Up‐regulation of ZFAS1 indicates dismal prognosis for cholangiocarcinoma and promotes proliferation and metastasis by modulating USF1 via miR‐296‐5p

Abstract LncRNAs has been demonstrated to modulate neoplastic development by modulating downstream miRNAs and functional genes. In this study, we aimed to detect the interaction among lncRNA ZFAS1 miR‐296‐5p and USF1. We explored the proliferation, migration and invasion of cholangiocarcinoma. The differentially expressed ZFAS1 was discovered in both tissues and cell lines by qRT‐PCR. The targeting relationship between miR‐296‐5p and ZFAS1 or USF1 was validated by dual‐luciferase assay. The impact of ZFAS1 on CCA cell proliferation was observed by CCK‐8 assay. The protein expression of USF1 was determined by Western blot. The effects of ZFAS1, miR‐296‐5p and USF1 on tumour growth were further confirmed using xenograft model. LncRNA ZFAS1 expression was relatively up‐regulated in tumour tissues and cells while miR‐296‐5p was significantly down‐regulated. Knockdown of ZFAS1 significantly suppressed tumour proliferation, migration, invasion and USF1 expression. Overexpressed miR‐296‐5p suppressed cell proliferation and metastasis. Knockdown of USF1 inhibited cell proliferation and metastasis and xenograft tumour growth. In conclusion, ZFAS1 might promote cholangiocarcinoma proliferation and metastasis by modulating USF1 via miR‐296‐5p.

Long non-coding RNAs (lncRNAs), identified as a wide category of transcripts that lack the potential of coding protein, are multifunctional members of the ncRNA family, which are greater than 200 nucleotides in length. [14][15][16][17] Despite lacking protein-coding potential, they are also associated with the progression of different types of cancers. Rapid advances of tumour genomics have highlighted the various function modes of lncRNAs in diverse human cancers, including CCA. For example, up-regulated lncRNA HOTAIR promotes glioblastoma cell cycle progression, HULC cooperates with MALAT1 to aggravate liver cancer stem cells growth, and overexpressed PANDAR contributes to the promotion of cholangiocarcinoma tumorigenesis. [18][19][20] Furthermore, emerging evidences have demonstrated that lncRNAs play crucial roles in the regulation of different biological processes, including regulation of mRNA transcription and post-transcription, alternative splicing modulation, oncogenesis promotion and many others. [21][22][23] Given to those evidences and oncogenic roles of variety, lncRNAs have been revealed to have the significant functions and a close relation with the progression of cancers.
Zinc finger antisense 1 (ZFAS1), located at chromosome 20, is antisense to the 5´end of ZNFX1 promoter. 24 Askarian-Amiri ME et al 25 discovered that ZFAS1 was host to three C/D box snoRNAs and highly regulated in the developing mouse mammary gland.
Furthermore, knockdown of lncRNA ZFAS1 could inhibit breast cancer cell proliferation and metabolic activity, which indicated lncRNA ZFAS1 a tumour suppressor. However, the following studies dramatically indicated its distinct role of cancer promotion, which refuted the tumour suppressor role of ZFAS1. For instance, lncRNA ZFAS1 is overexpressed in hepatocellular carcinoma and acts as oncogene by binding miR-150, ZFAS1 promotes gastric cancer cell growth by repressing KLF2 and NKD2 expression, ZFAS1 is an unfavourable prognostic factor for glioma patients and promotes tumour cell malignant progression by the activation of Notch signalling pathway, and many others. 24,26,27 Up to date, ZFAS1 has been demonstrated dysregulated and involved in several different cancers functions as oncogene; however, the possible biological role and prognostic capacity of ZFAS1 in CCA are still ambiguous.
In the present study, we investigated the pattern of lncRNA ZFAS1 in cholangiocarcinoma and evaluated the correlations between ZFAS1 expression, clinicopathological parameters and overall survival of CCA patients. Furthermore, we also explored the function of ZFAS1 in CCA cell proliferation, migration and invasion both in vitro and in vivo. Additionally, the effect of dysregulation of ZFAS1 on epithelial-mesenchymal transition (EMT) and the proliferation-related proteins were further detected.

| RNA extraction and quantitative real-time PCR
Total RNA was extracted from tissues or cells using TRIzol reagent (Invitrogen). The cDNA was synthesized using a reverse transcriptase kit in accordance with the manufacturer's recommendations. The RNA quality and concentration were detected using NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, Inc). Complementary DNA (cDNA) was reversely transcribed from RNA using a reverse transcriptase kit (Roche). The quantitative real-time PCR (qRT-PCR) was performed using the FastStart Universal SYBR Green Master (Roche, Germany) on a BIO-RAD C1000 Thermal Cycler and U6, and GAPDH was used as an internal control. The 2 −ΔΔCt method was performed to compute the relative expression levels. The qRT-PCR for primers is shown in Table 1.

| Wound healing and transwell assays
Cell migration was firstly measured by wound healing assays. Pipette tubes were used to make a scraped, acellular area. After 0 hours and 36 hours, photographs were taken to evaluate the motility of each transfected group. For transwell assay, the transfected cells were suspended in serum-free medium and plated into the transwell chambers with a pore size of 8 µm. Cell invasion was evaluated performing the Chamber matrigel invasion 24-well units (Costar). The assays were performed according to the manufacturer's instructions. Briefly, cells from each group were suspended in serum-free medium and were seeded into the upper chamber. The lower chamber was filled with medium containing 10% FBS. After incubation for 24 hours, the migrated/ invaded cells in the lower chamber (below the filter surface) were fixed in 4% paraformaldehyde, stained with crystal violet solution and counted under a microscope.

| Hoechst 33342 staining
Cells were seeded on sterile cover glasses placed in the four chambered slide. When they grew to approximately 70% confluence, cells were washed twice in ice-cold PBS. After washing, the cells were fixed with 4% paraformaldehyde in PBS for 30 minutes at 4℃, washed twice with PBS and stained with Hoechst 33342 (Thermo Fisher Scientific) at a final concentration of 10 mg/mL at room temperature for 5 minutes. Nuclear morphology was then examined using a fluorescent microscope.

| Tumour xenograft study
The tumour xenograft formation and evaluation were reviewed and approved by the Animal Care and Use Committee of Harbin Medical University, and animals were treated with euthanasia if inevitable. CCLP-1 cells were transfected with shZFAS1 or negative control. About 4 × 106 tumour cells were subcutaneously injected into either side of each female BALB/c nude mouse (6 weeks of age) after transfection for 48h. Tumour growth measurement was performed every 72 hours from the sixth day. The calculation for tumour volumes, euthanization, mice weight measurement and xenografts RNA evaluation were conducted as previously described. 28

| Protein extraction and western blot analysis
Total proteins were extracted from the cells with RIPA buffer and quantified by a BCA kit (Beyotime Biotechnology). About 50μg of extracted proteins were separated by SDS-PAGE and then transferred onto PVDF membranes (Merck Millipore). After soaking with 5% non-fat milk for 2h at 25°C and incubated with primary antibodies, including USF1, E-cadherin, vimentin, PCNA, Bax and GAPDH, respectively, the PVDF membranes were eventually incubated with secondary antibody (Cell Signaling Technology).

Gene name
Forward primer Reverse primer

| Statistical analyses
Each experiment was performed for at least three times. Unless otherwise noted, data were presented as the mean ± SD and analysed

| Up-regulated ZFAS1 transcription in CCA tissues is associated with patients' clinicopathological parameters and indicates an undesirable overall survival
To investigate the transcription level of ZFAS1 in paired CCA and adjacent normal tissues, quantitative real-time PCR was carried out to quantify ZFAS1 expression. The results showed that ZFAS1 was significantly overexpressed in CCA tissues with P-value < .05 in Figure 1A, and the expression level of ZFAS1 was 2.25-fold higher in the CCA samples than the paired normal tissues. And then, 64 patients were categorized into low (< the average value) and high (> the average value) groups according to the average expression level of the ZFAS1. As shown in Table 2, all data were calculated by Fisher's exact tests which indicated that the up-regulation of ZFAS1 was analysed to be associated with lymph node invasion (P = .042), TNM stage (P = .023) and postoperative recurrence (P = .035). However, no significance was found between ZFAS1 expression and other parameters. Kaplan-Meier analyses and logrank tests were performed to assess the relation between aberrant expression and overall survival (OS). Figure 1B indicated that relative high expressional ZFAS1 was relevant to worse overall survival.

| ZFAS1 is overexpressed in CCA cell lines and knockdown of ZFAS1 inhibits cell proliferation migration and invasion in vitro and in vivo
To  Figure 2D).
Interestingly, the activities of caspase-3 and caspase-9 were both higher in the ZFAS1 silence group than the negative control ( Figure 2E) and the Hoechst 33342 staining outcomes also presented severer apoptosis of cells resulted from ZFAS1 inhibition ( Figure 2F). To further investigate whether ZFAS1 expression could have impact on tumorigenesis in vivo, BALB/c nude mice (n = 10) were injected with transfected CCLP-1 cells (shZFAS1/ shCtrl). In accordance with previous in vitro outcomes, tumour growth of shZFAS1-transfected cell injection group showed a relative slower progress on tumour formation ( Figure 2G). Furthermore, tumour weights examination after inoculation for 18 days displayed that the weights of shZFAS1 transfected injection tumours were significantly lower than shCtrl group ( Figure 2H).
F I G U R E 1 ZFAS1 was up-regulated in cholangiocarcinoma (CCA) and indicated a poor survival. A, ZFAS1 was relatively upregulated in tumour tissues than adjacent normal tissues. B, Relatively high ZFAS1 expression group is associated with worse patient survival (OS)

| Knockdown of ZFAS1 inhibits cell migration and invasion by reversing epithelialmesenchymal transition
Given that ZFAS1 was related with lymph node invasion, we further explored the potential impact of ZFAS1 on migration and invasion by performing wound healing assays and transwell assays. Compared with si-NC, cells transfected either with si-ZFAS1-1 and si-ZFAS1-2 presented a dramatically weaker migration and invasion ability ( Figure 3A and B). Since epithelial-mesenchymal transition (EMT) has already been proved to be one crucial mechanism correlated with cell metastasis capability and protein PCNA, Bax are associated with cell proliferation and apoptosis regulation, respectively.
We then conducted the Western blot analyses to assess whether ZFAS1 could be involved into EMT and proliferation progression.
After transfected with si-ZFAS1-1, si-ZFAS1-2 or si-NC, we found Ecadherin and Bax were increased but the expression levels of others were decreased ( Figure 3C).

| Knockdown of ZFAS1 Increased the Expression of miR-296-5p and Decreased the Expression of USF1
Thanks to the help of computer-aided algorithms, the binging motif sequences were predicted as Figure 4A. Wild and mutant ZFAS1 sequences were inserted into pmirGLO reporter, and the luciferase activity in CCLP-1 and RBE cells were cotransfected with miR-296-5p mimics or miR-NC and pmirGLO-ZFAS1-WT or pmirGLO-ZFAS1-Mut.

| USF1 is up-regulated in CCA cells and knockdown of USF1 inhibits cell proliferation and migration
The qRT-PCR was performed to test the expression of ZFAS1 in cells. The siRNA to silence USF1 (si-USF1) and negative control (si-USF1-NC) were formed and found USF1 was down-regulated in CCLP-1 and RBE cells ( Figure 5A). Again, we performed CCK-8 assay, transwell assay to validate the role of USF1. The outcomes indicted that si-USF1 group induced a relative undesirable proliferation ( Figure 5B) and metastasis ( Figure 5C). The Hoechst 33342 staining also showed the same trend that si-USF1 transfected leaded to a severe cell apoptosis ( Figure 5D). The cell immunofluorescence results displayed that the expression of vimentin suppressed cell EMT progression detected by immunofluorescence of vimentin expression and cell morphology ( Figure 5E). The Western blot evaluated that despite E-cadherin, PCNA, Bax and vimentin were suppressed as a result of USF1 silence ( Figure 5F).

| USF1 induces CCA-related transcription by the prediction of binding to the promoter regions
Plentiful evidence has proved that transcription factors such as SP1 and USF1 could activate the transcription of targets by binding to specific region of the very promoter including lncRNAs. Although we have found ZFAS1 was overexpressed in CCA tissues and cell lines, the factors involved in ZFAS1 dysregulation remained unknown. In the present study, we predicted that the potential transcription factor USF1 could bind to ZFAS1 promoter region at two binding sites E1 (−1878 to −1872) and E2 (−1530 to −1524) with relative high scores by using JASPAR database ( Figure 6). After that, we assessed the binding motifs of several recently discovered CCA-related lncRNAs' promoters by JASPAR and found USF1 might bind some lncRNAs' promoters with relative high scores (Table 3) to activate oncogenic lncRNAs. peutic target for CCA. [29][30][31][32] LncRNA ZFAS1, antisense to the 5' end of ZNFX1 promoter, has been demonstrated aberrantly expressed in many cancers and equipped with different functional mechanisms, which we have previously described. As one of our previous works shows that a larger number of studies have indicated that the ZFAS1 is F I G U R E 4 ZFAS1 regulated USF1 expression by directly binding miR-296-5p as a ceRNA manner and affected tumour growth in vivo. A, The binding motif of miR-296-5p on ZFAS1 3′UTR predicted by TargetScan database. B, Relative luciferase activity between miR-296-5p and ZFAS1 was pulled up by shifting sequence using pmirGLO-ZFAS1-Mut. C, The miR-296-5p was significantly down-regulated in tumour tissues compared with adjacent normal tissues. D, Inhibition of miR-296-5p rescued the si-ZFAS1-1 transfected RBE and CCLP1 cell vitality. E, The predicted motif and shifted sequences of USF1 3′UTR. F, Relative luciferase activity between miR-296-5p and USF1 3′UTR was pulled up by shifting sequence using pmirGLO-USF1-Mut. G, Inhibition of miR-296-5p rescued the shZFAS1 transfected xenograft tumour growth on volumes and weights in vivo. H, Up-regulated ZFAS1 or inhibited miR-296-5p promoted USF1 expression overexpressed in many human cancers, such as breast cancer, gastric cancer, colorectal cancer hepatocellular cancer and many others, which is one important evidence to imply the potential role of ZFAS1 in the progression of human tumours. 33  Consequently, detecting whether miR-296-5p could be directly targeted by ZFAS1 became our concern. Online computer-aided algorithms were conducted and predicted the target binding sites and the luciferase assay verified the prediction. After evaluation with cell lines, miR-296-5p was finally selected. Interestingly, miR-296-5p was tested to be relatively down-regulated in the paired tissues from the same 64 patients, which as a result inspired our confidence to proceed. TargetScan was used for further prediction and found USF1 as one potential downstream target of miR-296-5p. In that case, we conducted experiments not only in vitro but also in vivo and found ZFAS1/miR-296-5p/USF1 regulation might work as one approach to influence tumour progression.
USF1 has been evaluated as transcription factor that could bind gene-specific binding motif in the previous research and activated gene expression. 34 Consequentially, we attempted with the help of JASPAR and discovered that USF1 might be an important transcription factor for ZFAS1. In addition to that results, the associative property of USF1 on the motifs of CCA-related lncRNAs was also evaluated. The currently found results indicated ZFAS1 as one oncogenic lncRNA might function in the way of ZFAS1/miR-296-5p/ USF1, and the feedback of USF1 might continuously activate oncogenic lncRNAs' transcription.
In conclusion, the results presented that overexpressed ZFAS1 is associated with dismal prognosis for cholangiocarcinoma patients and this aberrant modulates proliferation and metastasis via miR-296-5p/USF1, which might activate oncogenes expression.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.