Long non-coding RNA XLOC_000647 suppresses progression of pancreatic cancer and decreases epithelial-mesenchymal transition-induced cell invasion by down-regulating NLRP3

Three pairs of PC and adjacent tissues were selected for lncRNAs microarray detection. The patients enrolled did not receive any treatment before surgery, and had no underlying diseases such as diabetes and hypertension. lncRNAs microarray detection (H1602063, Arraystar_Human_LncRNA_8x60k v3.0 1-color) was performed and analyzed by KangChen Bio-tech Inc. (Shanghai, China), among which 2074 up-regulated and 2693 down-regulated of lncRNAs were found.

We collected PC and corresponding adjacent normal tissue samples from 48 patients who underwent pancreatic resection at the Pancreas Center, The First Hospital Affiliated to Nanjing Medical University, from Dec 2015 to Aug 2016. The protocol was approved by the Hospital Ethics Committee and all patients signed a written informed consent form before specimen collection. All PC and matched non-tumor specimens were diagnosed by pathology. None of the patients received radiotherapy and/or chemotherapy before surgery. The resected specimens were immediately frozen by liquid nitrogen until further use. The clinicopathologic characteristics of patients are detailed in Table 1. Follow-up data were collected for all subjects and the overall survival time was calculated from the date of surgery to the date of death or the end of follow-up (Jun 2017).

Human primary PC cell lines (MIA-PaCa-2, BxPC-3 and PANC-1) and human embryonic kidney cells (293 T) were purchased from American Type Culture Collection (ATCC, Manassas, USA), and the immortal human pancreatic duct epithelial cell line (HPDE6), which is considered as a normal pancreatic cell line, was obtained from Pancreas Institute, Nanjing Medical University. Cells were incubated at 37 °C in humidified air with 5% CO₂, maintained in RPMI1640 (Gibco, CA, USA) or Dulbecco’s modified Eagle’s medium (DMEM; Gibco, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco), 100 U/ml penicillin, and 100 mg/ml streptomycin, with or without 2.5% horse serum (Gibco).

For stable overexpression of XLOC_000647, the full-length XLOC_000647 cDNA was synthesized by GENEWIZ (Suzhou, China) and cloned into pBABE retroviral vector (RTV-001-PURO, Cell Biolabs, CA, USA), named as XLOC_000647-pBABE, which were confirmed by sequencing (BioSune, Shanghai, China). After transfection of Plat-A (Cell Biolabs) cells for 48 h, the retrovirus supernatants were collected. Retroviral particles were concentrated by using Retro-Concentin Virus Precipitation Solution (ExCell Bio, Shanghai, China) according to the manufacturer’s guidelines. Then cancer cells were infected with virus and polybrene overnight. Positive clones were screened with puromycin for 2–3 weeks to establish XLOC_000647 stable expression cell lines and corresponding negative control for further study.

The expression of NLRP3 was inhibited by small hairpin RNA (shRNA) interference. shNLRP3 and its corresponding negative control pSH-U6 were purchased from Vigene Biosciences (Shandong, China). All oligonucleotide sequences were listed in Table 2. The overexpression of NLRP3 was performed by gene transfection. The full-length NLRP3 cDNA was synthesized and cloned into pENTER plasmid by Vigene Biosciences, named as NLRP3-pENTER, which were validated by sequencing.

Cell proliferation was detected using a cell counting kit-8 (CCK-8, Dojindo, Japan) following the manufacturer’s protocol. Cells were plated on 96-well plates (1 × 10³ cells/well) and 10 μL of CCK-8 solution was added at the right time on days1 to 5. Then, the cells were incubated for 2 h at 37 °C, and finally, the cells were determined for absorbance by a fluorescent microplate reader at 450 nm. The assays were repeated in triplicate.

Cells’ migration and invasion ability were measured using transwell chambers (24-well insert, 8 μm, Millipore, USA). The chambers were coated with 1:8 diluted Matrigel Matrix (Invasion assay, Corning, USA) for 1 h at 37 °C, 5 × 10⁴ cells were placed in upper chamber with 200 μL serum-free medium. The lower chamber was filled with 600 μL of medium containing 10% FBS with or without 2.5% horse serum. After incubation for 48 h, the number of cells passing through the bottom membrane of the chamber was counted under a microscope in five random fields. For motility assays, 1 × 10⁴ cells were seeded in the upper chamber without Matrigel. Other procedures were similar to the invasion assay.

Male athymic BALB/c nude mice, 4–6 weeks old, were purchased from Vital River Laboratory Animal Technology Co. (Beijing, China). The animal care and experimental protocols were approved by the institutional guidelines of Jiangsu Province and by the Animal Care and Use Committee of Nanjing Medical University. 1 × 10⁷ PC cells were resuspended in 200 μL PBS medium and were subcutaneously injected into the flank of each nude mouse. The tumors were measured weekly and the tumor volume was calculated following the formula length × width²/2. The mice were killed at 6 weeks after inoculation.

Total RNA was extracted from cells or tissues using TRIzol (Invitrogen) according to the manufacturer’s instructions. For the mRNA quantitative assay, total RNAs were reverse transcribed using the reverse transcription kit (Vazyme, China) following the manufacturer’s protocol. qPCR was performed using SYBR Green Master Mix (Vazyme, China) and analyzed on a Roche LightCycler system (Roche, Switzerland). ACTB was selected as the internal reference. All the primer sequences were listed in Table 2. The results were normalized to the expression of ACTB and showed as the fold change (2^−ΔΔCT). To clarify the clinical significance of XLOC_000647, the tissues samples were divided into two groups (high expression group vs. low expression group) by using the median expression value of XLOC_000647. Each result was repeated three times.

Total protein was extracted from the cells using whole cell lysis assay kit (KeyGEN, Nanjing, China) according to the manufacturer’s protocol. Equivalent amounts of proteins from each sample were subjected to SDS-PAGE electrophoresis and then transferred to a PVDF membrane (Millipore), blocked in 5% fat-free milk for 1 h at room temperature, incubated with specific primary antibodies overnight at 4 °C with gentle shaking, and followed by detection with enhanced chemiluminescence system (SuperSignal West Femto trial kit, Pierce). Primary antibodies were as follows: E-cadherin (1:300, 3195, CST), Vimentin (1:1000, 5741, CST), NLRP3 (1:1000, ab16097, Abcam), and ACTB (1:5000, AT0001, CMCTAG).

NLRP3 and Ki67 were detected in both human PC tissue specimens and xenograft tumor specimens from nude mice, which were fixed in 4% paraformaldehyde and subsequently embedded in paraffin. After dewaxing and hydration, antigen retrieval and blocking, the 5 μm sections were incubated with specific primary antibodies overnight at 4 °C, and followed by the observation using 3,3-diaminobenzidine color kit (Dako,Denmark). Primary antibodies were as follows: NLRP3 (1:200, ab214185, Abcam) and Ki67 (1:500, ab92742, Abcam).

The dual luciferase reporter assay was employed to examine the XLOC_000647 and NLRP3 relationship. Luciferase reporter plasmid containing NLRP3 promoter sequence (NLRP3 promoter-pGL-3-Basic, referred to as NLRP3 promoter) and its corresponding negative control pGL-3-Basic were purchased from Vigene Biosciences (Shandong, China). 1 × 10⁴ 293 T cells were plated on 48-well plates with 200 μL culture medium. The target plasmid (pBABE or XLOC_000647 and pGL-3-Basic or NLRP3 promoter) and pRL-TK plasmid expressing Renilla luciferase (Promega, USA) were co-transfected into cells with 50–60% confluency. After incubation for 48 h, cells were harvested for luciferase reporter assay.

All quantitative data were presented as mean ± SD. The chi-square test (χ² test) and Fisher’s exact test were used for categorical variables, and Student’s t test or ANOVA for quantitative variables. Differences in patient survival were performed using the Kaplan-Meier method and analyzed by log-rank test. The relative risk for each factor was evaluated using univariate and multivariate Cox regression analysis. Correlation analysis was explored by Pearson’s correlation. Statistical analysis and graph presentation were performed using SPSS v.17.0 software (SPSS Inc., Chicago, IL) and GraphPad Prism 5 software (GraphPad, San Diego, CA). A P value of <0.05 was regarded as statistically significant.

Microarray results from our previous study determined that the expression of lncRNA XLOC_000647 was significantly decreased in PC tissues relative to adjacent tissues (Fig. 1a). In this study, the expression of XLOC_000647 was further assessed with paired PC and adjacent tissues from 48 patients by qRT-PCR. Compared with adjacent tissues, XLOC_000647 was remarkably decreased in PC tissues (Fig. 1b). And the expression level of XLOC_000647 in three PC cell lines (MIA-PaCa-2, BxPC-3, and PANC-1) was dramatically lower than in normal human pancreatic ductal epithelial cell line HPDE6 (Fig. 1c). Thus our data suggest that XLOC_000647 is decreased in PC tissues and cell lines.

We explored the clinical significance of XLOC_000647 in PC (Table 1). Our results showed that low expression of XLOC_000647 was significantly correlated with >advanced TNM staging, lymph node metastasis (Fig. 2a, b), and shorter survival time (log-rank test, P < 0.001, Fig. 2c). Moreover, the univariate analysis showed that high TNM stage, a high T stage, and a low expression of lncRNA were remarkably associated with an increased risk of cancer-related death (Table 3). Further multivariate analysis revealed that the T stage was a key prognostic factor (P = 0.005, Table 3). More importantly, the low expression of XLOC_000647 was found to be an independent prognostic factor associated with poor prognosis in PC (P = 0.004, Table 3). These data indicate that XLOC_000647 is significantly correlated with the TNM stage and lymph node metastasis and is also an independent factor for the prognosis of PC patients.

To investigate the effect of lncRNA on proliferation, invasion, and EMT of PC cells, we constructed a retroviral stable expression of XLOC_000647 in MIA-PaCa-2 and BxPC-3 cells. The levels of XLOC_000647 in these two cells were validated by qRT-PCR (Fig. 3a). Overexpression of XLOC_000647 decreased cell proliferation and invasion when compared with pBABE controls, as shown by the results of the CCK-8 assay and transwell chambers measurement (Fig. 3b and c). However, overexpression of XLOC_000647 had little impact on PC cells migration (data not shown). Furthermore, compared with pBABE controls, western blot results indicated that the expression of EMT-related epithelial marker E-cadherin (E-cad) was significantly increased and the mesenchymal marker Vimentin was dramatically decreased in the above two cells (Fig. 3d). These results indicate that XLOC_000647 play a key role in inhibition of proliferation and EMT-induced invasion of PC cells in vitro, but has no effect on migration of PC cells.

We then determined the inhibition of XLOC_000647 in tumor progression in vivo. Stably expressing XLOC_000647 of MIA-PaCa-2 and BxPC-3 cells and their corresponding pBABE control cells were subcutaneously injected into nude mice. Six weeks after injection, the tumor size and weight of stably expressing XLOC_000647 groups were all remarkably lessened than the control groups (Fig. 4a and b). Moreover, we verified the inhibition of XLOC_000647 in tumors from stably expressing XLOC_000647 groups using qRT-PCR (Fig. 4c). Furthermore, immunohistochemical results of tumors showed that positive rate of cell proliferation indicator Ki67 was decreased in stably expressing XLOC_000647 groups (Fig. 4d). Taken together, our data demonstrate that XLOC_000647 acts as a tumor suppressor gene in PC tumor growth both in vitro and in vivo.

Recent studies have shown that many lncRNAs can act as local regulators to regulate the expression of its nearby genes ‘in cis’ [19‐21]. Therefore, we investigated whether XLOC_000647 was involved in regulating its neighboring gene. Our microarray results showed that there are three nearby genes of lncRNA_XLOC_000647, including NLRP3, olfactory receptor family 2 subfamily B member 11 (OR2B11), zinc finger protein 496 (ZNF496). Our study mainly focused on the invasion and metastasis of pancreatic cancer. Compared with other two nearby genes, NLRP3 was reported to be involved in the metastasis of many tumors, which was in line with our research. Therefore, we finally identified NLRP3 as a candidate gene to study. NLRP3 was located at about 25 kb of the XLOC_000647 downstream. Immunohistochemistry (IHC) revealed that PC tissues exhibited an increased positive rate of NLRP3 than adjacent tissues (Fig. 5a). Then, the expression of NLRP3 was further examined with paired PC and adjacent tissues from 48 patients by qRT-PCR. Data showed NLRP3 levels of PC tissues were significantly higher than that of adjacent tissues (Fig. 5b). And similar results were detected with matched PC and HPDE6 cell lines (Fig. 5c). Furthermore, western blot and IHC results indicated that overexpression of XLOC_000647 resulted in a decreased expression of NLRP3 in PC cell lines and tumor tissues from nude mice compared with their corresponding control groups (Fig. 5d and e). More importantly, correlation analysis revealed NLRP3 mRNA levels negatively correlated with XLOC_000647 in 48 PC tissues (R² = 0.4407, P < 0.001, Fig. 5f). And the luciferase reporter assay showed that the activity of NLRP3 promoter was greatly inhibited followed by overexpression of XLOC_000647 in 293 T cells (Fig. 5g). These results suggested that the expression of NLRP3 was negatively regulated by XLOC_000647, which mediated the proliferation, invasion, and EMT of PC.

To explore the role of NLRP3 on proliferation, invasion, and EMT of PC, MIA-PaCa-2 and BxPC-3 cells were transfected with shNLRP3 for 48 h respectively. The levels of NLRP3 were verified by qRT-PCR (Fig. 6a). Results of CCK-8 assay and transwell chambers demonstrated that knockdown of NLRP3 suppressed cells proliferation and invasion when compared with pSH-U6 controls (Fig. 6b and c). Furthermore, compared with pSH-U6 controls, western blot showed that the expression of E-cad was remarkably up-regulated and the Vimentin was significantly down-regulated in the above two cells (Fig. 6d). These results established that NLRP3 is crucial for the proliferation, invasion, and EMT of PC cells in vitro.

Next, we investigated whether XLOC_000647 suppressed EMT induced cells invasion by down-regulating NLRP3. Firstly, stably expressing XLOC_000647 cells of MIA-PaCa-2 and BxPC-3 cells and their corresponding pBABE control cells were transfected with NLRP3-pENTER for 48 h respectively. The levels of NLRP3 were validated by western blot (Fig. 7a). The transwell assay showed that overexpression of NLRP3 restored cells invasion ability inhibited by XLOC_000647 overexpression (Fig. 7b). Moreover, compared with pENTER controls, western blot indicated that the expression of E-cad was significantly decreased and the Vimentin was remarkably increased in the above two cells of stable expressing XLOC_000647 (Fig. 7c). These data revealed that XLOC_000647 might indirectly or directly regulate the expression of NLRP3, which in turn affected EMT induced invasion of PC.