of c-Myc and c-Jun were downregulated in the pcDNA3.1-CASC2 group compared with that in the blank and negative groups (*p < 0.05).

Effect of CASC2 on cell apoptosis

After transfection for 48 h, the changes in cell apoptosis were detected by flow cytometry. The results showed that the numbers of apoptotic cells were obviously increased in the pcDNA3.1-CASC2 group, indicating that CASC2 can promote liver cancer cell apoptosis (Figure 4(a)–(c); *p < 0.05). Moreover, western blotting demonstrated that the protein expression level of Bax and caspase-3 was upregulated and Bcl2 expression was downregulated in the pcDNA3.1-CASC2 group compared with that in the blank and negative control groups (Figure 4(d)–(h); *p < 0.05).

Figure 4.

LncRNA CASC2 promotes cellular apoptosis in HCC cells. (a–c) Flow cytometry was performed to test the rate of apoptosis, displaying that CASC2 interference induced an obviously higher rate of apoptosis in the pcDNA3.1-CASC2 group than in the control groups (*p < 0.05). (d–h) Western blotting indicated that upregulated CASC2 led to the downregulated protein expression of Bcl2 and upregulated expression of Bax and caspase-3 (*p < 0.05).

Inhibitory role of CASC2 in cell migration and invasion

Following the verification of the inhibitory role of CASC2 in HCC cell viability, we further investigated whether this lncRNA participates in cell migration and invasion. Compared with HepG2 and HuH7 cells transfected with the negative vector, cells overexpressing CASC2 for 48 h showed significantly decreased ability in migration (Figure 5(a), (b) and (e); *p < 0.05). Subsequently, in the evaluation of cell invasion, HepG2 and HuH7 cells exhibited significant impairment of invasion ability after transfection with CASC2 for 48 h (Figure 5(a), (b) and (f); *p < 0.05). Western blotting showed that the protein expression levels of matrix metalloproteinase 2 (MMP2) and MMP9 were downregulated in the pcDNA3.1-CASC2 group (Figure 5(c), (d), (g) and (h); *p < 0.05).

Figure 5.

LncRNA CASC2 suppresses cellular migration and invasion in HCC cells. (a, b and e) The transwell assay was performed to assess the migration capability, indicating that upregulated CASC2 markedly suppressed the ability of migration compared with that in the two control groups (*p < 0.05). (a, b and f) The invasion capability was also tested by the transwell assay, showing that the upregulation of CASC2 significantly depressed the cell invasive potential compared with that in the blank and negative groups. (c, d, g and h) Western blotting indicated that the protein expression of MMP2 and MMP9 was downregulated in the pcDNA3.1-CASC2 group (*p < 0.05).

CASC2 inhibits HCC growth through the MAPK signalling pathway

We next attempted to investigate the underlying mechanism by which CASC2 regulated cell growth, apoptosis, migration and invasion. MAPKs, including extracellular signal–regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38, are mediators of the cellular responses to extracellular signals. In this study, we further assessed whether the expression levels of ERK, JNK and p38, which are crucial molecules involved in pathways associated with cancer pathogenesis, were altered when CASC2 was overexpressed. Our results indicated that ERK, JNK and p38 kinase phosphorylation was detected in CASC2 overexpressed cells in contrast to that in control cells, indicating that CASC2 may regulate cell growth, migration and invasion and induce apoptosis through the inactivation of the MAPK signalling pathway (Figure 6).

Figure 6.

Up-regulated expression of lnRNA CASC2 inhibits the activation of the MAPK pathway protein.(A,B) Western blotting was performed to detect the expression of total and the phosphorylation levels of P38, JNK and ERK protein.(C,F) Western blotting revealed that the total and the phosphorylation levels of P38 were not change in the pcDNA3.1-CASC2 group(P>0.05). (D,E,G,H))Western blotting revealed that the total and the phosphorylation levels of ERK and JNK were decreased in the pcDNA3.1-CASC2 group compared with those in the blank and negative groups (*P<0.05).

Discussion

Recent genome-wide studies have shown that a greater proportion of the genomic sequences are transcribed into lncRNAs than into protein-coding RNAs. Evidence has indicated that dysfunctional lncRNAs are involved in the pathogenesis of human cancer, including HCC.¹⁹ However, the functional and clinical significance of lncRNA and its molecular mechanisms are still not well known. With the rapid development of high-throughput detection technologies, an increasing number of HCC-related lncRNAs will be identified and characterised.^20,21 In this research, we aimed to explore deeply into the functions and mechanisms of the abnormally expressed lncRNAs.

CASC2 was shown to be expressed at a lower level in various tumour tissues such as endometrial cancer, glioma, non-small cell lung cancer, colorectal cancer and renal carcinoma tissues than that in normal tissues.^13–18 In U251 and U87 glioma cancer cell lines, CASC2 behaved as a tumour suppressor and inhibited cell proliferation, migration and invasion, as well as promoted apoptosis.¹⁵ In addition, recent studies have shown that the abnormal regulation of the MAPK signalling pathway was associated with aggressive progression and the poor prognosis of liver cancer.²² The MAPK signalling pathway regulates various cellular biological processes, such as gene expression, cell growth, differentiation, proliferation, apoptosis, invasion and metastasis.²³ The ERK signalling pathway possesses the core molecules of the MAPK pathway and the capacity to mediate cell proliferation, apoptosis and invasion once activated by inducing factors, differentiation signals and stimuli.^24,25 However, the function of JNK and p38 is mainly involved in the cell inflammatory response and stress reaction.²⁶ At present, many studies have confirmed that the deregulated MAPK signalling pathway is involved in the occurrence and development of HCC.²⁷ To elucidate the precise mechanism involved in CASC2-induced cell biological behaviour, the effects of CASC2 on MAPK activation or inactivation were examined.

Despite its importance in several malignant tumours, it remains unclear whether CASC2 expression is associated with the tumourigenesis and progression in primary liver cancer. First, qRT-PCR detection in a larger sample of tissues confirmed the significant downregulation of lncRNA CASC2, and the reduction of CASC2 expression was also observed in HCC cells, suggesting that CASC2 may act as a tumour suppressor in HCC. After demonstrating the regulatory role of CASC2 in HCC, we detected its influence on HCC cells. In a series of experiments on malignant cell behaviour, cancer cells overexpressing CASC2 exhibited significantly decreased proliferation, migration and invasion, as well as an increased apoptosis. The observed changes in the cancer cell behaviour provide evidence for the tumour-suppressing role of CASC2 and are consistent with our speculation.

The relationship between CASC2 and MAPK was also detected for the first time in this study. Therefore, we focused on whether MAPK was involved in the cell proliferation and apoptotic processes that were regulated by CASC2 expression. Western blot analyses confirmed that CASC2 overexpression significantly decreased ERK and JNK phosphorylation compared with that in blank cells and negative cells. The regulation of CASC2 suggests that CASC2 may inactivate the MAPK signalling pathway.

Although we demonstrated that CASC2 may act as a tumour suppressor gene to regulate cell biological behaviour by targeting the MAPK pathway, the relationship between the gene expression and prognosis of HCC remains unclear. More liver cancer samples and follow-up of the survival time are needed to further study the effects of gene expression on the prognosis of HCC. In addition, the complex regulatory mechanisms, including nuclear transcription and epigenetic inheritance, that may affect lncRNA expression remain unclear. Therefore, further studies using luciferase reporter and/or chromatin immunoprecipitation (ChIP) assays to elucidate the regulatory mechanisms controlling CASC2 expression are necessary. Moreover, the construction of a loss-of-function CASC2 model system in HCC cells would contribute to comprehensively evaluate its influence on HCC cell biology in subsequent studies. Future research is required to establish a special animal model of tumour growth to verify the actual effect of CASC2 in vivo. The detailed underlying mechanism remains to be further elucidated between CASC2 and the MAPK signalling pathway for CASC2 to be extensively applied in clinical target treatment. Many experiments remain to be performed to explain the roles of the lncRNA CASC2. In addition, the 5-ethynyl-2′-deoxyuridine (EdU) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) methods or cell cycle analysis can be used to study the effects on cell proliferation. Furthermore, the fluorescence double-labelling method and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay can be used to study apoptosis. We can also use the wound-healing test and real-time cell tracking method to study cell migration.

In conclusion, we have identified that CASC2 is downregulated in HCC cells and tissues. Our findings indicate that CASC2 inhibits cell proliferation, migration and invasion and promotes apoptosis by inactivation of the MAPK pathway. We primarily demonstrated the regulatory mechanism of CASC2 in HCC progression. Therefore, our findings indicate that CASC2 may act as a tumour suppressor in HCC growth and has the potential to be a therapeutic target for HCC.

Footnotes

Acknowledgements

Y.G. and N.H. contributed equally to this work.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by Natural Science Foundation of Hubei Province of China (No. 2012FKC143).

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