The Influence Of BARF1 On GES-1 And The Role Of CCND1 In Gastric Cancer

Objectives 1 To explore the effects of Epstein-Barr virus-encoded BARF1 on proliferation,migration and cell cycle distribution in human gastric epithelial cells（GES-1）.2 The expression of CCND1 gene in gastric cancer,and the relationship of CCND1 and immune cell infiltration,CCND1 and patients’prognosis were analyzed by bioinformatics to explore the role of CCND1 in gastric cancer.Methods 1 The plasmid of p IRES-EGFP-BARF1 and p IRES-EGFP were transformed into E.coil TOP 10 competent cells for amplification respectively,and verified by restriction endonuclease digestion,then transfected into human gastric epithelial cells GES-1.The GES-1 cells carrying p IRES-EGFP-BARF1 and p IRES-EGFP were obtained by G418 screened,and named them as GES-1/p IRES-EGFP-BARF1 and GES-1/p IRES-EGFP.2 The growth and morphological changes of GES-1/p IRES-EGFP-BARF1,GES-1/p IRES-EGFP and GES-1 cells were observed under inverted microscope.3 The expression of BARF1 gene in the transfected cells was detected by reverse transcription PCR（RT-PCR）and Immunohistochemistry（IHC）staining.4 The proliferation rate of GES-1/p IRES-EGFP-BARF1,GES-1/p IRES-EGFP and GES-1 cells was evaluated by cell counting and CCK8（Cell Counting Kit-8）method.5 The migration efficiency of GES-1/p IRES-EGFP-BARF1 and GES-1/p IRES-EGFP cells was analyzed by wound healing assay.6 Colony formation ability of the three group cells was evaluated by soft agar colony assay.7 The distribution of cell cycle was detected by flow cytometry.8 The expression of CCND1 in different groups was evaluated by IHC staining and Western Blot.9 The expression of CCND1 in human malignant tumors was analyzed by GEPIA and other bioinformatics analysis tools.10 The expression of CCND1 m RNA and protein in gastric cancer was analyzed by Ualcan,HPA and other bioinformatics analysis tools.11Chi-square test and Fisher’s exact test were used to evaluate the relationship between CCND1 m RNA expression and clinicopathological features of gastric cancer patients,respectively.12 The relationship between CCND1 and prognosis of gastric cancer patients was analyzed by GEPIA website.13 The significance of CCND1 in gastric cancer diagnosis was evaluated by ROC curve.14 Visual analysis of CCND1 genetic changes in gastric cancer patients was carried out through c Bio Portal website,and the relationship between the genetic changes and patients’prognosis was explored.15 The relationship between CCND1 methylation level and CCND1 gene expression in gastric cancer was evaluated by using spearman.16 The role of CCND1 in immune cell infiltration was evaluated by TISIDB,TIMER and other bioinformatics analysis tools.Results 1 The cells of GES-1/p IRES-EGFP-BARF1 were obtained.2 The GES-1/p IRES-EGFP-BARF1 cells became large and oval,with clear nuclear-plasmic boundary,strong refractive index,rapid cell proliferation and overlapping growth.3 The proliferation rate of GES-1/p IRES-EGFP-BARF1 group was higher than that of GES-1 group and GES-1/p IRES-EGFP group.4 The migration ability of GES-1/p IRES-EGFP-BARF1 group was significantly higher compared with that of GES-1/p IRES-EGFP group.5 It was found that GES-1/p IRES-EGFP-BARF1 cells formed cell clones in soft agar meanwhile GES-1 cells and the GES-1/p IRES-EGFP cells died through microscope examination.6 The S phase of GES-1/p IRES-EGFP-BARF1 group was significantly prolonged.7 BARF1 up-regulated the expression of CCND1,and it was highly expressed in nuclei of GES-1/p IRES-EGFP-BARF1 cells.8 CCND1 was abnormally high expression in gastric cancer,which was closely correlated with the history of reflux（P=0.036）,age（P=0.018）,Barrett’s esophagus（P=0.042）,and tissue type（P=0.031）,and which was no correlation with overall survival（Log rank P=0.2）and disease-free survival（Log rank P=0.65）.9 The sensitivity,specificity,positive predictive value and negative predictive value of CCND1 in gastric cancer diagnosis were 0.848,0.900,0.944 and 0.750,respectively.10 Abnormal amplification and mutation of CCND1 in gastric cancer patients were mostly found,and the genetic changes were not associated with patients’prognosis.11 The expression of CCND1 was correlated with the degree of methylation,and most of them showed negative correlation.12 ss GSEA analysis showed that CCND1 expression was significantly correlated with B lymphocytes,mast cells,plasmacytoid dendritic cells（p DC）,gamma-delta T cells（Tgd）,T follicular helper cell（Tfh）,NK CD56^bright cell,NK CD56^dim cell,Type 2 T helper cell（Th2）and dendritic cells（DC）in gastric cancer.13 TISIDB analysis showed that CCND1 was correlated with 17 immune cells,and most of them showed negative correlation.TIMER analysis showed that CCND1 expression was negatively correlated with immune infiltration of CD4 T cells（r=-0.165,P=1.51e-03）,macrophages（r=-0.255,P=6.95e-07）and DC cells（r=-0.112,P=3.09e-02）.Conclusions 1 BARF1 can promote the proliferation,migration,division and malignant transformation of human gastric epithelial cells GES-1.2 The abnormally high expression of CCND1 in gastric cancer is related to the history of reflux,age,histological classification,Barrett’s esophagus and the level of methylation,it has a certain value in the diagnosis of gastric cancer and plays an important role in tumor immune infiltration.Figure 19;Table 6;Reference 176...