lncRNA SNHG7 sponges miR‐425 to promote proliferation, migration, and invasion of hepatic carcinoma cells via Wnt/β‐catenin/EMT signalling pathway

Increasing evidence has indicated the important roles of long noncoding RNA small nucleolar RNA host gene 7 (SNHG7) in tumourigenesis as a potential oncogene. However, the function of SNHG7 in hepatic carcinoma remains unclear. In the present study, we found that SNHG7 expression was significantly upregulated in hepatic carcinoma tissues, especially in aggressive cases, and it was closely correlated with the poor prognosis. Furthermore, knockdown of SNHG7 inhibited the proliferation, migration, and invasion of hepatic carcinoma cell lines in vitro. Mechanistically, SNHG7 directly interacted with miR‐425 as a ceRNA. Moreover, knockdown of SNHG7 significantly inhibited the tumorigenic Wnt/β‐catenin/EMT pathway. SNHG7 regulated Wnt/β‐catenin/EMT pathway through sponging miR‐425 and played an oncogenic role in hepatic carcinoma progression. Together, our study elucidated the role of SNHG7 as a ceRNA in hepatic carcinoma, provided new potential diagnosis and therapeutic application in hepatic carcinoma progression. Significance of the study SNHG7 could promote proliferation and metastasis of hepatic carcinoma cell in vitro and in vivo, suggesting that SNHG7 exerts tumorigenic role in hepatic carcinoma progression. Further mechanism research revealed that SNHG7 exhibited the tumorigenic role through Wnt/β‐catenin/EMT pathway as a miR‐425 sponge. These findings provided new cues to understand the molecular signalling network in carcinogenesis of hepatic carcinoma, and it may provide new evidence for therapeutic application in hepatic carcinoma.

Recently, noncoding RNAs (ncRNAs) have caused great concerns for its complex and crucial role in cancer progression. 6,7 ncRNAs are mainly composed of small nucleolar RNAs (snoRNAs), micro-RNAs (miRNAs), and long ncRNAs (lncRNAs). miRNA is a small ncRNA molecule consists of about 22 nucleotides. miRNA inhibits translation of mRNA by binding to the 3′ untranslated region (UTRs) of the mRNA via its seed sequence, which in turn causes degradation of mRNA. lncRNA is defined as transcripts longer than 200 nucleotides that does not translated into protein but participates in various of biological processes, including transcription regulation, posttranscriptional regulation, protein modification, and tumourigenesis especially. [8][9][10] An increasing number of studies have demonstrated that lncRNA can interact with the miRNA as competing endogenous RNAs (ceRNAs) to sponges target mRNA, therefore to participate in the expression regulation of target genes that exhibit important role human diseases. 11,12 Small nucleolar RNA host gene 7 (SNHG7) is a long ncRNA with 2157 bp in length, located on chromosome 9q34.3. Recent studies have suggested that SNHG7 could promote proliferation, migration, and invasion and inhibit apoptosis in various of cancers, such as malignant pleural mesothelioma, breast cancer, colorectal cancer, and lung cancer. [13][14][15][16] However, the biological mechanisms and ceRNA role of SNHG7 in the tumourigenesis of hepatic carcinoma have not been elucidated. The Wnt/β-catenin is a classical signalling pathway in regulation process of cell proliferation, differentiation, and tumourigenesis. 17,18 Epithelial-mesenchymal transition (EMT) is regarded as the initiation phase of cancer metastasis for it involves the breakdown junctions between cells and generating individual cells with invasive capability and multiple mesenchymal attributes. 19 Studies have shown that ncRNAs can regulate proliferation and EMT in carcinogenesis by regulating Wnt/β-catenin signalling pathway. [20][21][22] However, the role of Wnt/β-catenin in hepatic carcinoma is unclear.
In present study, we first revealed that SNHG7 overexpression is a characteristic molecular change in hepatic carcinoma progression. SNHG7 knockdown in HepG2 and HCC-LM3 cells significantly inhibited cell proliferation, migration, and invasion. In-depth mechanism research revealed that SNHG7 acts as a ceRNA for hsa-miR-425-5p (miR-425), which sponged miR-425 and regulated the Wnt/β-catenin signalling pathway in hepatic carcinoma. Collectively, our data elucidated the crucial role of SNHG7 in hepatic carcinoma progression. Therefore, the SNHG7/miR-425 axis might be a potential therapeutic target for hepatic carcinoma treatment.

| Clinical specimens and cell lines
The paired hepatic carcinoma tissues and corresponding relative normal tissues (n = 40) were collected after surgical resection in the Second Affiliated Hospital of Nanchang University according to its ethical and legal standards. All samples were saved in liquid nitrogen immediately after extraction until RNA was extracted. All patients signed a written informed consent. A 36-month follow-up survival survey was conducted, and the overall survival (OS) was defined as the interval between resection and death or the last follow-up. The clinicopathological information of the 40 patients who received curative surgery   at the Second Affiliated Hospital of Nanchang University between   March 2011 and August 2014, including sex, age, tumour size, tumour   depth, differentiation, T stage, lymph node invasion, and distant metastasis, were recorded. HepG2 and HCC-LM3 cell lines used in this study were purchased from the Cell Bank of Type Culture Collection (CBTCC, Chinese Academy of Sciences, Shanghai, China) and cultured in RPMI-1640 medium (HyClone, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, NY), 1% penicillin, and streptomycin in 5% CO2 at 37°C.

| Dual-luciferase reporter assay
The pcDNA3.1 SNHG7 wild/mutant type with/without designed miR-425 binding sites were synthesized by GenePharma and cotransfected with miR-425-mimic or mimic negative control into HepG2 and HCC-LM3 cells; 24 hours after transfection, luciferase activities were measured with a dual-luciferase reporter gene assay system (Promega, USA).

| Cell proliferation assay
MTT assay was performed to assess cell viability of HepG2 and HCC-LM3 cells which were transfected with si-SNHG7 or si-NC. Briefly, cells were incubated in 96-well plates for appropriate time, 20 μL of 5 mg/mL MTT was added to each well, and then the plates were incubated at 37°C for additional 2 hours. The medium was subsequently removed; 150 μL of DMSO was added to dissolve the purple-coloured precipitates of formazan. Absorbance value was recorded at 490 nm using a Spectra MAX M5 microplate spectrophotometer (Molecular Devices, USA).
Colony formation assay was performed, briefly; HepG2 and HCC-LM3 cells cotransfected with si-SNHG7 or si-NC were seeded in sixwell plates (200-500 cells per well) and incubated for 10 days. Then, cells were fixed with methanol for 15 minutes and stained with 0.5% crystal violet solution for 20 minutes; the colonies (>50 cells) were counted manual with microscope (Zeiss, Germany).

| Cell apoptosis assay
Apoptosis assay was performed with apoptosis detection kit (BD, USA) as protocol. Cells (2 × 10 5 cells per well) were incubated in a six-well plate and received different treatments for 48 hours; then cells were harvested and washed twice with ice-cold PBS. Apoptosis detection kit was used to measure the levels of apoptosis according to manufacturer's instructions by FCM (BD, USA). Data were analysed by FlowJo.

| Cell migration and invasion assay
Migration and invasion assay were conducted with Transwell chambers (Corning, USA) according to general procedure. Briefly, cells (4 × 10 4 ) were culture in serum-free medium in the upper chamber with (migration) or without (invasion) matrigel precoated; subsequently, medium containing 10% FBS was added to lower chamber as induction factor. Then, cells were incubated for 24 hours; the migrated cells on the surface of lower membrane were fixed with 70% methanol and stained by 0.1% crystal violet. The capacity of invasion was evaluated with amounts of invasive cells under a microscope (Zeiss, Germany) in three different fields of each filter.

| Wound-healing assay
The invasive capacity of cells transfected with si-SNHG7 was assessed with wound-healing assay. Generally, cells were cultured into 12-well plates and incubated until 70% to 80% confluence. Then, artificial wound scratching was made with a sterile pipette tip. After incubated in serum-free medium for appropriate time, wounds were captured with microscope (Zeiss, Germany). Reverse: 5′-AACGGGAGUGAUCGUGUCAUUUU-3′.

| Quantitative real-time PCR
The GAPDH level was set as the normalization, and relative expression of SNHG7 and other mRNAs was calculated with the 2 −△△Ct method as standardized operation.

| Western blot analysis
Forty-eight hours after transfection, hepatic carcinoma cells were harvested and total protein was extracted with RIPA Lysate containing protease inhibitor (Beyotime, China). After the concentration of protein was determined and uniformized, protein was separated by 10% sodium dodecyl sulfate polyacrylamide gel. Subsequently, protein was transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, USA); PVDF membrane was blocked in 5% skim milk in TBST at 37°C for 1 hour. Subsequently, PVDF membranes were washed with TBST and incubated with primary antibodies (Abcam, UK) for β-catenin (ab32572), MMP-9 (ab38898), E-cadherin (ab15148), N-cadherin (ab76057), and β-actin (ab124964) at 4°C overnight. After incubation with the relevant secondary antibodies for additional 1 hour at 37°C, enhanced chemiluminescence kit (Amersham, NJ) was used to detect the reactive bands as protocol.

| In vivo tumour formation assay
Male BALB/c nude mice (4 wk old) from Huafukang Bioscience Co, Inc

| Immunohistochemistry assay
The tumour samples were resected from mice, fixed in 10% formaldehyde, embedded in paraffin, and sectioned (5 μm thickness). Immunohistochemical analyses was performed with anti-Ki-67 (ab15580) and anti-MMP-9 (ab38898) antibody obtained from Abcam. After stained with related secondary antibody for 1 hour at 37°C, slide was counterstained with haematoxylin for 20 to 30 seconds, then dehydrated and fixed; slide was sealed with the neutral gum. Images were taken with microscope (Zeiss, Germany).

| Statistical analysis
All experiments were performed at least three times, and data were expressed as mean ± standard deviation as indicated. Sample t test or one-way analysis of variance (ANOVA) were performed for statistical analysis, P < .05 was considered to be statistically significant.
The statistical analysis was graphed with GraphPad.

| Overexpressed SNHG7 in hepatic carcinoma tissues was associated with poor prognosis
To determine whether there was a difference of SNHG7 expressional profile in hepatic carcinoma and adjacent histologically normal hepatic tissues, 40 pairs of specimens were analysed with qRT-PCR. The result suggested that SNHG7 was significantly overexpressed in hepatic carcinoma tissue compared with adjacent normal tissue ( Figure 1A). Moreover, the SNHG7 expression level may be associated with the metastasis of hepatic carcinoma ( Figure 1B). Further, Kaplan-Meier survival analysis also showed that up-regulated SNHG7 was positive correlation with poor OS of patients with hepatic carcinoma ( Figure 1C). Furthermore, statistical analysis revealed that there is a significant association between overexpressed SNHG7 and bad clinical stage ( Figure 1D). Overall, these findings elucidated that the overexpressed SNHG7 may be associated with progression, metastasis, and poor prognosis of patients with hepatic carcinoma.

| Knockdown of SNHG7 suppressed cell proliferation, invasion, and migration and promoted apoptosis of hepatic carcinoma cell HepG2 and HCC-LM3 in vitro
RNA interference was performed to explore the biologic function of SNHG7 in hepatic carcinoma. siRNA targeting the coding region of SNHG7 (SNHG7-inhi) was transfected into HepG2 and HCC-LM3 cells, respectively. The knockdown efficiency of SNHG7-siRNA was confirmed by RT-PCR assay, and result showed that the expression level of SNHG7 in SNHG7-siRNA-transfected HepG2 and HCC-LM3 cells was drastically decreased by 70% (Figure 2A   Furthermore, silence of SNHG7 led to decreased level of metastasis related protein MMP-9 (matrix metallopeptidase 9), N-cadherin, and increased level of cell adhesion protein E-cadherin ( Figure 2N). Above result suggested that knockdown of SNHG7 could impede hepatic carcinoma cell proliferation and metastasis.

| SNHG7 sponging miR-425 as a ceRNA
To determine whether SNHG7 could interact with the miRNA as ceRNAs or natural miRNA sponge as previous report, targetscan was conducted with bioinformatic database (TargetScanHuman Release 7.2, Starbase v3.0 and miRcode). Bioinformatics analysis indicated that there are putative binding sites between SNHG7 and miR-425 ( Figure 3A). Then dual-luciferase reporter assay was conducted to validate the interaction between miR-425-5p and SNHG7 in HepG2 cells. Result revealed that luciferase activity of SNHG7-Wt was dramatically reduced by miR-425 mimic. In contrast, the luciferase activity of SNHG7-Mut experienced no statistical changes ( Figure 3B).
These results indicated that SNHG7 could sponge miR-425 as a ceRNA.   Figure 4D). Collectively, above findings were according with our results in vitro; knockdown of SNHG7 could impede hepatic carcinoma cell proliferation in nude mice.

| DISCUSSION
Numerous studies have confirmed that lncRNA exerted complex effects in progression of tumourigenesis and numerous overexpressed lncRNA has been found in hepatic carcinoma. 23,24 SNHG7 has been identified as an oncogene in breast cancer, lung cancer, and colorectal cancer. 13,14 In this work, a significant SNHG7 overexpression was observed in hepatic carcinoma tissues compared with adjacent histologically normal hepatic tissues. In addition, overexpressed SNHG7 in hepatic carcinoma patients was significantly associated with aggressiveness, OS rate, and clinical stage. Knockdown experiments identified the oncogenous function of SNHG7 in promoting cellular proliferation and metastasis, suppressing apoptosis in hepatic carcinoma. Furthermore, in vivo assay revealed that silence of SNHG7 could inhibit tumour growth. Meanwhile, immunohistochemical assay illuminated knockdown of SNHG7 promoted cell apoptosis in tumour tissues, as well as suppressed metastasis of hepatic carcinoma. ceRNA has emerged as an alternative function for lncRNAs. 25 lncRNA can interact with the miRNA directly to regulate the expression of target genes. Novel regulatory network has been identified in crosstalk between lncRNAs and mRNAs. 26 lncRNA SNHG7 has been recognized as a sponge of miR-216b to promote proliferation and liver metastasis of colorectal cancer via upregulating GALNT1. 14 Bioinformatics analysis was conducted to reveal putative binding miRNA of SNHG7; putative binding sites was found between SNHG7 and miR-425. Following that, it was proved that there was an endogenous interaction between miR-425 and SNHG7 by performing the dualluciferase assay. Subsequently, rescued miR-425 was observed when SNHG7 was silenced, which further confirmed SNHG7 sponging miR-425 as a ceRNA.
WNT/β-catenin pathway plays a crucial role in regulation process of cell proliferation, differentiaton, and tumourigenesis, and it has been recognized as a target of ncRNA in cancer proliferation and EMT of carcinogenesis. 17 Further molecular biology experiment FIGURE 3 SNHG7 sponges miR-425 to promote metastasis of hepatic carcinoma via Wnt/β-catenin EMT signalling pathway. A, The putative binding sites of SNHG7 to miR-425 sequence were shown. B, Luciferase activity of HepG2 cells cotransfected with luciferase reporters containing SNHG7-Wt/SNHG7-Mut and miR-425 mimic/miR-425 control were analysed with dual-luciferase reporter assays system. C, Expression of miR-425 was determined by qRT-PCR when HepG2 and HCC-LM3 cells were treated with SNHG7-siRNA. (D,E) Migration ability of miR-425-mimic treated cells was assessed by wound-healing assay. Scale bars = 50 μm. (F,G) Transwell migration assay was measured after cells were treated with miR-425-mimic. Scale bars = 50 μm. (H,I) Transwell invasion assay was measured after cells were treated with miR-425-mimic. Scale bars = 50 μm. (J) The levels of β-catenin, MMP-9, E-cadherin, and N-cadherin following miR-425-mimic treated in hepatic carcinoma cells were assessed through western blot. (K,L) Transwell migration assay was measured after different treatments in HepG2 cells. *P < .05 illustrated that the protein levels of β-catenin, MMP-9, and N-cadherin, which could promote carcinogenesis, were decreased markedly; by contrary, metastatic suppress protein E-cadherin was increased notably both in miR-425-mimic or SNHG7-siRNA transfected HepG2 and HCC-LM3 cells. The above results suggested that SNHG7 could promote proliferation and metastasis of hepatic carcinoma cell via Wnt/β-catenin/EMT pathway via sponging miR-425.
Collectively, SNHG7 could promoted proliferation and metastasis of hepatic carcinoma cell in vitro and in vivo, suggesting that SNHG7 exert tumorigenic role in hepatic carcinoma progression. Further mechanism research revealed that SNHG7 exhibited the tumorigenic role through Wnt/β-catenin/EMT pathway as a miR-425 sponge.
These findings provided new cues to understand the molecular signalling network in carcinogenesis of hepatic carcinoma, and it may provide new evidence for therapeutic application in hepatic carcinoma.

AVAILABILITY OF DATA AND MATERIALS
The data sets used or analysed in this study are available from the corresponding author on reasonable request.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE
Tissues and information of patients were gained with written  4 SNHG7 knockdown suppressed tumour growth in vivo. A, Tumours collected from mice. B, Tumour volume curve of mice injected with HepG2-Lv-SNHG7 or HepG2-Lv-NC cells was analysed. C, Tumour weight of mice was measured. D, Immunohistochemical staining of MMP-9 and Ki-67 were used to assess proliferation and metastasis