Long non-coding RNA CERS6-AS1 facilitates the oncogenicity of pancreatic ductal adenocarcinoma by regulating the microRNA-15a-5p/FGFR1 axis

The long non-coding RNA CERS6 antisense RNA 1 (CERS6-AS1) has critical regulatory roles in breast cancer progression. Here, we determined CERS6-AS1 expression in pancreatic ductal adenocarcinoma (PDAC) and the roles of CERS6-AS1 in PDAC carcinogenesis. The mechanisms underlying the regulatory actions of CERS6-AS1 in PDAC cells were elucidated in detail. CERS6-AS1 expression was evidently increased in PDAC tissues and cell lines. Patients with PDAC having high CERS6-AS1 expression had shorter overall survival periods than those having low CERS6-AS1 expression. Functionally, the knockdown of CERS6-AS1 attenuated the proliferation, migration, and invasion and stimulated apoptosis of PDAC cells in vitro. Additionally, CERS6-AS1 depletion decreased PDAC tumor growth in vivo. Mechanistically, CERS6-AS1 could competitively bind to microRNA-15a-5p (miR-15a-5p) and effectively work as a molecular sponge in PDAC cells, resulting in the upregulation of fibroblast growth factor receptor 1 (FGFR1), a direct target of miR-15a-5p. Rescue experiments revealed that miR-15a-5p downregulation or FGFR1 restoration rescued the effects of CERS6-AS1 knockdown on the behaviors of PDAC cells. In conclusion, CERS6-AS1 promoted the oncogenicity of PDAC by serving as a competing endogenous RNA to sequester miR-15a-5p and increase FGFR1 expression, which highlights the potential of the CERS6-AS1/miR-15a-5p/FGFR1 pathway as an effective target for cancer therapy.


INTRODUCTION
Pancreatic cancer significantly contributes to cancerassociated mortality worldwide, and it is characterized by aggressive local invasion and high metastatic potential [1]. Approximately 80% of pancreatic cancer cases are of the pancreatic ductal adenocarcinoma (PDAC) arising from pancreatic ductal epithelial cells [2]. Curative surgical excision is currently the most effective therapy for PDAC; however, approximately 80% of patients with PDAC are not candidates for this surgery owing to a delayed diagnosis, which prevents the management of the disease at an optimal time [3]. Despite continuous improvements in diagnostic methods and treatment strategies, the clinical outcomes of patients with PDAC remain poor [4]. In this regard, comprehensively elucidating the molecular events underlying tumor pathogenesis is vital for developing novel diagnostic and treatment strategies in PDAC.
MicroRNAs (miRNAs) represent one subgroup of small non-coding RNA molecules comprising 19-25 nucleotides [21]. miRNAs have emerged as regulatory transcripts and can negatively regulate genes expression [22]. Regarding the mechanism, a competing endogenous RNA (ceRNA) theory has been proposed that describes the crosstalk among lncRNAs, miRNAs, and mRNAs [23,24]. LncRNAs can adsorb certain miRNAs and prevent the inhibition of mRNAs targeted by miRNAs, thereby regulating the oncogenicity of cancer [25]. Thus, studying the lncRNAs and miRNAs that contribute to PDAC tumorigenesis may provide valuable information for the development of anticancer treatment options.
The lncRNA CERS6 antisense RNA 1 (CERS6-AS1) plays critical regulatory roles in breast cancer progression [26]. Yet, the involvement of CERS6-AS1 in PDAC oncogenicity has not been characterized. Herein, we attempted to measure CERS6-AS1 expression in PDAC and further explore the functions of CERS6-AS1 in PDAC carcinogenesis. The molecular events responsible for the regulatory roles of CERS6-AS1 in PDAC were elucidated too. The identified CERS6-AS1/miR-15a-5p/FGFR1 pathway may offer a foundation for the identification of alternative therapies for PDAC.

The siRNA-mediated knockdown of CERS6-AS1 exerts tumor-suppressing effects on PDAC cells
First, the expression profile of lncRNAs in PDAC was assessed employing Gene Expression Profiling Interactive Analysis (http://gepia.cancer-pku.cn/). Of these lncRNAs, CERS6-AS1 was one of the most notably highly expressed lncRNAs ( Figure 1A).
Consistently, CERS6-AS1 expression was higher in PDAC tissues relative to the adjacent non-tumor tissues ( Figure 1B). Additionally, the overexpressed CERS6-AS1 was also validated in all four tested PDAC cell lines ( Figure 1C). We then categorized PDAC patients into two groups based on median CERS6-AS1 expression within this patient cohort, and found that CERS6-AS1-high patients exhibited a significantly shorter overall survival relative to CERS6-AS1-low patients ( Figure 1D; P = 0.0310).

FGFR1 is a direct target of miR-15a-5p in PDAC cells
The roles of miR-15a-5p overexpression on PDAC cells were then evaluated. The increase of miR-15a-5p in PDAC cells was achieved by transfecting with miR-15a-5p mimic ( Figure 4A). Ectopic miR-15a-5p expression inhibited PANC-1 and SW1990 cell proliferation ( Figure 4B), facilitate cell apoptosis ( Figure 4C). In addition, transfection with miR-15a-5p AGING mimic caused an obvious impairment in cell migration ( Figure 4D) and invasion ( Figure 4D) in PANC-1 and SW1990 cells. We subsequently identified the downstream target of miR-15a-5p in PDAC cells. Two miR-15a-5p binding sites were observed within the 3'-UTR region of FGFR1 ( Figure 4E). The gene was chosen for further experimental confirmation owing to its wellknown contribution to the oncogenesis and progression of PDAC [27,28]. Luciferase reporter assay confirmed that miR-15a-5p mimic lowered the luciferase activity of FGFR1-wt (1 and 2; Figure 4F); nevertheless, this suppressive action was abrogated when the binding sites were mutated. Furthermore, the upregulation of miR-15a-5p reduced FGFR1 expression ( Figure 4G, 4H) in PANC-1 and SW1990 cells. In addition, the level of FGFR1 mRNA was higher in PDAC tissues, and presented an inverse association with miR-15a-5p level ( Figure 4J). Overall, these results identified FGFR1 as a direct miR-15a-5p target in PDAC.

DISCUSSION
Several studies have revealed that lncRNAs exhibit crucial functions in controlling complex cellular behaviors, and this has attracted considerable interest [29][30][31]. To date, a large number of lncRNAs have been validated as critical regulators of PDAC malignancy [32,33]. Therefore, elucidating the detailed mechanisms via which lncRNAs contribute to the genesis and progression of PDAC might be advantageous for the development of effective targets for cancer management. Herein, we assessed the expression and functional importance of the lncRNA CERS6-AS in PDAC cells. Our results identified the oncogenic CERS6-AS1/miR-15a-5p/FGFR1 pathway in PDAC.
CERS6-AS1 expression is overexpressed in breast cancer [26], presenting a negative correlation with the overall survival [26]. Functionally, CERS6-AS1 upregulation induces breast cancer cell proliferation and colony-forming abilities and suppresses cell apoptosis in vitro [26]. However, whether CERS6-AS1 is implicated in PDAC requires further investigation.
Here, a high CERS6-AS1 was validated in PDAC.
AGING PDAC patients expressing high levels of CERS6-AS1 exhibited shorter overall survival relative to patients expressing low levels of this lncRNA. Functional analysis demonstrated that CERS6-AS1 knockdown restricted the in vitro properties of proliferative, migratory, and invasive, facilitated cell apoptosis in vitro, and impaired tumor growth in vivo of PDAC cells. Accordingly, CERS6-AS1 worked as a   AGING cancer-facilitating lncRNA in PDAC, and it may be an effective target for PDAC diagnosis and management.
Regarding the mechanism of action of lncRNAs, growing research has confirmed the existence of extensive ceRNA networks [25]. lncRNAs can sequester certain miRNAs and prevent their binding to target mRNAs, thereby weakening the inhibitory effect of miRNAs on their target genes [34]. To determine whether CERS6-AS1 affects PDAC malignancy through this mechanism, its location in PDAC cells was first analyzed by lncLocator and cell cytoplasmic/nuclear fractionation assays. CERS6-AS1 was corroborated to be mostly distributed in PDAC cell cytoplasm. Hence, bioinformatics analysis was used to identify the potential miRNAs with complementarity to CERS6-AS1, and the prediction unveiled that miR-15a-5p interacts with CERS6-AS1. Next, CERS6-AS1 knockdown resulted in the overexpression of miR-15a-5p in PDAC cells.
Additionally, miR-15a-5p was underexpressed in PDAC and presented a negative correlation with the CERS6-AS1 level. Further investigations, including luciferase reporter assay and RIP, verified the direct binding between CERS6-AS1 and miR-15a-5p in PDAC.
It is well established that lncRNAs have miRNA response elements and sequester miRNAs to positively modulate the downstream target mRNAs of miRNAs [35]. After identifying that miR-15a-5p directly targets FGFR1, we next examined whether CERS6-AS1 regulates the expression of FGFR1 by decoying miR-15a-5p in PDAC. To this end, FGFR1 expression in CERS6-AS1-deficient PDAC cells was detected, and the data uncovered that CERS6-AS1 silencing reduced FGFR1 amounts in PDAC cells, whereas miR-15a-5p inhibition partially reversed this effect. Importantly, FGFR1 expression was increased in PDAC and positively correlated with CERS6-AS1. Together, these results confirmed a new ceRNA model in PDAC cells, comprising of CERS6-AS1, miR-15a-5p, and FGFR1.
Our study performed luciferase reporter assay, RT-qPCR, western blotting, and relationship analysis and confirmed that miR-15a-5p directly targeted FGFR1 in PDAC. However, ChIP assay was not implemented to confirm this interaction, and it was a limitation of our study.

CONCLUSIONS
CERS6-AS1 was upregulated in PDAC, and CERS6-AS1 knockdown clearly suppressed PDAC progression. CERS6-AS1 acted as an miR-15a-5p sponge in PDAC cells and thus increased FGFR1 expression, thereby performing critical roles in the tumorigenesis of PDAC (Figure 9). Altogether, our data offer new insights into the mechanisms of PDAC pathogenesis, and suggest that the CERS6-AS1/miR-15a-5p/FGFR1 axis may represent a viable therapeutic target in PDAC therapy.

Tissue specimen collection and cell culture
Human PDAC tissues and adjacent non-tumor tissues were acquired from 57 patients in The First Hospital of Jilin University. All patients had not received either local or systemic anticancer therapies before surgery. Approval was obtained from the Ethics Committee of The First Hospital of Jilin University (ECTFHJU.2015-0112). All patients signed informed consent at the initial stage of the study.

RT-qPCR
Using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.), total RNA was extracted, after which the RNA quantity and quality was measured with a Nano-Photometer spectrometer (Thermo Fisher Scientific, Inc.). The synthesis of complementary DNA (cDNA) was executed using a PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara, Dalian, China). Next, qPCR was performed to determine CERS6-AS1 and FGFR1 expression utilizing TB Green Premix Ex Taq (Takara). The internal control for CERS6-AS1 and FGFR1 expression was glycerol 3-phosphate dehydrogenase (GAPDH).
Small RNA was isolated from analyzed samples or cells using the RNAiso for Small RNA Kit (Takara). In order to determine miR-15a-5p expression, Mir-X miRNA First-Strand Synthesis Kit and Mir-X miRNA qRT-PCR TB Green® Kit (Takara) were used for, respectively, conducting reverse transcription and qPCR. U6 small nuclear RNA served as a normalization control. The data were analyzed using the 2 -ΔΔCt approach.

Cell cytoplasmic/nuclear fractionation
In order to separate the nuclear and cytoplasmic fractions of PDAC cells, a Nuclear/Cytosol Fractionation Kit (Cell Biolabs, CA, USA) was utilized, after which RT-qPCR was done to test CERS6-AS1 distribution.

CCK-8 assay
At 24 h post-transfection, 2 × 10 3 cells in 100-µl aliquots of culture medium were seeded into each well of 96-well plates. At different times after cell seeding, 10 µl of the CCK-8 reagent (Dojido, Kumamoto, Japan) was introduced into each well, after which were AGING cultivated for additional 2 h at 37° C. In the following step, a microplate reader (PerkinElmer, MA, USA) was utilized to read the absorbance at a 450 nm wavelength.

Flow cytometry analysis
Annexin V-FITC Apoptosis Detection Kit (BioLegend, CA, USA) was employed to assess PDAC cell apoptosis. Briefly, transfected cells were collected by trypsin (0.25%) digestion and resuspended in 100 μl binding buffer. Next, these cells were double-stained using 5 µl each of Annexin V-FITC and PI. After 20 min cultivation in the dark, a FACSCalibur Flow Cytometer was used to analyze cell apoptosis.

Transwell cell migration and invasion assays
To assess cell migration, cells were collected 48 h after transfection. Cells were then resuspended in FBS-free culture medium, and the cell concentration was adjusted to 5 × 10 5 cells/ml. Thereafter, the upper portion of transwell chambers (8-μm pore inserts; Corning, NY, USA) was loaded with a 100-µl cell suspension. Simultaneously, 700 µl of culture medium added with 20% FBS was introduced into the lower chamber as a chemoattractant. The transwell chambers were then allowed for 24 h incubation, after which the migrated cells were fixed and stained, respectively, utilizing 4% paraformaldehyde and 0.05% crystal violet. After washing and drying, the migrated cells in five random fields of view were counted. Cell invasion was quantified using the same protocol, with transwell inserts having been coated with Matrigel (BD Biosciences) prior to cell inoculation.

Xenograft assay
The Institutional Animal Care and Use Committee of The First Hospital of Jilin University (ACUCTFHJU.2015-0112) approved the present animal study. A lentivirus expressing a short hairpin RNA (shRNA) against CERS6-AS1 (sh-CERS6-AS1) and negative control shRNA (sh-NC) were designed and packaged by Genechem (Shanghai, China). SW1990 cells were injected with the lentivirus to generate a cell line with stable CERS6-AS1 knockdown. To establish a tumor xenograft model system, male BALB/c nude mice (4-week-old; Laboratory Animal Center of Shanghai Academy of Science, Shanghai, China) were subcutaneously injected with SW1990 cells (2 × 10 6 ) stably expressing sh-CERS6-AS1 or sh-NC (n = 6 each group). Tumor width (W) and length (L) were monitored every 6 days for a total of 30 days, and tumor volumes were calculated using the formula V = 0.5 × (L × W 2 ). In the end, all mice were euthanized, and tumors were collected, weighted, and imaged as appropriate.

Luciferase reporter assay
The wild-type (wt) CERS6-AS1 and FGFR1 fragments containing putative miR-15a-5p binding site were prepared and cloned into the psiCHECK-2 reporter vectors (Promega, Madison, WI, USA) to yield the CERS6-AS1-wt and FGFR1-wt reporter constructs. The mutated (mut) versions of the fragments were additionally prepared with the GeneTailor™ Site-Directed Mutagenesis System (Invitrogen), after which CERS6-AS1-mut and FGFR1-mut reporter constructs were generated as above. After growing until 70%-80% confluence, cotransfection of wt or mut reporter vectors and miR-15a-5p mimic or NC mimic was implemented using Lipofectamine 2000. Forty-eight h later, the Dual-Luciferase Reporter Assay System (Promega) was utilized for luciferase activity determination.

RIP assay
The possible interactions between miR-15a-5p and CERS6-AS1 were examined with a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Bedford, MA, USA). RIP lysis buffer was utilized to lyse PDAC cells, after which 10 μl of the cell lysate was retained as input and 100 μl of the cell lysate were probed overnight at 4° C using magnetic beads conjugated with either normal mouse IgG or human anti-Ago2 antibody (Millipore) suspended in 900 μl of RIP lysis buffer. Magnetic beads were then collected and treated with Proteinase K to digest the proteins. The immunoprecipitated RNA was evaluated via RT-qPCR to measure CERS6-AS1 and miR-15a-5p enrichment.

Western blot analysis
RIPA buffer containing protease and phosphatase inhibitor cocktails (Beyotime, Shanghai, China) was utilized to lyse cells. Then, protein concentrations were assessed with a Detergent Compatible Bradford Protein Assay Kit (Beyotime). Using 10% SDS-PAGE, the protein was equally separated and then transferred to PVDF membranes, followed by blocking for a period of 2 h at room temperature using 5% nonfat milk. Subsequently, the membranes were probed overnight with anti-FGFR1 (ab76464; Abcam, Cambridge, UK) and anti-GAPDH (ab128915; Abcam) primary antibodies at 4° C, rinsed with TBST thrice, and probed for 1 h with a secondary HRP-conjugated antibody (ab205718; Abcam). Protein was visualized with BeyoECL Plus (Abcam). GAPDH functioned as the endogenous reference.

Statistical analysis
All measured data were exhibited as the mean ± SD. Statistical analysis was executed using SPSS software 22.0 (SPSS, Chicago, IL, USA). Data analysis between groups was done utilizing Student's t-test and one-way analysis of variance. The correlations among CERS6-AS1, miR-15a-5p, and FGFR1 levels in PDAC tissues were tested via Pearson's correlation coefficient. Using the median CERS6-AS1 value, all PDAC patients were stratified into either CERS6-AS1-low or CERS6-AS1high groups, after which their survival analysis was conducted employing Kaplan-Meier method and the log-rank test. P < 0.05 indicated a statistical significance.

AUTHOR CONTRIBUTIONS
Zhennan Yun and Ping Zhang conceived and designed the study. All authors performed the experiments. Zhennan Yun and Ping Zhang wrote the paper. All authors reviewed and edited the manuscript. All authors read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved.