Adjacent Normal Gastric Tissues Research Articles

Methionine restriction promotes cisplatin sensitivity of gastric cancer resistant cells by down-regulating circ-CDK13 level

BackgroundMethionine restriction (MR) is a research direction in the treatment of gastric cancer (GC). The aim of this study was to investigate the molecular mechanism of MR on enhancing cisplatin (DDP) sensitivity of drug-resistant GC cells. MethodsTwenty pairs of GC tissues and adjacent normal gastric mucosa tissues were collected. DDP-resistant cell lines (KATO/DDP and MKN45/DDP), mouse model of GC and GC patient-derived organoid (PDO) models were established. Lentivirus-mediated METase overexpression was used for MR. Cell viability and apoptosis were detected by MTT assay and flow cytometry. Western blotting was used to detect multi-drug resistance-1 (MDR1), MDR-associated protein 1 (MRP1) eukaryotic initiation factor 4A-Ⅲ (EIF4A3), and METase protein expressions. The levels of circRNAs were detected by qRT-PCR. Tumor volume and weight were measured. The proliferation of tumor cells was detected by immunohistochemical staining. ResultsThe differentially expressed circRNAs of GC were screened in Gene Expression Omnibus database. MR in KATO/DDP and MKN45/DDP cells significantly down-regulated circ-CDK13 level. Overexpression of circ-CDK13 significantly inhibited apoptosis of sensitive cells (KATO III and MKN45). Interference with circ-CDK13 significantly promoted apoptosis of drug-resistant cells (KATO/DDP and MKN45/DDP). MR enhanced the DDP sensitivity of GC resistant cells, GC PDO and GC mice by down-regulating circ-CDK13. EIF4A3 binds to the downstream flanking sequence of circ-CDK13, and interference with EIF4A3 reduces circ-CDK13 levels, but does not affect CDK13. The expressions of circ-CDK13 and EIF4A3 in GC clinical samples were increased and positively correlated. Simultaneously overexpression of METase and EIF4A3 in resistant cells inhibited apoptosis, and further interference with circ-CDK13 reversed this effect. ConclusionMR inhibits circ-CDK13 level by down-regulating EIF4A3, thereby increasing the sensitivity of GC drug-resistant cells to DDP.

281. PROTEOMIC, TRANSCRIPTOMIC AND GENOMIC ANALYSIS OF ESOPHAGEAL ADENOCARCINOMA UNCOVERS CANDIDATE THERAPEUTIC TARGETS AND CANCER-SELECTIVE POST-TRANSCRIPTIONAL REGULATION

Abstract Background Addressing the poor prognosis associated with esophageal adenocarcinoma (EAC) has been hindered by the absence of biomarkers for early disease detection and therapeutic targets. Despite extensive research on the somatic mutations associated with EAC, the impact of genomic aberrations on protein expression and cancer phenotype remains unclear. Methods We conducted a quantitative proteomic analysis using tumour tissue and patient-matched adjacent normal esophageal and gastric tissues from 23 patients undergoing resection for EAC. We further examined the relationship between transcript and protein levels using patient-matched whole transcriptome RNA sequencing and proteomic data from 7 patients and integrated these findings with data from a cohort of 264 EAC patients with RNA sequencing and 454 patients with whole-genome sequencing, as well as external published datasets. Results We quantified protein expression from 5897 genes in EAC and normal tissues. Several potential biomarkers showing selective expression in EAC, such as the transmembrane protein GPA33, were identified. We validated the EAC-specific expression of GPA33 in an external cohort of 115 patients, suggesting its utility as a diagnostic and therapeutic target. Additionally, integrated analysis of protein and RNA expression in EAC and normal tissues revealed genes with discordant protein and RNA levels, indicating post-transcriptional regulation of protein expression. These outlier genes, including SLC25A30, TAOK2, and AGMAT, exhibited rare somatic mutations, suggesting post-transcriptional mechanisms driving this EAC-specific phenotype. AGMAT, as an example, was found to be overexpressed at the protein level in EAC compared to adjacent normal tissues, with a proposed EAC-selective, post-transcriptional mechanism regulating protein abundance. Conclusion Through quantitative proteomic analysis, we identified GPA33 as a candidate EAC biomarker. Integrated analysis of the proteome, transcriptome, and genome in EAC uncovered genes with tumor-selective post-transcriptional regulation of protein expression, offering potential exploitable vulnerabilities for therapeutic intervention.

Clinicopathological association of CD93 expression in gastric adenocarcinoma

AimsCD93 was recently identified as a promising therapeutic target for angiogenesis blockade in various tumors. Herein, we aimed to investigate the expression and clinicopathological significance of CD93 in gastric adenocarcinoma.MethodsThe gene expression of CD93 gastric adenocarcinoma was assessed using The Cancer Genome Atlas (TCGA) dataset. We then analyzed CD93 expression in 404 cases of gastric adenocarcinoma using immunohistochemistry. Clinicopathological associations and prognostic implications of CD93 expression were further investigated.ResultsUsing the TCGA dataset, we observed a significantly elevated CD93 gene expression in gastric adenocarcinoma compared to normal gastric tissues. The immunohistochemistry assay revealed a highly variable CD93 expression among patients with gastric adenocarcinoma, consistently demonstrating higher intratumor expression than in adjacent normal tissues. Notably, CD93 was predominantly expressed on the membrane of CD31+ vascular endothelial cells. Furthermore, patients with higher CD93 expression demonstrated significantly poorer overall survival. Accordingly, higher CD93 expression was associated with deeper invasion and a higher possibility of lymph node metastasis and developing tumor thrombus. Cox proportional hazards regression suggested CD93 expression was an independent predictor for the prognosis of patients with gastric adenocarcinoma.ConclusionsOur study revealed a significantly higher CD93 expression in gastric adenocarcinoma when compared with adjacent normal gastric tissues, and demonstrated its predominant expression on vascular endothelial cells. Our findings also highlighted the clinicopathological significance of CD93 in gastric adenocarcinoma, shedding light on a potential therapeutic target.

Multi-Omic Analysis of Esophageal Adenocarcinoma Uncovers Candidate Therapeutic Targets and Cancer-Selective Posttranscriptional Regulation

Efforts to address the poor prognosis associated with esophageal adenocarcinoma (EAC) have been hampered by a lack of biomarkers to identify early disease and therapeutic targets. Despite extensive efforts to understand the somatic mutations associated with EAC over the past decade, a gap remains in understanding how the atlas of genomic aberrations in this cancer impacts the proteome and which somatic variants are of importance for the disease phenotype. We performed a quantitative proteomic analysis of 23 EACs and matched adjacent normal esophageal and gastric tissues. We explored the correlation of transcript and protein abundance using tissue-matched RNA-seq and proteomic data from seven patients and further integrated these data with a cohort of EAC RNA-seq data (n = 264 patients), EAC whole-genome sequencing (n = 454 patients), and external published datasets. We quantified protein expression from 5879 genes in EAC and patient-matched normal tissues. Several biomarker candidates with EAC-selective expression were identified, including the transmembrane protein GPA33. We further verified the EAC-enriched expression of GPA33 in an external cohort of 115 patients and confirm this as an attractive diagnostic and therapeutic target. To further extend the insights gained from our proteomic data, an integrated analysis of protein and RNA expression in EAC and normal tissues revealed several genes with poorly correlated protein and RNA abundance, suggesting posttranscriptional regulation of protein expression. These outlier genes, including SLC25A30, TAOK2, and AGMAT, only rarely demonstrated somatic mutation, suggesting post-transcriptional drivers for this EAC-specific phenotype. AGMAT was demonstrated to be overexpressed at the protein level in EAC compared to adjacent normal tissues with an EAC-selective, post-transcriptional mechanism of regulation of protein abundance proposed. Integrated analysis of proteome, transcriptome, and genome in EAC has revealed several genes with tumor-selective, posttranscriptional regulation of protein expression, which may be an exploitable vulnerability.

Prognostic significance and relationship of SMAD3 phospho-isoforms and VEGFR-1 in gastric cancer: A clinicopathological study.

The TGF-β/SMAD3 and VEGFR-1 signaling pathways play important roles in gastric cancer metastasis. SMAD3 phosphorylation is a crucial prognostic marker in gastric cancer. To determine the prognostic value and relationship of SMAD3 phospho-isoforms and VEGFR-1 in gastric cancer. This was a single-center observational study which enrolled 98 gastric cancer patients and 82 adjacent normal gastric tissues from patients aged 32-84 years (median age 65) between July 2006 and April 2007. Patients were followed up until death or the study ended (median follow-up duration of 28.5 mo). The samples were used to generate tissue microarrays (TMAs) for immunohistochemical (IHC) staining. The expressions of TGF-β1, pSMAD3C(S423/425), pSMAD3L(S204), and VEGFR-1 in gastric cancer (GC) tumor tissue and normal tissue were measured by IHC staining using TMAs obtained from 98 GC patients. Prognosis and survival information of the patients was recorded by Outdo Biotech from May 2007 to July 2015. The relationship between TGF-β1, pSMAD3C(S423/425), pSMAD3L(S204), and VEGFR-1 protein expression levels was analyzed using Pearson's correlation coefficient. The relationship between protein expression levels and clinicopathological parameters was analyzed using the Chi-squared test. A survival curve was generated using the Kaplan-Meier survival analysis. TGFβ-1 and VEGFR-1 expression was significantly upregulated in gastric cancer tissue compared to adjacent non-cancerous tissue. The positive expression of phosphorylated isoforms of Smad3 varied depending on the phosphorylation site [pSMAD3C(S423/425): 51.0% and pSMAD3L(S204): 31.6%]. High expression of pSMAD3L(S204) was significantly correlated with larger tumors (P = 0.038) and later N stages (P = 0.035). Additionally, high expression of VEGFR-1 was closely correlated with tumor size (P = 0.015) and pathological grading (P = 0.013). High expression of both pSMAD3L(S204) and VEGFR-1 was associated with unfavorable outcomes in terms of overall survival (OS). Multivariate analysis indicated that high expression of pSMAD3L(S204) and VEGFR-1 were independent risk factors for prognosis in GC patients. VEGFR-1 protein expression was correlated with TGF-β1 (r = 0.220, P = 0.029), pSMAD3C(S423/425) (r = 0.302, P = 0.002), and pSMAD3L(S204) (r = 0.201, P = 0.047), respectively. Simultaneous overexpression of pSMAD3L(S204) and VEGFR-1 was associated with poor OS in gastric cancer patients. Co-upregulation of pSMAD3L(S204) and VEGFR-1 can serve as a predictive marker for poor gastric cancer prognosis, and pSMAD3L(204) may be involved in enhanced gastric cancer metastasis in a VEGFR-1-dependent manner.

Function of hsa_circ_0006646 as a competing endogenous RNA to promote progression in gastric cancer by regulating the miR-665-HMGB1 axis.

Mounting evidences indicate that circular RNAs (circRNAs) are a novel class of non-coding RNAs and play vital roles in the tumorigenesis and aggressiveness including gastric cancer (GC). Nevertheless, the precise functions and underlying mechanisms of circRNAs in GC remain largely unknown. The Gene Expression Omnibus (GEO) data set GSE163416 was analyzed to screen the key circRNAs in GC. hsa_circ_0006646 was chosen for further study. GC tissues and matched adjacent normal gastric mucosal epithelial tissues were obtained from the Fourth Hospital of Hebei Medical University. The expressions of hsa_circ_0006646 was detected using quantitative real-time polymerase chain reaction (qRT-PCR). hsa_circ_0006646 was knocked down to identify its effects on GC cells. Bioinformatics algorithms were analyzed to predict the microRNA (miRNAs) potentially sponged by hsa_circ_0006646 and its target genes. Fluorescence in situ hybridization (FISH) was conducted to determine the subcellular location of hsa_circ_0006646 and the predicted miRNA. Then, qRT-PCR, luciferase reporter assay, radioimmunoprecipitation assay, Western blotting, and miRNA rescue experiments were used to confirm the hsa_circ_0006646-related regulatory axis in GC. Cell Counting Kit-8 (CCK-8), colony formation, wound healing, and Transwell experiments were performed to determine the effect of the hsa_circ_0006646-related regulatory axis on GC cells' malignant behaviors in vitro. The xenograft tumor mouse model was established to evaluate the effect of hsa_circ_0006646 in vivo. hsa_circ_0006646 exhibited a high expression in GC tissues as compared to corresponding adjacent normal gastric mucosal epithelial tissues and its high expression was positively correlated with TNM stage, lymph node invasion and poor prognosis (P<0.05). Knockdown of hsa_circ_0006646 suppressed the proliferation, colony formation, migration, and invasion in GC cells (all P<0.05). hsa_circ_0006646 upregulated high mobility group box 1 (HMGB1) by sponging miR-665 in GC cells (P<0.05). The hsa_circ_0006646-miR-665-HMGB1 axis promoted malignant behaviors and epithelial-mesenchymal transition (EMT) in GC cells by activating the Wnt/β-catenin pathway (P<0.05). The existence of hsa_circ_0006646-miR-665-HMGB1 axis was confirmed in GC specimens (P<0.05). Consequently, down-regulated hsa_circ_0006646 inhibited the progression and EMT of GC cells in vivo (P<0.05). For the first time, we demonstrated that hsa_circ_0006646-miR-665-HMGB1 axis exerted its tumor-promoting effects in GC, which suggested that hsa_circ_0006646 could be potentially targeted for GC treatment.

RNU12 inhibits gastric cancer progression via sponging miR-575 and targeting BLID

Gastric cancer (GC) is one of the major causes of cancer deaths with 5-year survival ratio of 20%. RNU12 is one of long noncoding RNAs (lncRNAs) regulating the tumor progression. However, how RNU12 affecting GC is not clear. qRT-PCR was utilized for determining the RNU12 expression in cell lines, 113 cases of paired gastric cancer (GC) and their adjacent normal gastric tissues. The biofunction alterations of RNU12 were assessed by its overexpression or knockdown in GC cells. MTT and cloning assay were assayed for the cell proliferation, the flow cytometry for the detection of cell cycle and the wound healing assay (WHA) and transwell invasion assay (TIA) for examining the migration and invasion of cells. The expressions of a set of genes related proliferation and migration were investigated with the Western Blotting (WB). RNA immunoprecipitation (RIP), biotinylated RNA pull-down and dual luciferase reporter tests were used to detect the interactions of RNU12 with miR-575/BLID. The in vivo proliferation and migration ability of RNU12 infected cells were determined in zebrafish system. This study revealed that RNU12 inhibited proliferation, invasion and metastasis by sponging of miR-575 and regulating the downstream BLID and modulated EMT of GC cells. The RNU12/miR-575/BLID axis is likely to be the prognosis biomarkers and drug targets of GC.

Identification and validation of crucial lnc-TRIM28-14 and hub genes promoting gastric cancer peritoneal metastasis

BackgroundGastric cancer peritoneal metastasis (GCPM) is an important cause of cancer-related deaths worldwide. Long non-coding RNAs (lncRNAs) play a key role in the regulation of GCPM, but the underlying mechanisms have not been elucidated.MethodsHigh-throughput RNA sequencing (RNA-seq) was performed on four groups of clinical specimens (non-metastatic gastric cancer primary tumor, adjacent normal gastric mucosal tissue, gastric cancer primary tumor with peritoneal metastasis and adjacent normal gastric mucosal tissue). After sequencing, many lncRNAs and mRNAs were screened for further Weighted Gene Co-expression Network Analysis (WGCNA). GCPM-related hub lncRNAs and genes were identified by cytoHubba and validated by Quantitative real-time PCR (qRT-PCR), Receiver operating characteristic curve (ROC) analysis and Kaplan-Meier survival analysis. GO, KEGG and GSEA showed GCPM-related pathways. Correlation analysis revealed the potential relationship between hub lncRNAs and genes.ResultsBy analyzing lncRNA expression data by WGCNA, we found that blue module was highly correlated with GCPM (r = 0.44, p = 0.04) and six lncRNAs involved in this module (DNM3OS, lnc-MFAP2-53, lnc-PPIAL4C-4, lnc-RFNG-1, lnc-TRIM28-14 and lnc-YARS2-4) were identified. We then performed qRT-PCR validation of gastric cancer specimens and found that the expression of lnc-RFNG-1 and lnc-TRIM28-14 was significantly increased in gastric cancer tissues with peritoneal metastasis. Kaplan-Meier survival analysis showed shorter overall survival time (OS) for gastric cancer patients with high expression of lnc-TRIM28-14. Receiver operating characteristic curve (ROC) analysis showed that lnc-TRIM28-14 could improve the sensitivity and specificity of GCPM diagnosis. In addition, we identified three key mRNAs (CD93, COL3A1 and COL4A1) associated with gastric cancer peritoneal metastasis through WGCNA analysis and clinical specimen validation. Moreover, there was a positive correlation between lnc-TRIM28-14 and the expression of CD93 and COL4A1 in gastric cancer peritoneal metastasis, suggesting a regulatory relationship between them. Subsequent GO, KEGG and GSEA analysis suggested that ECM-receptor interaction and focal adhesion were the hub pathways of GCPM.ConclusionIn summary, lnc-RFNG-1, lnc-TRIM28-14, CD93, COL3A1 and COL4A1 could be novel tumor biomarkers and potential therapeutic targets for GCPM.

Overexpression of IL-8 and Wnt2 is associated with prognosis of gastric cancer.

This study is to detect the expression of inflammatory factor or neutrophil-activating factor IL-8 and Wnt2 in gastric cancer (GC) and investigate the involvement of IL-8 and Wnt2 expressions in the clinicopathological indexes and prognosis. We detected the expression of IL-8 and Wnt2 in 100 GC tissues and 40 normal gastric mucosae using immunohistochemistry. The relationships between the IL-8 and Wnt2 expression and the clinicopathological characteristics were explored. The relationship between IL-8 expression, Wnt2 expression, and prognosis of GC was analyzed by survival curve and survival regression. The expression of IL-8 and Wnt2 in GC tissue was 64% and 75% respectively, which was significantly higher than that in adjacent normal gastric mucosa tissues, moreover, expressions of IL-8 and Wnt2 were positively correlated. The positive rate of IL-8 and Wnt2 expressions were correlated with lymph node metastasis and TNM staging （P < 0.01, and Wnt2 was also correlated with infiltration depth (P = 0.021), but there was no difference with age, sex, and differentiation (P > 0.05). The 3-year survival analysis showed that the survival rates of IL-8- and Wnt2-positive patients were 20% and 24%, respectively, which were significantly lower than those of negative patients. Cox regression analysis showed that IL-8 and Wnt2 may be independent factors affecting the prognosis of GC. Our data demonstrated that the overexpression of IL-8 and Wnt2 could be isolated prognostic factors in patients with GC and, possibly, may present new targets for the treatment of GC.

A novel protein AXIN1-295aa encoded by circAXIN1 activates the Wnt/\u03b2-catenin signaling pathway to promote gastric cancer progression

BackgroundCircular RNA (circRNA), a subclass of non-coding RNA, plays a critical role in cancer tumorigenesis and metastasis. It has been suggested that circRNA acts as a microRNA sponge or a scaffold to interact with protein complexes; however, its full range of functions remains elusive. Recently, some circRNAs have been found to have coding potential.MethodsTo investigate the role of circRNAs in gastric cancer (GC), parallel sequencing was performed using five paired GC samples. Differentially expressed circAXIN1 was proposed to encode a novel protein. FLAG-tagged circRNA overexpression plasmid construction, immunoblotting, mass spectrometry, and luciferase reporter analyses were applied to confirm the coding potential of circAXIN1. Gain- and loss-of-function studies were conducted to study the oncogenic role of circAXIN1 and AXIN1-295aa on the proliferation, migration, invasion, and metastasis of GC cells in vitro and in vivo. The competitive interaction between AXIN1-295aa and adenomatous polyposis coli (APC) was investigated by immunoprecipitation analyses. Wnt signaling activity was observed using a Top/Fopflash assay, real-time quantitative RT-PCR, immunoblotting, immunofluorescence staining, and chromatin immunoprecipitation.ResultsCircAXIN1 is highly expressed in GC tissues compared with its expression in paired adjacent normal gastric tissues. CircAXIN1 encodes a 295 amino acid (aa) novel protein, which was named AXIN1-295aa. CircAXIN1 overexpression enhances the cell proliferation, migration, and invasion of GC cells, while the knockdown of circAXIN1 inhibits the malignant behaviors of GC cells in vitro and in vivo. Mechanistically, AXIN1-295aa competitively interacts with APC, leading to dysfunction of the “destruction complex” of the Wnt pathway. Released β-catenin translocates to the nucleus and binds to the TCF consensus site on the promoter, inducing downstream gene expression.ConclusionCircAXIN1 encodes a novel protein, AXIN1-295aa. AXIN1-295aa functions as an oncogenic protein, activating the Wnt signaling pathway to promote GC tumorigenesis and progression, suggesting a potential therapeutic target for GC.

Long Non-Coding RNA GRIK1-AS1 Inhibits the Proliferation and Invasion of Gastric Cancer Cells by Regulating the miR-375/IFIT2 Axis.

Long non-coding RNAs (lncRNAs) play important roles in various biological processes and human diseases, including cancer. In this study, we demonstrated a regulatory relationship between lncRNA GRIK1-AS1 and miR-375/IFIT2 axis in gastric cancer. Our results show a decreased expression of GRIK1-AS1 in gastric cancer tissues compared to adjacent normal gastric tissues. Gastric cell lines also have reduced levels of GRIK1-AS1 compared to gastric epithelial cell line GES-1. Ectopic expression of GRIK1-AS1 in gastric cancer cell lines significantly inhibits cellular viability, migration, and invasion. RNA-pull down and the luciferase activity assays show that GRIK1-AS1 mainly interacts specifically with miR-375. We further demonstrate a negatively regulatory relationship between lncRNA GRIK1-AS1 and miR-375. We discovered that IFIT2 was one of the direct key downstream target genes of miR-375, and established the important role of the GRIK1-AS1/miR-375/IFIT2 axis in the progression of gastric cancer. Taken together, our results revealed a novel mechanism of GRIK1-AS1 as a sponge to miR-375 that impacts gastric cancer progression via modulating target mRNA IFIT2 translation, and as a result, opens a new strategy to future GRIK1-AS1 based therapeutic development.

Gastrokine 2 Regulates the Antitumor Effect of JAK2/STAT3 Pathway in Gastric Cancer.

GKN2 (gastrokine 2) mainly plays a regulatory role in gastric mucosal defense and cell protection mechanisms, and its role in gastric cancer has not been thoroughly elucidated. Immunohistochemistry was used to detect GKN2 and TFF1 expressions in 90 gastric cancer tissues, 48 neoplastic resection margins, and 22 normal gastric mucosa epithelia. It showed that the downregulation of GKN2 and TFF1 expressions in gastric cancer tissues was significantly different from that in adjacent normal gastric tissues and distal gastric mucosal tissues. Nevertheless, correlation analysis showed that GKN2 expression in gastric cancer tissues was independent of TFF1 expression. After overexpression of GKN2 was constructed in human gastric cancer cell line MKN28 with the Ad-GFP-GKN2 transfected, cell viability was measured by CCK-8 assay, and migration and invasion ability were analyzed by transwell migration assay and transwell invasion assay. It indicated that overexpression of GKN2 significantly reduced the viability of MKN28 and SGC7901 cells. Overexpression of GKN2 could also inhibit the migration and invasion ability in MKN28 and SGC7901 cells. In addition, upregulation of GKN2 can inactivate the JAK2/STAT3 pathway. Our data suggest that GKN2 and TFF1 play the antitumor role in gastric carcinoma, and TFF1 may not interact or cooperate with GKN2. GKN2 overexpression can inhibit the growth and metastasis by downregulating the JAK2/STAT3 pathway in gastric cancer cells.

DEPDC1 upregulation promotes cell proliferation and predicts poor prognosis in patients with gastric cancer.

Previous studies revealed that DEP domain containing 1 (DEPDC1) is involved in the carcinogenesis and progression of several types of human cancer. However the role of DEPDC1 in gastric cancer has not been studied. The objective of this study was to study the expression and pathophysiological function of DEPDC1 in gastric cancer. DEPDC1 expression in gastric adenocarcinoma cells was examined with Western blot and qRT-PCR. Clinical pathological features of patients were determined by immunohistochemistry. The effect of DEPDC1 expression on cell proliferation was studied by in vitro cell proliferation assay; and cell cycle influence was assessed by flow cytometry. Survival curves were plotted using Kaplan-Meier. DEPDC1 was overexpressed in gastric adenocarcinoma tissues compared with the paired adjacent normal gastric tissues, in accordance with mRNA level downloaded from GEPIA database. DEPDC1 expression level was significantly associated with cancer metastasis and differentiation. DEPDC1 upregulation caused cell cycle accelerating from G1 to S phase, and it was correlated with poorer overall survival. Therefore, DEPDC1 upregulation in gastric adenocarcinoma is associated with tumor development and poor clinical outcomes of the patients, implying DEPDC1 might be a potential therapeutic target against gastric cancer.

High Expression of Interleukin-2 Receptor Subunit Gamma Reveals Poor Prognosis in Human Gastric Cancer.

Precision medicine for gastric cancer (GC) is still an unsolved issue, because most available target drugs are not specifically designed for GC. Exploring novel signaling molecules with target value for GC is in urgent need. This study aimed to reveal that interleukin-2 receptor subunit gamma (IL2RG) is such a key molecule in human GC progression. GC tissues and paracancerous gastric tissues were collected from 7 patients (5 males and 2 females) during tumor radical excision surgery. These tissues were used to identify the differentially expressed genes (DEGs) with RNA-seq and serial bioinformatics analyses including Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, gene expression profiling interactive analysis (GEPIA), and survival analysis. RT-qPCR and western blotting were performed to compare the mRNA and protein expression levels of IL2RG between GC tissues and adjacent normal gastric tissues as well as between GC cell line SGC-7901 and normal gastric epithelial cell line GES-1. Results showed striking elevations of IL2RG both in the mRNA and protein levels in GC tissues and human gastric cancer SGC-7901 cell line compared, respectively, with the adjacent normal gastric tissues and normal GES-1 cells, and higher IL2RG expression was associated with lower survival. Analyses on the GSE29272 and GSE15459 datasets from Gene Expression Omnibus verified that IL2RG was highly expressed in GC patients and was associated with poor overall survival. In addition, molecular docking showed that a small molecule, resatorvid (TAK 242), might be an inhibitor of IL2RG. We conclude that IL2RG is overexpressed in advanced GC and is associated with low survival. IL2RG may serve as a biomarker of GC progression and poor prognosis.

NOD-like receptor X1, tumor necrosis factor receptor-associated factor 6 and NF-κB are associated with clinicopathological characteristics in gastric cancer.

Gastric cancer (GC) is a common cancer worldwide and its precise mechanism is largely unknown. The aim of the present study was to evaluate the expression levels of NOD-like receptor X1 (NLRX1), tumor necrosis factor receptor-associated factor 6 (TRAF6) and NF-κB in GC and normal gastric tissue samples to determine the association with the clinicopathological features of GC. GC and adjacent normal gastric tissues from 60 patients with GC were analyzed using immunohistochemistry and western blotting analysis. In addition, the association between NLRX1, TRAF6 and NF-κB expression levels were investigated by Spearman's correlation analysis. The results revealed that NLRX1 protein expression levels were downregulated in the GC tissues compared with the normal gastric tissues (P<0.05). Conversely, TRAF6 and NF-κB protein expression levels were upregulated in the GC tissues compared with the normal gastric tissues (P<0.05). A significant difference was identified between GC patients with high and low expression levels of each protein in the tumor size, vascular invasion, neural invasion, lymph node metastasis, differentiation, gross stage and clinical stage. In addition, a negative correlation was observed between NLRX1 and TRAF6, and NLRX1 and NF-κB expression levels, while a positive correlation was observed between TRAF6 and NF-κB expression levels. In conclusion, NLRX1 expression levels were discovered to be downregulated in GC tissues. The expression levels of NLRX1, TRAF6 and NF-κB were also significantly associated with the clinicopathological characteristics of GC, and the aforementioned results indicated that NLRX1 may be a biomarker in assessing GC.

Wnt/β-Catenin Signaling Axis Is Required for TFEB-Mediated Gastric Cancer Metastasis and Epithelial-Mesenchymal Transition.

Gastric cancer remains the third leading cause of cancer-related death, and tumor metastasis is the main risk factor for poor prognosis of patients with gastric cancer. Transcription factor EB (TFEB) is a MiT family member and has been found to drive tumorigenesis in a number of tissues, whereas few studies were focused on investigating its prometastasis role and mechanism in gastric cancer. Here, we found TFEB was upregulated in gastric cancer tissues compared with adjacent normal gastric epithelial tissues. IHC analysis from gastric cancer tissue microarray revealed that TFEB in gastric cancer was correlated with depth of tumor invasion, lymph node or distant metastasis, tumor tumor-node-metastasis stage, and overall survival. Gastric cancer cells with TFEB overexpression presented an increased cell migration or invasion, and epithelial-mesenchymal transition (EMT). Furthermore, gene correlation analysis and gene set enrichment analysis enriched Wnt/β-catenin signaling pathway members in TFEB high-expression group, and the TOP/FOPflash assay verified the effect of TFEB on β-catenin transcription activity. Besides, we found that TFEB could trigger the aggregation of β-catenin in nucleus and activate its transcription, as well as facilitate the expression of Wnt/β-catenin target genes and EMT-related markers, which could be reversed by the Wnt/β-catenin inhibitor XAV-939. Collectively, TFEB enhances gastric cancer metastatic potential by activating Wnt/β-catenin signaling pathway and may become a promising therapeutic target for gastric cancer metastasis. IMPLICATIONS: Overexpressed TFEB predicts a higher rate of metastasis and worse survival in patients with gastric cancer. Mechanistically, TFEB activates Wnt/β-catenin signaling to fuel migratory and invasive activities of gastric cancer cells, as well as EMT.

Up-regulated ONECUT2 and down-regulated SST promote gastric cell migration, invasion, epithelial-mesenchymal transition and tumor growth in gastric cancer.

Gastric cancer is a common malignancy, with high metastasis and poor prognosis. Our purpose was to explore potential molecular mechanisms of gastric cancer. A total of 10 pairs of gastric cancer tissues and adjacent normal gastric tissues were collected for RNA sequencing (RNA-seq), followed by differential expression analysis. Combining qRT-PCR results, two novel genes were selected for in-depth analysis, including up-regulated ONECUT and down-regulated SST. To investigate the effects of ONECUT and SST on the biological behaviors of gastric cancer cells, gastric cancer cell lines were transfected by ONECUT2 knockdown and SST overexpression. Afterwards, cell migration and invasion were examined using transwell assays, and the expressions of epithelial-mesenchymal transition (EMT)-related proteins were measured by Western blot analysis. Furthermore, cell viability was detected by CCK-8 assay. Finally, tumorigenicity in nude mice was performed. Gastric cancer cell migration and invasion were inhibited in BGC823 cells transfected by shONECUT2. Similar results were observed in SST overexpression in MGC803 cells. Silencing ONECUT2 or overexpressing SST reduced the expressions of mesenchymal markers (N-cadherin and vimentin), STAT3, fibronectin, Wnt2, β-catenin and increased epithelial marker (E-cadherin), p-STAT3, smad2/3, α-catenin protein levels. In addition, inhibiting ONECUT2 or elevated SST suppressed tumor cell viability in vitro. Moreover, ONECUT2 silencing or elevated SST significantly inhibited tumor growth in vivo. Up-regulated ONECUT2 and down-regulated SST promote gastric cell migration, invasion, epithelial-mesenchymal transition and tumor growth in gastric cancer.

2,4-Diamino-Quinazoline, a Wnt Signaling Inhibitor, Suppresses Gastric Cancer Progression and Metastasis.

Gastric cancer (GC) is among the most treatment-refractory epithelial malignancies. Aberrant activation of Wnt/β-catenin-signaling has been implicated in a variety of human cancers, including gastric cancer. Here we report that the elevated expression of lymphoid enhancer binding factor 1 (Lef1) is associated with the TNM (tumor– node–metastasis) stage of gastric cancer. Subsequently, 2,4-diamino-quinazoline (2,4-DAQ), a selective inhibitor of Lef1, was identified to suppress the expression of Wnt/β-catenin target genes such as AXIN2, MYC and LGR5 and result in the suppression of gastric cancer cell growth through the apoptotic pathway. The 2,4-DAQ also exhibited an inhibitory effect on the migration/invasion of gastric cancer cells. Importantly, the treatment of human gastric tumor xenograft with 2,4-DAQ suppressed tumor growth in a nude mouse model. Furthermore, 2,4-DAQ appears effective on patient-derived organoids (PDOs). Transcriptome sequencing analysis also revealed that 2,4-DAQ are more effective on the gastric cancers that exhibit higher expression levels of Wnt-signaling pathway-related genes than their adjacent normal gastric tissues.

Activation of the LINC00242/miR-141/FOXC1 axis underpins the development of gastric cancer

BackgroundLong non-coding RNAs (LncRNAs) are a class of newly identified transcripts recognized as critical governors of gene expression during human carcinogenesis, whereas their tumor-suppressive or tumor-promoting effects on gastric cancer (GC) are required for further investigation. In the study, we identify the expression pattern of a novel lncRNA LINC00242 in GC and its possible permissive role in the development of GC.MethodsThe study included 68 pairs of GC and adjacent normal gastric tissue samples. The viability, migration, and invasion of cultured human GC cells HGC27 were evaluated by CCK-8 and Transwell chamber assays. In vitro tube formation of human brain microvascular endothelial cells (HBMVECs) in HGC27 cell coculture was detected. The regulatory network of LINC00242/miR-141/FOXC1 was verified using dual luciferase reporter gene assay and RNA immunoprecipitation (RIP) assay. Subcutaneous xenografts of HGC27 cells were performed in nude mice.ResultsLINC00242 was highly expressed in GC tissues and cells and contributed to poor prognosis. LINC00242 knockdown inhibited HGC27 cell viability, migration and invasion, and tube formation of HBMVECs. LINC00242 interacted with miR-141 and positively regulated FOXC1, a target gene of miR-141. LINC00242 knockdown was partially lost in HGC27 cells upon miR-141 inhibition or FOXC1 overexpression. The tumor-promoting effect of LINC00242 on GC was demonstrated in nude mice.ConclusionTaken together, the present study demonstrates the oncogenic role of the LINC00242/miR-141/FOXC1 axis in GC, highlighting a theoretical basis for GC treatment.

Expression, Location, Clinical Implication, and Bioinformatics Analysis of RNASET2 in Gastric Adenocarcinoma.

Background: In addition to exploiting its ribonuclease capacity, Ribonuclease T2 (RNASET2) has been reported to exert anti-angiogenic and anti-tumorigenic effects in several tumors. However, the role of RNASET2 in gastric adenocarcinoma (GAC) remains unclear. The purpose of this study was to explore the expression, location, and clinical implications of RNASET2 in GAC.Methods: Data of RNASET2 mRNA expression in GAC and normal gastric mucosa tissues were extracted from three GSE series and 388 TCGA samples and reanalyzed. Genome-wide CRISPR/Cas9 proliferation screening datasets were used to investigate cell growth changes after RNASET2 knockout in 19 GAC cell lines. The biological processes involved in RNASET2 were studied by the bioinformatics analysis. Furthermore, the corresponding experiments including immunohistochemical staining, clinicopathological features analysis, survival curve, microvessel density detection, cell viability assay, and colony formation assay were performed to validate the expression and function of RNASET2 in GAC.Results: An abundance of RNASET2 was present in the fundus glands and pylorus glands of the normal gastric mucosa. RNASET2 mRNA and protein were down-regulated in GAC compared with adjacent non-cancerous or normal gastric mucosa tissues. The expression of RNASET2 mRNA and protein in early GAC was higher than that in advanced GAC. 79/134 gene sets involved in the early GAC pathway were enriched in the RNASET2 mRNA high expression group. Genome-wide shRNA and CRISPR/Cas9 proliferation screening showed that knockdown or knockout of RNASET2 could not significantly promote GAC cell growth. AlamarBlue cell viability assay and colony formation assay in AGS cells further validated these results. Clinicopathologic features and survival analysis demonstrated that RNASET2 protein was significantly correlated with tumor cell differentiation, Lauren's classification, and TM4SF1 protein expression, but not correlated with lymph nodal metastasis and patient's prognosis. Microvessel density detection indicated that no significant correlation was found between the expression of RNASET2 protein and the angiogenesis of GAC.Conclusions: Down-regulation of RNASET2 in GAC was only the consequence of the GAC, instead of the driver. The expression of RNASET2 could be regarded as a good biomarker for identifying the early stage of GAC.