Long non-coding RNA PCED1B antisense RNA 1 promotes gastric cancer progression via modulating microRNA-215-3p / C-X-C motif chemokine receptor 1 axis

ABSTRACT Long non-coding RNAs (lncRNAs) emerge as vital modulators and tissue-specific biomarkers of multiple cancers, including gastric cancer (GC). Instead, the expression characteristics, biological function and molecular mechanism of lncRNA PCED1B antisense RNA 1 (PCED1B-AS1) in GC await more elaboration. In this study, 48 cases of GC tissues and matched non-cancerous tissues were collected, and PCED1B-AS1, microRNA-215-3p (miR-215-3p) and C-X-C motif chemokine receptor 1 (CXCR1) expression levels were detected by qRT-PCR. Besides, CCK-8, EdU, Transwell and Western blot assays were conducted to assess the impact of PCED1B-AS1 or miR-215-3p on cell growth, migration, invasion and epithelial-mesenchymal transition (EMT). The interaction between genes was verified by bioinformatics analysis, rna immunoprecitipation (RIP) and dual-luciferase reporter gene assays. We demonstrated that, PCED1B-AS1 expression level was raised in GC tissues and cell lines, and increased expression of PCED1B-AS1 was in association with tumor size, TNM stage and lymph node metastasis in GC patients. Additionally, PCED1B-AS1 overexpression promoted GC cells proliferation, migration, invasion and EMT, and miR-215-3p overexpression counteracted the biological effects of PCED1B-AS1. Mechanistically, PCED1B-AS1 specifically inhibited miR-215-3p expressions, thus up-regulating CXCR1 expressions. In conclusion, PCED1B-AS1 accelerates GC progression via adsorbing miR-215-3p and up-regulating CXCR1, indicating that PCED1B-AS1 is a novel therapeutic target for treating GC.


Introduction
Gastric cancer (GC) is a common malignancy, posing a threat to global public health [1]. GC is also one of the main causes of cancer-related deaths in China [2]. Although there is great progression in diagnosis and treatment in recent years, patients who suffered from advanced or metastatic GC still have poor prognosis [3,4]. Thus, searching for novel biomarkers and understanding the potential mechanism of GC are urgent for the improving the diagnosis and prognosis of GC.
Non-coding RNAs (ncRNAs) are widely expressed in human cells, including microRNAs (miRNAs) with approximately 22nt and long noncoding RNAs (lncRNAs) with exceeding 200nt, and they all feature prominently in posttranscriptional regulation [5]. LncRNA is at play in various biological activities of tumor cells, including proliferation, apoptosis, metastasis, and drug resistance [6][7][8]. To date, diverse abnormally expressed lncRNAs have been unveiled in GC [9]. LncRNA, as a competitive endogenous RNA (ceRNA), can modulate downstream target gene expressions via decoying miRNAs. For example, lncRNA LINC00511 represses miR-625-5p and up-regulates transducers and activators of transcription 3 (STAT3) to facilitate the progression of GC [10]. Another study reports that, lncRNA AL139002.1 promotes GC progression by sponging miR-490-3p to regulate hepatitis A virus cellular receptor 1 [11]. LncRNA PCED1B antisense RNA 1 (PCED1B-AS1) mediates macrophage apoptosis and autophagy via targeting the miR-155 axis in active tuberculosis [12]. Additionally, PCED1B-AS1 is a tumor-promoter, and for example, PCED1B-AS1 expression level is markedly raised in glioblastoma tissues and cell lines, which is in association with larger tumor size and higher grade [13]. Instead, the role of PCED1B-AS1 in GC is still unclear.
Chemokines, as a family of small proteins , which are divided into four classes (C, CC, CXC, and CX3C), can promote directional chemotaxis of leukocytes and play important roles in inflammation and cancers [14]. CXC chemokines bind to the G-protein-coupled receptors (GPCR) e.g. C-X-C motif chemokine receptor 1 (CXCR1) and C-X-C motif chemokine receptor 2 (CXCR2), to exert their biological effects [15,16]. CXCR1 is reported to be up-regulated in GC tissues, and its high expression indicates poor prognosis of GC patients; besides, CXCR1 expedites the growth, migration, and invasion of GC cells [17,18]. Instead, its regulatory mechanism in GC is not yet clear.
In this work, we supposed that PCED1B-AS1 could probably participate in GC progression. Our study was performed to investigate the expression characteristics, biological function and underlying mechanism of PCED1B-AS1 in GC. We demonstrated that, PCED1B-AS1 expression level was markedly up-regulated in GC tissues, which was closely associate with larger tumor size, higher TNM stage and lymph node metastasis. Also, PCED1B-AS1 strengthened cell viability, migration and invasion, and epithelial-mesenchymal transition (EMT) through the microRNA-215-3p (miR-215-3p)/CXCR1 axis.

Clinical samples collection
48 pairs of GC tissues and normal tissues adjacent to cancer were available from the First Affiliated Hospital of Kunming Medical University. Besides, this work was endorsed by the Ethics Committee of the First Affiliated Hospital of Kunming Medical University with informed consent available from patients. All volunteers did not receive any treatment before operation, and the clinicopathological characteristics of the sufferers are detailed in Table 1. Immediately after the removal, the tissue samples were positioned in liquid nitrogen and subsequently stored at −80°C.

qRT-PCR
The total RNA from GC tissues and cell lines was extracted by a RNAiso Plus kit (TaKaRa, Dalian, China) and reverse-transcribed into complementary DNA (cDNA) with a PrimeScript First Strand cDNA Synthesis Kit (Takara, Dalian, China). Next, qRT-PCR amplifications were accomplished by a SYBR® Premix ExTaq kit (TaKaRa, Dalian, China) on ABI PRISM 7300 system (Applied Biosystems, Foster City, CA, USA). Besdes, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was adopted as the internal reference for PCED1B-AS1 and CXCR1, and U6 snRNA as that for miR-215-3p. The relative expressions of the genes involved in this study were calculated by 2 −ΔΔCt . Specifically, the primer sequences are listed in Table 2. To identify the sub-cellular localization of PCED1B-AS1 in GC cells, nuclear/cytoplasmic separation of GC cells was performed with a PARIS™ Kit (Ambion, Austin, TX, USA) according to the manufacturer's instruction. After the cytoplasmic RNA and nuclear RNA were isolated, PCED1B-AS1 level was measured by qRT-PCR, with GAPDH as the cytoplasmic endogenous control and the U6 as the nuclear endogenous control.

Cell counting kit-8 (CCK-8) assay
GC cells were positioned in 96-well plates at 2 × 10 3 cells per well, and cultured. At the 24 th , 48 th , and 72 nd h, 10 μL of CCK-8 reagent (Dojindo Molecular Technologies, Japan) was added into each well respectively, and the cells were cultured at 37°C for 2 h. Then the OD 450 nm value was estimated by a microplate reader (Bio-Rad, Hercules, CA, USA).

EdU proliferation assay
Cell proliferation was examined with an EdU assay kit (RiboBio, Guangzhou, China). Cells were cultured with 50 μM EdU for 2 h, then were fixed in 4% paraformaldehyde and subsequently stained with Apollo staining solution away from light for 2 h at room temperature. Subsequently, the cells were stained with DAPI staining solution (Beyotime, Shanghai, China) away from light for 30 min at room temperature. Next, the cells were washed with phosphate buffer saline (PBS), the EdU positive cells were photographed under a fluorescence microscopy (Olympus, Tokyo, Japan) in five randomly selected fields, and counted.

Cell migration and invasion assay
48 h after transfection, cells in serum-free medium were transferred into the upper compartment of Transwell chambers (pore size: 8 μm, Millipore, Bedford, MA, USA). 600 μL of medium containing 10% FBS was dripped into the lower compartment. After the cells were cultured for 24 h, the cells which passed through membrane were fixed with 4% formaldehyde and stained with 0.5% crystal violet. Ultimately, cells were observed and the numbers were subsequently counted under the microscope (Olympus, Tokyo, Japan). Notably, Matrigel (Sigma-Aldrich, Louis, MO, USA) was used to cover the membrane to mimic the extracellular matrix in invasion analysis, but it was not used in migration assay.
HEK293T cells were inoculated in 96-well plate and cultured for 24 h, then co-transfected with the reporter plasmids and miR-215-3p mimics or miR-NC by Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). 48 h later, the luciferase activity was measured by the Dual-Luciferase Reporter System (Promega, Madison, WI, USA).

RNA immunoprecipitation (RIP) assay
A Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA) was utilized for RIP assay. Cell was lysed with RIP lysis buffer, and cell lysate was incubated with human anti-Argonaute 2 (Ago 2) antibody (Millipore, Billerica, MA, USA) or negative control mouse IgG (Millipore, Billerica, MA, USA) coupled with magnetic bead overnight at 4°C. Then the proteins in the immunoprecipitate were degraded with proteinase K and the immunoprecipitated RNA was then isolated and purified, followed by qRT-PCR detection.

Statistical analysis
SPSS 22.0 software (SPSS, Inc., Chicago, IL, USA) and GraphPad Prism 8 (GraphPad Software, Inc., La Jolla, CA, USA) were employed for statistical analysis. Each experiment was repeated for three times or more, with the data expressed as the mean ± standard deviation (SD). Additionally, student's t-test and one-way analysis of variance (ANOVA) were performed for comparisons. Chisquared test was executed to analyze the association between PCED1B-AS1 expression and the clinicopathological characteristics.
Besides, Spearman's correlation analysis was used to analyze the correlation between gene expressions in GC tissues. Statistically, P < 0.05 is significant.

Results
In the present study, with in vitro experiments, we investigate the expression pattern and biological functions of PCED1B in GC. We also investigated the regulatory effects of PCED1B-AS1 on the expression of miR-215-3p and CXCR1.

PCED1B-AS1 is highly expressed in GC tissues and cell lines
First of all, we searched GEPIA database (http:// gepia.cancer-pku.cn/) and observed that PCED1B-AS1 expression in GC patients was demonstrably higher than that in normal tissues (Figure 1a). Besides, Kaplan-Meier Plotter database (http:// kmplot.com/analysis/index.php?p=service) highlighted that the overall survival of patients with low PCED1B-AS1 expression was longer than that of patients with high PCED1B-AS1 expression (Figure 1b). qRT-PCR revealed that PCED1B-AS1 expression in GC tissues was significantly higher compared with that in normal tissues adjacent to cancer (Figure 1c). In addition, as against normal gastric epithelium cell line GES-1, PCED1B-AS1 expression in GC cell lines (HGC-27, KATO III, NCI-N87, and AGS) was dramatically increased (Figure 1d). Additionally, it was also revealed that higher PCED1B-AS1 expression in GC tissues was associated with larger tumor size, higher TNM stage and lymph node metastasis ( Table 1).

Effects of PCED1B-AS1 on GC cell viability, migration, invasion and EMT
As mentioned above, among the GC cells, PCED1B-AS1 expression was the highest in HGC-27 and the lowest in NCI-N87, so we transfected siRNA (si-PCED1B-AS1-1, si-PCED1B-AS1 -2, and si-PCED1B-AS1-3) and PCED1B-AS1 overexpression vector (pc-PCED1B-AS1) targeting PCED1B-AS1 into HGC-27 and NCI-N87, respectively (Figure 2a). CCK-8 and EdU assays revealed that the proliferation rate of GC cells with PCED1B-AS1 overexpression was higher, while PCED1B-AS1 knockdown worked oppositely (Figure 2b-c). Transwell assay revealed that the  A. The expression of PCED1B-AS1 in GC cells transfected with si-PCED1B-AS1 and pc-PCED1B-AS1 was detected by qRT-PCR.B-C. CCK-8 and EdU methods were used to detect the regulatory effect of PCED1B-AS1 on the proliferation of GC cells.D-E. Transwell assay was used to detect the regulatory effect of PCED1B-AS1 on migration and invasion of GC cells.F. Western blot assay was used to detect the regulatory effect of PCED1B-AS1 on protein levels of EMT markers (E-cadherin, N-cadherin and Vimentin) in GC cells.* P < 0.05, ** P < 0.01, and *** P < 0.001. metastatic potential of GC cells with PCED1B-AS1 overexpression was evidently stronger than that of the control group, and PCED1B-AS1 knockdown suppressed the migration and invasion of GC cells (Figure 2d-e). Additionally, Western blot assay depicted that PCED1B-AS1 overexpression promoted N-cadherin and Vimentin expressions and inhibited the protein expression of E-cadherin, while PCED1B-AS1 knockdown had the opposite effect (figure 2f).

PCED1B-AS1 works as a ceRNA via adsorbing miR-215-3p in GC
To expound the hidden regulatory mechanism of PCED1B-AS1 in GC, we first performed nucleocytoplasmic separation experiments and observed that PCED1B-AS1 was mainly distributed in the cytoplasm of GC cells (Figure 3a). DIANA database (http://carolina.imis.athena-innovation.gr/diana_ tools/web/index.php?r=lncbasev2/index-predicted) highlighted that miR-215-3p may be a promising A. The expression of PCED1B-AS1 in the nuclear fraction and cytoplasmic fraction of GC cells was detected by qRT-PCR. U6 and GAPDH were detected in nuclear and cytoplasmic fractions.B. Potential binding sites between PCED1B-AS1 and miR-215-3p were analyzed through the LncBase Predict v.2 database.C. RIP assay was used to validate the interaction between PCED1B-AS1 and miR-215-3p.D. The targeting relationship between PCED1B-AS1 and miR-215-3p was confirmed by dual-luciferase reporter gene assay. E. qRT-PCR was used to detect the expression of miR-215-3p in 48 cases of GC tissues and normal adjacent tissues.F. The expression of miR-215-3p in GES-1 cells and GC cell lines (HGC-27, KATO III, NCI-N87 and AGS) was detected by qRT-PCR.G. Spearman's correlation analysis was used to analyze the correlation between PCED1B-AS1 expression and miR-215-3p expression in tissues. H. qRT-PCR was used to detect the expression of miR-215-3p in GC cells, after PCED1B-AS1 was overexpressed or knocked down. *P < 0.05, ** P < 0.01, and *** P < 0.001. downstream target of PCED1B-AS1 (Figure 3b). In the 48 patients, low miR-215-3p expression was associated with larger tumor size, higher TNM stage and poor differentiation status of the tumor tissues (Table 3). RIP assay revealed that PCED1B-AS1 and miR-215-3p were specifically enriched in anti-Ago2 group, but not in anti-IgG group (Figure 3c). Dual-luciferase reporter gene assay depicted that the luciferase activity of HEK293T cells co-transfected with PCED1B-AS1-WT and miR-215-3p mimics was greatly lower than that of NC mimics group, but there was no significant change in PCED1B-AS1-MUT group (Figure 3d). Besides, miR-215-3p was markedly reduced in GC tissues and cell lines (Figure 3e-f). Furthermore, miR-215-3p expression was negatively correlated with PCED1B-AS1 expression in GC tissues (Figure 3g). Additionally, miR-215-3p was inhibited in GC cells with PCED1B-AS1 overexpression, but overexpressed in cell lines with PCED1B-AS1 knockdown (Figure 3h). These highlights that PCED1B-AS1, as a miRNA sponge, has a negative regulatory effect on miR-215-3p expression.

Discussion
Reportedly, lncRNA features prominently in regulating epigenetics, transcriptional and posttranscriptional processes to modulate gene expression. LncRNA, abnormally expressed in cancer cells, participates in regulating the progression of multiple cancers, including GC. For example, lncRNA cancer susceptibility 11 can promote GC development by facilitating cell cycle progression [19]. LncRNA SOX2 overlapping transcript is in high expression in GC, and it contributes to the growth and metastasis of GC cells [20]. There are few studies on the role of PCED1B-AS1 in tumors, specifically, PCED1B-AS1, as a tumor-promoter, partakes in modulating glioma cell proliferation and apoptosis [13]. Additionally, it also facilitates the progression of clear cell renal cell carcinoma and pancreatic cancer [21,22]. Here, we proved that PCED1B-AS1 expression was greatly raised in GC tissues, which was closely associated with larger tumor size, higher TNM stage and lymph node metastasis. Besides, functional experiments further confirmed that PCED1B-AS1 overexpression strengthened malignant biological behaviors of GC cells, while PCED1B-AS1 inhibition showed the opposite effects, implying that PCED1B-AS1 was a tumor-promoter in GC.
As reported, lncRNAs, as a ceRNA, can exert its function by sponging endogenous miRNA to inhibit mRNA translation, and this mechanism is involved in tumorigenesis [23,24]. For example, small nucleolar RNA host gene 5/microRNA-32/ Kruppel like factor 4 (KLF4) axis regulates GC cell migration, which contributes to improving the diagnosis and treatment of GC [25]. LncRNA TMPO antisense RNA 1 can sponge miR-140-5p and indirectly regulate SRY-box transcription factor 4 expression to accelerate the progression of GC [26]. Reportedly, PCED1B-AS1 is elevated in gliomas and head and neck squamous cell carcinoma [13,27], and PCED1B-AS1 modulates the multiplication and apoptosis of gliomas via serving as ceRNA and regulating the miR-194-5p/PCesterase domain containing 1B (PCED1B) axis [28]. In this work, we confirmed that miR-215-3p was a direct target of PCED1B-AS1. According to previous reports, miR-215-3p is pivotal in cancer biology as a tumor suppressor [29,30]. This study revealed that miR-215-3p level was dramatically declined in GC tissues and cell lines, and miR-215-3p and PCED1B-AS1 expression were negatively correlated in GC tissues; the transfection of miR-215-3p mimics partially impeded the promoting effect of PCED1B-AS1 overexpression on the malignant biological behaviors of GC cells. These  A-B. MiR-215-3p mimic or si-CXCR1 was co-transfected with pc-PCED1B-AS1 into NCI-N87 cells, and the expression of miR-215-3p and CXCR1 detected by qRT-PCR.C-D. CCK-8 and EdU assays were used to detect the proliferation of GC cells after transfection. E-F. Transwell assay was used to detect the migration and invasion of GC cells after transfection.G. Western blot assay was used to detect the protein levels of E-cadherin, N-cadherin, and Vimentin in GC cells after transfection.*P < 0.05, ** P < 0.01, and *** P < 0.001. data suggest that PCED1B-AS1 can probably function as a ceRNA to regulate miR-215-3p and its downstream genes.
In the present work, CXCR1 was identified as a target gene of miR-215-3p in GC cells. CXCR1 mediates the progression of multiple cancers, including GC [15,18]. For example, IL-8 promotes the migration of liver cancer cells through CXCR1 and CXCR2, and targeting CXCR1/2 may be a strategy for treating liver cancer [31]. Another study reports that miR-761 can impede osteosarcoma cell growth and invasion partly via targeting CXCR1 [32]. In colorectal cancer cells, miR-215-3p improves the 5-Fu sensibility of cancer cells via regulating CXCR1 expression [16]. In GC, CXCR1 regulates the activation of AKT and ERK1/2 signal pathways to modulate the malignant biological behaviors of cancer cells [18]. Besides, E-cadherin is a cell adhesion molecule involved in cell-cell and cell-matrix interactions and EMT process of cancer cells, and it reduces the aggressiveness of cancer cells [33]; reportedly, CXCR1 knockdown increases E-cadherin expression in GC cells [18]. Herein, our rescue experiments revealed that inhibition of CXCR1 demonstrably restrained N-cadherin and Vimentin expressions, increased the expression of E-cadherin, and reversed the promoting effect of PCED1B-AS1 overexpression on the malignant biological behaviors of GC cells. In short, these data suggest that PCED1B-AS1 can facilitate the growth, migration, invasion and EMT of GC cells via modulating the miR-215-3p/CXCR1 axis.

Conclusion
This study confirms the tumor-promoting effect of PCED1B-AS1 in GC. PCED1B-AS1 is highly expressed in GC cancer tissues and cell lines, and PCED1B-AS1 expression is associated with the clinicopathological characteristics of GC patients. Furthermore, PCED1B-AS1, as ceRNA, upregulates CXCR1 through competitively binding to miR-215-3p to promote the malignancy of GC cells, suggesting that PCED1B-AS1/miR-215-3p/ CXCR1 axis is a novel mechanism involved in GC progression.