LncRNA TP73‐AS1 enhances the malignant properties of pancreatic ductal adenocarcinoma by increasing MMP14 expression through miRNA ‐200a sponging

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an invasive and aggressive cancer that remains a major threat to human health across the globe. Despite advances in cancer treatments and diagnosis, the prognosis of PDAC patients remains poor. New and more effective PDAC therapies are therefore urgently required. In this study, we identified a novel host factor, namely the LncRNA TP73‐AS1, as overexpressed in PDAC tissues compared to adjacent healthy tissue samples. The overexpression of TP‐73‐AS1 was found to correlate with both PDAC stage and lymph node metastasis. To reveal its role in PDCA, we targeted TP73‐AS1 using LnRNA inhibitors in a range of pancreatic cancer (PC) cell lines. We found that the inhibition of TP73‐AS1 led to a loss of MMP14 expression in PC cells and significantly inhibited their migratory and invasive capacity. No effects of TP73‐AS1 on cell survival or proliferation were observed. Mechanistically, we found that TP73‐AS1 suppressed the expression of the known oncogenic miR‐200a. Taken together, these data highlight the prognostic potential of TP73‐AS1 for PC patients and highlight it as a potential anti‐PDAC therapeutic target.

understanding of the key PC drivers are therefore urgently required.
LncRNAs are dysregulated in a range of cancers. 4 LncRNA TP73 (TP73-AS1) is a member of TP53 family and a known posttranscriptional regulator of TP73 expression. 5,6 TP73-AS1 influences tumorigenesis through its activity as a competitive endogenous RNA (ceRNA). 7 That suppresses miR-200a to enhance hepatocellular carcinoma (HCC) cell proliferation, mediated through the HMGB1/RAGE axis. 8 TP73-AS1 also promotes colorectal cancer (CRC) development through its ability to regulate TGFα signalling, an effect achieved through the sponging of miR-194. 9 TP73-AS1 also targets miR-449a in lung cancer cells to enhance the expression of zeste homolog 2 (EZH2) and promote cancer development. 10 A role of TP73-AS1 in the development of PDAC has, however, not been fully defined.
MicroRNAs (miRNAs) are ~23 RNA molecules that are non-coding and regulate gene expression. 11 miR-200a in a range of important human malignancies including breast, colon, cervical, ovarian, colon and liver cancer. 12 MiR-200a is induced by a range of physiological stimuli, including IL-9, that enhances miR-200a/β-Catenin expression and subsequent cancer cell metastasis. 13 Low levels of miR-93 and miR-200a expression in cancer cells are also associated with a loss of differentiation in PDAC, 14 while its overexpression enhances chemo-resistance and MT1-MMP expression in PC cells. 15,16 MiR-200a can inhibit PC metastasis through its ability to suppress DEK. 17 In our previous studies, we confirmed that miR-200a is suppressed in PC stem cells 18 but the involvement of TP73-AS1 was not reported.
Here, we identify TP73-AS1 as overexpressed in PDAC tissues compared to adjacent healthy tissue samples. The overexpression of TP-73-AS1 was correlated with both PDAC stage and lymph node metastasis. To reveal its role in PDCA, we targeted TP73-AS1 using LnRNA inhibitors in PC cells. The inhibition of TP73-AS1 led to a loss of MMP14 expression in PC cells and significantly inhibited their metastatic capacity. TP73-AS1 was further found to up-regulate MMP14 expression through its suppression of miR-200a. These data provide new insight into the pro-oncogenic effects of the TP73-AS1/ miR-200a axis during PC development.

| Cell culture and samples
The PC cells PANC-1, miacapa-2, BxPC-3 and non-PC cells HPDE6C7 were purchased from the Shanghai Cell Bank. HPDE6C7, PANC-1 and BxPC-3 cells were cultured in DMEM plus 10% FBS (Invitrogen Biotech Co, Ltd) and 1% pen/strep at 5% CO 2 and 37°C, and Miacapa-2 cells were grown in DMEM supplemented with 5% horse serum. We obtained non-tumour and PC tissues from patients who received Pancreaticoduodenectomy in the Affiliated Hospital of Nantong University from 2013 to 2017. The study was approved by our ethics committee, and informed consent was provided by all participants. All tissue samples were snap-frozen and stored prior to use.

| Tissue microarray
The immunohistochemical staining rate was classified as 0 (negative), 1 (1%-25% positive tumour cells), 2 (26%-50%), 3 (51%-75%) and 4 (76%-100%). Staining intensity was classified as 0 (absence of stained cells), 1 (weak staining), 2 (moderate staining) and 3 (strong staining). The immunohistochemistry (IHC) score was calculated by multiplying the staining rate and intensity. The final expression scores of TP73 were calculated with the value of per cent positivity score multiplied by staining intensity score, which ranged from 0 to 4. The degree of protein staining was quantified using a two-level grading system, and when the sample was scored ≥2 point, we defined it as high expression, otherwise low expression or negative.
Absorbances were read at 450 nm on a microplate reader.

| Transwell invasion assay
Place the Transwell chamber in a 24-well plate in advance, spread a layer of Matrigel in the upper chamber and incubate it in a cell culture incubator for 5-6 hours. After the Matrigel has solidified, the steps are the same as the transwell migration assay.
Dual-Luciferase Reporter Assays were then performed as per the manufacturer's protocols (Promega).

| RT-qPCR
RNA was extracted from PC cells or tumour tissues via TRIzol lysis, and cDNA was synthesized using Rayscript Kits. RT-qPCRs were performed using a qPCR PreMix Kit on an ABI7500 RT-PCR System. U6 was assessed as a control to which miR-200a expression was normalized. TP73-AS1 and MMPs were normalized to GAPDH. The 2 -ΔΔCT method was used to quantify relative gene expression. The primers of the molecules used in the article are listed in Table 1. Primer sequences were synthesized by Generay Co Ltd.

| RNA FISH
A total of 58 frozen tissue samples harvested from PC patients were sectioned (~5 µm thick), washed in PBS and fixed in 3.7% formaldehyde for 10 minutes. Sections were then washed in 10% formamide and labelled with hybridization solution containing 10% formamide and 10% dextran sulphate (w/v) at 37°C overnight. Slides were then washed in 10% formamide and DAPI stained. TP73-AS1 was labelled with rhodamine probes. Sections were rinsed in 2 × sodium citrate buffer and assessed by confocal microscopy.

| Western blot
For WB analysis, PC cells were lysed in RIPA buffer and resolved by SDS-PAGE electrophoresis. Proteins were then transferred to PVDF membranes and blocked with 5% 2092 M XUE ET AL pure milk for 1 hour and labelled with primary antibodies to MMP14 and GAPDH (1:1000, CST, USA) at 4°C overnight. Membranes were then labelled with the appropriate conjugated secondary antibodies.

| Metastasis models in nude mice
To assess the in vivo roles of TP73-AS1, BALB/c nude mice (4-6 weeks old, n = 12) from the laboratory animal centre of Nantong University were divided into 4 groups (n = 3). Stably transfected Panc-1 and

| Immunohistochemistry
Nude mouse tumour tissues were fixed in 4% formaldehyde, paraffin embedded and sectioned. Sections were probed with Anti-MMP14 antibodies (Abcam) and counterstained with haematoxylin. Sections were then dehydrated, mounted and imaged.

| Statistics
All data are shown as mean ± standard deviation. Data were ana- t test, one-way analysis of variance or a -chi-square test was used to nalyse the differences between two or more groups. P <.05 was deemed a statistical difference.

| TP73-AS1 is overexpressed in PC cells
To explore the role of TP73-AS1 during PC progression, we analysed its expression in paracancerous samples (n = 24). The results demonstrated that TP73-AS1 is overexpressed in cancer tissues ( Figure 1A), PC cells (Panc-1, miacapa-2, Bxpc-3) and normal pancreatic epithelial cell ( Figure 1B). These data highlighted TP73-AS1 as a potential oncogene. For further verification, the expression of TP73-AS1 was detected in a tumour microarray containing 58 cancer tissues from patients ( Figure 1C). According to the basic clinical and pathological parameters of tumours, patients with pancreatic cancer are grouped according to age, sex, tumour TNM stage and lymph node metastasis. The results showed that the high expression of TP73-AS1 in pancreatic cancer tissue was related to tumour TNM stage (P =.001) and lymph node metastasis (P =.018), and the difference was statistically significant. There was no significant difference with the patient's gender, age (P >.05) ( Table 2).

| TP73-AS1 enhances PC metastasis by inhibiting MMP14 expression
To evaluate its role in PC, TP73-AS1 was silenced through si-TP73-AS1 transfection. RT-qPCR analysis confirmed effective TP73-AS1 silencing (Figure 2A). Following si-TP73-AS1 transfection, the invasion, migration, proliferation, cell cycle and cell apoptosis of PC cells were assessed by transwell, cck-8 and flow cytometry assays, respectively. We found that the invasion and migration of si-TP73-AS1 groups were lower than the NC group, but PC cell survival, cell cycle progression and cell apoptosis were unaffected ( Figure 2B-E). MMPs degrade the extracellular matrix and are up-regulated in many tumours. We detected MMP expression in Panc-1 and Miacapa-2 cells transfected with si-TP73-AS1 and NC by RT-qPCR. We found that MMP14 was down-regulated in si-TP73-AS1 groups vs NC groups ( Figure 2F). These data indicated that TP73-AS1 influenced the metastatic phenotypes of PC cells by inhibiting MMP14 expression.

F I G U R E 2
Down-regulation of TP73-AS1 inhibited the migration and invasion of PC cells. A, Inhibitory efficiency of si-TP-AS1. B, TP73-AS1 knock-down had no effect on cell viability of PC cells. C, TP73-AS1 knock-down had no effect on cell cycle progression of PC cells. D, TP73-AS1 knock-down had no effect on apoptosis of PC cells. E, TP73-AS1 knock-down inhibited migration and invasion of PC cells. F, MMPs expressions were assessed via RT-qPCR and WB. N = 3, mean ± SD. (NS: No significance, *P <.05, **P <.01, ***P <.001)

| TP73-AS1 regulates PC metastasis in vivo
To

| D ISCUSS I ON
Pancreatic cancer (PC) is a malignant tumour that despite advances in surgical resection, radiotherapy, chemotherapy and immunotherapy, has one of the lowest 5-year survival rates. Due to the lack of early diagnostics, PC is often diagnosed as late and advanced stages leading to a dismal prognosis for afflicted patients. 19 By 2030, PC is predicted to be the 2nd leading cause of US cancer-associated deaths. 2 PC has a similarly poor prognosis in European countries with close to 1 000 000 thought to have died from PC. 20 New and effective diagnostics are therefore urgently required. In this study, we found that lnc-TP73-AS1 is highly expressed in PC patients and cells and is The LncRNA TP73-AS1 is a ceRNA of miRNA-103 that modulates hepatoma cell proliferation and promotes gastric cancer (GC) cell cisplatin resistance. 25,26 Given this knowledge, this study investigated the contribution of TP73-AS1 to PC development.
In 24 PC patients, TP73-AS1 was found to be significantly over- In summary, we have identified TP73-AS1 was an important oncogene in the metastasis of PC through its ability to regulate MMP14 and miR-200a expression in PC cells. As a result, the TP73-AS1/ miR200a/MMP14 axis represented a promising target for PC therapeutics.

ACK N OWLED G EM ENT
This work was financially supported by the National Natural Science Planning (QA2020040).

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest. Funding acquisition (equal); Methodology (equal); Resources (equal).

DATA AVA I L A B I L I T Y S TAT E M E N T
All data which support these findings of the study are contained in this article.