Long non-coding RNA FEZF1-AS1 promotes the proliferation and metastasis of hepatocellular carcinoma via targeting miR-107/Wnt/β-catenin axis

Hepatocellular carcinoma (HCC) is a public health problem around the world, with the molecular mechanisms being still incompletely clear. This study was carried out to explore the role and mechanism of long-noncoding RNA (lncRNA) FEZF1-AS1 in HCC progression. RNA sequencing and quantitative real time polymerase chain reaction (qRT- PCR) were applied to identify differently expressed lncRNAs in HCC tissues and adjacent normal tissues. CCK8 assay was adopted to test cell proliferation and flow cytometry was taken to detect cell apoptosis. Wound healing assay and transwell experiment were performed to determine cell migration and invasion. To validate the function of lncRNA FEZF1-AS1 in vivo, tumor-burdened models were established. The results showed that lncRNA FEZF1-AS1 level was prominently enhanced in HCC tumor specimens and overexpression of FEZF1-AS1 promoted the proliferation, migration and invasion of HCC cells. In mechanism, overexpression of FEZF1-AS1 reduced the expression of miR-107 which inhibited the activation of Wnt/β-catenin signaling. Overexpression of β-catenin promoted cell proliferation, migration and invasion which were inhibited by FEZF1-AS1 downregulation. In conclusion, our study demonstrated that FEZF1-AS1 promoted HCC progression through activating Wnt/β-catenin signaling by targeting miR-107, which provided a novel target for the therapy of HCC.


INTRODUCTION
Hepatocellular carcinoma (HCC) is a leading cause of cancer death. It is the main type of primary liver cancer [1,2]. Although HCC therapy is in fast development, such as tissue resection, liver translation, chemotherapy, radiotherapy and biotherapy, patients with HCC still present with unfavorable prognosis and low 5-year survival rates [3,4]. In recent years, the molecular mechanisms underlying HCC have attracted more and more attentions, and many genes play a critical role in HCC development [5]. However, it's still a long road to elucidate fully the molecular mechanisms of HCC. It is of significance to find novel biological markers for the diagnosis and treatment of HCC.
Evidence has identified that miRNAs are frequently deregulated in cancers and involve in cancer development by altering the expression of target genes [16,17]. For instance, miR-143 was found to be downregulated in bladder cancer [18] and breast cancer [19]. LncRNAs have been shown to usually act as sponges of miRNAs and then contribute to the development of cancers [20]. Using the bioinformatics methods, miR-107 is shown to be a predict target of lncRNA FEZF1-AS1, but their interaction in HCC waits to be verified.
We aimed to detect the regulatory pattern of FEZF1-AS1 in HCC in this study.

RESULTS
The HCC tumorigenesis always accompanied with alteration of genes, and growing evidence suggested that lncRNAs are critical for cancer procession [21]. To explore the expression profiles of lncRNAs in HCC, the Arraystar Human lncRNA Microarray was performed in 3 paired HCC tissues and the neighboring normal tissues. A prominent alteration in FEZF1-AS1 level was found in the lncRNA profile ( Figure 1A, 1B). The expression level of FEZF1-AS1 was found to be upregulated in HCC tissues as compared to the adjacent normal liver tissues by qRT-PCR ( Figure 1C). We then detected the FEZF1-AS1 expression level in HCC cell lines. FEZF1-AS1 showed a higher expression pattern in SUN-182 cells and a lower expression pattern in Hep3b cells among the 4 cells, SNU-182, SNU-398, SNU-449 and Hep3b ( Figure 1D).
To investigate the role of FEZF1-AS1 in HCC, it was overexpressed and downregulated in SNU-398 and SNU-449 because these two cells showed moderate level of EFZF-AS1 among the 4 cells ( Figure 1D). qRT-PCR was used to detect the FEZF1-AS1 expression ( Figure 2A) and CCK8 assay was used to assess the cell proliferation. Overexpression or downregulation of FEZF1-AS1 promoted or suppressed cell proliferation of SNU-398 and SNU-449 cells ( Figure 2B, 2C). The flow cytometry demonstrated that FEZF1-AS1 overexpression suppressed the apoptosis of SNU-398 and SNU-449 cells ( Figure 2D-2F). The results of transwell chamber and wound healing assays showed that FEZF1-AS1 overexpression significantly enhanced the invasion ( Figure 2G-2I) and migration ( Figure 3A, 3B) of HCC cells, and downregulation of FEZF1-AS1 caused opposite results. It is believed that EMT is a main cause of cancer cell migration and invasion. Therefore, we assessed FEZF1-AS1 role in HCC cell EMT. Results of western blot showed that FEZF1-AS1 overexpression induced EMT in HCC cells with increased expression levels of ICAM1 and Vimentin and decreased expression level of E-cadherin ( Figure  3C, 3D), while downregulation of FEZF1-AS1 decreased ICAM1 and Vimentin expression and increased E-cadherin expression.
To verify the function of FEZF1-AS1 in HCC in vivo, SUN-398 cells with FEZF1-AS1 stable downregulation and/or β-catenin stable overexpression were transplanted into nude mice. Figure 7A showed the histopathological changes of the mice tissues and no cachexia was found after the nude mice were sacrificed. Downregulation of FEZF1-AS1 inhibited tumor growth with smaller volumes and less weights as compared with the sh-NC or normal saline group, whereas overexpression of βcatenin rescued the reduction of the tumor growth caused by shFEZF1-AS1 ( Figure 7B, 7C). Additionally, FEZF1-AS1, wnt3a, β-catenin, ICAM1 and Vimentin expressions were repressed and E-cadherin and miR-107 levels were increased in the sh-FEZF1-AS1 group, whereas these tendencies were neutralized by β-catenin overexpression ( Figure 7D, 7E).

DISCUSSION
HCC is a malignant cancer which is characterized by low survival rate [24]. Although great progress has been made in improving the therapeutic method of HCC in the past years, the efficacy is still unsatisfied. It's important to investigate the oncogenic mechanisms in HCC. We proved in this study that FEZF1-AS1 had a high expression level in HCC. Also, we found that FEZF1-AS1 contributed to the malignant alterations of HCC via targeting miR-107/Wnt/β-catenin axis, as well as EMT, which is a main cause of cancer metastasis [25].
It was believed that lncRNAs played vital roles in HCC metastasis [26]. EZF1-AS1 is dysregulated in several cancers and contributes to carcinogenesis by targeting miRNAs or specific signaling pathway [13,[27][28][29]. Previous studies demonstrated that FEZF1-AS1 had an impact on the proliferation and invasion of HCC cells [15,30]. Herein, we confirmed a high expression pattern of FEZF1-AS1 in HCC and FEZF1-AS1 overexpression promoted proliferation, migration and invasion of HCC cells. Further, we found miR-107 was another target of FEZF1-AS1 in HCC. From the luciferase gene reporter and qRT-PCR assays, we observed that FEZF1-AS1 could bind MiRNAs play vital roles in controlling cell proliferation, apoptosis, migration and invasion. Dysfunction of miRNAs contributes to the occurrence and development of cancers [31]. MiR-107, a widely studied miRNA, was believed to be implicated in the development of many kinds of cancers. However, miR-107 function is controversial. For instance, some researched identified that miR-107 was a tumor suppressor in gastric cancer, NSCLC, breast cancer and AGING bladder cancer [32][33][34][35], but some found that miR-107 was an oncogene in gastric cancer, bladder cancers and breast cancer by targeting different pathway [36][37][38]. Wang et al. [39] found that miR-107 could inhibit cell proliferation in HCC by binding to the 3'-untranslated region of HMGA2. In contrast, some studies demonstrated that miR-107 promoted HCC cell proliferation [40]. We found that miR-107 level was downregulated in HCC. miR-107 upregulation leaded to a significant inhibition in Wnt/β-catenin signaling with decreased expressions of β-catenin and wnt3a and an increased level of p-GSK-3β. However, miR-107 was identified to inhibit cell proliferation, colony-forming ability and tumorigenicity in osteosarcoma, with increased expression level of β-catenin [23]. We speculate that different cell contents maybe a main cause of the different roles of miR-107 plays in regulating β-catenin expression. AGING Furthermore, we explored whether Wnt/β-catenin signaling was regulated by FEZF1-AS1 in HCC. We found that β-catenin overexpression greatly enhanced cell proliferation and metastasis in HCC which were inhibited by FEZF1-AS1 downregulation. This result suggested that downregulation of FEZF1-AS1 inhibited HCC progression through inhibiting the Wnt/β-catenin pathway. However, miR-107 role in AGING this process is not clear and we intend to clarify it in our further study.
As a whole, our study identified that FEZF1-AS1 accelerated the malignant behavior of HCC via miR-107/Wnt/β-catenin axis.

Tissue obtainment
HCC tissues and the adjacent normal liver tissues were harvested from HCC patients in Shanghai Jiao Tong University Affiliated Sixth People's Hospital between 2013 and 2015. Patients participated in this study received no other therapeutic method before surgery. This study was conducted in accordance with the World Medical Association Declaration of Helsinki and was approved by the Research Ethics Committee of Shanghai Jiao Tong University. Informed consent was written by each patient.

Cell growth and apoptosis assay
The cell proliferation was tested by CCK8 kit (Beyotime, Beijing, China). 96-well plates were used to seed the cells at a density of 2000 cell/cm 2 and the cells were transfected/infected with plasmids/lentivirus after AGING 24 hours of incubation at 37C. Following 24,48,72,96 or 120 hours of incubation after inoculation, 10 μl CCK8 solution was then added into each well. The cells were incubated for 2 hours. Then, the absorbance was measured by the reader (model 680; Bio-Rad, Hertfordshire, UK) at 450 nm. Flow cytometry was used to determine the apoptosis rates of cells which were transfected/infected with sh-FEZF1-AS1, OE-FEZF1-AS1, sh-NC, OE-NC, mimic-NC, and mimic-miR-107. Annexin V(FITC)/PI Apoptosis Detection Kit (BioLengen, CA, USA) was used according to the instruction.

Wound healing assay and transwell assay
Cells were seeded into six-well plates in a density of 3000 cells/cm 2 . After cell transfection/infection with plasmids/lentivirus, wounds were scraped by tips. The distance of the wound was captured after 0 hour and 24 hours of incubation at 37C by DM2500 bright field microscope (LEICA, Wetzlar, Germany). The migration distance was measured by Image J. Upper chamber was coated with matrigel. Cells were seeded in the density of 3000 cells/cm 2 . Bottom chamber was filled with DMEM with 10% FBS and 1% penicillin/streptomycin. 4% paraformaldehyde (PFA) was used to fix the invaded cells and crystal violet was used to stain the cells. The amount of invaded cells was counted by image J.

In vivo assay
A mouse xenograft model was established using male BALB/c nude mice (Shanghai Slac Laboratory Animal Company (Shanghai, China) at an age of six weeks. Animals were housed in a controlled environment (50% humidity, 23  2C, 12-h light-dark cycle) with free access to water and food. 2 × 10 6 SUN-398 cells were stably transfected with sh-FEZF1-AS1 and/or OE-βcatenin and resuspended in 100 μL normal saline. Then, the cells were then injected subcutaneously in flanks of mice. Cells in normal saline served as negative control. Four weeks later, all animals were euthanized and tumor xenografts were harvested. Weight of tumors were measured. All experiments regarding animal handling were approved by the Animal Care and Use Committee of Shanghai Jiao Tong University Affiliated Sixth People's Hospital and performed following the Guide for the Care and Use of Laboratory Animals.

Statistical analysis
All experiments were performed in triplicate and repeated three times. Data were analyzed by the SPSS software (version 24.0) and shown as mean  standard deviation. Student's t-test and one-way analysis of variance (ANOVA) were used for statistical AGING comparisons between two groups or among multiple groups, respectively. A value of p < 0.05 was considered statistically significant.