EBV-miR-BART19-3p Promotes Gastric Cancer Cell Proliferation Through Inhibiting GADD45B

Background and purpose: Epstein-Barr virus associated gastric cancer(EBVaGC)is a malignant tumor associated with EBV infection and is one of the molecular subtypes of gastric cancer defined by TCGA,accounting for about 1.3-30.9% of gastric cancers.Micro RNAs encoded by EBV Bam-HI-A rightward transcripts(EBV Bam-HI-A rightward transcripts miRNAs,EBV BART miRNAs)are currently recognized as key factors in the pathogenesis of EBV-related tumors,encoding 25 pre-miRNAs precursor and 44 mature miRNAs.The research group performed miRNA microarray screening on the mouse model of EBV-related human lymphoma and found that EBV-miR-BART19-3p was significantly up-regulated,but the role of EBV-miR-BART19-3p in EBV-related gastric cancer is still unclear.clear.This study intends to use clinical samples and data of patients with EBVrelated gastric cancer to clarify the expression of EBV-miRBART19-3p in EBV-related gastric cancer.Biological functions and mechanisms of related gastric cancer.Chapter 1 Expression of EBV-miR-BART19-3p in human gastric cancer tissues and gastric cancer cell linesMethods: The paired cancer and normal gastric mucosa tissues of gastric cancer patients were collected,and the clinical data were collected.EBER-ISH was used to detect EBV infection.DNA was extracted from the paired cancer and normal gastric mucosa to detect EBV virus index EBNA1 by PCR gel electrophoresis.EBV-positive patients were screened.RNA was extracted from EBV-related gastric cancer,and the expression level of EBV-miR-BART19-3p in EBVrelated gastric cancer was detected by RT-q PCR.EBNA1 was detected in EBV-negative gastric cancer cell strain AGS,gastric mucosal immortalized cell strain GES1,and EBV-positive cell strain AGS-EBV,and the expression level of EBV-miR-BART19-3p in the above cells was detected by RT-q PCR.Results: In our study,gastric cancer tissues and paired gastric mucosal tissues were collected from 95 gastric cancer patients.EBER-ISH confirmed that 5 patients were EBVaGC,accounting for 5.26%(5/95 cases)of the collected gastric cancers.DNA PCR agarose gel Electrophoresis showed that 5 of the 95 gastric cancer patients were positive for EBNA1.EBV-miR-BART19-3p was significantly higher expressed in the cancer tissues of EBV-related gastric cancer patients than in normal gastric mucosa by RT-q PCR,and there was a statistical difference.DNA PCR agarose gel electrophoresis of cell strain showed that EBNA1 was negative in AGS and GES1 cell strains;EBNA1 was positive in AGS-EBV cell strain.EBV-miR-BART19-3p was not expressed in EBV-negative gastric cancer cell strain AGS and immortalized gastric mucosa epithelial cell strain GES1,but was highly expressed in EBV-positive gastric cancer cell strain AGS-EBV by RTq PCR assasy.Chapter 2 Effects of EBV-miR-BART19-3p on the proliferation,invasion and migration of gastric cancer and gastric mucosal immortalized epithelial cellsMethods: EBV-negative gastric cancer cells AGS,gastric mucosal immortalized cells GES1 and EBV-positive gastric cancer AGS-EBV were used as in vitro cell model research objects,and NC/BART19-3p mimics transient transfection and lentivirus stable infection techniques were used to treat EBV-negative gastric cancer strain AGS and gastric mucosal immortalized cell strain GES1 to construct a cell model overexpressing EBV-miR-BART19-3p.Using NC/BART19-3p antagomir transient transfection and lentivirus stable infection technology,the EBV positive gastric cancer line AGS-EBV was constructed to knock down EBV-miR-BART19-3p cell model,the following in vitro experiments were performed: CCK8,clone formation,Ed U were used to study the relationship between overexpression/knockdown of EBV-miR-BART19-3p and gastric cancer cell proliferation;scratch assay,Transwell migration and invasion assay To study the relationship between overexpression/knockdown of EBV-miR-BART19-3p and migration and invasion of gastric cancer cells;to study the relationship between overexpression/knockdown of EBV-miR-BART19-3p and immortalized cell cycle and cell cycle of gastric cancer and gastric mucosa by flow cytometry relationship to apoptosis.BALB/C nude mice were used as the in vivo experimental research objects.According to the intervention measures,they were divided into three groups: AGS-LV-NC group,AGS-LV-miR-BART19-3p group,and AGS-LVmiR-BART19-3p+antagomir group.Subcutaneous tumorigenesis was used to construct an in vivo model and intratumoral injection of antagomir was used to intervene.The expression levels of Ki67 and PCNA in subcutaneous tumorigenic tissues of three groups of nude mice were detected by immunohistochemistry.Result:1.LV-BART19-3p/BART19-3p mimics were significantly higher than LV-NC/NC mimics by RT-q PCR assasy,which proved that the overexpression of EBV-miR-BART19-3p cell strain was successfull.The inhibition of LV-BART19-3p was significantly lower than that of LV-NC by RT-q PCR,indicating that the knockdown EBV-miRBART19-3p cell strain was successfully constructed.2.The results of CCK8,clone formation and Ed U showed that the cell proliferation activity of AGS and GES1 increased after overexpression of EBV-miR-BART19-3p.The number of cell clones increased and the DNA replication increased.After EBV-miR-BART19-3p knocked down,the proliferation and proliferation activity of the AGS-EBV cells decreased.The cell clone colonies decreased and the DNA replication decreased.3.Scratch assay and Transwell migration and invasion results showed that the migration and invasion ability of AGS and GES1 increased after overexpression of EBV-miR-BART19-3p.The migration and invasion ability of AGS-EBV cells decreased after knockdown of EBVmiR-BART19-3p.4.Flow cytometry results showed that overexpression of EBV-miRBART19-3p could inhibit G2/M arrest of AGS and GES1 cell cycle.Knockdown of EBV-miR-BART19-3p could lead to G2/M arrest of AGS-EBV cell cycle.Flow cytometry showed that overexpression of EBV-miR-BART19-3p had no significant effect on the apoptosis of AGS and GES1 cells.5.The tumor volume growth curve and tumor weight of nude mice showed that the tumor volume and weight of the mice in the overexpression EBV-miR-BART19-3p group were significantly increased compared with the NC group.Intratumoral injection of BART19-3p antagomir could partially restore EBV-miR-Growth promotion of gastric cancer tumors by BART19-3p.6.The immunohistochemical results of subcutaneous tumors in nude mice in three groups showed that the expression levels of Ki67 and PCNA in LV-BART19-3p group were ++~++++,Ki67 and PCNA in LV-NC group were-~+,and LV-BART19 The expression levels of Ki67 and PCNA in the-3p+antagomir group were +~++.Chapter 3 The mechanism of EBV-miR-BART19-3p promoting the proliferation of gastric cancer cells and immortalized gastric mucosal epithelial cellsMethods: After the AGS-EBV was treated with NC antagomir/BART19-3p antagomir knocking EBV-miR-BART19-3p,the differential gene list was obtained by high-throughput transcript sequencing technology,and the differential gene list was analyzed by combining R language and bioinformatics,and obtained Five candidate target genes downstream of EBV-miR-BART19-3p were further screened and verified by RT-q PCR,dual-luciferase reporter gene,and Western Blot detection,and the target gene GADD45 B of EBV-miRBART19-3p was verified.The cell cycle functional phenotype was obtained by KEGG pathway and GO enrichment analysis.According to the literature reports that GADD45 B is related to cell cycle G2/M arrest,this project further constructed the pc DNA3.1-GADD45 B overexpression vector,and simultaneously overexpressed GADD45 B in the LV-BART19-3p overexpressed and stably transfected cell strain.Formation and Ed U experiments were used to observe the effect of GADD45 B restoring EBV-miR-BART19-3p on cell proliferation;Western Blot detected the effect of overexpression GADD45 B restoring EBV-miR-BART19-3p on cellular protein PCNA,Relationship between miR-BART19-3p and the expression levels of cyclin B and CDK1 in G2/M phase of cells.Result:1.Transcript high-throughput sequencing found that after knockdown of EBV-miR-BART19-3p,206 up-regulated differentially expressed genes and 99 down-regulated differentially expressed genes were obtained.KEGG pathway enrichment analysis showed that upregulated differential genes were enriched in cell cycle and Pathway in cancer.Five potential target genes,GADD45 B,FOSB,RASD1,ATF3 and CCDC80,were screened from the differential gene list by bioinformatics analysis.RT-q PCR and dual-luciferase reporter gene results confirmed that EBV-miR-BART19-3p directly targets GADD45B.Western Blot results showed that EBV-miR-BART19-3p could inhibit the expression of GADD45 B protein.2.CCK8,clone formation and Ed U recovery experiments showed that overexpression of GADD45 B could restore the promoting effect of EBV-miR-BART19-3p on the proliferation of gastric cancer cells.Western Blot results showed that overexpression of GADD45 B could restore the effect of EBV-miR-BART19-3p on proliferation.The upregulation of markers PCNA and Ki67 protein;overexpression of GADD45 B can restore the up-regulation of EBV-miR-BART19-3p on G2/M phase protein cyclin B1 in gastric cancer cells,but does not affect the expression of CDK1 protein.Conclusion:1.EBV-miR-BART19-3p was highly expressed in cancerous tissue samples from EBV-associated gastric cancer patients,but not in matched normal gastric mucosa.2.EBV-miR-BART19-3p promotes the proliferation,migration and invasion of gastric cancer cells,and promotes the cell cycle.3.EBV-miR-BART19-3p directly targets the GADD45 B 3’UTR and inhibits the expression of GADD45 B.EBV-miR-BART19-3p promotes gastric cancer cell proliferation by inhibiting GADD45B;EBV-miR-BART19-3p reduces gastric cancer cell G2/M phase arrest by inhibiting GADD45 B.