scholarly journals High-throughput sequencing reveals circular RNA hsa_circ_0000592 as a novel player in the carcinogenesis of gastric carcinoma

2019 ◽  
Vol 39 (6) ◽  
Author(s):  
Min Liang ◽  
Zhaoyu Liu ◽  
Hai Lin ◽  
Boyun Shi ◽  
Ming Li ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
T Cells ◽  
Epithelial Cells ◽  
Gastric Carcinoma ◽  
Cell Lines ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Gastric Epithelial Cells ◽  
Environmental Carcinogen ◽  
Functional Roles

Abstract Background/Aim: Gastric cancer is one of the most common malignant tumors, and its complex pathogenesis has not been fully elucidated. Circular RNAs (circRNAs) are involved in various biological processes and human diseases. However, their exact functional roles and mechanisms of action remain largely unclear. We previously discovered the differential expression of non-coding RNAs (ncRNAs) during the malignant transformation of human gastric epithelial cells. In this study, we investigated the functional roles of a significantly up-regulated circRNA (hsa_circ_0000592) in gastric cancer. Methods:N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)-induced malignant-transformed gastric epithelial cells (GES-1-T) and normal gastric epithelial cells (GES-1-N) were analyzed by high-throughput circRNA sequencing. The top 15 up-regulated circRNAs in high-throughput sequencing results were further confirmed by qRT-PCR in different gastric epithelial cell lines. The function of the most significant circRNA (hsa_circ_0000592) was investigated by using RNA interference (RNAi) assays, fluorescence in situ hybridization analysis (FISH), and bioinformatics prediction methods. Results: A total of 1509 genes were up-regulated and 3142 genes were down-regulated in GES-1-T cells when compared with GES-1-N cells. When compared with GES-1-N cells, hsa_circ_0000592 was obviously up-regulated in GES-1-T cells, as well as in other gastric cancer cell lines. The silencing of hsa_circ_0000592 mRNA led to a decrease in cell proliferation, cell cycle arrest at the G0/G1 phase, an increased rate of apoptosis, and a reduction in cell migration. Furthermore, FISH showed that hsa_circ_0000592 was mainly located in the cytoplasm, and a bioinformatics analysis suggested that hsa_circ_0000592 might function by sponging multiple miRNAs, and most notably four conserved miRNAs, including miR-139-3p, miR-200, miR-367-3p, and miR-33a-3p. Conclusion: This study is the first to identify hsa_circ_0000592 as a novel circRNA with a critical role in MNNG-induced gastric cancer. Due to the essential role of hsa_circ_0000592 in gastric carcinoma cells, it may be considered as a potential biomarker for use in diagnosing gastric carcinoma. Our findings provide a new insight into the function of circRNAs in environmental carcinogen-induced gastric cancer.

scholarly journals Identification of Functional Interactome of Gastric Cancer Cells with Helicobacter pylori Outer Membrane Protein HpaA by HPLC-MS/MS

2020 ◽  
Vol 2020 ◽  
pp. 1-10
Author(s):  
Ruyue Fan ◽  
Xiurui Han ◽  
Di Xiao ◽  
Lihua He ◽  
Yanan Gong ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Helicobacter Pylori ◽  
Epithelial Cells ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Cell Lines ◽  
Outer Membrane Protein ◽  
Regulation Of Transcription ◽  
Gastric Epithelial Cells ◽  
H Pylori

HpaA as an outer membrane protein of Helicobacter pylori (H. pylori) plays a significant role in the adhesion to the human stomach, but the functional relation between HpaA and gastric epithelial cells is still not clear. To screen the interaction between HpaA and cellular proteins in gastric epithelial cells, the HpaA protein from H. pylori 26695 fused with a tag (6× His) was expressed and purified successfully, the secondary structure was estimated by the Circular Dichroism (CD) spectrum, and the purified recombinant protein was used to perform the pull-down assays with gastric cancer cell lines (AGS and SGC-7901) lysates, respectively. The pull-down proteins were identified by high-performance liquid chromatography tandem mass spectrometry system (HPLC-MS/MS). A total of 9 and 13 proteins related were analyzed from AGS and SGC-7901 cell lysates, respectively. ANXA2 was considered as putative HpaA functional partner discovered from lysates of both cell lines with high score and coverage. It is hypothesized that HpaA may be involved in the biological process of regulation of transcription and nucleic acid metabolism during the adhesion of H. pylori to human gastric epithelial cells, and HpaA-binding proteins also be used as targets for the development of antiadhesion drugs against H. pylori.

scholarly journals GTPBP4 Promotes Gastric Cancer Progression via Regulating P53 Activity

2018 ◽  
Vol 45 (2) ◽  
pp. 667-676 ◽  
Author(s):  
Li Li ◽  
Xunlei Pang ◽  
Zuan Zhu ◽  
Lili Lu ◽  
Jun Yang ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cell Proliferation ◽  
Epithelial Cells ◽  
Cancer Cells ◽  
High Throughput Sequencing ◽  
Health Concern ◽  
High Morbidity ◽  
Gastric Epithelial Cells ◽  
Gastric Cancer Cells ◽  
Cancer Tissues

Background/Aims: gastric cancer is a serious health concern with high morbidity and mortality. Therefore, it is urgent to find novel targets for gastric cancer diagnosis and treatment. Methods: qRT-PCR and immunohistochemistry assays were used to detect GTPBP4 expression in gastric cancer tissues, and gastric cancer and gastric epithelial cells. Lentivirus infection was used to construct GTPBP4 stable knockdown cells. Annexin V/PI apoptosis, CCK8, EdU incorporation and cell clone formation analysis were performed to evaluate the effects of GTPBP4 on gastric cancer cell proliferation and apoptosis. Further RNA-based high-throughput sequencing and co-IP assays were constructed to explore the related mechanisms contributing to GTPBP4-mediated effects. Results: GTPBP4 expression was significantly increased in gastric cancer tissues compared with that in adjacent normal tissues, and positively correlated with gastric cancer stages. Meanwhile, GTPBP4 level was markedly upregulated in gastric cancer cells than in gastric epithelial cells. Additionaly, stable knockdown of GTPBP4 inhibited cell proliferation and promoted cell apoptosis. Mechanistically, p53 and its related signaling were significantly activated in GTPBP4 stable knockdown cells. And GTPBP4 interacted with p53 in gastric cancer cells. Conclusions: our results provide insights into mechanistic regulation and linkage of the GTPBP4-p53 in gastric cancer, and also a valuable potential target for gastric cancer.

Epigenetic regulation and functional roles of SHP1 in gastric carcinoma cell lines.

2015 ◽  
Vol 33 (3_suppl) ◽  
pp. 61-61
Author(s):  
Moon Kyung Joo ◽  
Jong-Jae Park ◽  
Hyo Soon Yoo ◽  
Yong Jeoung ◽  
Jiwon Kim ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Gastric Carcinoma ◽  
Cell Lines ◽  
Carcinoma Cell ◽  
Promoter Hypermethylation ◽  
Migration And Invasion ◽  
Gastric Carcinoma Cell ◽  
Functional Roles ◽  
Carcinoma Cell Lines ◽  
Gastric Carcinoma Cell Lines

61 Background: The SH2-containing protein tyrosine phosphatase 1 (SHP1) is an important negative regulator in cytokine-mediated signal transduction and cell cycling. Recent studies demonstrated that promoter methylation of SHP1 is frequently observed in gastric adenocarcinoma tissues. We tried in this in vitrostudy to reveal promoter hypermethylation and to investigate the effects of SHP1 in gastric carcinoma cell lines. Methods: We performed RT-PCR, Western blot, methylation specific PCR (MSP) and bisulfite pyrosequencing to demonstrate promoter hypermethylation of SHP1. To evaluate functional roles of SHP1, we transfected SHP1 plasmid and analyzed WST-1 assay, wound closure assay and Matrigel invasion assay. Results: We observed that both gene and protein expression of SHP1 were negative in 8 of 10 gastric cancer cell lines (SNU-1, SNU-5, SNU-16, SNU-638, SNU-719, MKN-28, MKN-45, AGS). MSP showed methylation-specific band only in all 10 gastric cancer lines. Bisulfite pyrosequencing revealed 96.5% and 97.3% of methylation frequency in AGS and SNU-719 cells. When treating SNU-719, MKN-28 and AGS cells with 5-Aza-2’-deoxycytidine (5-Aza-dc), SHP1 was re-expressed in all three cells. SHP1 expression is known to be correlated with Janus kinase (JAK) and signal transducers and activators of transcription (STAT) signaling pathway in epithelial cells. When introducing exogenous SHP1 in SNU-719 and MKN-28 cells by transient transfection, protein expression of constitutive phospho-JAK2 (Tyr 1007/1008) and phospho-STAT3 (Tyr 705) were substantially down-regulated both, which in turn decreased target gene expression of STAT3, including cyclin D1, MMP-9, VEGF and survivin. Induction of SHP1 significantly inhibited cell proliferation, migration and invasion in SNU-719 and MKN-28 cells. Conclusions: Epigenetic silencing of SHP1 is frequently caused by promoter hypermethylation in gastric carcinoma cell lines. Overexpression of SHP1 down-regulates JAK2/STAT3 pathway to modulate various target genes and inhibit cell proliferation, migration and invasion in gastric cancer cells.

scholarly journals CDK10 Regulates Gastric Cancer Metastasis By Inhibiting lncRNA-C5ORF42-5 Via Phosphorylation Level of AKT/ERK

2021 ◽  
Author(s):  
Zi-Jian Deng ◽  
Dong-Wen Chen ◽  
Xi-Jie Chen ◽  
Jia-Ming Fang ◽  
Liang Xv ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cancer Cells ◽  
Cell Line ◽  
High Throughput ◽  
Cancer Metastasis ◽  
High Throughput Sequencing ◽  
Gastric Cancer Cells ◽  
Related Proteins

Abstract Background: Gastric cancer is the fourth most common malignant disease. Both CDK10 and long noncoding RNAs (lncRNAs) have been found to exert biological functions in multiple cancers. However, it is still unclear whether CDK10 represses tumor progression in gastric cancer by reducing potential targeting lncRNAs.Methods: The functions of CDK10 and lncRNA-C5ORF42-5 in proliferation, invasion and migration were assessed by MTS assays, colony formation assays, cell cycle and apoptosis assays, Transwell assays, wound healing assays and animal experiments. We used high-throughput sequencing to confirm the existence of lncRNA-C5ORF42-5 and quantitative real-time PCR was used to evaluate lncRNA expression. Then, with RNA-seq sequencing as well as GO function and KEGG enrichment analysis, we identified the signaling pathways in which lncRNA-C5ORF42-5 was involved in gastric cancer. Finally, western blotting was used to identify the genes regulated by lncRNA-C5ORF42-5.Results: Our results showed that CDK10 is expressed at relatively low levels in gastric cancer cell lines and inhibits the progression of gastric cancer cells both in vitro and in vivo. Next, based on high-throughput sequencing, we identified a novel lncRNA, lncRNA-C5ORF42-5, in the stable CDK10-overexpressing cell line compared with the CDK-knockdown cell line and their controls. Additionally, we confirmed that lncRNA-C5ORF42-5 acts as an oncogene to promote metastasis in gastric cancer in vitro and in vivo. We then ascertained that lncRNA-C5ORF42-5 is a major contributor to the function of CDK10 in gastric cancer metastasis by upregulating lncRNA-C5ORF42-5 to reverse the effects of CDK10 overexpression. Finally, we explored the mechanism by which lncRNA-C5ORF42-5 overexpression affects gastric cancer cells to elucidate whether lncRNA-C5ORF42-5 may increase the activity of the SMAD pathway of BMP signaling and promote the expression of EMT-related proteins, such as E-cadherin. Additionally, overexpression of lncRNA-C5ORF42-5 affected the phosphorylation levels of AKT and ERK.Conclusion: Our findings suggest that CDK10 overexpression represses gastric cancer tumor progression by reducing lncRNA-C5ORF42-5 and hindering activation of the related proteins in metastatic signaling pathways, which provides new insight into developing effective therapeutic strategies in the treatment of metastatic gastric cancer.

Fusion of MSC With Gastric Epithelial Cells Increases Invasion and Metastasis of Gastric Cancer

2011 ◽  
Vol 140 (5) ◽  
pp. S-39
Author(s):  
Hanchen Li ◽  
Calin Stoicov ◽  
Jian Hua Liu ◽  
Jean Marie Houghton
Keyword(s):  
Gastric Cancer ◽  
Epithelial Cells ◽  
Gastric Epithelial Cells ◽  
Invasion And Metastasis

Regulation of p14ARF by Siva1 in H. pylori-infected gastric epithelial cells.

2021 ◽  
Vol 39 (3_suppl) ◽  
pp. 243-243
Author(s):  
Manikandan Palrasu ◽  
Elena Zaika ◽  
El-Rifai Wael ◽  
Richard Peek ◽  
Alexander Zaika
Keyword(s):  
Gastric Cancer ◽  
Tumor Suppressor ◽  
Epithelial Cells ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Bacterial Degradation ◽  
Tumor Suppressor Protein ◽  
Gastric Epithelial Cells ◽  
H Pylori ◽  
Oncogenic Stress

243 Background: Helicobacter pylori ( H. pylori) is the strongest known risk factor for gastric cancer. Bacterial degradation of tumor suppressor proteins affect the host microbe’s interactions and host cellular response, which contribute to tumorigenesis. p14ARF, a crucial tumor suppressor protein that activates p53 protein under oncogenic stress plays a major role in oncogenic stress response (OSR) regulation. However, little is known about the mechanism of ARF and OSR regulation in H. pylori-infected gastric epithelial cells. Methods: The expression of p14ARF and cytotoxin-associated gene A (CagA) were analyzed in gastric cells co-cultured with H. pylori strains isolated from high-gastric risk and low-gastric risk areas by immunoblotting. To investigate the potential role of CagA in regulation of p14ARF, we employed isogenic cagA− and cagE− H. pylori mutants in gastric epithelial cells, and C57BL/6 mice (n = 10). We also analyzed the expression of Siva1 in human individual infected with cagA-positive (n = 13) and cagA-negative (n = 13) bacteria as well as uninfected human subjects (n = 6). siRNA was used to inhibit activity of Siva1 protein. Results: In this study, H. pylori strains expressing high levels of CagA virulence factor and associated with a higher gastric cancer risk more strongly suppress p14ARF compared with low-risk strains in vivo and in vitro. We found that degradation of p14ARF induced by CagA is mediated by E3 ubiquitin ligase Siva1, which works in concert with another E3 ubiquitin ligase TRIP12. Decreased expression of Siva1 protein and consequent up-regulation of p14ARF was also found in gastric mucosa of H. pylori-infected mice and human individuals. Tumorigenic strain 7.13 was more potent in upregulation of Siva1 and downregulation of p14ARF than non-tumorigenic strain B128. Inhibition of p14ARF protein by H. pylori causes inhibition of autophagy in infected cells. Conclusions: Our results provide first evidence that carcinogenic H. pylori strains significantly alter the host tumor suppressor protein p14ARF, leading to suppression of host OSR and autophagy, which may affect host-bacteria interactions and tumorigenic alteration in the stomach.

scholarly journals T cell neoepitope discovery in colorectal cancer by high throughput profiling of somatic mutations in expressed genes

Gut ◽  
2015 ◽  
Vol 66 (3) ◽  
pp. 454-463 ◽  
Author(s):  
Daniele Mennonna ◽  
Cristina Maccalli ◽  
Michele C Romano ◽  
Claudio Garavaglia ◽  
Filippo Capocefalo ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Cell Epitope ◽  
Patient Specific ◽  
Cancer Genes ◽  
Healthy Donors

ObjectivePatient-specific (unique) tumour antigens, encoded by somatically mutated cancer genes, generate neoepitopes that are implicated in the induction of tumour-controlling T cell responses. Recent advancements in massive DNA sequencing combined with robust T cell epitope predictions have allowed their systematic identification in several malignancies.DesignWe undertook the identification of unique neoepitopes in colorectal cancers (CRCs) by using high-throughput sequencing of cDNAs expressed by standard cancer cell cultures, and by related cancer stem/initiating cells (CSCs) cultures, coupled with a reverse immunology approach not requiring human leukocyte antigen (HLA) allele-specific epitope predictions.ResultsSeveral unique mutated antigens of CRC, shared by standard cancer and related CSC cultures, were identified by this strategy. CD8+and CD4+T cells, either autologous to the patient or derived from HLA-matched healthy donors, were readily expanded in vitro by peptides spanning different cancer mutations and specifically recognised differentiated cancer cells and CSC cultures, expressing the mutations. Neoepitope-specific CD8+T cell frequency was also increased in a patient, compared with healthy donors, supporting the occurrence of clonal expansion in vivo.ConclusionsThese results provide a proof-of-concept approach for the identification of unique neoepitopes that are immunogenic in patients with CRC and can also target T cells against the most aggressive CSC component.

scholarly journals High-Throughput miRNA Sequencing Reveals a Field Effect in Gastric Cancer and Suggests an Epigenetic Network Mechanism

2015 ◽  
Vol 9 ◽  
pp. BBI.S24066 ◽  
Author(s):  
Monica B. Assumpção ◽  
Fabiano C. Moreira ◽  
Igor G. Hamoy ◽  
Leandro Magalhães ◽  
Amanda Vidal ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
High Throughput ◽  
Field Effect ◽  
High Throughput Sequencing ◽  
Gastric Carcinogenesis ◽  
Field Cancerization ◽  
P Value ◽  
Primary Tumors ◽  
Field Effects ◽  
Network Mechanism

Field effect in cancer, also called “field cancerization”, attempts to explain the development of multiple primary tumors and locally recurrent cancer. The concept of field effect in cancer has been reinforced, since molecular alterations were found in tumor-adjacent tissues with normal histopathological appearances. With the aim of investigating field effects in gastric cancer (GC), we conducted a high-throughput sequencing of the miRnome of four GC samples and their respective tumor-adjacent tissues and compared them with the miRnome of a gastric antrum sample from patients without GC, assuming that tumor-adjacent tissues could not be considered as normal tissues. The global number of miRNAs and read counts was highest in tumor samples, followed by tumor-adjacent and normal samples. Analyzing the miRNA expression profile of tumor-adjacent miRNA, hsa-miR-3131, hsa-miR-664, hsa-miR-483, and hsa-miR-150 were significantly downregulated compared with the antrum without tumor tissue ( P-value < 0.01; fold-change < 5). Additionally, hsa-miR-3131, hsa-miR-664, and hsa-miR-150 were downregulated ( P-value < 0.001) in all paired samples of tumor and tumor-adjacent tissues, compared with antrum without tumor mucosa. The field effect was clearly demonstrated in gastric carcinogenesis by an epigenetics-based approach, and potential biomarkers of the GC field effect were identified. The elevated expression of miRNAs in adjacent tissues and tumors tissues may indicate that a cascade of events takes place during gastric carcinogenesis, reinforcing the notion of field effects. This phenomenon seems to be linked to DNA methylation patterns in cancer and suggests the involvement of an epigenetic network mechanism.

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