LncRNA ZFAS1 promotes pancreatic adenocarcinoma metastasis by sponging miR-3924 via RHOA/ROCK2 pathway CURRENT STATUS: UNDER REVIEW

Background : the mortality and morbidity rate of pancreatic adenocarcinoma has been increasing during the past two decades, mechanisms in pancreatic adenocarcinoma progression are in urgent need of research. LncRNA ZFAS1 has been demonstrated as an oncogene in some cancers, but its function and mechanism in pancreatic adenocarcinoma still remain unclear. Methods : ZFAS1 expression level was predicted in pancreatic adenocarcinoma by bioinformatic analysis, the expression level of ZFAS1 in pancreatic adenocarcinoma tissues and cell lines were further investigated by qRT-PCR and ISH. The functions of ZFAS1 on pancreatic adenocarcinoma in vitro and in vivo were investigated according to bioinformatic analysis. Dual luciferase report assays investigated the binding of ZFAS1/miR-3924 and miR-3924/ROCK2, rescue assays further investigated the underlying mechanism. Results: ZFAS1 was predicted and further experimental verified to be over-expressed in pancreatic adenocarcinoma. ZFAS1 silence showed the inhibition to pancreatic adenocarcinoma metastasis in vitro and in vivo. The competing endogenous RNAs mechanism of ZFAS1 was also demonstrated. Conclusions : Our results demonstrated the promotion of ZFAS1 to pancreatic adenocarcinoma metastasis and suggested its candidacy as a novel regulator of ROCK2. Total cell RNAs were exacted by using Trizol reagent (invitrogen, USA) -according to the standard protocol. cDNA and qRT-PCR were performed by u-sing the Reverse Transcriptase Kit (Takara, Japan) and SYBR Green Mixture (Takara, Japan) in the ABI 7500 System. Data were compared by using the 2-ΔΔCt method. The sequences were as follows: ZFAS1, forward, 5'-GCGAAAGCCATCTTTGGTTA-3', reverse, 5'-GGGCAGGACAATAGCGTATG-3'; GAPDH, forward, 5'-GGACCTGACCTGCCGTCTAG-3', reverse, 5'-GTAGCCCAGGATGCCCTTGA-3'. suggested to metastasis in PAAD. relative function assays demonstrated that ZFAS1 as an oncogene PAAD metastasis in vitro and in vivo.


Abstract
Background : the mortality and morbidity rate of pancreatic adenocarcinoma has been increasing during the past two decades, mechanisms in pancreatic adenocarcinoma progression are in urgent need of research. LncRNA ZFAS1 has been demonstrated as an oncogene in some cancers, but its function and mechanism in pancreatic adenocarcinoma still remain unclear.
Methods : ZFAS1 expression level was predicted in pancreatic adenocarcinoma by bioinformatic analysis, the expression level of ZFAS1 in pancreatic adenocarcinoma tissues and cell lines were further investigated by qRT-PCR and ISH. The functions of ZFAS1 on pancreatic adenocarcinoma in vitro and in vivo were investigated according to bioinformatic analysis. Dual luciferase report assays investigated the binding of ZFAS1/miR-3924 and miR-3924/ROCK2, rescue assays further investigated the underlying mechanism.
Results: ZFAS1 was predicted and further experimental verified to be over-expressed in pancreatic adenocarcinoma. ZFAS1 silence showed the inhibition to pancreatic adenocarcinoma metastasis in vitro and in vivo. The competing endogenous RNAs mechanism of ZFAS1 was also demonstrated.
Conclusions : Our results demonstrated the promotion of ZFAS1 to pancreatic adenocarcinoma metastasis and suggested its candidacy as a novel regulator of ROCK2.

Background
Pancreatic adenocarcinoma (PAAD) is one of the most malignant tumors with increasing mortality and a 5-years survival rate less than 8% (1). In views of the situation that current treatment of PAAD including surgery, chemotherapy and immunotherapy could not improve patient prognosis obviously, more deeply and widely researches on exploring the molecular mechanism of PAAD progression are needed.
After the Human Genome Project completed in 2003, more than 90% of the long non-coding RNAs (lncRNAs) that longer than 200 nts were considered as useless molecules because of the non-protein coding properties (2), but after a growing number of evidences suggest that lncRNAs could regulate gene expression on epigenetic and other levels (3,4), scholars are paying more attention to the correlation between lncRNAs and cancers. LncRNA ZFAS1, the antisense transcript of gene ZNFX1, is 3 expressed abnormally in many cancers. It was first found to be a tumor suppressor gene in breast cancer (5), but following studies in other cancers drew the opposite conclusion(6) , (7). Though there are already studies on ZFAS1 in other cancers, few ZFAS1 related study in PAAD was found, the expression level and underlying molecular mechanism of ZFAS1 in PAAD also remain unknown.
In this study, microarray data were downloaded from Gene Expression Omnibus (GEO) database to investigate the expression level of ZFAS1 and other differentially expressed lncRNAs in PAAD. The expression level and clinical significance of ZFAS1 in PAAD were further investigated by other databases and our microarray. Furthermore, we validated the function of ZFAS1 to PAAD and its underlying mechanism according to the result of Gene Set Enrichment Analysis (GSEA) aiming to find a novel potential therapeutic target for PAAD.

Metastatic model in vivo
Sh-NC/sh-ZFAS1 sw1990 cells (1 × 10 6 cells/mouse) were injected in two groups of four weeks old nude BALB/c mice (HFK Biotechnology, China), Nude mice were sacrificed after four weeks and tumors in livers and lungs were observed after HE staining. This assay was approved by the Department of Laboratory Animal Science of China Medical University. The approval number was CMU2019201.

Statistical analysis
Data were presented as mean ± SD and analyzed by SPSS 24, Graphpad Prism 7, and MedCal 19. The differences were calculated by the student t-test. Survival curve was calculated by the Kaplan-Meier method and estimated using the log-rank test. Fisher exact test was used to analysis the correlation between ZFAS1 expression and PAAD clinicopathological factors. P value less than 0.05 was taken as statistically significant (*p < 0.05).

Results 1 Prediction of ZFAS1 over-expression in PAAD by bioinformatic analysis
To investigate the differential expression level of ZFAS1 and other lncRNAs in PAAD, nine GEO datasets were normalized and background adjusted to make differential expression analysis. 44 lncRNAs including ZFAS1 were identified to be up-regulated in PAAD (table 1)  Genome Atlas (TCGA), showed the correlation between ZFAS1 expression level and patients' gender, tumor grade and drinking habits (Fig. 1D-F), patients also showed survival differences when considering the combined effect of ZFAS1 level and drinking habit (Fig. 1G). However, no significant expression or survival difference of ZFAS1 were found according to TCGA based results alone (Fig. 1H-I). It is noteworthy that once the addition of GTEx data increased the sample size of TCGA-PAAD normal tissues (from 4 to 171), the significance of ZFAS1 expression level difference in PAAD was improved immediately (Fig. 1J).

Identification of ZFAS1 expression level and clinical correlation in PAAD by experimental analysis
A microarray with 170 points (71 paired and 28 single PAAD tissues) was obtained from Outdo Biotech (Shanghai, China). The expression level of ZFAS1 in PAAD tissues and its subcellular distribution were investigated by ISH, the expression score were collected by calculating the intensity*area. Result showed that ZFAS1 was significantly higher expressed in cancer than normal tissues and existed both in cytoplasm and nucleus ( Fig. 2A-C). According to ZFAS1 expression value, patients were divided into two groups, Furthermore, patients with lower ZFAS1 level have significant longer overall survival time ( Fig. 2D) and the AUC of ROC curve was 0.747 (Fig. 2E). However, ZFAS1 expression level showed no correlation with PAAD clinicopathological factors ( Table 2). ZFAS1 expression in PAAD cell lines were also detected by qRT-PCR, results showed that ZFAS1 expression level was also significantly upregulated in PAAD cell lines (bxpc-3, sw1990 and panc1) than the normal cell line hpde6c7 (Fig. 2F).
With highest ZFAS1 expression level, sw1990 and bxpc3 cell lines were selected for the following assays.

ZFAS1 knockdown inhibits cell metastasis in PAAD
To investigate the function of ZFAS1 in vitro, the synthesized small interfering RNAs (siRNAs) were respectively transfected into sw1990 and bxpc3 for 48 h. Wound healing (Fig. 3A-B) and transwell migration assays (Fig. 3C) showed the metastasis of bxpc3 and sw1990 were inhibited after ZFAS1 knockdown. The transfection efficiency of si-ZFAS1 was detected with qRT-PCR (Fig. 3D). Thus, ZFAS1 9 knockdown was suggested to inhibit PAAD metastasis in vitro.

ZFAS1 functions as a sponge of miR-3924
Bioinformatic tools DIANA lncbase, Starbase and RNAhybrid were used to explore the target miRNAs of ZFAS1 (Fig. 4A). According to the intersection of Starbase and Diana tool, miR-3924 was chosen and the binding site was predicted by RNAhybrid ( Fig. 4B-C), dual luciferase report results showed that miR-3924 mimic could inhibit the luciferase activity of ZFAS1-wt but not of ZFAS1-mut (Fig. 4D).
To further demonstrate whether ZFAS1 played its role through miR-3924, we established bxpc3 and sw1990 cell lines stable ZFAS1 silenced by using shRNA. By inhibiting miR-3924 expression level in ZFAS1 stable silence bxpc3 and sw1990 cell lines, wound healing ( Fig. 4E-F) and transwell migration assays (Fig. 4G) showed the reversed results of both two cell lines, indicating the antagonistic effect of ZFAS1 and miR-3924. Besides, in view of few miR-3924 related studies has been reported, we also investigated the function of miR-3924 in PAAD cells, results showed that the over-expression of miR-3924 could inhibit the metastasis of bxpc3 and sw1990 (Fig. 4H-J). The sh-ZFAS1 transfection efficiency was detected with qRT-PCR (Fig. 4K). Taken together, ZFAS1 functions as a sponge of miR-3924.

ROCK2 functions as the target of ZFAS1/miR-3924
GSEA was made according to the merged GEO data, the results showed that high ZFAS1 expression level was most correlated with focal adhesion (Table 3) (Fig. 5A), by the intersection of GSEA results and miR-3924 target mRNAs prediction, ROCK2 was selected as the mRNA target ( Fig. 5B-C). Dual luciferase report showed that the luciferase activity of ROCK2-wt/miR-3924 mimic group was significantly lower than ROCK2-Wt group, furthermore, no difference between ROCK2-Mut group and ROCK2-Mut/miR-3924 mimic group was found (Fig. 5D). Western blot results showed the expression level of FAK, RHOA and ROCK2 were decreased after ZFAS1 knockdown or miR-3924 over-expression ( Fig. 5E-H). Taken together, ROCK2 was suggested to function as the target of ZFAS1/miR-3924.

ZFAS1 silence inhibited tumor metastasis in vivo
To investigate the biological function of ZFAS1 in vivo, stable sh-ZFAS1 and sh-NC transfected sw1990 cells were selected by puromycin and tumor metastasis models were established by intravenous injection. Results showed that comparing with NC group, ZFAS1 silence could significantly inhibit liver but not lung metastasis in vivo (Fig. 6).

Discussion
Over the past two decade of data, not like the rapid decline of prostate cancer or breast cancer mortality rate, the incidence and mortality rate of PAAD have been growing all the time, so it is urgent to study the mechanisms of PAAD progression. Recently, considerable researches have reported that lncRNAs play important regulatory roles in the development of tumor process including PAAD. For example, the silencing of lnc958 inhibited the PAAD progression by the inhibition of PAX-8 (16). The down-regulation of TUG1 was connected with the inhibition of tumor growth and invasion in PAAD (17).
HULC was also found to promote PAAD cell progression by down-regulating miR-15a (18). ZFAS1, as a novel star lncRNA candidate after HOTAIR and MALAT1, expressed abnormally in various cancers, however, few ZFAS1 related studies on PAAD was found.
To avoid results with low reliability which are caused by small sample size and high false positive rate of single chip, we started with the differential expression analysis in PAAD by analyzing the merged nine datasets from GEO database, ZFAS1 were found to be significantly over-expressed in PAAD, data from ONCOMINE but not TCGA bioinformatically supplemented the GEO results. According to the significant change brought by the increased normal sample number in TCGA-PAAD, results of simply TCGA-PAAD based bioinformatic analysis may need further consideration (Fig. 1H, J).
The over-expression of ZFAS1 in PAAD and its clinical correlation were further experimentally verified based on bioinformatic analysis. UALCAN results showed that ZFAS1 expression level was significantly correlated with PAAD grade (Grade 1 vs Grade 3, p = 0.04). Besides, focal adhesion, extracellular matrix receptor interaction and mTOR signaling pathway were correlated with ZFAS1 high expression according to GSEA results. Considering these metastasis related pathways and the correlation between ZFAS1 and PAAD grade, ZFAS1 was suggested to regulate metastasis in PAAD. In line with the prediction, relative function assays demonstrated that ZFAS1 functions as an oncogene by regulating PAAD metastasis in vitro and in vivo.
Though do not code protein, lncRNAs regulate biologic processes at different levels in multiple ways, one of the major mechanism is by competitive binding target miRNAs and further regulate functional mRNA expression as competing endogenous RNAs (ceRNAs) (19,20). The binding of ZFAS1/miR-3924 and miR-3924/ROCK2 were predicted by bioinformatic analysis and validated by dual luciferase reports. Additionally, miR-3924 showed opposite effect to ZFAS1 in transwell and wound healing assays. Besides, miR-3924 inhibition could further reverse the effect caused by ZFAS1 inhibition.
Moreover, the silencing of ZFAS1 and the over-expression of miR-3924 could both down regulate the expression level of ROCK2, RHOA and FAK. Taken together, ZFAS1 was suggested to promote PAAD metastasis by sponging miR-3924 via RHOA/ROCK2 pathway. It is worth noting that the expression level of focal adhesion protein FAK, RHOA and ROCK2 in sw1990 and bxpc3 cell lines were different ( Fig. 5E, 5G), sw1990 showed significantly higher focal adhesion protein level than bxpc3, meanwhile sw1990 showed significantly higher migration rate in wound healing assays than bxpc3. Whether the correlation between focal adhesion protein level and migration ability in PAAD exists need further study, but if this correlation does exist, it could be another novel evidence that focal adhesion pathway proteins could promote metastasis in PAAD.
Rho-associated coiled-coil kinases (ROCKs) are major effectors of the small GTPase RHOA, the important role of ROCK in cancer progression including cell metastasis has been demonstrated by thousands of studies (21)(22)(23). Moreover, ROCK inhibitors such as Y-27632 (24) or fasudil (25) have also been widely studied in various cancer cell lines and animal models and showed significant benefits when used along (26)(27)(28) or combined with other chemotherapy drugs (29)(30)(31)

Conclusions
In summary, our data demonstrated the over-expression of ZFAS1 in PAAD and its clinical significance with tumor grade and prognosis. Its promotion to cell metastasis by regulating miR-3924/ROCK2 axis was also demonstrated. We hope our study could provide new idea for solving pancreatic cancer problems and translating ZFAS1/miR-3924/ROCK2 signaling knowledge into anti-cancer therapies. The ethics approval (PDF file) is available from the corresponding author on reasonable request.  figure 1H and 1J is log2(TPM+1).