LINC00978 promotes hepatocellular carcinoma carcinogenesis partly via activating the MAPK/ERK pathway

Abstract Objective: To study the role of long non-coding RNA (lncRNA) LINC00978 in hepatocellular carcinoma (HCC) carcinogenesis. Materials and methods: LINC00978 expression level was measured by reverse transcription quantitative real-time PCR (RT-qPCR) in HCC tissues and adjacent healthy liver tissues from 49 HCC patients. MTT assay, colony forming assay, and flow cytometry were performed to evaluate the effects of shRNA-mediated LINC00978 knockdown on HCC cell proliferation, cell cycle progression, and apoptosis in vitro. Xenograft tumor model was performed to determine the effects of LINC00978 knockdown on HCC tumor growth in vivo. Western blot was used to assess the activation of signaling molecules in the apoptosis and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway. Results: LINC00978 expression was significantly up-regulated in human HCC tissue relative to adjacent normal tissue, and LINC00978 high expression was correlated with poor HCC overall survival. LINC00978 was up-regulated in HCC cell lines. ShRNA-mediated LINC00978 knockdown significantly decreased HCC cell proliferation, and induced HCC cell cycle arrest and apoptosis in vitro. LINC00978 knockdown led to significant decrease in tumor xenograft size in vivo. Western blots revealed LINC00978 inhibition decreased ERK, p38, and c-Jun N-terminal kinase (JNK) phosphorylation in HCC cells. Conclusions: LINC00978 is highly expressed in human HCC tissue and correlates with poor HCC prognosis. LINC00978 promotes HCC cell proliferation, cell cycle progression, and survival, partially by activating the MAPK/ERK pathway. Our findings partially elucidated the roles of LINC00978 in HCC carcinogenesis, and identified a therapeutic target for HCC.


Introduction
Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver, and the most common cause of death in people with chronic liver disease and cirrhosis [1]. In contrast with the overall decline for most major cancers, the death rates of HCC rose by 1.6-2.7% per year from 2011 to 2015 for both women and men [2], and it is expected to continue to rise in the coming years [3]. Currently, HCC is the second leading cause of cancer deaths worldwide [4,5]. In China, 466100 HCC cases are newly diagnosed each year, accounting for over 50% of global incidences [6,7]. The incidence of HCC is higher in Asia and Africa comparing with other regions, which is largely due to the high prevalence of hepatitis B infection and exposure to aflatoxin. More recently, there have been steady increases in HCC in the developed countries due to an increase in hepatitis C infection [2]. Current treatment for HCC is limited to surgical resection and liver transplantation, with no effective systemic therapies. The overall outcome is poor, with more than 50% of patients suffering from recurrence and metastasis, as well as death within 3-6 months. Therefore developing novel, efficacious diagnostic and therapeutic strategies for HCC becomes a major public health demand.
Long non-coding RNAs (lncRNAs) participated in carcinogenesis, and offered valuable information in early diagnosis and prognosis [8][9][10]. lncRNAs are a diverse class of non-protein-coding RNA molecules with over 200 nucleotides in length. The human transcriptome is enriched for lncRNAs, containing nearly 30000 different transcripts that far outnumber protein coding transcripts [10]. LncRNAs can be classified into five subtypes based upon their genomic organization: intergenic, intronic, antisense, bidirectional, and enhancer lncRNAs [10]. Several mechanisms that govern how lncRNAs regulate cellular functions have been described [11,12], including directly affecting the chromatin architecture, or binding to regulatory RNAs or proteins. The aberrant expression of several lncRNAs have been identified in HCC, including HOTAIR [13,14], MALAT1 [15], HULC [16,17], XIST [18], MEG3 [19], HEIH [20,21], and PANDA [22], some of which have displayed potential values in prognosis or a causal role in tumorigenesis [10]. However, to date, the vast majority of HCC-relevant lncRNAs have not been characterized in detail.
Recently, LINC00978 (also known as MIR4435-2HG, AGD2, or MORRBID) was identified as a novel lncRNA that promotes lung cancer growth [23] and acts as a biomarker for gastric [24] and breast cancer patients [25]. LINC00978 transcription was correlated with stem cell differentiation [26], and its murine ortholog, Morrbid, promotes myeloid cell lifespan [27], suggesting a pro-survival role for this lncRNA. However, the functional significance of LINC00978 in HCC has never been studied.
Here we showed that LINC00978 plays a critical role in HCC carcinogenesis in vitro and in vivo. LINC00978 expression was up-regulated in HCC tissue compared with normal liver tissue, and it was associated with poor HCC prognosis. LINC00978 promoted HCC cell proliferation, inhibited apoptosis in vitro, and enhanced tumor growth in vivo, partially by activating the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway. Our study suggests that LINC00978 may be an oncogene in HCC, and could offer potential prognostic value for HCC patients.

Patients and samples
Liver tumor tissue and matched adjacent healthy liver tissues were from 49 HCC patients that underwent surgeries at Hebei University between February 2010 and July 2011. All tissues were snap-frozen in liquid nitrogen immediately following excision during the surgery and stored at −80 • C until analysis. Two pathologists confirmed pathological diagnosis of all exercised tissues. Clinicopathological parameters of all recruited HCC patients were documented. The protocols were approved by the Ethics Committee of the Hebei University. Written informed consent was obtained from all patients from whom tissue samples were collected.

Proliferation assay
HCC cell proliferation was measured by colony formation and MTT assays. Colony formation assay was performed in triplicate on six-well plates. In brief, transfected HCC cells were re-seeded at 500 per well and allowed to grow for 2 weeks. Afterward, colonies were fixed with 4% paraformaldehyde for 5 min, and stained with 1% Crystal Violet for 10 min at room temperature. For MTT, assays were performed at 24-h intervals on transfected HCC cells seeded at 3 × 10 3 per well on 96-well plates. For each well, 20 μl of MTT (5 mg/ml, Sigma, CA, U.S.A.) was added and incubated at 37 • C for 2 h. The optical density (OD) values were determined at 570 nm with a microplate reader (Tecan Sunrise, Männedorf, Switzerland).

Human xenograft tumor model
Twenty-four male BALB/c nude mice were obtained from Laboratory Animal Centre of Hebei University and maintained under aseptic barrier conditions. At 6-week-old, Huh7 and Hep3B cells were each transfected with Lenti-shLacZ or Lenti-shLINC00978, and 4 × 10 5 cells per transfection were subcutaneously injected into the back-skin area of each mouse. Tumors were measured with a vernier caliper every 4 days, and tumor volume was estimated by the formula: volume = 0.5 × (length × width 2 ) [28]. Tumor weight was measured after excision from killing mice on a digital scale. The animal protocols were approved by the Committee on Animal Welfare of The Affiliated Hospital of Hebei University.

Statistical analysis
Statistical analysis was performed on SPSS (SPSS, Inc., Chicago, IL, U.S.A.) or GraphPad Prism 7.0 (GraphPad Software, CA, U.S.A.). Data were presented as mean + − standard deviation (SD) of at least three independent experiments. Student's t test and one-way ANOVA were used for comparing differences between two groups or among three or more groups, respectively. Kaplan-Meier's method and the log-rank test were performed to compare survival rate. P<0.05 was considered to be statistically significant. III-IV 32 16 16 Abbreviations: AFP, α-fetoprotein; TNM, tumor-node-metastasis. *P<0.05 presented as significant difference.

LINC00978 is up-regulated in HCC tissues and correlates with poor prognosis
We first examined LINC00978 expression in 49 pairs of tumor tissues and adjacent non-cancerous tissues from HCC patients using RT-qPCR. The clinicopathologic data of all patients were presented in Table 1. LINC00978 expression was significantly higher in HCC tumor tissue compared with adjacent normal tissue ( Figure 1A). Moreover, LINC00978 expression was significantly higher in HCC patients with advanced clinical stages (Edmondson grading III-IV) than that in HCC patients with early stages (Edmondson grading-II) ( Figure 1B). To explore the clinical relevance of LINC00978 expression in HCC, we searched for a correlation between LINC00978 expression and clinicopathological characteristics in HCC patients (Table 1). We set the median LINC00978 expression as a cut-off value and divided 49 HCC patients into LINC00978 high expression group (n=26 cases) and low expression group (n=23 cases) ( Figure 1C).
Five-year overall survival was significantly poorer in LINC00978 high expression group than that of low expression group ( Figure 1D). In addition, LINC00978 expression was significantly associated with higher tumor size, lymph node metastasis, Edmondson grading, and tumor-node-metastasis (TNM) stage (Table 1). There was no significant association with gender, age, α-fetoprotein level, HCC histological grade, or macrovascular invasion. Taken together, these results showed that LINC00978 was up-regulated in HCC tissue, and high LINC00978 was correlated with poor prognosis for HCC patients.

LINC00978 is up-regulated in HCC cell lines
We next examined LINC00978 expression in multiple HCC cell lines including SK-Hep1, Bel-7404, Huh7, Hep3B, and HepG2. Consistent with the results in tissue of HCC patient, LINC00978 showed three-to six-fold higher expression in HCC cell lines than that in normal human hepatic cell line LO2 (Figure 2). The results in HCC tissue and HCC cell lines indicated a positive role of LINC00978 in HCC carcinogenesis. Based on the expression profiles, we chose two HCC cell lines Huh7 and Hep3B, which had the highest LINC00978 expression, for subsequent studies.

LINC00978 increases HCC cell proliferation
To examine the role of LINC00978 in HCC cells, we transfected Huh7 and Hep3B cells with either non-targeting shRNA (sh-NC) or shRNA against LINC00978 (sh-LINC00978). RT-qPCR showed significant reduction in LINC00978 expression by sh-LINC00978 comparing with sh-NC ( Figure 3A,B). We found Huh7 and Hep3B cells that were transfected with sh-LINC00978 showed significantly reduced proliferation capacity than sh-NC, which was measured by the MTT assay ( Figure 3C,D) and the colony formation assay (Figure 3E-H). These results suggest that LINC00978 promotes HCC cell proliferation.

LINC00978 promotes HCC cell cycle progression and inhibits apoptosis
We next examined the role of LINC00978 in regulating HCC cell cycle and apoptosis by flow cytometry. In both Huh7 and Hep3B cells, LINC00978 knockdown induced cell cycle arrest, which was demonstrated by a significant increase in the proportion of cells in G 1 phase (P<0.05) and decrease in proportion of cells in S phase (P<0.01) at 48 h after shRNA-transfection ( Figure 4A-D). In both Huh7 and Hep3B cells, LINC00978 knockdown significantly increased the percentage of apoptotic cells compared with sh-NC (P<0.01, Figure 4E,F). Consistent with the increase in apoptosis by sh-LINC00978, Western blot showed that LINC00978 knockdown significantly increased expression of pro-apoptotic protein Bax, and decreased expression of anti-apoptotic protein Bcl-2 compared with sh-NC ( Figure  4G-K).

LINC00978 promotes HCC tumor growth in vivo
In order to test whether LINC00978 regulates HCC carcinogenesis in vivo, we compared Lenti-shLacZ and Lenti-shLINC00978-treated HCC tumor growth in vivo, using a subcutaneous xenograft tumor model in mice. Huh7 and Hep3B cells that transfected with Lenti-shLacZ or Lenti-shLINC00978 were injected into nude mice subcutaneously, and the volume of xenografts were measured over time. Both of the HCC xenografts developed into visible tumors. We found that the volume of Lenti-shLINC00978-treated HCC xenografts was significantly smaller than that of Lenti-shLacZ-treated tumors ( Figure 5). This difference in xenograft growth strongly supports that LINC00978 promotes HCC carcinogenesis in vivo.

LINC00978 activates MAPK/ERK signaling pathway in HCC cells
In order to investigate the mechanism between LINC00978 and HCC carcinogenesis, we examined activation of signaling proteins in the MAPK/ERK pathway, which is essential to the proliferation, survival, and differentiation of many cancer types including HCC [29]. We confirmed that LINC00978 is inhibited in Lenti-shLINC00978-treated HCC xenografts than that in Lenti-shLacZ-treated tumors by RT-PCR ( Figure 6A). Then the MAPK/ERK signaling pathway was found to be activated ( Figure 6B). In both Huh7 and Hep3B cells, LINC00978 knockdown lead to decreased levels of phospho-ERK (p-ERK), phospho-p38 (p-p38) and p-JNK ( Figure 6C,D). These data suggest that LINC00978 may partially induce HCC carcinogenesis via activating the MAPK/ERK signaling pathway.

Discussion
The major result of our study is that lncRNA LINC00978 promotes HCC carcinogenesis. LINC00978 was up-regulated in HCC patient tissues and predicted poor overall HCC survival. LINC00978 promoted HCC cell proliferation and cell cycle progression, inhibited apoptosis in vitro, and increased HCC tumor growth in vivo. Several signaling molecules in the ERK/MAPK signaling pathway was inhibited by LINC00978 knockdown in HCC cells. Thus, the present data provide strong functional evidence for the regulatory role of a novel lncRNA LINC00978 in HCC pathogenesis.
Given the limited therapeutic choices for HCC, there is an urgent need for developing early diagnostic and prognostic tools. Non-coding RNAs have been recognized as excellent biomarkers for the diagnosis and prognosis of various cancer types including HCC [8,10,30,31]. A previous study found that high LINC00978 expression in breast cancer is associated with poor disease-free survival [25]. Our results suggest the potential use of LINC00978 as an early HCC prognostic marker. However, studies with larger sample sizes are required to further assess its sensitivity and specificity.
A previous study has found that LINC00978 was dysregulated in breast cancer [25]. However, whether LINC00978 dysregulation was causal or simply a marker for tumors was unclear. Fu et al. [24] showed that LINC00978 was a biomarker and an oncogene that promotes gastric cancer progression. Our results are concordant with that conclusion and indicate that LINC00978 is an HCC biomarker as well as a causal factor that extensively involved in promoting a proliferative, pro-survival phenotype essential to HCC carcinogenesis. Notably, the causal role of LINC00978 in  tumorigenesis has been reported in other cancer types [23,24,32], suggesting that LINC00978 could have a broad impact on the progression of various cancer types.
The regulatory function of lncRNA depends on lncRNA-DNA, lncRNA-RNA, or lncRNA-protein interactions [33]. However, the target interactors for LINC00978 in HCC are unknown. The murine ortholog of LINC00978, Morrbid, was found to regulate the expression of a pro-apoptotic gene Bcl2l11 in mouse myeloid cells and promote myeloid cell lifespan [27]. While a similar mechanism for LINC00978 in HCC would be concordant with its anti-apoptotic role, i.e., the function of lncRNA is highly cell type-specific and displays vast tissue variation [34]. For example, others found that LINC00978 knockdown inhibited the TGF-β/SMAD pathway in gastric cancer, but we found that LINC00978 knockdown inhibited the MAPK/ERK pathway in HCC cells. Illuminating the exact regulatory target of LINC00978 in HCC will require unbiased transcriptome profiling such as RNA sequencing after RNA depletion of LINC00978, and biochemical studies are necessary to directly capture lncRNA interactions in HCC tissues or cell lines [35].
Recent preclinical data showed that blocking particular signaling molecules in the MAPK/ERK pathway could be beneficial for patients with advanced HCC [36][37][38], which is regarded as one of the most promising approaches for targeted HCC therapy [39][40][41]. We found that LINC00978 knockdown inhibited the phosphorylation of several signaling molecules in the MAPK/ERK pathway. Similarly, in colorectal cancer, Ouyang et al. [42] demonstrated that LINC00978 was involved in the P38/MAPK pathway by performing functional annotation based on Gene set enrichment analysis, and they suggested that LINC00978 might promote colorectal cancer cell growth, metastasis, and poor survival via P38/MAPK pathway. ERK, p38, and JNK have been shown to have profound effects on HCC cell differentiation, proliferation, survival, and invasion [40,[43][44][45]. These data suggest that inhibiting LINC00978 could be a desirable therapeutic direction for HCC. However, the mechanisms by which LINC00978 activates MAPK/ERK pathway requires further evaluation.
Our results have a few limitations. First, the HCC tissue was collected from Chinese patients with high prevalence of hepatitis B infection. Given the difference in HCC etiology, whether our findings are applicable to non-Asian HCC patients remains to be verified. Second, our clinical study was based on limited samples, and would have to be validated by larger cohorts with more heterogeneous populations. Third, it is conceivable that LINC00978 may have a wide range of functions in HCC tumorigenesis and may involve additional signaling pathways, which are not identified by the present study.

Conclusion
Overall, current work identified the role of a novel lncRNA LINC00978 in HCC carcinogenesis. LINC00978 is up-regulated in HCC tissue and it correlates with poor HCC prognosis. LINC00978 promotes the proliferation and survival of HCC in vitro, and increases HCC tumor growth in vivo, partially by activating the MAPK/ERK pathway.