Long non-coding RNA SPRY4-IT1 promotes development of hepatic cellular carcinoma by interacting with ERRα and predicts poor prognosis

Hepatocellular carcinoma (HCC) has become one of the most common leading causes of cancer-related deaths worldwide. This study investigates the role of lncRNA, SPRY4-IT1 in the development of HCC. Quantitative real-time PCR (qRT-PCR) was performed and the results showed that SPRY4-IT1 expression was up-regulated in HCC tissues and high expression of SPRY4-IT1 was associated with poor 5-year overall survival in the HCC patient cohort. Clinicopathological analysis showed that the expression of SPRY4-IT1 was significantly correlated with TNM stage in HCC patients. In vitro CCK-8 assay, colony formation assay, cell invasion and migration assays demonstrated that knock-down of SPRY4-IT1 suppressed cell proliferation, colony formation, cell invasion and migration in HCC cells. Flow cytometric analysis showed that knock-down of SPRY4-IT1 induced cell cycle arrest at G0/G1 phase and induced apoptosis. In addition, knock-down of SPRY4-IT1 also suppressed the mRNA and protein expression of estrogen-related receptor α (ERRα). Similarly, knock-down of ERRα inhibited cell proliferation, colony formation, cell invasion and migration in HCC cells. More importantly, ERRα overexpression antagonized the effects of SPRY4-IT1 knock-down on cell proliferation, colony formation, cell invasion and migration in HCC cells. Taken together, our data highlights the pivotal role of SPRY4-IT1 in the tumorigenesis of HCC.

Hepatocellular carcinoma (HCC) has become one of the most common leading causes of cancer-related deaths worldwide 1 . Despite advancement in the treatment modalities for HCC such as surgery, liver transplantation, chemotherapy, and radiotherapy, the 5-year overall survival rate of HCC patients has hardly improved 2,3 . The high mortality and poor prognosis of HCC are attributed to the incomplete understanding of the molecular mechanisms underlying the development and progression of HCC. In this regard, further understanding the molecular mechanisms is of great clinical significance for developing novel therapeutic targets for HCC treatment.
Recently, numerous researches have been focused on the long non-coding RNAs (lncRNAs), which are non-protein transcripts with the length more than 200 nt 4 . The lncRNAs are found to involve in a diverse biological processes including transcriptional regulation, splicing, chromatin modification et al. 5,6 . LncRNAs have been suggested to serve as diagnostic and prognostic biomarkers because studies have found that dysregulation of lcnRNAs were associated with cancer development in various types of cancers. LncRNAs such as TUG1, Linc00152 and CCAT1 were found to involve in the pathogenesis in colorectal cancer [7][8][9] . Further,XIST, GAS5-AS1, HOTAIR and MALAT1 are found to play key roles in the development and progression of lung cancer [10][11][12][13] . In HCC, an increasing number of lncRNAs have been reported to be associated with cancer development. PVT1 has been found to serve as a biomarker for HCC prognosis 14 . HULC promotes tumorigenesis and metastasis via epithelial-mesenchymal transition in HCC 15 . HOTAIR was found to promote human liver cancer stem cell growth through down-regulation of SETD2 16 . The expression of lncRNA, SPRY4 intronic transcript 1 (SPRY4-IT1), transcribed from an intron, is found to be up-regulated in various types of cancers including colorectal cancer, esophageal squamous cell carcinoma, glioma, and bladder cancer [17][18][19][20] . However, the role of SPRY4-IT1 in other types of cancers remains unknown, particularly in HCC.
The estrogen-related receptor α (ERRα) belongs to the nuclear receptor family. Because there is no natural ligand of ERRα has been identified to date, ERRα is regarded as an "orphan" receptor. It regulates the target genes in a ligand-independent way 21 . ERRα is found to involve in various physiological processes such as innate immunity, energy metabolism and osteoblast differentiation as well as bone formation [22][23][24][25] . Recently, ERRα has been suggested to be associated with the development and progression of various types of cancers. Knock-down of ERRα inhibited the tumor development on uterine endometrial cancer; 26 ERRα is a marker of tamoxifen response and survival in triple-negative breast cancer; 27 ERRα was also found to coordinate colon cancer cell proliferation and tumorigenic capacity with energy metabolism; 28 WNT11 expression is induced by ERRα and beta-catenin, and acts in an autocrine manner to increase cancer cell migration 29 . However, whether ERRα plays a role in HCC development is still unclear.
In this study, we identified the up-regulation of SPRY4-IT1 in HCC tissues, which was associated with poor prognosis in HCC patients. We also found that high level of SPRY4-IT1 promoted proliferation, cell cycle progression and suppressed cell apoptosis of HCC. Furthermore, SPRY4-IT1 was found to function in the progression of HCC via interacting with ERRα.

Materials and Methods
Human tissues samples. A total of 82 patients with HCC were included in this study. All the patients had undergone routine hepatic resection at the Fifth Affiliated hospital of Guangzhou Medical University from 2012 to 2015, and none of the patients had received chemotherapy or radiotherapy prior to surgical resection. This study was approved by the Ethics Committee of the Fifth Affiliated hospital of Guangzhou Medical University. All patients provided written informed consent for the use of tissue samples for clinical research. The histological diagnosis and differentiation of tumors were evaluated by pathologists according to the criteria of WHO classification system. The clinicopathological features are shown in Table 1. All the tissue samples were snap-frozen in liquid nitrogen immediately following surgical resection, and stored in −80 °C for further experimentation.   cells. Data are shown as mean ± SD, significant differences were marked as *P < 0.05, **P < 0.01, ***P < 0.001.
Western blotting assay. The HepG2 and SMMC7721 cells were lysed with denaturing SDS-PAGE sample buffer using standard methods. Protein lysates were separated on a 10% SDS-PAGE gel and transferred to the nitrocellulose membranes. The membranes were blocked with 5% skimmed non-fat milk for 1 h at room temperature, and then the membrane were incubated with rabbit polyclonal anti-ERRα antibody (Abcam, Cambridge, USA) at 4 °C overnight. After primary antibody incubation, the membranes were then incubated with HRP-conjugated anti-IgG at room temperature for 2 h. Signal was detected by an ECL system (Amersham Pharmacia, Piscataway, USA).   Statistical analysis. GraphPad Prism software was used for data analysis. All the data are shown as mean ± SD, and data were analyzed by t-test (for comparing two groups) or by one-way ANOVA followed by Turkey's multiple comparison tests (for comparing more than two groups). Results were considered statistically significant at P < 0.05.

SPRY4-IT1 mRNA is up-regulated in HCC tissues and is correlated with poor prognosis in HCC patients.
We evaluated the RNA levels of SPRY4-IT1 in 82 pairs of human primary HCC tissues and the respective adjacent normal non-cancerous liver tissues using qRT-PCR. We found that the expression of SPRY4-IT1 in HCC tissues were significantly higher than that examined in the respective adjacent normal non-cancerous liver tissues (Fig. 1A, P < 0.001). Clinical analysis demonstrated that SPRY4-IT1 RNA levels were correlated with TNM stage and metastasis (Table 1, P < 0.05). However, we have not observed significant correlation between SPRY4-IT1 expression levels and other clinical characteristics such as age, gender, serum AFP level, HBsAg status, tumor size, liver cirrhosis and histological differentiation (Table 1). To understand the prognostic role of SPRY4-IT1 in HCC, we examined the correlation between SPRY4-IT1 expression and HCC patients' overall survival. The results showed that high level of SPRY4-IT1 was significantly correlated with poor 5-year overall survival rate in HCC patients (Fig. 1B, P < 0.05). SPRY4-IT1 knock-down inhibits cell proliferation, colony formation, cell invasion and migration in HCC cell lines. The expression of SPRY4-IT1 was further examined in vitro in one normal liver cell line (HL7702) and four HCC cell lines (MHCC97H, HCCLM6, HepG2 and SMMC7721) by qRT-PCR. We found that the relative expression of SPRY4-IT1 in HCC cell lines were higher than that in normal liver cell line, with the highest expression in HepG2 and second highest in SMMC7721 cell lines ( Fig. 2A, P < 0.05). In the following study, HepG2 and SMMC7721 cell lines were chosen for further functional investigation in vitro. We transfected the siRNA targeting SPRY4-IT1 to knock-down SPRY4-IT1 in HepG2 and SMMC7721 cell lines. The SPRY4-IT1 siRNA transfection in HepG2 and SMMC7721 cells significantly reduced the expression levels of SPRY4-IT1 (Fig. 2B, P < 0.05). To examine the role of SPRY1-IT1 knock-down in cell proliferation, CCK-8 assay was performed at 0 h, 24 h, 48 h and 72 h after SPRY1-IT1 transfection. In comparison with scrambled siRNA (Control) transfected HepG2 and SMMC7721 cells at 48 or 72 h, HepG2 and SMMC7721 cells transfected with SPRY4-IT1 siRNA had significantly lower proliferative ability ( Fig. 2C and D, P < 0.05). To further investigate the effect of SPRY4-IT1 knock-down on cell growth, we performed colony formation assay. The results showed that the number of colonies in SPRY4-IT1 transfected HepG2 and SMMC7721 cells were significantly lower than that in scrambled siRNA transfected cells (Fig. 2E, P < 0.05). To examine the effect of SPRY4-IT1 knock-down on cell invasive and migratory abilities, we performed the cell invasion and cell migration assays. As shown in Fig. 2F and G, the number of invaded and migrated cells in SPRY4-IT1 transfected HepG2 and SMMC7721 cells were significantly reduced when compared to scrambled siRNA transfected cells ( Fig. 2F and G, P < 0.05).

SPRY4-IT1 knock-down suppresses ERRα expression, and ERRα knock-down suppressed cell proliferation, colony formation, cell invasion and cell migration. To investigate if SPRY4-IT1 had
an interaction with ERRα, we performed qRT-PCR and Western blotting to examine the RNA and protein expression of ERRα in SPRY4-IT1 siRNA or scrambled siRNA transfected HCC cell line. As shown in Fig. 4A and B, SPRY4-IT1 siRNA transfection HepG2 cells significantly suppressed the mRNA and protein expression levels of ERRα ( Fig. 4A and B). Furthermore, we examined the role of ERRα in HCC cell proliferation, colony formation, cell invasion and migration. ERRα inhibitory transfection significantly suppressed the mRNA and protein expression of ERRα in HepG2 cells ( Fig. 4C

Discussion
LncRNAs have become increasingly recognized as important regulators of gene expressions, as lncRNAs can regulate protein-coding genes at epigenetic, transcriptional, and post-transcription levels 5 . Recently studies have shown that a lot of lncRNAs are frequently aberrantly expressed in various types of cancers, and the dysregulation of lncRNAs have been suggested to be associated with tumor pathogenesis and metastasis. They are also useful in tumor diagnosis and prognosis 6 . Therefore, it is necessary to understand the molecular mechanisms of lncRNAs in cancer development and progression.
In this study, we detected the up-regulation of SPRY4-IT1 RNA in the HCC tissues when compared to adjacent non-cancerous liver tissues. Furthermore, the expression of SPRY4-IT1 was significantly correlated with TNM stage and tumor metastatic status. This was consistent with a previous study which also showed that SPRY4-IT1 was up-regulated in HCC tissues and was correlated with tumor differentiation, tumor size, and TNM stage 30 . Furthermore, we performed the loss-of-function experiments to look into the in vitro molecular mechanisms of SPRY4-IT1 in HCC development. The in vitro results showed that knock-down of SPRY4-IT1 suppressed the cell proliferation, colony formation, cell invasion and migration in HCC cell lines, and the results were in agreement with previous studies showing that knock-down of SPRY4-IT1 suppressed the cell proliferation, colony formation, cell invasion and migration in several types of cancers including colorectal cancer, esophageal squamous cell carcinoma, prostate cancer, glioma, gastric cancer, bladder cancer and breast cancer [17][18][19][20] . Thus, our results may suggest the oncogenic role of SPRY4-IT1 in the pathogenesis of HCC. In addition, the flow cytometry was performed to investigate the mechanistic role of SPRY4-IT4 in cell cycle and cell apoptosis. We found that knock-down of SPRY4-IT1 induced G 0 /G 1 cell cycle arrest and also increased the apoptotic rate of HCC cell lines. Similarly, in other types of cancers such as esophageal squamous cell carcinoma, breast cancer, lung cancer, melanoma, knock-down of SPRY4-IT1 also induced G 0 /G 1 cell cycle arrest and cell apoptosis 18,[31][32][33] . Therefore, these results indicate that knock-down of SPRY4-IT1 inhibited HCC progression by inducing cell cycle arrest as well as cell apoptosis.
The roles of ERRα in cancer development have been revealed in recent studies. In breast cancer, ERRα has been extensively studied, in which dysregulation of ERRα not only contributes to the progression of breast cancer, but also closely associated with the chemo-resistance of breast cancers 34 . In addition, ERRα was also found to play an important role in prostate cancer. ERRα augments HIF-1 signaling by directly interacting with HIF-1α in normoxic and hypoxic prostate cancer cells 35 . However, the role of ERRα in HCC is still unclear. The elevated levels of ERRα are associated with the increased cell proliferation and migration in breast cancer and prostate cancer cells 36,37 . In the present study, we demonstrated that knock-down of EERα significantly inhibited cell proliferation, colony formation, cell invasion and migration. Moreover, we examined whether SPRY4-IT1 had an interaction with ERRα, and the transfection study showed that SPRY4-IT1 knock-down also suppressed the expression of ERRα in HCC cells. More importantly, ERRα overexpression antagonized the effects of SPRY4-IT1 knock-down on HCC cell progression. These results may suggest that SPRY4-IT1 regulated HCC progression via interacting with ERRα. However, our results only showed the preliminary findings about the interaction between SPRY4-IT1 and ERRα. In order to find out more detailed molecular mechanisms underlying these interactions, more experiments should be performed in the future studies.
In conclusion, the present study showed that SPRY4-IT1 was up-regulated in HCC tissues and was associated with poor prognosis in HCC patients. Knock-down of SPRY4-IT1 inhibited cell proliferation, colony formation, cell invasion and migration at least via interacting with ERRα. These results suggest that SPRY4-IT1 may serve as a novel target for the management of HCC or as a biomarker for HCC diagnosis or prognosis, though the detailed molecular mechanisms of SPRY4-IT1 on HCC development may require further investigation.