PITPNA-AS1 Activated by H3K27ac Sponged miR-98-5p to Regulate Cisplatin Resistance in Gastric Cancer

To evaluate the expression of PITPNA-AS1 and miR-98-5p in gastric cancer tissues as well as their association with progression of gastric cancer, and investigate the role of PITPNA-AS1 and miR-98-5p in developing platinum resistance. RNA sequencing was used to identify candidate lncRNAs and microRNAs related to local recurrence of gastric cancer. qRT-PCR was used to investigate the expression of PITPNA-AS1 and miR-98-5p. CCK-8 and caspase3/7 activity were used to evaluate the cell proliferation and apoptosis rate. Dual luciferase reporter gene assay and RNA pull down were used to evaluate the cross talk between PITPNA-AS1 and miR-98-5p. PITPNA-AS1 and miR-98-5p could regulate cell proliferation and inhibit apoptosis in gastric cancer cell lines. Cisplatin and lobaplatin could signicantly suppress the expression of PITPNA-AS1, which interacted with negatively regulated miR-98-5p expression. PITPNA-AS1 overexpression impaired the effect of platinum, which was partially reversed by downregulation of miR-98-5p knock down. In gastric cancer, PITPNA-AS1 and miR-98-5p could regulat cell growth, apoptosis and platinum resistance. They have the potential to be biomarkers and curative therapeutic targets. However, further research on molecular mechanisms are needed. present study experiments conducted in accordance with relevant and which consistent

Despite the remarkable progressive surgical and medical techniques, prognosis of patients with GC remains relatively poor mainly due to recurrence and metastasis 3 . Neoadjuvant chemotherapy improves survival in comparison to best supportive care or surgery alone. 5-uorouracil combinated with cisplatin has been convincingly proved that it is survival bene t for HER-2-positive patients [4][5][6] . As the main chemotherapy treatment for postoperative GC patients, the e cacy of platinum has been largely limited due to the chemo-resistance 7 . Laboratory studies illustrated that resistance to platinum is almost multifactorial, including damaged cellular uptake of platinum 8 , reinforced endocellular detoxi cation by glutathione and metallothionein systems 9 , modi ed patterns of DNA platination, enhanced tolerance to DNA damage 10 and rising restore of DNA damage 8,11 .
Long non-coding RNAs (LncRNAs) are a class of non-coding RNAs longer than 200 nt without protein coding potential. Several lncRNAs were con rmed as biotargets for modulating cisplatin resistance in cancer through the cell cycle, apoptosis and Wnt pathways 12 , which acting as a competing endogenous RNA or directly binding to mRNAs or proteins and regulating their expression and functions 13   PITPNA-AS1 expression was correlated with local recurrence in gastric cancer patients PITPNA-AS1 was highly expressed in 153 gastric cancer tissues compared with para-cancer tissues, and miR-98-5p expression was lower in cancer tissues ( Fig. 2a and 2b). In addition, survival analysis demonstrated that PITPNA-AS1 overexpression was combination with worse cancer speci c survival (Fig.   2c). On the contrary, miR-98-5p led to reduced risk of death shown in the analysis of cancer speci c survival (Fig. 2d). Furthermore, PITPNA-AS1 negatively correlated with miR-98-5p expression (Fig. 2e). Besides, PITPNA-AS1 expressed higher in local recurrent gastric cancer patients than non-recurrent ones, while miR-98-5p was down regulated ( Fig. 2f and 2g).
PITPNA-AS1/miR-98-5p regulated cell proliferation and inhibits apoptosis in gastric cancer cell lines We found that PITPNA-AS1 over-expressed in human gastric cancer cell lines MKN45 and AGS than that in normal gastric mucosal cell line GES-1 (Fig. 3a), besides, miR-98-5p expressed lower in MKN45 and AGS than that in GSE-1 cell line (Fig. 3b). Then we knocked down the expression of PITPNA-AS1 and found that MKN45 and AGS cell proliferation rate was inhibited ( Fig. 3c and 3d), however, the apoptosis rate was increased ( Fig. 3e and 3f). Moreover, overexpression of miR-98-5p brought decreased cell proliferation rate and enhanced cell apoptosis rate ( Fig. 3g-3j); PITPNA-AS1 negatively regulated the expression of miR-98-5p We found that knocking down PITPNA-AS1 resulted in overexpression of miR-98-5p ( Fig. 4a and 4b).

PITPNA-AS1 expression can be suppressed by cisplatin in gastric cancer cell lines
The half maximal inhibitory concentration IC50 of CDDP in MKN45 is 0.52 ug/mL (Fig. 5a); and the IC50 in AGS is 0.59 ug/mL (Fig. 5b). We used CDDP (0.52 ug/mL) to treat MKN45 and CDDP (0.59 ug/mL) to treat AGS cells for 24 hours and found that PITPNA-AS1 expression can be signi cantly suppressed ( Fig.  5c and 5d).
In the meantime, CDDP induced the expression of miR-98-5p in MKN45(0.52 ug/mL) and in AGS cells (0.59 ug/mL) ( Fig. 5e and 5f). We then noticed that H3K27ac expression was suppressed when treated with CDDP in MKN45 and AGS cells (Fig. 5g).

Discussion
Collectively, in this study, we discovered the role of PITPNA-AS1 and miR-98-5p in gastric cancer through gain and loss-of-function assays and analyzed the mechanism by which PITPNA-AS1 regulates apoptosis and drug resistance through the miR-98-5p targeting axis.
Gastric cancer is one of the leading public health problems worldwide because of its high incidence, morbidity and mortality rate 29 . Currently, lacking of screening methods and early symptom, patients are most often diagnosed at advanced stages, with metastatic at distant sites and somber prognosis (median overall survival is 10-12 months) 30,31 . For locally advanced disease, adjuvant or neoadjuvant therapy which recognized as the optimal therapeutic option is usually introduced with surgery owing to its curability 30 . Fluoropyrimidine plus oxaliplatin doublet is considered as the preferred rst-line regimen due to its comparable survival bene ts and lower toxicity 32 . Overcoming resistance is still a challenge in GC chemotherapy.
LncRNAs are associated with the tumor recurrence and poor prognosis,and abnormal expression has been observed in various tumors 33 . Mounting evidence elucidated that lncRNAs could act as oncogenes or tumor suppressors by modulating the gene expression and function in tumorigenesis 34 , which possibly induce signi cant in uence on the alterations of cell proliferation, metastasis, autophagy and apoptosis 35,36 . Our study indicated that lncRNA PITPNA-AS1 was highly expressed in gastric cancer patients and was associated with poor prognosis. Alteration of gene expression is correlated with the cancer speci c survival of patients. PITPNA-AS1 was over-expressed in MKN45 cell line while knocking down PITPNA-AS1 resulted in inhibiting cell proliferation rate and increasing apoptosis rate. We rst time inspected the role of PITPNA-AS1 in GC, which founding the basis for further exploration.
Next, we investigated the potential mechanism underlying PITPNA-AS1. Biased on current study, mechanism assays unveiled that PITPNA-AS1 targeted miR-98-5p. Dual luciferase reporter gene assay, RNA pull-down assay and RIP consequence provided powerful evidence that PITPNA-AS1 could interact with miR-98-5p. Moreover, knocking down PITPNA-AS1 resulted in decreased expression of miR-98-5p, which con rmed this discovery again. The anti-tumor function of miR-98-5p has been documented yet.
For instances, Fu Y, et al. recognized miR-98-5p underexpression as biomarkers for predicting poor prognosis in pancreatic ductal adenocarcinoma (PDAC) patients because miR-98-5p inhibits proliferation and metastasis via targeting MAP4K4 24 . Acting as a tumor suppressor, miR-98 could decelerate cancer aggressiveness by inhibiting TWIST expression in non-small cell lung cancers 37 . In hepatocellular carcinoma (HCC), miR-98-5p could restrain cell proliferation and induce cell apoptosis via inhibition of its target gene IGF2BP1 38 . As for colon cancer, miR-98 play the role of tumor suppressor gene and inhibits Warburg effect by targeting HK2 (HK2 involves in miR-98-mediated suppression of glucose uptake, lactate production, and cell proliferation, whose expression were negatively correlated with miR-98) in colon cancer cells, which provided promising therapeutic candidate for clinical treatments 39 .
In our study, miR-98-5p was shown to be downregulated in GC. Overexpression of miR-98-5p led to decreased cell proliferation rate and ascended apoptosis rate. Moreover, inhibition of miR-98-5p partially reversed the inhibitory effects of PITPNA-AS1 on GC cell proliferation and apoptosis. Thus, we draw the conclusion that PITPNA-AS1 exerts its tumor-promotion effect in GC via negatively modulating the expression of miR-98-5p. Laboratory ndings were consistent with literature reports. Guo Q, et al. revealed that lncRNA PITPNA-AS facilitate the cervical cancer progression on the proliferation, cell cycle and apoptosis by targeting the miR-876-5p/c-MET axis 40 .
Aberrant lncRNA expression is certi ed to strongly implicate in drug resistance in some cancers 41,42  Conjointly, these results suggest that miR-98-5p could be used as a novel prognostic factors and critical therapeutic strategy in GC by enhancing chemo-sensitivity for platinum treatment against GC. However, downstream signal molecule and other biological processes requires further investigation.

Gastric cancer patients
The GC tissues and local recurrence GC tissues, as well as the corresponding para-cancer tissues were collected from the patients who were diagnosed as GC by surgical resection at the First A liated Hospital of Xi'an Jiao Tong University. All of the samples were pathologically diagnosed and stored in liquid nitrogen. All of the patients had signed a written informed consent. The present study gained approval from the Ethics Committee of The First A liated Hospital of Xi'an Jiao tong University. And all experiments were conducted in accordance with relevant guidelines and regulations, which consistent with the Declaration of Helsinki regulations.

Cell culture
The human GC cell line MKN45 and AGS were purchased from Shanghai Gaining Biological Technology Co., Ltd (Shanghai, China), and the human gastric epithelial cell line GES-1 was obtained from American Type Culture Collection (Virginia, USA). All the cells were cultured in DMEM medium (HyClone, USA) containing 10% fetal bovine serum (Gibco, USA) and 1% penicillin-streptomycin (HyClone, USA) in a 37℃ and 5% CO2 incubator. The GC cells were then treated with continuous low-dose of cisplatin in a stepwise manner to developed cisplatin resistant GC (MKN45-CDDPR ) cells.

Cell transfection
The PITPNA-AS1 knockdown and miR-98-5p overexpression plasmids were purchased from Genechem (Shanghai, China). The above plasmids were delivered into MKN45 and AGS cell lines by using the Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) reagent according to the manufacturer's instruction.

RNA sequencing
The RNA sequencing process was guided and supported by Genechem(Shanghai, China). In brief, total RNA was extracted from 3 GC patients' tissues and corresponding normal tissues by using TRIzol (Invitrogen, Carlsbad, CA). And the RNA puri cation was performed by using the RNA Clean XP Kit qRT-PCR Total RNA was extracted from GC tissues and cell lines by using Trizol reagent (Invitrogen, Carlsbad, CA, USA). The cDNA was generated by using the First-strand cDNA synthesis kit (Tiangen Biotech, Beijing, China). The expression levels of PITPNA-AS1 was tested by conducting qRT-PCR using SYBR® Premix Dimer Eraser kit (Takara Shiga, Japan). And β-actin was used as the inner reference. The miScript microRNA RT PCR kit (Qiagen, Toronto, ON, Canada) was used for cDNA synthesis and qRT-PCR process for miR-98-5p expression. U6 was used as the internal reference. ABI 7500 Real-Time PCR system (Applied Biosystems, Carlsbad, CA, USA) was conducted to perform the qRT-PCR process. The expression level was calculated by 2-ΔΔCt method.

Western blot
The total proteins were extracted from GC cells by using RIPA lysis buffer (Sigma-Aldrich, Darmstadt, Germany) and were quanti ed by BCA Protein Assay Kit (Beyotime, Shanghai, China). Then proteins were diverted onto PVDF membranes (Millipore, USA) after separated by SDS-PAGE. The transferred PVDF membranes were blocked by using 5% skim milk, and then were incubated overnight at 4°C with primary antibodies, which including H3K27ac (1:1000, CST, Shanghai, China) and Histone H3 (1:2000, CST, Shanghai, China). Subsequently, the membranes were incubated with the secondary antibody (1:10000, Beyotime, Shanghai, China). Then the enhanced chemiluminescence (ECL, Beyotime, Shanghai, China) was used to quantify the protein expression levels.

Cell proliferation
In order to evaluate the proliferation and cisplatin resistance of GC cells, the cell counting kit-8 (CCK-8) kit (AbMole, USA) was used according to the manufacturer's protocol. In brie y, GC cells were seeded in 96well plates with a density of 5×10 3 cells per well, then 10 μL of CCK-8 reaction solution was supplemented into each well every 24h followed by incubation for 2h. Then the optical density (OD) values of GC cells at 450 nm were detected to assess cell proliferation of GC.
Cell apoptosis assay The Caspase 3/7 Activity Apoptosis Assay Kit (Invitrogen) was used to detect the apoptosis rate of GC cells. According to the manufacturer's instruction, GC cells were plated into the 96-well plate overnight at 20000 cells per well. Then 50 μL of Caspase 3/7 Substrate (Component A) was added into 10 mL of Assay Buffer (Component B) to make caspase 3/7 assay loading solution. GC cells were incubated in a a 37°C, 5% CO2, incubator for 6 h after treated with camptothecin to induce apoptosis. Then GC cells were added with 100 μL/well of caspase 3/7 assay loading solution, followed by supplemented with the assay loading solution at room temperature under dark conditions for 1 h of incubation. Finally, GC cells were centrifuged at 800 rpm for 2 min, then the uorescence intensity at Ex/Em=490/525 nm was monitored to evaluate cell apoptosis rate.

RNA pull-down assay
Biotin-labeled miR-98-5p-WT and miR-98-5p-Mut were synthesized by GeneCreate (Wuhan, China) and were transfected into GC cells which were incubated with lysis buffer (Ambion, Austin, Texas, USA). Then the GC cell lysates were incubated with the streptavidin Dynabeads (Invitgen, USA) precoated with RNasefree bovine serum albumin (BSA) and yeast tRNA (Sigma-Aldrich, USA) overnight at 4 °C. After washed with washing buffer, the bound RNA was puri ed by using Trizol. Finally, the enrichment of PITPNA-AS1 was identi ed and estimated by performing qRT-PCR.

Statistics
The SPSS 18.0 software and the Graphpad Prism 8.2 software were used to analyze and visualize the data involved in this study. The Limma package were used for RNA sequencing analysis. A paired Student's t-test was used to evaluate the statistical differences between two groups. And one-way ANOVA was applied for multiple groups comparison. The Kaplan-Meier survival analysis was used to estimate the prognosis of GC patients. Each assay was independently repeated at least three times and all the statistical results presented in this work were expressed as Mean±Standard Deviation (SD). A p value of <0.05 was indicative of statistically signi cant difference.

Declarations
Author contributions Y.P.L. designed and performed the experiments in this study; and completed the writing of the manuscript. S.X.H. and J.S.W. supervised the progression of the study. X.Z. completed the statistical analyses of the data and contributed to the manuscript editing. Y.N.C. plotted the statistic graphs. G.G. helped to edit the manuscript. All authors gave nal approval of the version to be published, and agreed to be accountable for all aspects of the work.