Long non-coding RNA DEPDC1-AS1 promotes proliferation and migration of human gastric cancer cells HGC-27 via the human antigen R–F11R pathway

Objective Long non-coding (lnc) RNAs are critical regulators in carcinogenesis. The novel lncRNA DEPDC1 antisense RNA 1 (DEPDC1-AS1) was recently associated with poor prognosis in triple-negative breast cancer and lung adenocarcinoma. However, its role in regulating the malignant progression of gastric cancer (GC) and its molecular mechanism are unclear. We herein explored the functions of DEPDC1-AS1 in GC progression. Methods DEPDC1-AS1 expression and prognosis in GC tissues were examined by bioinformatics analysis and quantitative reverse transcription polymerase chain reaction. The DEPDC1-AS1 function in GC cells was explored by the cell counting kit-8 assay, colony formation assay, Transwell assay, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling, 5-ethynyl-2′-deoxyuridine-incorporation, and the xenograft tumor model. The DEPDC1-AS1 and human antigen (Hu)R interaction was determined by RNA pull-down and RNA immunoprecipitation. Results DEPDC1-AS1 was overexpressed in GC tissues and cell lines, and associated with a worse prognosis in GC patients. In vitro and in vivo assays showed that DEPDC1-AS1 promoted HGC-27 cell proliferation and migration. Mechanistically, DEPDC1-AS1 served as a scaffold by combining with HuR to target the specific mRNA F11R. Conclusion DEPDC1-AS1 plays a crucial role in GC development and progression and is a potential biomarker for the early detection or prognosis of GC.


Introduction
Gastric cancer (GC) was the fifth most commonly diagnosed cancer type and the fourth most common cause of cancer-related deaths worldwide in 2020. 1 In recent years, GC incidence rates have increased in Eastern Asia, including China, Japan, South Korea, and North Korea. 2 Indeed, GC has become the third leading cause of malignant tumor deaths in China, with 478,508 estimated new cases diagnosed in 2020, according to data from the World Health Organization. 1 The 5-year survival rate of GC is very low in China because most patients are diagnosed at an advanced stage. 3 Moreover, despite recent advances in screening, diagnosis, and treatment, the prognosis of patients with GC is still poor. 4 This is mainly attributed to the low accuracy of serum tumor biomarkers for GC, such as pepsinogen I (PGI), PGII, and carcinoembryonic antigen. 5 Therefore, more effective biomarkers for early GC detection and the prediction of prognosis are warranted.
Recent technological advances in molecular biology, especially in transcriptome sequencing and bioinformatics, have revealed a complex and diverse range of RNA molecules. Thousands of non-coding RNAs (ncRNAs) have been identified, including small ncRNAs such as microRNAs and long ncRNAs (lncRNAs). 6 LncRNAs are transcripts longer than 200 nucleotides, which are transcribed by RNA polymerase II but not translated into proteins. 7 They act as key regulators in many biological processes such as the cell cycle, cell proliferation, metabolism, apoptosis, and differentiation. 8 Human antigen R (HuR, also known as ELAVL1) belongs to the human/embryonic lethal abnormal vision (Hu/ELAVL1) RNA binding protein family. 9,10 It interacts with and stabilizes mRNAs through binding to U-and AU-rich elements in their 3 0untransalted regions (UTRs). 11 HuR is highly expressed in numerous malignant tumors, 12 where it functions as an mRNA stability factor in response to various cancer-associated stressors, and binds to specific target mRNAs and translocates to the cytoplasm. 13 Recently, HuR was shown to interact with different lncRNAs, 14 as seen in the lncRNA-RMST interaction in which HuR enhanced DNA methyltransferase 3 mRNA expression. 15 We previously demonstrated that lncRNA LINC00707 interacted with HuR to increase the stability of F11R mRNA, which is associated with GC cell proliferation and metastasis, 16 indicating that HuR-F11R is a critical pathway involved in GC malignant progression.
In the current study, we identified and characterized a novel lncRNA, DEPDC1 antisense RNA 1 (DEPDC1-AS1), which is considered to be a prognosis predictor for triple-negative breast cancer 17 and lung adenocarcinoma. 18 A comprehensive analysis of The Cancer Genome Atlas (TCGA) database and paired GC and healthy tissues demonstrated that DEPDC1-AS1 was markedly elevated in human GC tissues, while DEPDC1-AS1 overexpression was associated with poor prognosis in patients with GC. Functional assays revealed that DEPDC1-AS1 silencing inhibited GC cell proliferation and migration. Mechanistically, HuR was confirmed as a target of DEPDC1-AS1 that enhanced F11R mRNA stability. This study offers a better understanding of DEPDC1-AS1-associated regulatory modes in the development and progression of GC.

Human tissues
Paired GC tissues and healthy adjacent tissues were obtained from 24 patients who underwent GC surgery at The Affiliated Suzhou Hospital of Nanjing Medical University between March 2019 and May 2021. Tissue specimens were stored in liquid nitrogen. This study was approved by the Ethics Committee of The Affiliated Suzhou Hospital of Nanjing Medical University (GSKY0233). Patients provided their written informed consent to participate in the study.

Cell culture and transfection
Human GC cell lines (HGC-27, Hs746T, NCI-N87, and AGS) and the normal human gastric mucosal epithelial cell line GES-1 were obtained from the Chinese Academy of Cell Collection (Shanghai, China). Cells were cultured in RPMI-1640 medium (Invitrogen, CA, USA) supplemented with 10% fetal bovine serum at 37 C with 5% (v/v) CO 2 in a watersaturated atmosphere.

RNA extraction and RT-qPCR assays
Total RNA was extracted from HGC-27, Hs746T, NCI-N87, AGS, and GES-1 cells using TRIzol reagent (Invitrogen). Firststrand cDNA was synthesized from total RNA using the PrimeScript RT reagent (Takara, Dalian, China) according to the manufacturer's instructions. SYBR Taq (Takara)-based quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis was carried out on a 7500 Real-Time PCR instrument (Applied Biosystems, CA, USA) under the following conditions: 95 C for 5 minutes, then 40 cycles of 95 C for 10 seconds and 58 C for 30 seconds, with a final step at 60 C for 5 minutes. Results were normalized to 18s rRNA expression. The following primers were used: DEPDC1-AS1, forward 5 0 -CGC TCCTCATAGCGAGTCTG-3 0 and reverse

CCK-8 and colony formation assays
HGC-27 cells were inoculated into 96-well plates (3000 cells/well). Cell viability was assessed every 24 hours using a Cell Counting Kit-8 (Beyotime, Nantong, China). For colony formation assays, 3 Â 10 3 HGC-27 cells were plated per well of six-well plates and grown for 2 weeks, during which the medium of each well was changed every 3 days. Cells were then harvested and fixed with methanol for 10 minutes before incubating with 0.1% crystal violet staining solution (Beyotime). Visible colonies were counted using ImagePro Plus software (Media Cybernetics, San Diego, CA, USA).

Transwell assays
A total of 2.5 Â 10 4 HGC-27 cells per well were seeded in serum-free medium (Invitrogen) into the upper culture chamber of a 24-well plate with an 8-mm pore size (Millipore, Billerica, MA, USA). Lower chamber wells contained a medium supplemented with 20% fetal bovine serum. After incubation for 48 hours, cells on the upper membrane were removed, whereas those on the lower membrane surface were harvested and stained with 0.1% crystal violet.

5-ethynyl-2 0 -deoxyuridine (EdU) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) experiments
EdU experiments were carried out using an EdU labeling/detection kit (Ribobio, Guangzhou, China) following the manufacturer's protocol. In brief, transfected cells were incubated with 50 mM EdU labeling medium for 2 hours, then fixed with 4% paraformaldehyde, and permeabilized using 0.2% Triton X-100. Subsequently, cells were reacted with an anti-EdU working solution. Finally, EdU-positive cells were observed and counted under a Zeiss LSM800 confocal laser microscope (Zeiss, Oberkochen, Germany).
The TUNEL assay was performed using an apoptosis detection kit (Vazyme, Nanjing, China), as described previously. 19 Fluorescence microscopy was used to observe and count TUNEL-positive cells.

In vivo assays
Female athymic BALB/c nude mice (4 weeks old) were maintained under specific pathogen-free condition and manipulated according to protocols approved by the Animal Care and Use Committee of Nanjing Medical University. For the cell proliferation assay, 100 ml of suspended HGC-27 cells transfected with sh-DEPDC1-AS1 or sh-NC at a concentration of 1 Â 10 7 cells/mL were injected subcutaneously into the flanks of athymic BALB/c nude mice (n ¼ 6). Tumors were measured every 3 days, and tumor volumes were calculated by the formula: In vitro transcription, RNA pull-down assay, and western blotting analysis In vitro transcription assays were performed using the Ribo TM RNAmax-T7 Biotin RNA Labeling Kit (Ribobio). In brief, DEPDC1-AS1 was transcribed using T7 RNA polymerase and labeled with biotin. RNA pull-down experiments were performed using the Pierce TM Magnetic RNA-Protein Pull-Down Kit following the manufacturer's instructions (Thermo Scientific, Waltham, MA, USA). Pulldown products were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by western blotting using an anti-HuR antibody (Proteintech, Chicago, IL, USA), as described previously. 20,21 The 3 0 UTR of the androgen receptor RNA provided by the manufacturer was used as the positive control ,22 and the antisense strand of DEPDC1-AS1 was used as the negative control.

RNA immunoprecipitation (RIP) assays
RIP experiments were performed using a Magna RIP TM RNA-Binding Protein Immunoprecipitation Kit (Millipore) according to the manufacturer's protocol. In brief, HGC-27 cells at 80% to 90% confluency were harvested and lysed in a RIP lysis buffer. The cell extract was incubated with magnetic beads conjugated with the anti-HuR antibody or control IgG overnight at 4 C. Precipitated RNA was purified and subjected to RT-qPCR analysis to evaluate the interactions between HuR and DEPDC1-AS1.

Immunofluorescence
Paraffin-embedded tissues were dewaxed, hydrated, and then subjected to antigen retrieval in 10 mM citrate for 15 minutes. Sections were blocked with 1% bovine serum albumin for 2 hours and then incubated with the anti-Ki67 antibody (Abcam, Cambridge, MA, USA) at a dilution of 1:200 overnight at 4 C and the corresponding Alexa-Fluor secondary antibodies (Thermo Scientific) for 1 hour at 37 C. Nuclei were stained with 4 0 ,6-diamidino-2phenylindole (Beyotime), and images were captured using a Zeiss laser confocal microscope (Carl Zeiss, Oberkochen, Germany).

Statistical analysis
The Student's t-test (two-tailed) and oneway analysis of variance were used to analyze differences between groups. Survival curves were drawn by a Kaplan-Meier survival plot and tested with log-rank tests. Gene set enrichment analysis (GSEA) analysis of pathways associated with DEPDC1-AS1 was based on TCGA dataset. All statistical analyses were conducted using SPSS 17.0 software (SPSS Inc., Chicago, IL, USA). P values less than 0.05 were recognized as statistically significant.

Results
Overexpression of DEPDC1-AS1 is correlated with GC malignant progression and poor prognosis DEPDC1-AS1 levels were evaluated in 375 human GC and 32 healthy tissues from the TCGA database, and shown to be significantly overexpressed in GC tissues (P < 0.01; Figure 1a). GSEA revealed that DEPDC1-AS1 expression was significantly positively correlated with the cell cycle and DNA replication in GC tissues (P ¼ 0.01; Figure 1b). Furthermore, overall survival and disease-free interval curves showed that DEPDC1-AS1 overexpression was associated with significantly worse prognosis in patients with GC (P < 0.05; Figure 1c and d). DEPDC1-AS1 expression was also significantly up-regulated in GC tissues compared with healthy adjacent tissues (P < 0.05; Figure 1e), and significantly higher in GC cell lines than in the GES-1 cell line (P < 0.05; Figure 1f). Because DEPDC1-AS1 expression was highest in HGC-27 cells, we used this cell line for subsequent study.
To further explore the reason for compromised viability in DEPDC1-AS1 knockdown cells, we performed TUNEL staining and EdU incorporation experiments to test cell proliferation and apoptosis, respectively. TUNEL staining revealed a significant increase in the proportion of TUNELpositive cells in the interference group compared with the control group (P < 0.05; Figure 3a and b). In contrast, EdU incorporation showed significantly compromised proliferative activity in DEPDC1-AS1knockdown cells (P < 0.05; Figure 3c and d). Taken together, our data suggested that DEPDC1-AS1 is an oncogene in GC.

DEPDC1-AS1 knockdown suppresses GC growth in vivo
To evaluate whether DEPDC1-AS1 could influence HGC-27 cell tumorigenesis in vivo, we injected HGC-27 cells transfected with sh-DEPDC1-AS1 or its control group (empty vector) into nude mice. In the course of xenograft tumor development, tumor growth was significantly slower in the sh-DEPDC1-AS1 group compared with the control group (P <0.05; Figure 4a). At 15 days postinjection, xenograft tumors grown from sh-DEPDC1-AS1 cells were significantly smaller and lighter than those from control cells (P <0.05; Figure 4b and c). RT-qPCR analysis found that DEPDC1-AS1 expression levels were significantly decreased in tumor tissues generated from sh-DEPDC1-AS1 cells (P < 0.01; Figure  4d). Furthermore, immunofluorescence staining revealed significantly fewer Ki67-positive cells in the tumors of DEPDC1-AS1 knockdown cells (P < 0.01; Figure 4e and f).

DEPDC1-AS1 interacts with the mRNA-stabilizing protein HuR
To further investigate the molecular mechanisms of DEPDC1-AS1 in GC progression, we employed RPISeq to predict the interaction between DEPDC1-AS1 and RNAbinding proteins. DEPDC1-AS1 was found to potentially bind HuR, as both the RF Classifier and SVM Classifier scores were greater than 0.5 (Figure 5a). Subsequently, RNA pull-down and RIP experiments confirmed the binding between DEPDC1-AS1 and HuR (Figure 5b and c). In GC, HuR improves the stability of mRNA F11R by interacting with LINC00707. 16 However, whether DEPDC1-AS1 regulates the HuR-F11R axis is unclear. Therefore, the correlation between DEPDC1-AS1 and the HuR-F11R axis was explored. F11R mRNA expression was significantly downregulated after DEPDC1-AS1 or HuR knockdown in HGC-27 cells (P < 0.001; Figure 5d). Notably, the interaction between F11R mRNA and HuR was significantly attenuated when DEPDC1-AS1 was silenced (P < 0.05; Figure 5e). Additionally, correlation analysis showed that F11R expression levels were significantly positively correlated with DEPDC1-AS1 expression levels in GC tissues (P < 0.001; Figure 5f). We therefore conclude that DEPDC1-AS1 is involved in the HuR-F11R co-regulatory effects on GC progression.

F11R is involved in the oncogene function of DEPDC1-AS1
We conducted rescue experiments to investigate whether F11R was involved in DEPDC1-AS1-induced GC cell proliferation and migration. CCK8 and colony formation assay data revealed that co-transfection of DEPDC1-AS1 siRNA and pcDNA-F11R vector largely rescued DEPDC1-AS1 knockdown-induced compromised cell viability (P < 0.01; Figure 6a-c), and cell migration was also significantly rescued after co-transfection (P < 0.001; Figure 6d and e).

Discussion
GC is one of the most common malignancies worldwide, and several oncogenic pathways that may contribute to GC carcinogenesis have been reported in recent years. 23,24 Multiple lncRNAs act as oncogenes or tumor suppressor genes during carcinogenesis and can also serve as prognostic markers of GC. [25][26][27] LncRNA-mediated regulation of signaling pathways in cancer has been shown to be involved in various mechanisms, which are mainly attributed to their interaction with  1) targeting specific DNA binding proteins to reduce their access to DNA recognition elements, 28 (2) miRNA targeting of lncRNAs to trigger their decay, 29 and (3) the selective regulation of mRNA stability of specific genes by interacting with target proteins. 28 DEPCE-AS1, a novel lncRNA, was recently associated with the prognosis of triple-negative breast cancer 17 and lung adenocarcinoma. 18 However, changes in the expression of DEPDC1-AS1 in GC are poorly understood. Here, we found that DEPDC1-AS1 was overexpressed in GC tissues and cell lines, while loss-of-function assays verified that excessive DEPDC1-AS1 promoted HGC-27 cell line proliferation and migration and that this function was attributed to its interaction with HuR. These findings indicate that DEPDC1-AS1 acts as an oncogene in GC development, and can be used as a potential biomarker of GC prognosis.
F11R, also known as junctional adhesion molecule-A, is a member of the immunoglobulin superfamily originally identified from the surface of human platelets, and is a crucial regulator of tight junction assembly in epithelia. 30,31 Various studies have focused on its role in cancers. These include the hypoxic induction of lncRNA NEAT1, which leads to the retention of F11R within the nucleus and correlates with poor survival in patients with breast cancer. 31 Moreover, Zhang et al. revealed that overexpression of F11R mRNA correlated with non-small cell lung cancer progression. 32, 33 We previously reported an interaction between LINC00707 and HuR in which HuR increased the stability of F11R mRNA, and verified the role of the HuR-F11R pathway in GC malignant progression. 16 This earlier study found that the 3 0 -UTR of F11R interacted with HuR, while RIP analysis demonstrated the high binding capacity of F11R for HuR protein. 16  The current study confirmed that DEPDC1-AS1 binds HuR, and showed that it functions as a scaffold to regulate F11R at the transcriptional level. Bioinformatics and mRNA expression analysis revealed a positive correlation between DEPDC1-AS1 and F11R, which was demonstrated to be a strong binding relationship by RNA pull-down and RIP assays, while in vitro loss-of-function assays showed that F11R regulation is potentially involved in the oncogene function of DEPDC1-AS1.
In conclusion, our results reveal GCassociated DEPDC1-AS1 to be an oncogenic lncRNA that promotes the proliferation and invasion of HGC-27 cells by serving as a scaffold, and combines with HuR to target F11R mRNA. Our findings support a role for DEPDC1-AS1 in GC development and progression and suggest that it could be used as an effective biomarker for the early detection or prognosis of GC.