The Biological Function And Clinical Significance Of IncRNA BCYRN1 In Gastric Cancer

Part 1 The expression and biological function of lncRNA BCYRN1 in gastric cancerObjective:To investigate the IncRNA BCYRN1 expression status in gastric cancer(GC)tissues and its correlation with clinicopathologic parameters of GC patients;to handle IncRNA BCYRN1 expression levels in vitro,perform relative functional experiments and confirm its downstream target gene,and investigate functional role and regulatory mechanism of IncRNA BCYRN1 in GC.Methods:1.Expression level of IncRNA BCYRN1 was detected in 56 paired GC tissues and adjacent tissues by qRT-PCR.The personal and clinicopathologic data were collected and the association between BCYRN1 and clinicopathologic parameters in GC patients was estimated.2.BGC-823 and SGC-7901 cells were transiently transfected with pcDNA-BCYRN1 or empty vector,while AGS was transfected with si-BCYRN1 or si-NC.qRT-PCR was used to detect transfection effiencies.CCK-8 assay,clone formation assay,transwell assay and flow cytometry in cell cycle and apoptosis were carried out to assess the change in cell proliferation,migration and apoptosis after transfection.3.The expression of EpCAM in 56 GC tissues was detected by qRT-PCR to evaluate the correlation between EpCAM and BCYRN1.Expression of EpCAM in both mRNA and protein were determined by qRT-PCR and western blot in BCYRN1 reexpressed GC cells.Results:1.Expression level of IncRNA BCYRN1 was found to be significantly up-regulated in GC tissues compared with adjacent tissues(P<0.0001).IncRNA BCYRN1 expression level was significantly related with TNM,but didn't show evident association with other clinicopathologic parameters such as age,sex,distant metastasis.2.Relative BCYRN1 expression in BGC-823 and SGC-7901 cells transfected with pcDNA-BCYRN1 was higher than that transfected with empty vector(all P<0.0001),while the level of BCYRN1 in AGS cells transfected with si-BCYRN1 was lower than negative control(P<0.01).Compared with negative control group,the proliferation ability of cells in pcDNA-BCYRN1 group was significantly increased 48h,72h after transfection(all P<0.05).The proliferation ability of si-BCYRN1 group was inhibited 48h,72h posttransfection(all P<0.05).In clone formation assay,the number of colony in pcDNA-BCYRN1 group was boost(BGC-823:P<0.01,SGC-7901:P<0.05)and colonies in si-BCYRN1 was decreased obviously(P<0.01).Promotion of BCYRN1 expression led to a boost of cells in S and G2 phases(BGC-823:P<0.01,SGC-7901:P<0.05)and a reduction of cells in GO and G1 phases(BGC-823:P<0.001,SGC-7901:P<0.01).Transfection of si-BCYRN1 promoted G0/G1 phase arrest of AGS cells compared with transfection of si-NC(P<0.01).Pretreatment of AGS with si-BCYRNI resulted in the promotion of apoptosis followed by overnight serum starvation(P<0.01).Meanwhile,up-regulation of BCYRN1 inhibited apoptosis(BGC-823:P<0.001,SGC-7901:P<0.01).When treated with pcDNA-BCYRN1,the numbers of migrated cells in BGC-823 and SGC-7901 were significantly higher than those treated with empty vector(all P<0.05).The migration ability was significantly reduced following inhibition of BCYRN1 in AGS(P<0.01).3.There was significant positive linear correlation between lncRNA BCYRNI and EpCAM mRNA(r=0.468,P<0.01).The RNA expression of BCYRN1 and EpCAM both significantly enhanced in the experimental group transfected with pcDNA-BCYRN1,compared with the negative control group.Inhibited BCYRN1 expression with si-BCYRN1 could reduce the expression of EpCAM in AGS.In the meantime,western blot also showed a rising tendency of EpCAM protein.Conclusion:lncRNA BCYRN1,correlated with the expression of EpCAM,is an oncogenic lncRNA which promotes cell proliferation and migration,suppresses apoptosis in GC.Part 2 Detection of long noncoding RNA BCYRN1 in serum and its diagnostic and prognostic value for gastric cancerObjective:To detect serum level of long noncoding RNA(IncRNA)BCYRN1 in gastric cancer(GC)patients,and investigate its relationship with clinical features,and evaluate its diagnostic value for GC.Methods:A case-control study was performed.From November 2014 to July 2015,serum levels of lncRNA BCYRN1 were detected by real-time quantitative polymerase chain reaction in 124 patients with GC,41 patients with atrophic gastritis and 59 normal controls who were hospitalized in Qilu Hospital of Shandong University.Meanwhile,serum carcinoembryonic antigen(CEA)and carbohydrate antigen 72-4(CA72-4)were detected by electrochemical luminescence immunoassay.Serum levels lncRNA BCYRN1 before and 3,7,10,30,100 days after radical operation in another 31 patients with GC were determined.The sensitivity and specificity of serum lncRNA BCYRN1,CEA and CA72-4 were analyzed using the receiver operating characteristic(ROC)curve.The comparison between two groups was performed with Mann-Whitney U test and the comparison among many groups was conducted with Kruskal-Wallis H test.Results:Serum levels of IncRNA BCYRN1 in GC patients with stage I and II[1.041(0.794,1.462)]and stage ? and ?[1.290(0.978,1.794)]were significantly higher than those in patients with precancerous lesion[0.969(0.699,1.219)]and normal controls[0.801(0.556,1.599)](H=54.68,P<0.0001).Compared with pre-operation[1.120(0.859,1.663)],the serum BCYRN1 levels decreased significantly in 10 days[0.903(0.724,1.182)](U=55.0,P<0.0001),30 days[0.759(0.671,1.037)](U=299.0,P=0.0261),100 days[0.478(0.378,0.635)](U=41.0,P<0.0001)after surgery.The area under the receiver operating characteristics curve(AUC)of serum IncRNA BCYRN1 was 0.865 in GC diagnosis,significantly higher than that of serum CA72-4(AUC=0.699)or CEA(AUC=0.807).The AUC of combined detection of three tests was 0.934.Conclusion:Serum IncRNA BCYRN1 levels are significantly increased in GC patients,which may be used as a potential biomarker in GC diagnosis and monitoring.