The Association Between Long Non-coding RNA-H19 And The Activation Of Gastric Cancer-associated Fibroblast

ObjectiveBy primarily culturing mouse gastric mucosa fibroblast(MGMF)and establishing its vitro cell activation model,mimicking the biological process of activation of fibroblast in gastric cancer microenvironment,we try to investigate the correlation between long non-coding RNA-H19(lncRNA-H19)and the activation of gastric cancer-associated fibroblast(GCAF).MethodsThe MGMF were isolated and cultured from gastric mucosa fragments of C57BL/6 mice with explant culture technique,and were identified according to morphological characteristics and immunofluorescence staining.To mimic the biological process of activation of fibroblast in gastric cancer microenvironment,the primary culture MGMF were cultured with the mouse forestomach carcinoma cell(MFC)and were managed by a intermittent hypoxic model.Then,to assure the activation of GCAF,we used ELISA assay to detect the secretion of relevant activation factors and Real-time Quantitative PCR(qPCR)to analyze the expression of mRNA of α-SMA and FAP.The ability of proliferation,migration and apoptosis were determined by CCK-8 assay,Transwell migration assay and flow eytometry,respectively.Then,MGMF and GCAF were respectively cultured with MFC,and the proliferation of MFC were compared by CCK-8 assay.After MGMF were successfully translated into GCAF,to investigate the correlation between the activation of GCAF and lncRNA-H19,we compared the relative expression of lncRNA-H19 between MGMF and GCAF using qPCR assay.In addition,to further certify this correlation,siRNA-mediated gene silencing was used to inhibit the expression of lncRNA-H19 of GCAF,and then we analyzed the secretion of relevant activation factors using ELISA assay and the changes of the ability of proliferation,invasion and apoptosis using CCK-8 assay,matrigel invasion assay and flow eytometry,respectively.Independent t test or One-way ANOVA were used for the statistical analyses.ResultsThe cells’ morphological features were fusiform or spindle-like shaped,in radiating patterns and uniform in size,while immunofluorescence assay showed the expression of Vimentin,and there were no expression of Desmin,which accord with the characteristics of fibroblast.ELISA test showed that GCAF had higher secretion levels of IL-6、TGF-β1、HGF and CXCL12,and higher relative expression of mRNA of α-SMA and FAP than MGMF(P<0.05).Cellular function tests showed that the ability of proliferation and migration were enhanced,while apoptosis were reduced compared to MGMF(P<0.05).Meanwhile,GCAF can significantly promote the proliferation of MFC compared to MGMF(P<0.05).QPCR assay showed the expression of lncRNA-H19 in GCAF was significantly up-regulated compared to MGMF(2.10±0.14 VS.1.00±0.09,P<0.001).After lncRNA-H19 was silenced,the secretion levels of IL-6、TGF-β1、HGF and CXCL12 of GCAF were reduced significantly(P<0.05).In addition,the GCAF’s ability of proliferation and invasion were reduced,and apoptosis were enhanced than control group(P<0.05).ConclusionlncRNA-H19 is highly associated with the activation of GCAF,and inhibition of its expression can suppress the ability of proliferation and invision of GCAF,as well as promote the apoptosis of this cell.It suggests that lncRNA-H19 may be a potential target for the treatment of gastric cancer.So,it is necessary to carry out further studies to investigate the specific mechanism of the lncRNA-H19 mediated-activation of GCAF.