LncRNA ZEB1-AS1 Accelerates The Development Of Gastric Cancer Through Regulating ZEB1 Expression

Backgroud:Gastric cancer(GC)is the most common digestive tract malignant tumor in the world.According to the global cancer(GLOBOCAN)statistical report of the world health organization and the international agency for research on cancer(IARC)in 2018,there are 1.04 million new cases of gastric cancer in the world every year,ranking the 5th in the number of new malignant tumor patients,780,000 in the number of deaths,and the 3rd in the number of malignant tumor deaths.The incidence of gastric cancer is highest in eastern Asia(especially in Korea,Mongolia,Japan and China)and central Europe and South America.There are about 410,000 new cases of stomach cancer and about 290,000 deaths in China each year,according to the national cancer registry(NCCR).At the same time,gastric cancer is also characterized by poor prognosis and high mortality,and its five-year survival rate is only about thirty percent.With the gradual improvement of the diagnosis and treatment methods and strategies for gastric cancer and the deepening understanding of its molecular mechanism,the overall incidence and mortality of gastric cancer show a declining trend.Nevertheless,due to the presence of distant metastasis and recurrence,it still faces great challenges.Therefore,it is necessary to further explore the molecular mechanism of gastric cancer progression so as to find new effective therapeutic strategies for patients.LncRNAs are a class of endogenous RNAs with larger than 200 nucleotides in length.So far,it has been found that IncRNA plays an important role in many biological processes,such as cell proliferation,differentiation and carcinogenesis.In recent years,a growing number of lncRNAs reported dysregulated in gastric cancer tissues,including ZEB1-AS1,PVT1,MALAT1,HOTAIR,NEAT1,CCAT1,ANRIL and PANDAR.ZEB1 is the protein 1 gene of box E zinc finger,and zebl-asl is the antisense protein product of the protein 1 gene of box E zinc finger,ZEB1-AS1 is located on the human chromosome 10 brokeback zone 11,zone 22(chr10p 11.22).The ZEB1 intron region contains single exons of the reverse chain of ZEB1-AS1 gene,as a member of the IncRNA family,has been found to be highly expressed in a variety of cancers,such as hepatocellular carcinoma,osteosarcoma,glioma and esophageal cancer.In HCC,the expression of ZEB1-AS1 in the cancer tissue is higher than that in the normal liver tissue,and the expression in the metastatic tumor tissue is higher than that of the primary tumor tissue.The expression of ZEB1-AS1 in the squamous cell carcinoma of the esophagus is higher than that in the para cancerous tissue,and it is also associated with the tumor classification and depth of invasion.In addition,the expression of ZEB1-AS1 expression in osteosarcoma tissues and cell lines is associated with tumor size,Enneking staging and tumor metastasis,and the high expression in the tissue specimens of glioma is closely related to the clinical stage of WHO.It is noteworthy that the high expression of this ZEB1-AS1 is closely related to the poor prognosis of the patients:the high expression of ZEB1-AS1 has a poor recurrence survival rate in the patients with liver cancer;ZEB1-AS1 overexpression is associated with the poor recurrent survival and total survival of the patients with esophageal squamous cell carcinoma and osteosarcoma;the expression of ZEB1-AS1 is higher in glioma.It can significantly reduce the overall survival of the patient.These results suggest that high expression of ZEB1-AS1 may play an adverse role in the pathogenesis of human cancer.However,the clinical significance and molecular mechanism of ZEB1-AS1 in gastric cancer have not been studied.Epithelial mesenchymal transition(EMT)is a complex pathological process in cancer metastasis,characterized by the potential of epithelial cells to migrate and transform into invasive mesenchyme cells.ZEB1 is a key transcription factor in this process.It can accelerate the invasion and metastasis of cancer cells by promoting the EMT process.Genomic studies show that ZEB1 is the protein 1 gene of box E zinc finger,and ZEB1-AS1 is the antisense protein product of the protein 1 gene of box E zinc finger,ZEB1-AS1 is located on the human chromosome 10 short arm zone 11,zone 22(chr10p11.22).the introns of the ZEB1 contain a single exon of the ZEB1-AS1 gene,The latter can enhance the activity of ZEB1 promoter in human cancer cells as an enhancer.In addition,ZEB1-AS1 can play a key regulatory role in tumor cell migration,invasion and EMT by regulating ZEB1 activity.Based on previous studies,we hypothesized that ZEB1-AS1 also plays an important role in regulating gastric cancer cell migration,invasion and EMT by regulating ZEB1 gene.Objectives:On the basis of previous reports,a number of experiments were carried out to solve the following questions:how is the expression level of the ZEB1-AS1 gene related to the occurrence and progression of gastric cancer?Secondly,the molecular mechanism of ZEB1-AS1 gene and its related gene ZEB1 regulating the development of gastric cancer is studied,and a new theoretical basis is provided for prognosis and target therapy for gastric cancer patients with abnormal ZEB1-AS1 expression.object and method:object:124 patients with gastric cancer and 20 patients with gastritis were randomly selected from January 2009 to December 2015 in shandong cancer hospital affiliated to shandong university.method:1.Surgical removal of metastatic lymph nodes from gastric cancer tissues and surrounding normal gastric tissues and some metastatic patients;And the inflammatory tissue in gastritis.Quantitative PCR was used to detect the expression level of ZEB1-AS1 gene in different tissues,in order to determine the potential relationship between the expression level of ZEB 1-AS1 and the occurrence of gastric cancer.2.According to the median expression level of ZEB1-AS1 in gastric cancer tissues,the 124 patients were divided into two groups:high expression and low expression(62 cases for each group).The relationship between ZEB1-AS1 expression and patients’ sex,age,tumor depth(T1-T2 and T3-T4),histological differentiation,clinical stage(I-II and III-IV),and lymph node metastasis(no more than 6 and more than 6)were evaluated respectively.The relationship between the distant metastasis and the distant metastasis was compared.The relationship between the expression of ZEB1-AS1 and the whole life period of the patients was analyzed by Kaplan-Meier test.The relationship between the expression of ZEB1-AS1 and the clinicopathological features and the prognosis was evaluated by the single variable and multivariable Cox regression analysis.3.RT-PCR and immunohistochemistry were used to detect the expression of ZEB1,on mRNA and protein levels,in ZEB1-AS1-high expression and low expression groups,and the correlation between them was evaluated.4.The expression levels of ZEB1-AS1 in human gastric cancer cell lines NCI-N87,SNU-1,AGS,MKN-45 and human normal gastric epithelial cell line GES-1 were detected by quantitative PCR.The two gastric cancer cell lines with the highest and lowest ZEB1-AS1 expression(A and B cells)were identified.The expression of ZEB1-AS1 was disturbed in the A cell line,and the expression of ZEB1 on the level of mRNA and protein was observed.ZEB1-AS1 was overexpressed in B cells to observe the changes in the expression of ZEB1,so as to further determine whether ZEB1-AS 1 was involved in the regulation of the expression of ZEB1.5,Boyden test and Transwell assays were used respectively to observe the changes of cell migration and invasion ability in the cells in the presence of ZEB1-AS1.The protein expression patterns of epithelial marker ZO-1 and E-Cadherin as well as mesenchymal markerVimentin and N-Cadherin were detected to determine the effect of ZEB 1-AS 1 on gastric cell migration,invasion and EMT.6.Resued functional studies were performed to verify the relationship between ZEB1-AS1 and 6whether the inhibition of migration,invasion and EMT caused by ZEB1-AS1 interference could be rescued by ZEB1 overexpression;similarly,pcDNA-ZEB1-AS1 and si-ZEB1 were co-transfected to determine if the influence of ZEB1-AS1 on migration,invasion and EMT could be compromised.Results:1.Quantitative PCR results showed that the expression of ZEB1-AS1 in gastric cancer tissues was higher than that in adjacent normal gastric tissues and gastritis tissues(P<0.001).At the same time,we observed that the expression of ZEB1-AS1 in metastatic lymph nodes was much higher than that in primary gastric cancer tissues.In addition,we evaluated the expression of ZEB1-AS1 in gastric cancer cells and human normal gastric epithelial cells.The expression level of ZEB1-AS1 in gastric cancer cell lines(NCI-N87,SNU-1,AGS and MKN-45)was significantly increased(P<0.001)compared with the normal gastric epithelial cell GES-1.This indicates that the expression of ZEB1-AS1 in the process of fracture healing is obviously down regulated.ZEB1-AS1 was the lowest expressed in NCI-N87 cells and the highest in MKN-45.2.The correlation between the expression level of ZEB1-AS1 and clinicopathological parameters in 124 cases of gastric cancer was analyzed.Statistical analysis showed that there was no significant correlation between ZEB1-AS1 expression and sex(P =0.067),age(P =0.584)and tumor depth(P=0.854).On the contrary,the expression of ZEB1-AS1 was significantly related to the histological type(P =0.031),clinical stage,lymph node metastasis,and distant metastasis,and the P value of the latter three was less than 0.001.Furthermore,Kaplan-Meier analysis showed that there was a strong statistical correlation between ZEB1-AS1 expression and overall survival rate of gastric cancer patients(P<0.001).Univariate and multivariate Cox regression analysis was used to detect the adverse prognostic factors in patients with gastric cancer.By single factor analysis,the high expression of ZEB1-AS1 was an important factor(P<0.001)in the patients with gastric cancer,regardless of the clinical stage,tumor depth,lymph node metastasis,and distant metastasis.In addition,based on multivariate analysis,we also confirmed that high expression of ZEB1-AS1 was an independent prognostic factor for patients with gastric cancer(HR=2.363,95%CI:1.410-3.962,P = 0.001).3.ZEB1-AS1 is located on Chr10p11.22,and the intron of ZEB1 contains a single exon of ZEB1-AS1 gene in reverse orientation.qRT-PCR assay showed that ZEB1-AS1 and ZEB1 mRNA expression were positively correlated in gastric cancer tissues(R =0.812,P<0.001).In addition,immunohistochemistry showed that the expression of ZEB1 protein was positively correlated with that of ZEB1-AS1(r=0.455,P=0.004).4.The expression of ZEB1-AS1 in the MKN-45 cell line was significantly lower than that of the control group(P<0.001),while ZEB1 mRNA and protein were significantly increased in the ZEB1-AS 1-overexpressed NCI-N87 cells(P<0.001)than the control group.This therefore suggested that the expression level of ZEB1 was positively regulated by ZEB1-AS1 at both mRNA and protein levels.5.Interfering ZEB1-AS1 expression inhibited gastric cancer cell migration,while overexpression of ZEB1-AS1 stimulated migration(P =<0.001).Similarly,the expression of ZEB1-AS1 suppressed the invasion of gastric cancer cells,whereas an increase in ZEB1-AS1 expression promoted gastric cancer cell invasion compared with the control group,with a significant difference.In addition,a decrease in ZEB1-AS1 increased the expression of ZO-1 and E-Cadherin,whilst reducing the expression of mesenchymal markers Vimentin and N-cadherin.On the other hand,up-regulation of ZEB1-AS1 inhibited the expression of epithelial markers ZO-1 and E-cadherin,and promoted Vimentin and N-Cadherin protein expression.6.Notably,ZEB1 overexpression rescued si-ZEB1-AS1-mediated inhibition of migration,invasion and EMT when pcDNA-ZEB 1-AS1 and si-ZEB1 were co-transfected into NCI-N87 cells,whereas ZEB1 interference compromised the ZEB1-AS-mediated promotion of migration,invasion and EMT process of gastric cancer cells.Conclusion:In aggregate,our experiment demonstrated that ZEB1-AS1 expression in gastric cancer tissue was higher,compared to adjacent normall tissues and gastritic tissues.The expression was also related to the histological type,clinical stage,lymph node metastasis,distant metastasis and prognosis of gastric cancer.In addition,ZEB1-AS1 plays a carcinogenic role in gastric cancer by regulating ZEB1 and thus promoting cell migration,invasion and EMT.