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Long noncoding RNA SNHG8 promotes chemoresistance in gastric cancer via binding with hnRNPA1 and stabilizing TROY expression

Open AccessPublished:March 27, 2022DOI:https://doi.org/10.1016/j.dld.2022.02.011

      Abstract

      Aims

      To determine SNHG8’s function and potential mechanisms in gastric cancer (GC) chemoresistance.

      Methods

      We assessed SNHG8 expression in GC cell lines, GC/CDDP cell lines (cell lines treated with cisplatin), and 42 GC tissues and SNHG8 levels in the lncRNA microarray analysis of AGS/CDDP and AGS cell lines. We also examined GC cell viability in vivo and in vitro and its apoptosis level with Flow cytometry assays. SNHG8 was localized in subcells using fluorescence in situ hybridization (FISH) and cell fraction assays, hnRNPA1’s link to SNHG8 was determined utilizing RNA immunoprecipitation (RIP) and FISH assays, gene expression profiles were assessed employing RNA transcriptome sequencing, and hnRNPA1’s relationship with TROY was ascertained with the RIP assay.

      Results

      SNHG8 increased significantly in GC cell lines and GC tissues. However, a decrease in its expression promoted sensitivity to chemotherapy and inhibited DNA damage repair in vitro and in vivo. SNHG8 appeared to regulate TROY expression via linking with hnRNPA1. Reducing TROY levels considerably stimulated GC cell chemosensitivity, whereas heightening them partially rescued the rate of chemoresistance caused by downregulating SNHG8.

      Conclusion

      In summary, the “SNHG8/hnRNPA1-TROY” axis is crucial to GC chemoresistance.

      Keywords

      1. Introduction

      Gastric cancer (GC) is among the primary causes of cancer-related deaths worldwide [
      • Van Cutsem E
      • Ducreux M.
      Colorectal and gastric cancer in 2015: The development of new agents and molecular classifications.
      ]. Early diagnosis and treatment can improve the prognosis of patients with the disease; however, most GC patients tend to present in advanced stages [
      • Dassen AE
      • et al.
      Changes in treatment patterns and their influence on long-term survival in patients with stages I-III gastric cancer in The Netherlands.
      ], which makes chemotherapy the first-line treatment, with its provision of a good curative effect especially important.
      Chemotherapy has been a vital treatment approach for GC over the past several decades, improving survival rates among patients over time. The significant progress in GC treatment owes much to the enhancement of chemotherapy regimens [
      • Choi AH
      • Kim J
      • Chao J
      Perioperative chemotherapy for resectable gastric cancer: MAGIC and beyond.
      ]. However, because of acquired or primary resistance, chemotherapy's efficacy in inhibiting tumor growth, metastasis, and recurrence is often restricted. The potential mechanism of chemoresistance must, therefore, be established to resolve this issue.
      Long non-coding RNAs (lncRNAs) – measuring more than 200 nucleotides in length – are a group of non-coding RNAs [
      • Chi Y
      • et al.
      Long non-coding RNA in the pathogenesis of cancers.
      ] that have attracted enormous attention from researchers in recent years and have been shown to play a variety of key roles in cancer biology, including drug resistance [
      • Chen X
      • et al.
      Long noncoding RNA LBCS inhibits self-renewal and chemoresistance of bladder cancer stem cells through epigenetic silencing of SOX2.
      ,
      • Han P
      • et al.
      The lncRNA CRNDE promotes colorectal cancer cell proliferation and chemoresistance via miR-181a-5p-mediated regulation of Wnt/beta-catenin signaling.
      ,
      • He W
      • et al.
      MSC-regulated lncRNA MACC1-AS1 promotes stemness and chemoresistance through fatty acid oxidation in gastric cancer.
      ]. For example, lncRNA MACC1-AS1 promotes stemness and chemoresistance in gastric cancer through the regulation of mesenchymal stem cells and fatty acid oxidation [
      • He W
      • et al.
      MSC-regulated lncRNA MACC1-AS1 promotes stemness and chemoresistance through fatty acid oxidation in gastric cancer.
      ], and lncRNA HOXA11-AS amplifies human lung adenocarcinoma (LUAD) cell resistance to cisplatin by acting as a ceRNA [
      • Zhao X
      • et al.
      LncRNA HOXA11-AS drives cisplatin resistance of human LUAD cells via modulating miR-454-3p/Stat3.
      ]. Many studies have suggested that lncRNAs’ ability to modulate protein, DNA, and RNA expressions and functions makes their involvement in various cellular processes critical [
      • Mercer TR
      • Dinger ME
      • Mattick JS.
      Long non-coding RNAs: insights into functions.
      ,
      • Noh JH
      • et al.
      Cytoplasmic functions of long noncoding RNAs.
      ,
      • Yu B
      • Shan G
      Functions of long noncoding RNAs in the nucleus.
      ]. LncRNAs also participate in countless cellular processes by interacting with DNA, RNA, or RNA-binding proteins (RBPs) [
      • Liu SJ
      • Lim DA.
      Modulating the expression of long non-coding RNAs for functional studies.
      ]. While much has been done to identify their functions and mechanisms, their role in chemoresistance remains largely undetermined.
      Recent facts have demonstrated that SNHG8 is dysregulated in several human cancer types [
      • Song H
      • et al.
      SNHG8 is upregulated in esophageal squamous cell carcinoma and directly sponges microRNA-411 to increase oncogenicity by upregulating KPNA2.
      ,
      • Song Y
      • et al.
      LncRNA SNHG8 promotes the development and chemo-resistance of pancreatic adenocarcinoma.
      ,
      • Dong J
      • et al.
      lncRNA SNHG8 promotes the tumorigenesis and metastasis by sponging miR-149-5p and predicts tumor recurrence in hepatocellular carcinoma.
      ], suggesting that it performs critical roles in the carcinogenesis and development of cancers. For instance, lncRNA SNHG8 is dysregulated and promotes pancreatic adenocarcinoma chemoresistance [
      • Song Y
      • et al.
      LncRNA SNHG8 promotes the development and chemo-resistance of pancreatic adenocarcinoma.
      ]. However, SNHG8’s role in GC chemoresistance is undefined. Therefore, in this study, we aimed to evaluate the expression status of SNHG8 in GC drug-resistant cells and decipher its clinical value to patients. We also explore SNHG8’s functions and potential mechanisms in GC chemoresistance.

      2. Materials and methods

      2.1 Clinical specimens

      We obtained 42 GC tissue samples from patients diagnosed with GC at the Union Hospital (Wuhan, China) based on the original histopathological reports from August 2014 to August 2015. The Institutional Ethics Committee at Huazhong University of Science and Technology approved all conducted clinical experiments, and participants signed informed consent forms.

      2.2 Cell lines and culture

      CDDP cells were cultured as described previously [
      • Gao J
      • Tian Y
      • Zhang J.
      Overexpression of interferon regulatory factor 1 enhances chemosensitivity to 5-fluorouracil in gastric cancer cells.
      ]. Human GC cell lines (MKN45, AGS, SGC7901 and GES1) were purchased from the Bena Culture Collection (Beijing, China) and cultured in an RPMI 1640 medium (Gibco, NY, USA) + 10% fetal bovine serum (Gibco) at 37°C and 5% CO2.
      2.3. RNA isolation of nuclear and cytoplasmic fractions
      We isolated and collected cytosolic and nuclear RNA fractions using the Nuclear and Cytoplasmic Extraction Reagents Kit (Norgen Biotek) per the manufacturer's protocol and analyzed the RNA levels of SNHG8, U6 (as the nuclear reference control), and GAPDH (as the cytosol reference control) utilizing qRT-PCR.

      2.4 Western blotting

      Western blotting was conducted as described previously [
      • Gao J
      • et al.
      IRF-1 transcriptionally upregulates PUMA, which mediates the mitochondrial apoptotic pathway in IRF-1-induced apoptosis in cancer cells.
      ]. Protein was prepared using a RIPA buffer (Sigma-Aldrich, Darmstadt, Germany) containing 1% PMSF and phosphatase inhibitors and analyzed with rabbit anti-hnRNPA1 polyclonal antibody (Proteintech, 11426-1-AP, 1:1000), TROY monoclonal antibody (Cell Signaling, #2775, 1:1000), γH2A (abcam, ab2893, 1:1000), cleaved PARP (abcam, ab32561, 1:1000), cleaved caspase-3 (abcam, ab2302, 1:1000) and GAPDH (Proteintech, 10494-1-AP, 1:5000); GAPDH was used as a loading control.

      2.5 Quantitative reverse transcription-polymerase chain reaction (RT-PCR)

      We used the Trizol Reagent (Takara, Dalian, China) to extract total RNA from cells or tissues per the manufacturer's instructions and performed Quantitative PCR to determine RNA levels under the following PCR cycle conditions: 95°C 10 min, 95°C 15 s, and 60°C 1 min for a total of 40 cycles. Primers for all the genes were synthesized by Sangon Biotech (Shanghai China); their sequences are listed in Supplemental Table 1.

      2.6 Small interfering RNAs and transfection

      We transfected small interfering RNAs (siRNAs) designed and produced by RiboBio (Guangzhou, China) into GC cells per the manufacturer's instructions and harvested the cells or culture supernatants 48 h later for subsequent experiments. All siRNA sequences are listed in Supplemental Table 1.

      2.7 Cell counting kit-8 (CCK-8) assay

      We used a commercial CCK-8 kit (Dojindo, Kyushu, Japan) to assess GC cell chemoresistance per the manufacturer's instructions. We incubated cells with the CCK-8 reaction solution for 4 h and measured their optical density (OD) values at a wavelength of 450 nm using the Gemini EM microplate reader (Thermo Fisher Scientific).

      2.8 Immunofluorescence

      We soaked MKN-45 and AGS cells in cold PBS, suspended them in PBS, and fixed them with 4% paraformaldehyde for 15 min, dipping the slide in cold PBS for 3 min each time. Next, we permeabilized the cells with 0.5% Triton X-100 at room temperature for 20 min, incubated them with a diluted primary antibody (γH2A (ab2893)) overnight at 4°C, washed them three times with a washing buffer (PBS, 0.1%Tween-20), incubated them with a diluted FITC-labeled goat secondary antibody at 37°C for 1 hour, washed them with PBST, covered them in a 20 μL antifade reagent with DAPI, and observed and imaged them with a fluorescence microscope.

      2.9 Flow cytometry assay

      We determined cell apoptosis ratio using the FITC-Annexin V apoptosis Detection Kit (YEASEN Corporation, China) per the manufacturer's protocol. In a few words, we digested GC cells with trypsin, washed them with PBS twice, added the Annexin V-FITC staining solution to them, incubated them at room temperature for 15 min in the dark, and then used flow cytometry (FACSVantage SE, BD, USA) to determine cell apoptosis ratio.

      2.11 RNA sequencing

      We assessed the transcriptome of parental AGS cells, AGS/CDDP cells, NC-shRNA AGS cells, and SNHG8-shRNA AGS cells using the Illumina Hiseq 2000 system (GM, Shanghai, China): three different samples from each group of cells were sequenced.

      2.11 RNA pull-down assay

      We used the Magnetic RNA Protein Pull-Down Kit (Thermo Fisher Scientific, USA) for the RNA pull-down assay per the manufacturer's protocol. Biotin-labeled SNHG8 or antisense RNA was co-incubated with the protein extract of GC cells (obtained as described above) and magnetic beads, and the retrieved protein was assayed with western blotting (using GAPDH as the reference control), separated with SDS-PAGE, and isolated for whole proteomic mass spectrometry (MS) analysis.

      2.12 RNA immunoprecipitation (RIP) assay

      The RIP assay was conducted using the RNA-Binding Protein Immunoprecipitation Kit (Millipore, Bedford, MA) per the manufacturer's protocol. Cell lysates extracted from GC cells were incubated with a RIP buffer containing magnetic beads conjugated with the hnRNPA1 antibody or negative control IgG (Millipore), and relative SNHG8 and TROY levels in immunoprecipitated RNA were evaluated with real-time PCR.

      2.13 RNA fluorescence in situ hybridization

      The RNA fluorescence in situ hybridization (FISH) assay was performed using the Fluorescent In Situ Hybridization Kit (RiboBio, China) per the manufacturer's protocol. Cells were soaked in 4% formaldehyde for 15 min, suspended in cold PBS containing 0.5% Triton X-100 for 30 min, and pre-hybridized in a pre-hybridization solution at 37°C for 30 min. Cy3-labeled SNHG8 antisense probe 1 and a cy3-labeled SNHG8 sense probe (both designed and synthesized by RiboBio, China) were added to the pre-hybridized cell solution, the mixture was incubated overnight at 37°C in the dark, and the cells were counterstained with DAPI and imaged the following morning.

      2.14 The mouse model

      The xenograft tumor formation assay was established as described previously [
      • Yuan J
      • et al.
      Interferon regulatory factor-1 reverses chemoresistance by downregulating the expression of P-glycoprotein in gastric cancer.
      ]. Severe combined immunodeficient (SCID) mice (HFK Bio-Technology, Beijing, China.) were injected subcutaneously with stable SNHG8-depleted MKN-45 cells or control cells (3 × 106 cells). Two weeks later, the mice were injected intraperitoneally with PBS containing CDDP(5mg/kg). The Institutional Animal Care and Use Committee of the Academic Medical Centre at Huazhong University of Science and Technology approved all the procedures for the animal study, which conformed to the legal mandates and federal guidelines for the care and maintenance of laboratory animals.

      2.15 Bioinformatics analysis

      We used UALCAN [
      • Tang Z
      • et al.
      GEPIA: a web server for cancer and normal gene expression profiling and interactive analyses.
      ] (http://ualcan.path.uab.edu) to analyze mRNA expressions in primary GC tissues and their association with clinicopathological parameters, the GEPIA [
      • Chandrashekar DS
      • et al.
      UALCAN: a portal for facilitating tumor subgroup gene expression and survival analyses.
      ] (http://gepia.cancer-pku.cn/) database to examine the correlation between the mRNA expressions of SNHG8 and TROY, and the Kaplan-Meier Plotter (www.kmplot.com) to evaluate the prognostic value of TROY mRNA expression.

      2.16 Data analysis and statistics

      All statistical analyses and data graphs were generated using the Graphpad Prism program (GraphPad Software). Results are reported as the mean ± SEM of three independent experiments. Outcomes were compared using the two-tailed paired Student's t-test. P < 0.05 was considered statistically significant: P-values are represented as ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

      3. Results

      3.1 SNHG8 is upregulated in human GC tissues, cell lines, and CDDP cell lines

      Our analysis of the expression of SNHG8 in human GC tissues using the TCGA GC data repository revealed upregulated SNHG8 levels in GC tissues compared to the corresponding adjacent non-tumorous tissues (Fig. 1A). Per the TCGA data, the higher the cancer stage, the higher the levels of SNHG8 (Fig. 1B). SNHG8 was also significantly higher in GC cell lines than in GES1 (Fig. 1C). Additionally, SNHG8 levels increased in 42 cases of GC tissues compared to normal tissues (Fig. 1D) and in late-stage GC cases (stages III + IV) (Fig. 1E) compared to early-stage GC cases (stages I + II).
      Fig 1
      Fig. 1SNHG8 was upregulated in human GC tissues, cell lines, and drug-resistant cell lines. (A) TCGA database-determined SNHG8 mRNA expression in GC. (B) TCGA database-determined SNHG8 mRNA expression at various GC stages. (C) qRT-PCR-determined SNHG8 expression in normal gastric epithelial cell lines (GES1) and GC cells. (D) Comparison of SNHG8 mRNA levels between GC tumor tissues and pair-matched normal tissues. (E) Relative SNHG8 expression in GC patients at early (I/II) and advanced stages (III/IV). (F) Heatmap of the lncRNA transcript expression in AGS and AGS/CDDP cell lines. (G) qRT-PCR-determined SNHG8 mRNA expression in CDDP cell lines and parent gastric cancer cell lines. (H) SNHG8 mRNA expression in CDDP-treated MKN-45 cells and AGS cells at different time points. * P < 0.05, ** P < 0.01, and *** P < 0.001.
      Our assessment of SNHG8 expression in the lncRNA microarray analysis of AGS/CDDP and AGS cell lines revealed amplified SNHG8 levels in AGS/MDR cells (Fig. 1F), and Quantitative reverse-transcription PCR (RT-qPCR) analysis established heightened SNHG8 amounts in CDDP cell lines compared to their parent GC cells (Fig. 1G). Per the evaluation of SNHG8 expression response to cisplatin, SNHG8 expression increased with prolonged treatment time (Fig. 1H).

      3.2 SNHG8 promotes chemotherapy resistance and DNA damage repair in vitro

      Because SNHG8 was upregulated in CDDP cell lines, we sought to understand the impact of this rise on chemotherapeutic efficacy. To do that, we knocked down SNHG8 expression with si-RNA (Fig. 2A), which resulted in a significant upsurge in the degree of PARP cleavage, caspase-3 cleavage, and levels of γ-H2AX compared to cells transduced with control siRNA (Fig. 2B). Consistent with our previous finding that DNA damage repair contributes to GC chemotherapy resistance [
      • Tan L
      • et al.
      Interferon regulatory factor-1 suppresses DNA damage response and reverses chemotherapy resistance by downregulating the expression of RAD51 in gastric cancer.
      ], we determined here, using immunofluorescence staining, that the frequency of DNA breaks increased substantially (Fig. 2C and D). Knocking down SNHG8 in MKN45/CDDP cells markedly reduced cell survival rates after treatment with cisplatin for 24 h, with MKN45/CDDP cell growth inhibited dose-dependently (Fig. 2E). Similar results were obtained with the AGS/CDDP cell lines (Fig. 2F).
      Fig 2
      Fig. 2Downregulating SNHG8 expression promoted GC sensitivity to chemotherapy in vitro. (A) RT-PCR-determined SNHG8 expression in MKN-45 and AGS cells transfected with control (si-NC), si-SNHG8#1, si-SNHG8#2, or si-SNHG8#3. (B)Western blotting-determined Cleaved-PARP, Cleaved-Caspase-3, and γ-H2AX expressions in MKN-45 and AGS cells transfected with control (si-NC) or si-SNHG8. (C and D) Representative immunofluorescence images of γ-H2AX expression in MKN-45 and AGS cells transfected with control (si-NC) or si-SNHG8. (E) Cell viability assay-determined cell survival rates of MKN-45/CDDP cells transfected with control (si-NC) or si-SNHG8 after treatment with various CDDP concentrations. (F) Cell viability assay-determined cell survival rates of AGS/CDDP cells transfected with control (si-NC) or si-SNHG8 after treatment with various CDDP concentrations. (G) Flow cytometry assay-determined SNHG8 knockdown-instigated cellular apoptosis. * P < 0.05, ** P < 0.01, and *** P < 0.001.
      In line with these findings, flow cytometry revealed that cisplatin treatment instigated an increase in the proportion of cell apoptosis among SNHG8-knockdown cells (Fig. 2G and H). These findings suggest that SNHG8 promotes chemoresistance and DNA damage response in GC.

      3.3 A decrease in SNHG8 expression promotes GC sensitivity to chemotherapy in vivo

      To explore the impact of downregulating SNHG8 in vivo, we transfected shRNA-SNHG8 and shRNA-NC into MKN-45 cells and injected the transfected cells subcutaneously into the shoulders of SCID mice. Consistent with results from our in vitro studies, SNHG8 knockdown decreased chemoresistance considerably (Fig. 3A, B, and C). RT-PCR results also revealed significantly downregulated SNHG8 expressions in shRNA-SNHG8 xenografts (Fig. 3D). PARP and caspase-3 cleavage were notably amplified in shRNA-SNHG8 xenografts as well, as were γ-H2AX levels (Fig. 3E and F). These results suggest that SNHG8 promotes chemoresistance and the DNA damage repair of GC cells.
      Fig 3
      Fig. 3Downregulating SNHG8 expression promoted GC sensitivity to chemotherapy in vivo. (A) Representative images of tumors in different groups after chemotherapy and graphs depicting the tumor volumes (B) and weights (C) of the different groups after the injection of MKN45/sh-NC and MKN-45/sh-SNHG8 cells into nude mice. (D) RT-PCR-determined SNHG8 expression in xenografts. (E) Western blotting-determined Cleaved-PARP, Cleaved-Caspase-3, and γ-H2AX expressions in xenografts. (F) Immunohistochemistry-determined Cleaved-PARP and Cleaved-Caspase-3 expression. Scale bars, 100 µm. * P < 0.05, ** P < 0.01, and *** P < 0.001.

      3.4 SNHG8 binds with heterogeneous nuclear ribonucleoprotein A1

      Based on the above results, we sought to identify the regulatory mechanism of SNHG8 in its promotion of chemoresistance in GC. First, we performed the FISH and subcellular fractionation assays to localize SNHG8 in subcells, demonstrating that SNHG8 was more prominent in the cytoplasm than in the nucleus but that it formed clusters in the nucleus (Fig. 4A and B).
      Fig 4
      Fig. 4SNHG8 binds with hnRNPA1. (A) qRT-PCR-determined SNHG8 expression in the cytoplasm and nuclei of MKN-45 and AGS cells (using GAPDH as the cytosol reference control and U6 as the nuclear reference control). (B) Representative FISH images showing the subcellular location of SNHG8 in AGS and SGC-7901 cells (red). Nuclei were stained with DAPI (blue). (C) RNA pull-down assay-determined interaction between SNHG8 and hnRNPA1 in MKN-45 and MKN-45 cells. (D) RNA immunoprecipitation in MKN-45 and AGS cells and qRT-PCR analysis of the co-precipitated RNA to examine SNHG8 expression. The fold enrichment of SNHG8 in hnRNPA1 RIP matched its corresponding IgG control. (E) Representative FISH images showing the co-localization of SNHG8 and hnRNPA1 in AGS and SGC-7901 cells. * P < 0.05, ** P < 0.01, and *** P < 0.001.
      Next, we hypothesized that SNHG8 could bind with certain co-factors and sought to identify these co-factors in GC cells using the RNA antisense purification-mass spectrum (RAP-MS). We performed the RNA pull-down assay using biotin-labeled SNHG8 as bait to isolate SNHG8-interacting proteins in AGS GC cells and used an SNHG8 pull-down sample to run a PAGE gel, which we analyzed with mass spectrometry, highlighting heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), an RNA binding protein (Fig. 4C).
      Furthermore, the RIP assay showed that SNHG8 interacted with hnRNPA1 in GC cell lines (Fig. 4D), while Immunofluorescence staining established that SNHG8 and hnRNPA1 were co-localized in GC cell lines (Fig. 4E), suggesting that hnRNPA1 binds with SNHG8.

      3.5 SNHG8 promotes TROY expression through binding with hnRNPA1

      To explore the downstream genes of SNHG8, we transfected SNHG8-shRNA (using NC-shRNA as control) into AGS cells to knock down SNHG8 and then performed RNA transcriptome sequencing. As a result, we uncovered 673 differentially expressed genes (fold change > 2, p < 0.05) (Supplemental Table 2 & Fig. 5A and B). The TROY gene showed a high fold change in SNHG8-depleted AGS and MKN-45 cells (Fig. 5C and D).
      Fig 5
      Fig. 5SNHG8 promoted TROY expression through its binding with hnRNPA1. (A, B) Altered transcripts in AGS cells treated with scrambled sh-NC or sh-SNHG8 (three replications each). (C) RT-PCR-determined TROY expression in MKN-45 and AGS cells transfected with control (si-NC) or si-SNHG8. (D) Western blotting-determined TROY expression in MKN-45 and AGS cells transfected with control (si-NC) or si-SNHG8. (E) Spearman-Pearson correlation between SNHG8 and TROY expressions in GC and normal gastric tissues (Spearman r = 0.2461, P < 0.001). (F) GEPIA database-determined Spearman-Pearson correlation between SNHG8 and TROY expressions in GC tissues (Spearman r = 0.21, P = 0.0021). (G) Bioinformatics-determined prediction of the interaction probabilities between TROY mRNA and hnRNPA1 (http://pridb.gdcb.iastate.edu/RPISeq/). Predictions with probabilities > 0.5 were considered “positive”. (H) RNA immunoprecipitation in MKN-45 and AGS cells and qRT-PCR analysis of the co-precipitated RNA to determine TROY mRNA expression. The fold enrichment of TROY mRNA in hnRNPA1 RIP matched its corresponding IgG control. (I) Western blotting-determined TROY expression in MKN-45 and AGS cells transfected with control (si-NC) or si-hnRNPA1. * P < 0.05, ** P < 0.01, and *** P < 0.001.
      Because TROY has been demonstrated to play key roles in cancers, we further examined the mRNA expression levels of SNHG8 and TROY in GC tissues to understand the relationship between the two. The TROY transcript expression correlated with SNHG8 mRNA levels (Fig. 5E). We obtained the same result in our database inquiry (Fig. 5F).
      To know if SNHG8 directly regulates TROY after binding with hnRNPA1, we predicted the binding probabilities between HNRNPA1 and TROY using bioinformatics methods. The binding probabilities between hnRNPA1 and TROY were significant (Fig. 5G), an outcome validated by the findings from the RIP assay (Fig. 5H). Silencing hnRNPA1 decreased TROY's stability (Fig. 5I). Therefore, per these findings, TROY could be an important downstream gene of SNHG8, with SNHG8 potentially enhancing its stability by binding to HNRNPA1.

      3.6 TROY participates in SNHG8-induced GC cell chemoresistance

      To assess TROY expression in CDDP cells further, we scrutinized results from the microarray analysis and established that TROY levels increased in AGS/CDDP cells (Fig. 6A). Western blotting produced similar findings (Fig. 6B).
      Fig 6
      Fig. 6TROY participated in SNHG8-induced GC cell chemoresistance. (A) Heatmap of TNFRSF1A, TROY, and TNFRSF11B transcript expressions in AGS and AGS/CDDP cell lines. (B) Western blotting-determined TROY expression in cell lines treated with CDDP and parent gastric cancer cell lines. (C) RT-PCR-determined TROY expression in AGS cells transfected with control (si-NC), si-TROY#1, si-TROY#2, or si-TROY#3. (D) Western blotting-determined TROY, Cleaved-PARP, Cleaved-Caspase-3, and γ-H2AX expressions in AGS cells transfected with control (si-NC) and, si-TROY. (E) Western blotting-determined TROY, Cleaved-PARP, Cleaved-Caspase-3, and γ-H2AX expressions in AGS cells transfected with si-SNHG8 or si-SNHG8 plus TROY. (F) Cell viability assay-determined cell survival rates of AGS CDDP cells expressing si-NC and si-TROY. (G) Cell viability assay-determined cell survival rates of AGS CDDP cells expressing si-SNHG8 and si-SNHG8 plus TROY. (H) Flow cytometry assay demonstration of the inhibition of cellular apoptosis levels after SNHG8 knockdown. * P < 0.05, ** P < 0.01, and *** P < 0.001.
      To determine TROY's role in GC cell chemoresistance, we knocked down its expression in AGS cells with siRNA (Fig. 6C and D) and used RT-PCR and western blotting to evaluate its expression levels. PARP and Caspase-3 cleavage and γ-H2AX levels increased in TROY-depleted cells (Fig. 6D). However, TROY-overexpression partially rescued the depletion of Cleaved-PARP, Cleaved-Caspase-3, and γ-H2AX levels caused by the decrease in SNHG8 (Fig. 6E). Per the CCK-8 assay, knocking down TROY significantly inhibited GC cell chemoresistance (Fig. 6F). These findings indicate that TROY promoted GC cell chemoresistance.
      To determine whether the SNHG8-mediated promotion of GC cell chemoresistance depends on the expression of TROY, we designed a rescue experiment in which we transfected pcDNA-TROY into AGS/SNHG8-shRNA to validate TROY's role. Per the CCK-8 assay, TROY overexpression partially reversed the downregulated SNHG8-induced reduction in chemoresistance (Fig. 6G), and, according to the Flow cytometry assay, the rate of GC cell apoptosis increased considerably after SNHG8 expression was silenced (Fig. 6H).

      4. Discussion

      lncRNAs are one of three major categories of non-coding transcripts that have been studied extensively by scholars across the globe [
      • Adams BD
      • et al.
      Targeting noncoding RNAs in disease.
      ], with multiple studies demonstrating in recent years how critical their role is to the development and progression of numerous human diseases, including cancers [
      • Anastasiadou E
      • Jacob LS
      • Slack FJ.
      Non-coding RNA networks in cancer.
      ,
      • Esteller M.
      Non-coding RNAs in human disease.
      ]. SNHG8 is a lncRNA that is overexpressed in various tumors and associated with cancer cell growth and metastasis of tumors, such as colorectal cancer, gastric carcinoma, nasopharyngeal carcinoma, endometrial carcinoma, ovarian carcinoma, and breast cancer [
      • Zhen Y
      • et al.
      Knockdown of SNHG8 repressed the growth, migration, and invasion of colorectal cancer cells by directly sponging with miR-663.
      ,
      • Liu J
      • et al.
      Knockdown of the lncRNA SNHG8 inhibits cell growth in Epstein-Barr virus-associated gastric carcinoma.
      ,
      • Tian X
      • et al.
      lncRNA SNHG8 promotes aggressive behaviors of nasopharyngeal carcinoma via regulating miR-656-3p/SATB1 axis.
      ,
      • Yang CH
      • et al.
      LncRNA SNHG8 participates in the development of endometrial carcinoma through regulating c-MET expression by miR-152.
      ,
      • Miao W
      • et al.
      LncRNA SNHG8 induces ovarian carcinoma cells cellular process and stemness through Wnt/beta-catenin pathway.
      ,
      • Fan D
      • et al.
      LncRNA SNHG8 promotes cell migration and invasion in breast cancer cell through miR-634/ZBTB20 axis.
      ]. It is upregulated in many cancers, including GC, whose analyses have shown that SNHG8 levels are amplified more in GC tissues than in normal gastric tissues. While SNHG8 overexpression in GC correlates with the proliferation, invasion, and poor prognosis of the disease [
      • Zhang P
      • et al.
      LncRNA SNHG8 promotes proliferation and invasion of gastric cancer cells by targeting the miR-491/PDGFRA axis.
      ,
      • Lin Y
      • et al.
      The fasting blood glucose and long non-coding RNA SNHG8 predict poor prognosis in patients with gastric carcinoma after radical gastrectomy.
      ,
      • Liu J
      • et al.
      Knockdown of the lncRNA SNHG8 inhibits cell growth in Epstein-Barr virus-associated gastric carcinoma.
      ], the underlying mechanism of its upregulation and the role of lncRNA, in general, in GC and other cancers’ resistance to drugs must undergo rigorous research.
      Because SNHG8 promotes pancreatic cancer development and chemoresistance [
      • Song Y
      • et al.
      LncRNA SNHG8 promotes the development and chemo-resistance of pancreatic adenocarcinoma.
      ], and numerous critical lncRNAs influence GC chemoresistance [
      • YiRen H
      • et al.
      Long noncoding RNA MALAT1 regulates autophagy associated chemoresistance via miR-23b-3p sequestration in gastric cancer.
      ,
      • He W
      • et al.
      MSC-regulated lncRNA MACC1-AS1 promotes stemness and chemoresistance through fatty acid oxidation in gastric cancer.
      ,
      • Raziq K
      • et al.
      Competitive endogenous network of lncRNA, miRNA, and mRNA in the chemoresistance of gastrointestinal tract adenocarcinomas.
      ], we focused our investigation on the impact of SNHG8 whose function and potential mechanisms in GC chemoresistance are yet undetermined. Our analysis of the expression of SNHG8 using a lncRNA microarray analysis and QRT-PCR established higher SNHG8 levels in CDDP cells than in their parent cells. Downregulating SNHG8 significantly increased GC cell sensitivity to cisplatin and stifled DNA damage repair, the latter finding consistent with previous reports that DNA damage contributes to chemoresistance by various cancers [
      • Abad E
      • Graifer D
      • Lyakhovich A
      DNA damage response and resistance of cancer stem cells.
      ,
      • Sakthivel KM
      • Hariharan S
      Regulatory players of DNA damage repair mechanisms: role in cancer chemoresistance.
      ]. SNHG8 is, therefore, arguably associated with GC chemoresistance.
      To determine the underlying molecular mechanisms of SNHG8 in GC, we localized it first: it was expressed in the nucleus and cytoplasm, clustering together into several foci. Next, we proved that SNHG8 partners with hnRNPA1, an RNA-binding protein from the Heterogeneous nuclear ribonucleoproteins (hnRNPs) family, which has been shown to participate in complex and diverse biological processes, including gene expression regulation, RNA maturation, and protein processing [
      • Roy R
      • et al.
      Emerging roles of hnRNPA1 in modulating malignant transformation.
      ]. Reportedly, lncRNAs binds with related modification enzymes or transcription factors to affect chromatin structure and gene transcription [
      • Kopp F
      • Mendell JT.
      Functional classification and experimental dissection of long noncoding RNAs.
      ]. For instance, Ai-lncRNA EGOT binds with RNA-binding protein hnRNPH1, and the resulting EGOT/hnRNPH1 complex directly promotes ITPR1 expression, which sensitizes paclitaxel cytotoxicity in human cancer [
      • Xu S
      • et al.
      Ai-lncRNA EGOT enhancing autophagy sensitizes paclitaxel cytotoxicity via upregulation of ITPR1 expression by RNA-RNA and RNA-protein interactions in human cancer.
      ]. Numerous hnRNPA1 target genes have been identified, and because hnRNPA1 acts as an oncogenic factor in cancers, many hnRNPA1-regulated genes are also recognized as tumor suppressor genes. Additionally, HNRNPA1 is a marker of poor prognosis in GC and a potential therapeutic target for GC [
      • Chen Y
      • et al.
      High expression of hnRNPA1 promotes cell invasion by inducing EMT in gastric cancer.
      ,
      • Zhou B
      • et al.
      The long noncoding RNA colon cancer-associated transcript-1/miR-490 axis regulates gastric cancer cell migration by targeting hnRNPA1.
      ]. Here, we found that SNHG8 regulated the expression of target mRNAs through binding with hnRNPA1, subsequently promoting GC cell chemoresistance.
      We concluded our investigation by demonstrating that SNHG8 acts as a molecular scaffold to regulate the expression of TROY by binding with hnRNPA1. TROY, also known as tumor necrosis factor receptor superfamily member 19 (TNFRSF19), is a type I cell surface receptor protein in which the cytoplasmic domain is a TNF receptor-associated factor (TRAF)-binding sequence and the extracellular domain contains highly conserved TNFR cysteine-rich motifs [
      • Hu S
      • et al.
      Characterization of TNFRSF19, a novel member of the tumor necrosis factor receptor superfamily.
      ]. TROY activates the Wnt/β-catenin, JAK1-STAT3, and NF-κB signaling pathways, thereby promoting GBM cell invasion and resistance and the development of colorectal tumors [
      • Schon S
      • et al.
      beta-catenin regulates NF-kappaB activity via TNFRSF19 in colorectal cancer cells.
      ,
      • Qiu W
      • et al.
      Tumor necrosis factor receptor superfamily member 19 (TNFRSF19) regulates differentiation fate of human mesenchymal (stromal) stem cells through canonical Wnt signaling and C/EBP.
      ,
      • Ding Z
      • et al.
      TROY signals through JAK1-STAT3 to promote glioblastoma cell migration and resistance.
      ]. TROY's expression and function have been observed predominantly in human glioma tissues; however, some recent studies have earmarked TROY for a prognosis marker role in gastric and colon cancers [
      • Wilhelm F.
      • et al.
      Troy is expressed in human stomach mucosa and a novel putative prognostic marker of intestinal type gastric cancer.
      ,
      • Nishioka M
      • et al.
      TROY is a promising prognostic biomarker in patients with colorectal cancer.
      ]. Knowledge of its underlying molecular mechanism in GC is, therefore, critical. Our analysis of TROY expression using the microarray analysis indicated that TROY was overexpressed in AGS/CDDP cell lines, as did the western blotting assay in CDDP cells. TROY reportedly contributes to DNA damage repair [
      • Shao L
      • et al.
      The inherited variations of a p53-responsive enhancer in 13q12.12 confer lung cancer risk by attenuating TNFRSF19 expression.
      ]. Here, our downregulation of TROY reduced the ability of GC cells to repair DNA damage and considerably promoted GC cell chemosensitivity, whereas, its overexpression partially reversed the reduced chemoresistance caused by the knocking down SNHG8.
      In summary, we determined that the “SNHG8/hnRNPA1-TROY” axis is crucial to GC chemoresistance, with SNHG8 levels upregulated in CDDP cells, GC tissues, and cell lines. Inhibiting SNHG8 decreased GC cell resistance to chemotherapy and suppressed DNA damage repair. SNHG8 possibly promotes chemoresistance partially by binding with hnRNPA1 and regulating the expression of TROY.
      While our research has revealed key starting points for further discoveries and provided a new molecular mechanism for the generation of GC chemoresistance, more in-depth investigations must be conducted to broaden our understanding of the biological function of SNHG8.

      Conflicts of interest

      There are no conflicts of interest to declare.

      Funding

      This study was supported by the National Natural Science Foundation of China.

      Appendix. Supplementary materials

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