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

ObjectiveThe purpose is to investigate the expression and clinical significance of long-chain non-coding RNA XIST(lncRNA-XIST)in gastric cancer tissues and plasma.The human gastric mucosa fibroblast(HGMF)was primarily cultured,and was stimulated its activation during hypoxia.On this basis,it was verified whether lncRNA-XIST could promote the activation of Gastric cancer-associated fibroblast(GCAF).Meanwhile,possible differentially expressed genes and signaling pathways were seeked,which might be associated with RNA-XIST promoting the activation of GCAF.MethodsSurgical resected cancer tissues together with paracancerous tissue were collected in 40 gastric cancer patients,while plasma samples were collected from 90 gastric cancer patients and 90 healthy subjects.lncRNA-XIST levels were detected by using real-time quantitative RT-PCR(qRT-PCR).In addition,the nonparametric test was used for statistical analysis,which revealed the relationship between the expression level of lncRNA-XIST and the clinicopathological parameters related to gastric cancer(age,sex,TNM stage,tumor size,lymph node metastasis,differentiation,Ki-67 positive rate).Furthermore,the efficacy of lncRNA-XIST in the diagnosis of gastric cancer was evaluated by plotting the receiver operating characteristic(ROC)curve.Normal gastric mucosal tissue in patients with gastric cancer was collected,and the HGMF were isolated and primary cultured using explant culture technique.After successful culture,the morphological characteristics of HGMF were identified under optical microscope.Meanwhile,the cell source and purity were also identified using immunofluorescence staining.Besides,the HGMF were cultured with the human forestomach carcinoma cell(SGC-7901 and MGC-803),which were used to mimic the biological process of GCAF’s activation by an intermittent hypoxic model.ELISA assay was adopted to detect the expression of α-SMA and FAP.The mimicked process was verified through checking the ability of proliferation,migration and apoptosis,which was implemented by utilizing CCK-8 assay,Transwell migration assay and flow eytometry,respectively.To verify whether the lncRNA-XIST was associated with the activation of GCAF,the changes of expression of lncRNA-XIST after GCAF’s activation was detected using qPCR.Furthermore,we use siRNA to interfer the expression of lncRNA-XIST and adopt ELISA assay to detect the interfered expression level of related activation factors.In addition,the ability of proliferation,invasion and antiapoptotic after interference was detected using CCK-8 array,Transwell invasion assay and flow eytometry,respectively.The results demonstrated that the lncRNA-XIST does have relationship with the activation of GCAF.Finally,transcriptome sequencing technology(RNA Sequencing,RNA-seq)was used to detect and analyze the differential genes and related pathways after the expression of lncRNA-XIST was interfered by siRNA so as to find out the specific mechanisms of lncRNA-XIST associated involvement in GCAF’s activation.ResultsCompared with paracancerous tissues,the expression of lncRNA-XIST significantly increased in gastric carcinoma tissues.Plasmatic lncRNA-XIST expression in gastric cancer group was significantly higher than that in healthy group,which was with significant difference in statistic(P < 0.05).The level of lncRNA-XIST in plasma and tissues of gastric carcinoma was related to gastric cancer staging,lymph node metastasis and differentiation(P < 0.05).It was observed under the optical microscope that the cells adhered and were with the consistent size.Cells were in the shape of a long strip or fusiform,and cell orientation was almost consistent.All observations were consistent with the morphological characteristics of fibroblasts.Immunofluorescence assay showed the expression of Desmin was negative,while that of Vimentin was positive,which was consistent with the protein characteristics of HGMF and demonstrated the success of primary culture.After establishing the activation model of GCAF,the expression level of CXCL12,IL-6,TGF-β1 and HGF were significantly higher than that in the control group(HGMF),whose differences were of statistical significance(P<0.05).In addition,the expression of α-SMA and FAP related mRNA were also significantly higher(P<0.05).The ability of cell proliferation,migration and apoptosis after GCAF’s activation improved significantly and the expression of lncRNA-XIST of GCAF was significantly higher compared with that of HGMF(P<0.05).After the lncRNA-XIST was silenced,the expression levels of CXCL12、IL-6、TGF-β1 and HGF were all lower than the blank group and the negative control group(P<0.05),and the ability of proliferation,migration and anti-apoptosis decreased(P<0.05).Furthermore,genes like,H19,PTP4A1,EIF3 E,KLF5,ALB,CYP24A1,were differentially expressed,mainly involved in biological processes,e.g.the regulation of tissue cell composition,and mitotic cell cycle.The participating cell tissues included vesicles,cytoskeleton,membrane tissue and so on.The related signaling pathways were adhesion spot kinase and FcγR mediated phagocytosis of macrophages.ConclusionsThe expression of lncRNA-XIST in gastric cancer tissues and plasma increased and the activation process of GCAF could be promoted.Interfering the expression of lncRNA-XIST could inhibit the proliferation and migration of GCAF and accelerate its apoptosis,which suggests that lncRNA-XIST might be a new promising tumor marker and therapeutic target for gastric cancer.