LncRNA ADAMTS9-AS2 inhibits gastric cancer (GC) development and sensitizes chemoresistant GC cells to cisplatin by regulating miR-223-3p/NLRP3 axis

The role of LncRNA ADAMTS9-AS2 in the regulation of chemoresistance of gastric cancer (GC) is largely unknown. Here we found that LncRNA ADAMTS9-AS2 was low-expressed in GC tissues and cells compared to their normal counterparts. In addition, LncRNA ADAMTS9-AS2 inhibited miR-223-3p expressions in GC cells by acting as competing endogenous RNA, and the levels of LncRNA ADAMTS9-AS2 and miR-223-3p showed negative correlations in GC tissues. Of note, overexpression of LncRNA ADAMTS9-AS2 inhibited GC cell viability and motility by sponging miR-223-3p. In addition, the levels of LncRNA ADAMTS9-AS2 were lower, and miR-223-3p was higher in cisplatin-resistant GC (CR-GC) cells than their parental cisplatin-sensitive GC (CS-GC) cells. LncRNA ADAMTS9-AS2 overexpression enhanced the cytotoxic effects of cisplatin on CR-GC cells, which were reversed by overexpressing miR-223-3p. Furthermore, LncRNA ADAMTS9-AS2 increased NLRP3 expressions by targeting miR-223-3p, and upregulation of LncRNA ADAMTS9-AS2 triggered pyroptotic cell death in cisplatin treated CR-GC cells by activating NLRP3 inflammasome through downregulating miR-223-3p. Finally, the promoting effects of LncRNA ADAMTS9-AS2 overexpression on CR-GC cell death were abrogated by pyroptosis inhibitor Necrosulfonamide (NSA). Collectively, LncRNA ADAMTS9-AS2 acted as a tumor suppressor and enhanced cisplatin sensitivity in GC cells by activating NLRP3 mediated pyroptotic cell death through sponging miR-223-3p.


INTRODUCTION
Gastric cancer (GC) is a common malignancy in the digestive system [1,2], although the number of inpatient admissions for GC have decreased over the past decades, the healthcare burden and cost related to it has increased significantly [3]. Currently, surgical resection [4], radiotherapy [5], chemotherapy [6] and combined therapy [7] remain the primary therapies for GC treatment in clinic. Cisplatin is the first-line chemotherapeutic drug for GC treatment [8,9], and cisplatin induces GC cell death by triggering DNA damage response (DDR) [10,11]. However, long-term cisplatin treatment stimulates cancer stem cells (CSCs) to differentiate into heterogeneous GC cells [12][13][14], as a result, chemoresistant GC cells appears and seriously limits the therapeutic efficacy of cisplatin for GC treatment [12][13][14]. Additionally, cell pyroptosis is a type of programmed cell death characterized by NLRP3 inflammasome activation and inflammatory cytokines secretion [15,16]. Recent study found that cisplatin induced pyroptotic cell death in lung cancer cells by AGING targeting Caspase-3/Gasdermin E (GSDME) signaling cascade [17], nevertheless, it is still unclear whether cisplatin induced GC cell pyroptosis.
Based on the above literatures, we aimed to investigate whether LncRNA ADAMTS9-AS2 regulated GC pathogenesis and cisplatin induced GC cell pyroptosis, and uncover the potential molecular mechanisms. This study will give some insights into the role of LncRNA ADAMTS9-AS2/miR-223-3p/NLRP3 pathway in the regulation of GC progression and chemoresistance to cisplatin.

RESULTS
LncRNA ADAMTS9-AS2 and miR-223-3p were aberrantly expressed in GC tissues and cells The GC tissues and their paired normal adjacent tissues (N = 45) were collected, and Real-Time qPCR was conducted to determine the expressions of LncRNA ADAMTS9-AS2 and miR-223-3p in the tissues. The results showed that LncRNA ADAMTS9-AS2 was lowexpressed ( Figure 1A), while miR-223-3p was highexpressed ( Figure 1B) in GC tissues compared to their corresponding normal tissues. The expression levels of LncRNA ADAMTS9-AS2 and miR-223-3p were negatively correlated in GC tissues ( Figure 1C), which were validated by the online Pan-cancer correlation analysis (http://hopper.si.edu/wiki/mmti/Starbase) for 372 specimens from the patients with stomach adenocarcinoma (STAD) ( Figure 1D). In addition, LncRNA ADAMTS9-AS2 was low-expressed, and miR-223-3p was high-expressed in GC tissues from the patients with tumor size (> 3), lymphatic invasion (yes) and TNM stage (III/IV), but did not correlate with other clinical parameters, such as patient age and gender (Table 1). Furthermore, the Kaplan-Meier survival analysis suggested that patients with low-expressed LncRNA ADAMTS9-AS2 ( Figure 1E) and highexpressed miR-223-3p ( Figure 1F) had a worse prognosis and shorter survival time. Next, we investigated the above results in vitro by using the human gastric epithelial cell line GES-1 and GC cell lines (SGC7901, MKN74, NUGC-4, HGC-27 and BGC-823), which also showed negative correlations ( Figure 1G, 1H). The results showed that the levels of LncRNA ADAMTS9-AS2 were lower ( Figure 1G), but miR-223-3p were higher ( Figure 1H) in GC cells comparing to the GES-1 cells.

Differential expression status of LncRNA ADAMTS9-AS2 and miR-223-3p in CS-GC and CR-GC cells
Since both LncRNA ADAMTS9-AS2 and miR-223-3p participated in the regulation of resistance of cancer cells to chemotherapeutic drugs [25,37], further experiments were conducted to explore the role of LncRNA ADAMTS9-AS2/miR-223-3p axis in the modulation of cisplatin-resistance in GC cells. The Real-Time qPCR results showed that the levels of LncRNA ADAMTS9-AS2 were lower ( Figure 4A), while miR-223-3p were higher ( Figure Figure 4C) according to the previous study [41].
Further results validated that we had successfully AGING inducted ACR-GC cells, which were resistant to highdose cisplatin stimulation ( Figure 4D). In addition, long-term low-dose cisplatin stimulation decreased LncRNA ADAMTS9-AS2 levels ( Figure 4E), while increased miR-223-3p levels in GC cells ( Figure 4F). The above results suggested that continuous low-dose cisplatin stimulation altered the expression patterns of LncRNA ADAMTS9-AS2 and miR-223-3p in GC cells, which might render GC cells chemoresistance to highdose cisplatin.

LncRNA ADAMTS9 triggered cisplatin-induced CR-GC cell pyroptosis by targeting miR-223-3p
Based on the above results, we next investigated whether LncRNA ADAMTS9/miR-223-3p/NLRP3 axis induced pyroptotic cell death in CR-GC cells treated with high-dose cisplatin. To achieve this, the CR-GC cells were pre-transfected with LncRNA ADAMTS9 overexpression vectors and miR-223-3p mimic, and subsequently treated with high-dose cisplatin for 24 h. The pyroptosis associated proteins (NLRP3, ASC, IL-18 and IL-1β) and caspase-1 activity were determined in cells and supernatants, respectively. The results showed that high-dose cisplatin alone did not affect the above proteins, while overexpression of LncRNA ADAMTS9 significantly increased the expression levels of NLRP3 and ASC in cisplatin treated CR-GC cells ( Figure 7A-7D). Consistently, cisplatin or LncRNA ADAMTS9 overexpression alone had little effects on caspase-1 activity ( Figure 7E), IL-1β ( Figure 7F) and IL-18 ( Figure 7G) expressions in CR-GC cells and their supernatants, which were promoted by cisplatin plus LncRNA ADAMTS9 overexpression treatment. Furthermore, pyroptosis was accompanied by pores formation in the membrane, which led to the release of intracellular contents, such as lactate dehydrogenase (LDH), and the results showed that LncRNA ADAMTS9 overexpression induced LDH release from CR-GC cells treated with cisplatin ( Figure 7H). Of note, LncRNA ADAMTS9 triggered pyroptotic cell death in high-dose cisplatin treated CR-GC cells were abrogated by overexpressing miR-223-3p ( Figure 7A-7H). The in vitro results were also validated by our in vivo experiments. Specifically, overexpression of LncRNA ADAMTS9 promoted LDH release (Figure 7I), and increased NLRP3 and ASC expression levels in cancer tissues ( Figure 7J, 7K), and promoted IL-18 and IL-1β expressions in mice serum ( Figure 7L). Additionally, the activity of caspase-1 was increased by LncRNA ADAMTS9 overexpression in mice tumor tissues ( Figure 7M).

DISCUSSION
Although recent study reported the involvement of LncRNA ADAMTS9-AS2 in the regulation of GC progression [24], the detailed molecular mechanisms are still not fully delineated. In this study, analysis of clinical samples showed that LncRNA ADAMTS9-AS2 was low-expressed, while miR-223-3p was highexpressed in GC tissues compared to the normal tissues, and patients with higher LncRNA ADAMTS9-AS2 and lower miR-223-3p tended to have a favorable prognosis. Further in vitro gain-and loss-function experiments validated that LncRNA ADAMTS9-AS2 inhibited GC cell proliferation and mobility by sponging miR-223-3p, which were also AGING verified in the previous study [23]. Specifically, LncRNA ADAMTS9-AS2 acted as a tumor suppressor in GC pathogenesis [23], and miR-223-3p overexpression promoted GC development [34]. Of note, LncRNA ADAMTS9-AS2 inhibited miR-223-3p expressions in lung cancer by serving as a competitive endogenous RNA [40]. Therefore, we demonstrated that LncRNA ADAMTS9-AS2 inhibited GC progression by sponging miR-223-3p.
Long-term cisplatin stimulation rendered GC patients with resistance to this chemotherapeutic drug [50,51], which seriously limited its therapeutic efficacy in clinic. Previous literature reported that LncRNA ADAMTS9-AS2 regulated chemoresistance in clear cell renal cell carcinoma [25], glioblastoma [26] and breast cancer [27], but no publications reported the involvement of LncRNA ADAMTS9-AS2 in the regulation of cisplatin resistance in GC cells. Additionally, miR-223-3p promoted cisplatin resistance of GC cells via targeting F-box and WD repeat domain containing 7 (FBXW7) [37]. Based on this, we found that LncRNA ADAMTS9-AS2 was low-expressed, while miR-223-3p was high-expressed in CR-GC cells and ACR-GC cells, and continuous low-dose cisplatin stimulation significantly decreased LncRNA ADAMTS9-AS2, and increased miR-223-3p levels in GC cells, which suggested that the expression patterns of LncRNA ADAMTS9-AS2 and miR-223-3p were changed by cisplatin treatment. Further experiments validated that overexpressed LncRNA ADAMTS9-AS2 enhanced the inhibiting effects of high-dose cisplatin on CR-GC cell viability, which were reversed by upregulating miR-223-3p. The above results suggested that LncRNA ADAMTS9-AS2 sensitized CR-GC cells to cisplatin by targeting miR-223-3p.
Cisplatin inhibited cancer progression by triggering autophagic cell death [42], apoptosis [43], pyroptosis [17], ferroptosis [44] and necroptosis [45]. Hence we investigated the underlying mechanisms of LncRNA ADAMTS9-AS2 overexpression induced cell death in CR-GC cells treated with high-dose cisplatin. The results showed that only the inhibitors for apoptosis and pyroptosis, instead of the inhibitors for other types of cell death, rescued cell viability in cisplatin treated CR-GC cells transfected with LncRNA ADAMTS9-AS2 overexpression vectors, which suggested that LncRNA ADAMTS9-AS2 enhanced the cytotoxic effects of cisplatin on CR-GC cells by triggering apoptotic and pyroptotic cell death. Of note, previous study found that cisplatin induced lung cancer cell pyroptosis through apoptotic stimulation [17], however, future work are still needed to investigate the underlying mechanisms. In addition, we found that LncRNA ADAMTS9-AS2 activated NLRP3 inflammasome in GC cells by downregulating miR-223-3p, which were in accordance with the previous studies [47,49]. Further results validated that LncRNA ADAMTS9-AS2 triggered cell pyroptosis in cisplatin treated CR-GC cells by regulating miR-223-3p/NLRP3 axis in vitro and in vivo.
Collectively, LncRNA ADAMTS9-AS2 overexpression inhibited GC progression and sensitized CR-GC cells to cisplatin by regulating miR-223-3p/NLRP3 axis mediated cell pyroptosis (Figure 8). This study will give AGING some insights into the molecular mechanisms of the chemoresistance generated by GC cells to cisplatin and provide new therapeutic agents for GC treatment in clinic.

Clinical specimens
The GC tissues and their paired normal adjacent tissues were collected from patients (N = 45) diagnosed as GC in the First Affiliated Hospital of Harbin Medical University from 2012 to 2014. These GC patients did not receive any treatment before surgical resection. The clinicopathological features of the above patients were summarized in Table 1. All the clinical experiments were approved by the Institutional Ethics Committee of the First Affiliated Hospital of Harbin Medical University, and all the participants involved in this study signed the informed consent form.

Cell culture
The

Vectors transfection
The vectors for LncRNA ADAMTS9-AS2 overexpression and silencing were obtained from YRBIO (Changsha, China). The miR-223-3p mimic and inhibitor were designed and synthesized by Sangon Biotech (Shanghai, China). The above vectors were delivered into GC cells by using the Lipofectamine 3000 reagent purchased from Invitrogen (CA, USA) according to the manufacturer's instruction.

Western blot
The radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China) was used to extract the total protein from cancer tissues and cells. Western Blot was conducted to determine the expression levels of proteins involved in this study according to the previous study [18]. The primary antibodies against NLRP3 (1:1000), β-actin (1:1000) and ASC (1:1500) were purchased from Abcam (UK). The protein bands were visualized by using the electro-chemiluminescence (ECL) Western Blot detection kit (GE Healthcare Bio-science, USA) in keeping with the manufacturer's instruction and quantified by Image J software.

Cell counting kit-8 (CCK-8) assay
The cell proliferation abilities of GC cells were measured by using the commercial CCK-8 kit (AbMole, USA) according to the manufacturer's instruction. Briefly, the cells were incubated with CCK-8 reaction solution for 4 h and the Gemini EM microplate reader (Molecular Devices, USA) was used to measure optical density (OD) values at the wavelength of 450 nm to evaluate cell proliferation.

Cell counting assay by Trypan blue staining
The GC cells were harvested and stained with Trypan blue (Sigma, USA) according to the protocol. After that, the cells were counted under optical microscope, the cells stained with blue were regarded as dead cells. The cell viability was calculated by using the following formula: cell viability (%) = live cells/total cells * 100 %.

Transwell assay
The GC cells were seeded into the upper floor of Transwell chambers (BD Biosciences, USA) with serum-free medium at the density of 5 × 10 4 cells per well, and the lower chambers were added with culture medium containing 20 % fetal bovine serum (FBS). After 24 h incubation under the standard conditions, the invasive cells were fixed in methanol and stained with 0.1 % crystal violet to visualize the cells. The cells were observed and photographed under optical microscope.

Colony formation assay
The GC cells were harvested and cultured in 6-well plates at the density of 500 cells per well for 14 days. After that, the cells were stained with crystal violet (Beyotime, China) based on the protocol provided by the manufacturer. The cells were photographed under optical microscope (ThermoFisher Scientific, USA) and the colonies containing at least 10 cells were counted.

Flow cytometry (FCM)
The Annexin V-FITC/Propidium Iodide (PI) doublestain kit (BD Bioscience, USA) was employed to detect cell apoptosis ratio according to the manufacturer's instruction. After that, the Flow Cytometry (FCM) (ThermoFisher Scientific, USA) was employed to measure cell apoptosis ratio.

Enzyme linked immunosorbent assay (ELISA)
The supernatants for GC cells and mice serum were collected, the ELISA kit was used to measure the expression levels of IL-1β and IL-18 according to the protocol provided by the producer. The HRP-labeled goat anti-rabbit IgG antibodies were used as secondary antibody. The absorbance values were detected by using the microplate reader (Molecular Devices, USA) at the wavelength of 450 nm.

Caspase-1 activity detection
The activity of caspase-1 was measured by using the Caspase-1 Activity Assay Kit (Solarbio, China) in keeping with the manufacturer's instruction. The specimens from tissues and cells were lysed by the lysis buffer. The contents of total proteins were evaluated by Bradford method and a microplate reader was employed to examine the optical density (OD) values at the wavelength of 405 nm, which was utilized to represent caspase-1 activity according to the previous study [52].

Lactate dehydrogenase (LDH) release assay
The LDH cytotoxicity assay kit (Beyotime, China) was purchased to measure LDH release based on the protocol provided by the manufacturer. Briefly, the GC cells were administered with different treatments, and the supernatants were harvested. The LDH reagent was used to detect LDH release at the wavelength of 490 nm according to the previous study [52].

Dual-luciferase reporter gene system
The wild-type (Wt) and mutant (Mut) LncRNA ADAMTS9-AS2 and 3'UTR regions of NLRP3 mRNA were synthesized and cloned into the psiCHECK-2 vectors (Promega, USA). The above vectors were cotransfected with miR-223-3p mimic and miR-NC (Sangon Biotech, China) into SGC7901 and BGC-823 cells, respectively. After 48 h incubation, the dualluciferase reporter gene system (Promega, USA) was employed to measure relative luciferase activity based on the manufacturer's protocol.

Pull-down assay
The biotin-labeled probes for LncRNA ADAMTS9-AS2 and 3' UTR regions of NLRP3 mRNA were designed and synthesized by Sangon Biotech (Shanghai, China). The cells were fixed, lysed and sonicated. After centrifugation, the supernatants were used as input, and the rest part of SGC7901 and BGC-823 cells were incubated with the streptavidin Dynabeads (Invitrogen, USA) mixture containing the above probes at 30 °C overnight. After that, the lysis buffer and Proteinase K were employed to reverse crosslinking and release miR-223-3p, which were quantified by Real-Time qPCR.

Xenograft models
The nude mice (N = 18, age 6-8 weeks) were purchased from the Experimental Animal Center of the First Affiliated Hospital of Harbin Medical University. The CR-GC cell line (SGC7901/DDP) were subcutaneously injected into the back flank of each mouse, the LncRNA ADAMTS9-AS2 overexpression vectors were also injected into the tumor formation sites. The above mice were equally divided into 3 groups, including control group, cisplatin alone group and cisplatin plus overexpressed LncRNA ADAMTS9-AS2 group, respectively. Until tumor volume reached 1 mm 3 , the high-dose cisplatin (10 μg/ml) were administered for 2 weeks, the cancer tissues and serum were collected from the mice for further investigation. All the animal experiments were approved by the Animal Management Center of the First Affiliated Hospital of Harbin Medical University.

Statistical analysis
All the data involved in this study were collected and represented as Mean ± Standard Deviation (SD). The SPSS 18.0 software was used to analyze the data. Specifically, student's t-test method was used to compare the differences between two groups. The oneway Analysis of Variance (ANOVA) method was employed to compare the differences among multiple groups. The Pearson correlation analysis was used to analyze the correlation of LncRNA ADAMTS9-AS2 and miR-223-3p in GC tissues. The prognosis for GC patients was analyzed by Kaplan-Meier survival AGING analysis. Each experiment in this study repeated at least 3 times. The analysis results were visualized by using the Graphpad Prism 5 software. "NS" indicated "No statistical significance", *P < 0.05 and **P < 0.01.