A novel antisense lncRNA NT5E promotes progression by modulating the expression of SYNCRIP and predicts a poor prognosis in pancreatic cancer

Abstract A novel antisense lncRNA NT5E was identified in a previous microarray that was clearly up‐regulated in pancreatic cancer (PC) tissues. However, its biological function remains unclear. Thus, we aimed to explore its function and clinical significance in PC. The lncNT5E expression was determined in PC specimens and cell lines. In vitro and in vivo studies detected the impact of lncNT5E depletion on PC cell proliferation, migration and invasion. Western blotting investigated the epithelial‐mesenchymal transition (EMT) markers. The interaction between lncNT5E and the promoter region of SYNCRIP was detected by dual‐luciferase reporter assay. The role of lncNT5E in modulating SYNCRIP was investigated in vitro. Our results showed that lncNT5E was significantly up‐regulated in PC tissues and cell lines and associated with poor prognosis. LncNT5E depletion inhibited PC cell proliferation, migration, invasion and EMT in vitro and caused tumorigenesis arrest in vivo. Furthermore, SYNCRIP knockdown had effects similar to those of lncNT5E depletion. A significant positive relationship was observed between lncNT5E and SYNCRIP. Moreover, the dual‐luciferase reporter assays indicated that lncNT5E depletion significantly inhibited SYNCRIP promoter activity. Importantly, the malignant phenotypes of lncNT5E depletion were rescued by overexpressing SYNCRIP. In conclusion, lncNT5E predicts poor prognosis and promotes PC progression by modulating SYNCRIP expression.


| INTRODUC TI ON
Pancreatic cancer (PC) is one of the most aggressive and leading lethal malignancies and has a very poor prognosis. 1 It is the seventh leading cause of cancer-related death in both men and women globally. 2 In the United States, PC is the fourth leading cause of cancer-related death 3 and is projected to rise to become the second leading cause within the next decade. 4 Despite advances in molecular mechanism research and therapeutic approaches for PC during the past few decades, the 5-year survival rate of approximately 9% remains dismal, the lowest rate among any malignant tumours. 3 This extremely poor prognosis is mainly influenced by extensive local invasion and early distant metastasis. Radical surgery in combination with chemotherapy and radiotherapy is the only potentially curative treatment strategy, but fewer than 20% of patients are eligible for resection 1 because of rapid progression and late diagnosis. Thus, it is urgent to explore the underlying molecular mechanisms of PC tumorigenesis and progression and identify innovative diagnostic biomarkers and therapeutic targets for PC.
Thus, lncRNAs are likely to represent a new class of potential diagnostic biomarkers and therapeutic targets for cancers. Within these lncRNAs, a particular group are natural antisense transcripts from the opposite strand of protein-coding transcripts, termed antisense lncRNAs. Antisense lncRNAs, located either in the nucleus or in the cytoplasm, are functionally diverse. They can modify the expression of neighbouring genes in cis or more distant genes in trans through various mechanisms to exert a wide variety of biological functions. 25 In our previous study, we performed microarrays to explore the expression profiles of lncRNAs and associated mRNAs in PC. A large number of differentially expressed lncRNAs and mRNAs were identified between PC and adjacent normal tissues, among which three markedly down-regulated lncRNAs (lncPCTST, XLOC_000647 and KCNK15-AS1) in PC tissues were reported to be correlated with the progression and prognosis of PC. [26][27][28] A novel antisense lncRNA NT5E (ENST00000421594), identified in microarrays, was also significantly up-regulated (fold change: 10.78) in PC tissues. LncNT5E, located on human chromosome 6q14.3, is a broadly expressed and evolutionarily non-conservative lncRNA with a length of 554 base pairs (bp). However, the biological function and clinical value in PC progression have not been fully elucidated so far.
In the current study, we evaluated the expression levels of lncNT5E in PC tissues and cell lines and investigated its clinical significance. Then, we assessed the biological functions of lncNT5E on PC cell oncogenic phenotypes, including cell proliferation, migration, invasion and epithelial-mesenchymal transition (EMT), both in vitro and in vivo. Finally, we explored the potential molecular mechanism of lncNT5E in PC progression. Thus, this study sheds light on lncNT5E as a promising potential novel molecular biomarker and therapeutic target for PC.

| Patients and clinical samples
A total of 45 paired PC and matched adjacent normal tissue specimens were collected from patients who underwent pancreatic resection at the Pancreas Center, the First Affiliated Hospital of Nanjing Medical University (Nanjing, China), from February 2016 to February 2017. The protocol was approved by the Hospital Ethics Committee, and written informed consent was signed from all patients before surgery. No patients had distant metastasis or received anticancer treatment before surgery. All specimens were evaluated and confirmed to be pancreatic ductal adenocarcinoma (PDAC) by a certified pathologist. All samples were snap-frozen in liquid nitrogen and then stored at −80°C until required. Complete follow-up data were acquired. Overall survival (OS) was defined as the time from the date of surgery to the date of death or the end of follow-up (November 2019), and progression-free survival (PFS) was the time interval between the date of surgery and the date of objective tumour progression or death or the end of follow-up.

| Cell lines and cell culture
Human PC cell lines (CFPAC, and human embryonic kidney cells (293T) were procured from the American Type Culture Collection (ATCC). The normal human pancreatic duct cell line HPNE was obtained from Pancreas Institute, Nanjing Medical University. All cells were cultured at 37°C in a humidified atmosphere with a 5% CO2 incubator and maintained in Dulbecco's modified Eagle's medium (DMEM; KeyGen BioTECH) or RPMI-1640 medium (Gibco) supplemented with 10% foetal bovine serum (FBS, Gibco), 100 U/mL penicillin and 100 mg/mL streptomycin (Gibco).

| Construction of the lncNT5E stable expression cell line
A lentiviral pGLV2-U6-Puro vector containing lncNT5E shRNA and a negative control lentivirus were purchased from GenePharma (GenePharma). They were cotransfected into 293T cells using EndoFectin Lenti transfection reagent according to the manufacturer's instructions. After culturing for 48 hours, the lentiviral particles in the supernatant were harvested, filtered and then used to transfect CFPAC cells. To select stably transduced cells, the cells were resuspended and cultured in 2 µg/mL puromycin (Meilunbio) for 2 weeks; qRT-PCR was performed to determine the level of lnc-NT5E expression.

| RNA extraction and quantitative real-time PCR (qRT-PCR)
Total RNA samples from cells and tissue specimens were extracted using TRIzol Reagent (Invitrogen) following the manufacturer's in-

| Western blotting
Tissues or cells were homogenized and lysed with RIPA lysis buffer.
After determining the protein concentration using a BCA assay (Thermo Fisher Scientific), equal amounts of proteins were separated via sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE; 10% gel) and then transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore). The membranes were blocked with 5% skim milk for 1 hour at room temperature and incubated with specific primary antibodies overnight at 4°C with gentle shaking. After incubation in a secondary antibody for 2 hours at room temperature, the immunoreactive bands were visualized by an en-

| Cell migration and invasion assays
Cell migration and invasion ability were evaluated using Transwell chambers (

| Dual-luciferase reporter assay
The promoter region of SYNCRIP (from −1391 to 138) was chosen to design primer and then amplified by PCR and inserted into the pGL3-Basic vector (GenePharma Company). In this study, luciferase reporter plasmids included SYNCRIP promoter-pGL3-Basic (referred to as pGL3-promoter) and pGL3-Basic (negative control). 293T cells Renilla luciferase activity was used for normalization. The luciferase activity was expressed in terms of the ratio of firefly luciferase activity to Renilla luciferase activity.

| Tumour formation assay in vivo
Briefly, 6-week-old female athymic nude mice (BALB/c) were purchased from Vital River Laboratory Animal Technology Co. A total of 5 × 10 6 CFPAC cells stably expressing sh-ctrl or shLncNT5E were resuspended in 200 µL medium (100 µL PBS + 100 µL Matrigel) and then subcutaneously inoculated into the flank of each nude mouse. The tumour size was measured with a calliper every 2 days, and the tumour volume was calculated using the following formula: length × (width) 2 /2. The weight of xenograft tumours was measured when harvested 18 days after inoculation. The harvested samples were fixed in 4% formaldehyde, embedded in paraffin, sectioned (5 µm) and subjected to haematoxylin and eosin (HE) and immunohistochemical (IHC) staining.
All animal experiments were approved by the Animal Care and Use Ethics Committee of Nanjing Medical University and conducted following the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines.
The staining score for PC cells and adjacent normal cells was analysed separately, and multiple regions were recorded. The percentage of immunostaining and the staining intensity (0, negative; 1+, weak; 2+, moderate; and 3+, strong) were assessed. A histochemistry score (H-score) ranging from 0 to 300 was recorded using the following formula 29 :

| Statistical analysis
All values are presented as the mean ± standard deviation (SD).
The quantitative data were analysed by Student's t test or one-

| LncNT5E is aberrantly up-regulated in PC tissues and cell lines
We used microarray detection (H1602063, Arraystar Human LncRNA 8 × 60 k v3.0 1-color) to study lncRNAs in three pairs of PC and adjacent normal tissues. We identified 17 up-regulated lncRNAs in PC tissues (fold change > 2, P < .05, and a false discovery rate < 0.05) by microarray ( Figure 1A) and then validated these results in PC tissues and cell lines. The qRT-PCR results indicated the lncRNA NT5E (ENST00000421594) had good repeatability and specificity (fold change = 10.78, P = .014). Thus, we chose lncNT5E for further analysis. LncNT5E is mapped to human chromosome 6, is 554 bp in length and is an antisense lncRNA (https:// lncip edia.org).
In this study, we further examined the expression of lncNT5E in 45 pairs of PC and adjacent normal tissues by qRT-PCR. Compared with normal tissues, lncNT5E was markedly increased in PC tissues ( Figure 1B). Then, we validated that the expression level of lncNT5E in four PC cell lines (CFPAC, COLO-357, PANC-1 and BXPC-3) was dramatically higher in PC cell lines, especially CFPAC and COLO-357, than in the normal human pancreatic ductal epithelial cell line HPNE ( Figure 1C).

| LncNT5E is associated with the clinicopathological features and prognosis of PC patients
We investigated the clinical significance of lncNT5E in PC patients.
As shown in Table 1, the results indicated that high expression of lncNT5E was significantly correlated with advanced TNM stage and lymph node metastasis. In addition, Kaplan-Meier survival analysis revealed that PC patients with high lncNT5E expression had significantly shorter OS (log-rank test, P = .021, Figure 2A) and PFS (logrank test, P = .016, Figure 2B)

| LncNT5E promotes the proliferation and EMT of pancreatic cancer cells
We examined the potential role of lncNT5E in regulating PC progression in vitro. The lncNT5E siRNA was transfected into CFPAC and COLO-357 cell lines, and the transfection efficiency was confirmed by qRT-PCR ( Figure 3A). The CCK-8 assay demonstrated that lnc-NT5E depletion in two PC cell lines resulted in significant tumour growth inhibition ( Figure 3B). Furthermore, we performed EdU staining assay to confirm that lncNT5E depletion down-regulated the proliferation of the PC cells. The results showed that the positive proportion of EdU staining nuclei was significantly decreased in si-LncNT5E group compared to that in si-ctrl group ( Figure 3C and D).
Moreover, we investigated the expression of apoptosis-related proteins (cleaved caspase-3, cleaved caspase-7 and cleaved caspase-9) via Western blotting. The results showed that the expression levels of cleaved caspase-3, cleaved caspase-7 and cleaved caspase-9 were not different under silencing of lncNT5E ( Figure 3E). These results indicated that lncNT5E depletion really inhibited proliferation rather than increased cell death.
In addition, the migration and invasion capacities were evaluated by Transwell assays. The results showed that invasion was inhibited in CFPAC and COLO-357 cells with lncNT5E depletion ( Figure 4A and B). We also detected the expression of several EMT markers via Western blotting. LncNT5E depletion led to increased levels of the  epithelial marker E-cadherin and decreased levels of the mesenchymal markers vimentin and N-cadherin ( Figure 4C). Therefore, these data suggest that lncNT5E can promote the proliferation and EMT of PC cells.

| LncNT5E depletion inhibits tumour growth in vivo
To further determine the function of lncNT5E in vivo, CFPAC cells with stable lncNT5E knockdown were subcutaneously injected into nude mice. The volume and weight of xenograft tumours in the stable lncNT5E knockdown group were markedly smaller than those in the control group ( Figure 5A-C). Moreover, immunohistochemical staining showed that the cell proliferation indicator Ki67 of tumours was decreased in the stable lncNT5E knockdown group ( Figure 5D).
These results suggest that lncNT5E promotes the tumorigenicity of PC cells in vivo.

| LncNT5E promotes transcription of SYNCRIP
Some lncRNAs are reported to activate the transcription of closely located genes in cis [30][31][32] by binding to the promoter. By using microarray results combined with bioinformatics analysis, we identified that the neighbouring coding gene of lncNT5E with SYNCRIP expression in pancreatic cancer tissues ( Figure 6C).
Next, we examined whether lncNT5E promoted SYNCRIP expression via increasing its promoter activity. We constructed a luciferase reporter containing the SYNCRIP promoter, and the results of luciferase reporter assays showed that lncNT5E knockdown significantly reduced luciferase activity ( Figure 6D). These results confirm that lncNT5E promotes the activity of SYNCRIP promoter to induce its transcription.

| SYNCRIP is overexpressed in PC and is associated with poor prognosis
We selected SYNCRIP as a candidate gene for further study. First, we detected the expression of SYNCRIP mRNA in 45 pairs of PC and normal tissues by qRT-PCR. The results showed that SYNCRIP levels were significantly higher in PC tissues than in adjacent normal tissues ( Figure 7A). Similar results were found in matched PC and HPNE cell lines ( Figure 7B). Furthermore, immunohistochemistry (IHC) and H-score showed that the positive rate of SYNCRIP was higher in PC tissues than in normal tissues ( Figure 7C). In addition, we identified SYNCRIP as differentially expressed genes using Gene Expression Profiling Interactive Analysis (GEPIA, http://gepia.cance r-pku.cn). The expression of SYNCRIP was clearly up-regulated in PC tissues ( Figure 7D). Moreover, survival

F I G U R E 4 LncNT5E promotes PC cells migration, invasion and EMT in vitro.
A and B, The migration (A) and invasion (B) capacity of the CFPAC and COLO-357 cells transfected with lncNT5E siRNA by Transwell assays. C, Analysis of the E-cad, N-cad and vimentin protein levels in the CFPAC and COLO-357 cells (si-lncNT5E#1 or si-lncNT5E#2 and si-ctrl), as detected by Western blotting. Results are represented as protein intensity relative to GAPDH.*P < .05, **P < .01 analysis showed that the high expression of SYNCRIP was associated with poor overall survival (OS) ( Figure 7E) and disease-free survival (DFS) ( Figure 7F). These results verify that SYNCRIP expression is significantly higher in PC tissues than normal tissues and is associated with poor prognosis.

| SYNCRIP is involved in lncNT5E-mediated proliferation and EMT of PC cells
Some studies have reported that SYNCRIP expression plays an important role in cancers. 33 We verified the function of SYNCRIP in PC cells. We confirmed that the SYNCRIP expression level was really down-regulated by si-SYNCRIP via Western blotting ( Figure 8C).
The CCK-8 assays demonstrated that silencing SYNCRIP inhibited the proliferation of CFPAC cells ( Figure 8A). Transwell assays indicated that silencing SYNCRIP inhibited PC cell invasion and migration ( Figure 8B). Furthermore, SYNCRIP depletion resulted in increased levels of E-cadherin and decreased levels of vimentin and N-cadherin ( Figure 8C).
Then, we evaluated whether the phenotypes associated with lncNT5E expression would be reversed by the restoration of SYNCRIP expression. We cotransfected the SYNCRIP overexpression plasmid into CFPAC cells with lncNT5E depletion.
Overexpression of SYNCRIP was confirmed by Western blotting ( Figure 8D).  Figure 8D). These findings suggest that SYNCRIP is involved in lncNT5E-mediated proliferation and EMT in pancreatic cancer cells.

| D ISCUSS I ON
Emerging evidence has demonstrated that lncRNAs are aberrantly expressed in various cancers, including PC, and play a vital role in cancer. [34][35][36] Although a large number of differentially expressed lncRNAs and mRNAs were identified between PC and corresponding adjacent normal tissues in our microarrays (H1602063, KangChen Bio-tech Inc), only a few have been experimentally verified and functionally annotated. [26][27][28] In the present study, we demonstrated that a novel antisense lncRNA, NT5E, which was identified in the microarray analysis, was markedly up-regulated in PC tissues and cell lines. Importantly, the overexpression of lnc-NT5E was positively associated with TNM stage and lymph node metastasis. Moreover, the overexpression of lncNT5E was significantly correlated with a poor prognosis in PC patients, indicating that lncNT5E acted as a potential prognostic biomarker for PC.
In addition, we showed that lncNT5E depletion inhibited PC cell F I G U R E 5 Stable lncNT5E depletion significantly inhibits tumour growth in vivo. A, Nude mice were killed 18 d after subcutaneous injection of CFPAC cells stably transfected with sh-lncNT5E or sh-ctrl, and tumour nodules were exhibited. B, Tumour volume curve of the sh-lncNT5E or sh-ctrl treatment groups was measured and analysed every 2 d. C, Tumour weights of sh-lncNT5E or sh-ctrl groups were measured when the tumours were collected. D, HE (haematoxylin and eosin) and IHC (immunohistochemical) staining of sections from subcutaneous xenograft tumours (×100 and ×200).
The H-score showed that stable lncNT5E depletion decreased the proliferation index of Ki67. Data are shown as the mean ± SD. **P < .01, ***P < .001 proliferation, migration and invasion in vitro and in vivo. We also found that lncNT5E could induce EMT to promote invasion and metastasis in PC, and its nearby protein-coding gene SYNCRIP might be involved. These results suggested that lncNT5E might be a novel therapeutic target for PC.
Pancreatic cancer is a highly malignant tumour and has become one of the leading causes of cancer-related mortality worldwide because of the lack of early diagnosis and effective treatments. In our study, lncNT5E was initially identified to be significantly up-regulated in PC tissues. Moreover, the high expression of lncNT5E was closely correlated with TNM stage and lymph node metastasis in PC patients. In addition, patients with high lncNT5E expression displayed worse OS and PFS. More importantly, lncNT5E was an independent prognostic factor for PC patients. Notably, perineural invasion also served as an independent risk factor for prognosis, which was consistent with the results of previous studies. 37,38 The results demonstrated that lncNT5E may play an imperative role in the tumorigenesis and progression of PC.
We further identified and explored the biological function of LncRNAs have been proposed to exert diverse functions, such as transcriptional regulation in cis or trans, organization of nuclear F I G U R E 8 SYNCRIP is involved in lncNT5E-mediated proliferation and EMT of PC cells. A-C, In CFPAC cells transfected with SYNCRIP siRNA, the SYNCRIP expression level down-regulated by si-SYNCRIP was confirmed by Western blotting (C), the proliferation activity was determined by CCK-8 assay (A), the migration and invasion capacity was detected by Transwell assays (B), and changes in the E-cad, N-cad and vimentin protein levels were verified by Western blotting (C). D-F, Inhibition of proliferation, migration, invasion and changes in EMT-related markers in lncNT5E-depleted PC cells was reversed by SYNCRIP overexpression. D, The protein levels of SYNCRIP were upregulated, and EMT-related marker expression was rescued after transfection with the SYNCRIP overexpression plasmid into CFPAC cells with lncNT5E depletion, as detected by Western blotting. E and F, Restoration of SYNCRIP expression significantly ameliorated the lncNT5E depletion-induced suppression of CFPAC cell proliferation (E), invasion and migration (F). The results are shown as the protein intensity relative to that of GAPDH. Values are presented as the mean ± SD. *P < .05, **P < .01, ***P < .001 domains and regulation of proteins or RNA molecules. 39 Recent evidence has verified that lncRNAs can regulate local chromatin structure and/or neighbouring gene expression in cis. [30][31][32]40 For example, lncXLOC_000647 down-regulates the expression of its neighbouring gene NLRP3 to suppress the progression of pancreatic cancer. 27 Similarly, lncPCTST inhibits the progression of pancreatic cancer by down-regulating TACC-3. 26 Specifically, antisense lncRNAs act as significant modulators of the expression of their neighbouring genes in cis. Therefore, we predicted that lncNT5E might promote oncogenic activity by regulating its local protein-coding gene expression.
Based on this theory, we found that SYNCRIP is a protein-coding gene near lncNT5E. Additionally, the expression of SYNCRIP was decreased in PC cell lines upon lncNT5E down-regulation, which confirmed our hypothesis. Moreover, the inhibition of invasion capacity in lncNT5E-down-regulated PC cells was rescued by SYNCRIP overexpression. Similarly, lncNT5E depletion-induced EMT was also reversed by SYNCRIP overexpression. Thus, our data revealed the significance of the interaction between lncNT5E and SYNCRIP in PC tumorigenesis and progression, given that lncNT5E exerted its oncogenic function partly via positive regulation of SYNCRIP in PC cells.
In conclusion, our work identified a novel lncRNA, NT5E, which is up-regulated in PC tissues and cell lines and associated with poor prognosis. The results showed that lncNT5E exerted strong oncogenic functions by promoting PC cell proliferation, migration and invasion both in vitro and in vivo. LncNT5E accelerated EMT and promoted the progression of PC possibly through the up-regulation of SYNCRIP expression. Taken together, our findings provide insight into lncNT5E as a promising therapeutic target that could regulate SYNCRIP in PC.

CO N FLI C T O F I NTE R E S T
The authors declare no potential conflicts of interest.