Studies On The Effect And The Functional Mechanism Of Long Non-coding RNA ASNR On The Biological Function In Gastric Cancer

Gastric cancer(GC) is one of the leading causes of cancer-related deaths worldwide,and it is also a major global health problem,especially in Asia.With the development of the fields of molecular biology and epigenetics,finding sensitive and specific diagnostic biomarkers and molecular targeted therapeutic targets is a hot and difficult point in cancer research today.It is helpful to explore the regulatory genes of gastric cancer.For the precise diagnosis and treatment of gastric cancer,it improves the prognosis of patients and prolongs the survival time.Long non-coding RNAs(lncRNAs)are a new class of transcripts that have received great attention in the current research environment,such as carcinogenesis.Recently,a new mechanism of interaction between RNAs is proposed,namely,competitive endogenous RNA(ce RNA),which means that transcripts such as lncRNA,m RNA and pseudogene transcripts pass through miRNA response elements(MREs).Competition to combine the same miRNA to regulate gene expression,thereby affecting cell function.The principle is that previous studies have revealed that miRNA can cause gene silencing by binding to m RNA,while ce RNA regulates gene expression by competitively binding miRNA to make it ineffective.ASNR(apoptosis suppressing-noncoding RNA)is a factor related to apoptosis,which has not been reported in GC.This study analyzes the role and mechanism of ASNR in the development of GC through cell experiments and clinical specimen detection,and then provides a research basis for finding important molecules that effectively regulate the development of GC to guide clinical diagnosis and develop molecular targeted drugs.PartⅠStudy on the expression level of lncRNA ASNR in gastric cancer and its relationship with clinicopathological featuresObjective:To verify the expression level of ASNR in gastric cancer tissue,adjacent tissue,human gastric mucosa epithelial cell lines and human gastric cancer cell lines with different degrees of differentiation,as well as its relationship with various clinicopathological characteristics of gastric cancer patients.Methods:1.GEPIA2 was used to analyze ASNR expression in gastric cancer and relationship with five-year survival.2.RT-qPCR technology was used to detect the expression level of 76pairs of ASNR in gastric cancer tissues and adjacent tissues.2.Analyze the relationship between ASNR expression level and various clinicopathological parameters of gastric cancer patients.3.RT-qPCR technology was used to to detect the expression level of ASNR in 4 human gastric cancer cell lines with different degrees of differentiation(MKN28,AGS,MKN45,HGC27)and immortalized human gastric normal mucosal cell lines(GES-1).Results:1.The results of bioinformatics analysis showed that ASNR was highly expressed in gastric cancer tissues,and ASNR expression affected the overall survival rate and disease-free survival rate of patients(P<0.05).2.The results of RT-qPCR showed that the expression level of ASNR in gastric cancer tissues was significantly higher than that of adjacent tissues,and the difference was statistically significant(P<0.05).3.The analysis of expression level and clinicopathological characteristics showed that the up-regulation of ASNR expression level was not related to the age,gender,tissue differentiation,and distant metastasis of gastric cancer patients,but is significantly related to tumor size,Lauren classification,tissue differentiation,infiltrating depth,nodal status and TNM staging(P<0.05).4.The results of univariate survival analysis showed that overall survival(OS)was significantly correlated with tumor size,Lauren classification,tissue differentiation,infiltrating depth,TNM stage and ASNR expression level(P<0.05).Multivariate Cox regression analysis showed that ASNR expression level and tissue differentiation were independent prognostic factors for patients.Survival analysis showed that the OS of patients with high ASNR expression level was shorter than that of patients with low ASNR expression level(P<0.05).5.RT-qPCR results showed that:Compared with GES-1 cell lines,ASNR expression levels in HGC27,MKN45,MKN28,AGS cell lines were significantly increased,and the difference was statistically significant(P<0.05).Summary:The lncRNA ASNR has the characteristics of proto-oncogene and affects the prognosis of gastric cancer patients,and may become one of the potential gastric cancer tumor markers.PartⅡ Study on the effect of lncRNA ASNR on the biological function of gastric cancer cellsObjective:1.To study the effect of overexpression of ASNR on the proliferation,invasion and migration of human gastric cancer cell line MKN28.2.To study the effect of inhibiting ASNR on the proliferation,invasion and migration of MKN45.3.To study the effect of stable inhibition of ASNR on the tumorigenesis ability and tumor growth of human gastric cancer cell line AGS in nude mice,and explore its role in the development of gastric cancer through in vivo experiments in nude mice.Methods:1.Construct a pc DNA3.1(+)vector plasmid that can effectively amplify ASNR,and transfect the plasmid into the MKN28 cell line.RT-qPCR was performed to detect the upregulation effect on ASNR.2.MTT assay was used to study the effect of overexpression of ASNR on the proliferation of MKN28 cells.3.RT-qPCR and Western blot technology was used to detect the effect of overexpression of ASNR on PCNA expression level and related protein expression.4.Flow cytometry was used to study the effect of overexpression of ASNR on the cell cycle of MKN28,and RT-qPCR and Western blot technology was used to detect the effect of overexpression of ASNR on the expression of cell cycle-related proteins.5.Cell scratch and Transwell experiments was used to study the effect of overexpression of ASNR on the migration and invasion of MKN28 cells,and RT-qPCR and Western blot technology was used to detect the effect of overexpression of ASNR on the expression of invasion and migration-related proteins.6.RT-qPCR and Western blot technology was used to detect the effect of overexpression of ASNR on the epithelial-mesenchymal transition(EMT)-related proteins of the MKN28 cell line.7.Construct a small interfering RNA that can effectively inhibit ASNR and transfect it into the MKN45 cell line.RT-qPCR was performed to detect the downregulation effect on ASNR.8.MTT assay was used to study the effect of inhibiting ASNR on the proliferation of MKN45 cells.9.RT-qPCR and Western blot technology was used to detect the effect of inhibiting ASNR on PCNA expression level and related protein expression.10.Flow cytometry was used to study the effect of inhibiting ASNR on the cell cycle of MKN45,and RT-qPCR and Western blot technology was used to detect the effect of inhibiting ASNR on the expression of cell cycle-related proteins.11.Cell scratch and Transwell experiments was performed to study the effect of inhibiting ASNR on the migration and invasion of MKN45 cells,and RT-qPCR and Western blot technology was used to detect the effect of inhibiting ASNR on the expression of invasion and migration-related proteins.12.RT-qPCR and Western blot technology was used to detect the effect of inhibiting ASNR on the epithelial-mesenchymal transition(EMT)-related proteins of the MKN45 cell line.13.Morphological observation,H&E staining and immunohistochemistry were used to investigate the effect of down-regulation of ASNR on tumorigenesis of AGS cells in nude mice.Results:1.RT-qPCR results showed that:Compared with Control group(Blank control)and Vector group(Negative control)cell lines,the expression level of ASNR in pc DNA3.1-ASNR transfection group(Transferred group)cell lines was significantly increased,and the effect was most obvious when the concentration was 2μg,and the difference was statistically significant(P<0.05).2.MTT results revealed that compared with the Control group and the Vector group,the OD value of the MKN28 cell line 48 h was obviously increased after overexpression of ASNR(P<0.05).3.The results of RT-qPCR and Western blot showed that:Compared with the Control and the Vector group,the PCNA expression in MKN28 cell line was obviously increased after overexpression of ASNR(P<0.05).4.The results of flow cytometry revealed that compared with the Control and the Vector group,the percentage of MKN28 cells in G0/G1 phase was significantly reduced after ASNR overexpression(P<0.05),and the percentage of MKN28 cells in S phase and G2/M phase was significantly increased(P<0.05),and it was the most increased in the G2/M phase.5.RT-qPCR and Western blot results revealed that the expression levels of Cyclin A,Cyclin D1 and Cyclin E in the overexpression ASNR transfection group were obviously higher than those in the Control and the Vector group(P<0.05),while the expression levels of P21 and P27 were higher than those in the Control group.6.The results of the cell scratch test showed that compared with the Control and the Vector group,the in-plane migration activity of the MKN28cell line was significantly enhanced after overexpression of ASNR(P<0.05).7.The results of the Transwell cell invasion experiment showed that compared with the Control and the Vector group,the number of cells passing through the Transwell chamber matrigel of the MKN28 cell line after overexpression of ASNR was significantly increased(P<0.05).8.RT-qPCR and Western blot results demonstrated that the expression levels of MMP2,MMP9 and ICAM1 in the overexpression ASNR transfection group were obviously higher than those in the Control and Vector groups(P<0.05).Compared with the Control and the Vector group,the expression levels of TIMP1,TIMP2,and NM23 in the transfection group were obviously lower(P<0.05).9.RT-qPCR and Western blot results indicated that the expression levels of N-cadherin,Snail,ZEB1,Twist and Vimentin in the overexpression ASNR transfection group were obviously higher than those in the Control and Vector groups(P<0.05).While the expression level of E-cadherin in the transfection group was significantly lower than that in the Control and Vector groups(P<0.05).10.The RT-qPCR results showed that:Compared with the Control group(Blank control)and si-NC group(Negative control)cell lines,the si-ASNR transfection group(Transferred group)cell lines had significantly lower ASNR expression levels,and the effect of si RNA3 was the most obvious,and the difference was statistically significant(P<0.05).11.The results of MTT assay indicated that compared with the Control group and the si-NC group,the OD value of the MKN28 cell line at 48 h was significantly reduced after ASNR inhibition(P<0.05).12.RT-qPCR and Western blot results showed that compared with the Control group and the si-NC group,the PCNA expression level of the MKN45cell line was obviously reduced after the inhibition of ASNR(P<0.05).13.The results of flow cytometry demonstrated that compared to the Control group and the si-NC group,the percentage of MKN45 cells in G0/G1phase was significantly increased after inhibiting ASNR(P<0.05),the percentages in S phase and G2/M phase were significantly decreased(P<0.05),and the percentage in G0/G1 phase was most decreased.14.The results of RT-qPCR and Western blot indicated that the expression levels of Cyclin A,Cyclin D1,and Cyclin E in the inhibitied ASNR transfection group were obviously lower than those in the Control and si-NC group(P<0.05),while the expression levels of P21 and P27 were higher.15.The results of the cell scratch test showed that compared with the Control and si-NC group,the in-plane migration activity of the MKN45 cell line was significantly weakened after inhibition of ASNR(P<0.05).16.The results of the Transwell cell invasion experiment showed that compared with the Control group and the si-NC group,the number of cells passing through the Transwell chamber matrigel of the MKN45 cell line after inhibition of ASNR was significantly reduced(P<0.05).17.The results of RT-qPCR and Western blot indicated that the expression levels of MMP2,MMP9 and ICAM1 in the ASNR transfection group were obviously lower than those in the Control group and si-NC group(P<0.05).Compared with the Control and Vector group,the expression levels of TIMP1,TIMP2,and NM23 in the transfection group was obviously higher(P<0.05).18.The results of RT-qPCR and Western blot indicated that the expression levels of N-cadherin,Snail,ZEB1,Twist and Vimentin in the ASNR transfection group were significantly lower than those in the Control and si-NC groups(P<0.05).While the expression level of E-cadherin in transfection group was significantly higher than that in Control group and si-NC group(P<0.05).19.The results of morphological observation showed that the average volume of transplanted tumors in nude mice in the sh-NC groupand sh-ASNR group were(1058.4±120.0 mm~3)and(596.4±100.0 mm~3),respectively.The average weights of transplanted tumors were(1226.6±124.7 mg)and(602.4±87.0 mg),and the growth rate and weight of transplanted tumors in nude mice in the sh-ASNR group were obviously lower than those in the sh-NC group(P<0.05).20.The results of H&E staining exhibited that compared with the sh-NC group,the transplanted tumor tissue necrosis of nude mice in the sh-ASNR group was obviously increased.21.Immunohistochemistry evaluation revealed that compared with the sh-NC group,the positive rate of Ki-67 in the transplanted tumor tissue of nude mice in the sh-Lnc＿ASNR group was significantly reduced.22.Western blot evaluation indicated that compared with the sh-NC group,the expression levels of PCNA,MMP2,MMP9,and ICAM1 in the transplanted tumor tissues of nude mice in the sh-ASNR group were significantly lower than those in the Control group and the si-NC group.And the expression level of NM23 was significantly higher than that of Control group and Vector group.Summary:1.ASNR effectively overexpressed human gastric cancer cell line MKN28 was successfully constructed.2.Overexpression of ASNR could significantly enhance the cell proliferation ability of MKN28 cell line,which could be associated with the up-regulation of PCNA expression level.3.Overexpression of ASNR could enhance the proliferation ability of MKN28 cells,which may be related to the up-regulation of Cyclin A,Cyclin D1 and Cyclin E expression levels and the down-regulation of P21 and P27expression levels.4.Overexpression of ASNR could obviously enhance the migration and invasion ability of the MKN28 cell line,which could be associated with the up-regulation of the expression levels of MMP2,MMP9 and ICAM1 and the down-regulation of the expression levels of TIMP1,TIMP2 and NM23.5.Overexpression of ASNR could obviously reduce the expression level of E-cadherin in the MKN28 cell line,and the expression of N-cadherin,Snail,ZEB1,Twist,and Vimentin protein were significantly increased.Therefore,overexpression of ASNR may promote the occurrence of EMT in MKN28 cell line.6.ASNR effectively inhibited human gastric cancer cell line MKN45 was successfully constructed.7.Inhibition of ASNR could significantly reduce the cell proliferation ability of MKN45 cell line,which could be associated with the down-regulation of PCNA expression.8.Inhibition of ASNR could reduce the proliferation ability of MKN45cells,which may be related to the down-regulation of Cyclin A,Cyclin D1 and Cyclin E expression levels and the up-regulation of P21 and P27 expression levels.9.Inhibition of ASNR could obviously reduce the migration and invasion ability of MKN45 cell line,which could be associated with the down-regulation of the expression levels of MMP2,MMP9,ICAM1 and the up-regulation of the expression levels of TIMP1,TIMP2 and NM23.10.Inhibiting ASNR could obviously increase the expression of E-cadherin in the MKN45 cell line and the expression levels of N-cadherin,Snail,ZEB1,Twist and Vimentin protein were significantly reduced,so overexpression of ASNR may inhibit the occurrence of EMT in the MKN45cell line.11.ASNR stably inhibited human gastric cancer cell line AGS was successfully constructed.12.Inhibiting ASNR could markedly decrease tumor volume and mass in nude mice,and the tissue necrosis of transplanted tumors in nude mice was significantly increased,which suggested that inhibition of ASNR could reduce the tumorigenic ability of AGS cell line in nude mice and inhibit tumor growth.Thus,the expression levels of PCNA,MMP2,MMP9 and ICAM1 were down-regulated and the expression level of NM23 was up-regulated.PartⅢ Study on the molecular mechanism of the effect of lncRNAASNR on gastric cancer cellsObjective:1.To study the interaction between ASNR and miR-519e-5p and FGFR2in gastric cancer cells,clarify the mechanism of ASNR,and reveal the potential clinical application value of ASNR/miR-519e-5p/FGFR2 in the development of gastric cancer.2.To verify the expression levels of miR-519e-5p and FGFR2 in gastric cancer tissues,adjacent tissues,human gastric normal epithelial cell lines,and human gastric cancer cell lines with different degrees of differentiation.3.To study the effect of ASNR/miR-519e-5p/FGFR2 on the invasion and migration of human gastric cancer cell lines.Methods:1.Bioinformatics analysis predicts the potential binding sites between ASNR and miR-519e-5p,miR-519e-5p and FGFR22.Dual luciferase reporter gene was used to detect the targeted regulation between ASNR and miR-519e-5p,miR-519e-5p and FGFR2.3.RT-qPCR technology was used to detect the expression level of miR-519e-5p in 60 pairs of gastric cancer tissues and adjacent tissues.4.RT-qPCR technology was used to detect the expression level of FGFR2 in 60 pairs of gastric cancer tissues and adjacent tissues.5.RT-qPCR technology was used to detect the expression level of FGFR2 in four human gastric cancer cell lines with different degrees of differentiation(MKN28,AGS,MKN45,HGC27)and immortalized human gastric normal mucosal cell lines(GES-1).6.RT-qPCR technology was used to detect the expression level of miR-519e-5p in MKN28 cell line overexpressing ASNR and MKN45 cell line inhibiting ASNR.7.RT-qPCR and Western blot technology was used to detect the changes in the expression levels of miR-519e-5p,FGFR2 and their proteins in the MKN28 cell line transfected with miR-519e-5p mimics.8.RT-qPCR and Western blot technology was used to detect the expression levels of miR-519e-5p,FGFR2 and their proteins in the MKN45cell line transfected with miR-519e-5p inhibitor.9.RT-qPCR and Western blot technology was used to detect changes in the expression level of FGFR2 and its protein in MKN28 and MKN45 cell lines after affecting the ASNR/miR-519e-5p/FGFR2 pathway.10.Cell scratch and Transwell experiments was used to study the effect of ASNR/miR-519e-5p/FGFR2 pathway on the migration and invasion of gastric cancer cells.Results:1.miRDB(http://mirdb.org/)and Targetscan(http://www.targetscan.org/)website analysis results showed that there were binding sites between ASNR and miR-519e-5p,miR-519e-5p and FGFR2.2.The results of the dual luciferase reporter gene showed that the luciferase activity of the ASNR-wild group was significantly reduced in the MKN45 cell line transfected with miR-519e-5p mimics(P<0.05),while the luciferase activity of ASNR-mut group showed no significant change.In the MKN45 cell line transfected with miR-519e-5p mimics,the luciferase activity of the FGFR2-wild group was significantly reduced(P<0.05),while the luciferase activity of the FGFR2-mut group did not change significantly.3.The results of RT-qPCR demonstrated that the expression of miR-519e-5p was markedly lower in gastric cancer tissues than in surrounding tissues(P<0.05).4.The results of RT-qPCR demonstrated that the expression of FGFR2was markedly higher in gastric cancer tissues than in surrounding tissues(P<0.05).5.RT-qPCR results showed that compared with GES-1 cell lines,FGFR2expression levels in HGC27,MKN45,MKN28,AGS cell lines were markedly increased(P<0.05).6.RT-qPCR results showed that the expression level of miR-519e-5p in the MKN28 cell line overexpressing ASNR was markedly reduced(P<0.05),and the expression level of miR-519e-5p in the MKN45 cell line that inhibited ASNR was markedly increased(P<0.05).7.RT-qPCR and Western blot results showed that the expression level of miR-519e-5p in the MKN45 cell line transfected with miR-519e-5p mimic was markedly increased(P<0.05),while the expression level of FGFR2 and its protein markedly reduced(P<0.05).8.RT-qPCR and Western blot results showed that the expression level of miR-519e-5p in the MKN28 cell line transfected with miR-519e-5p inhibitor was markedly reduced(P<0.05),while the expression level of FGFR2 and its protein was markedly increased(P<0.05).9.RT-qPCR and Western blot results showed that the expression levels of FGFR2 and its protein in the MKN28 cell line transfected with pc DNA3.1-ASNR were significantly increased(P<0.05);However,the up-regulation of FGFR2 and its protein expression in MKN28 cells co-transfected with pc DNA3.1-ASNR+miR-519e-5p mimic was abolished.The expression level of FGFR2 and its protein in the MKN45 cell line transfected with si-ASNR was markedly decreased(P<0.05);while the expression levels of FGFR2 and its proteins were restored in MKN45 cells co-transfected with si-ASNR+miR-519e-5p inhibitor.10.The results of the cell scratch test showed that compared with the Control group and the Vector group,the in-plane migration activity of the MKN28 cell line was significantly enhanced after overexpression of ASNR(P<0.05),while the enhanced migration activity of the MKN28 cell line co-transfected with pc DNA3.1-ASNR+miR-519e-5p mimic was counteracted.11.The results of the Transwell cell invasion experiment showed that compared with the Control group and the Vector group,the number of cells of the MKN28 cell line through the Transwell chamber matrigel was significantly increased after the overexpression of ASNR(P<0.05),while the number of cells in the MKN28 cell line co-transfected with pc DNA3.1-ASNR+miR-519e-5p mimic through the Matrigel in the Transwell chamber had no difference compared with the Control group and the Vector group.12.The results of the cell scratch experiment indicated that compared with the Control group and the si-NC group,the in-plane migration activity of the MKN45 cell line was significantly weakened after ASNR inhibition(P<0.05).However,the number of cells in the MKN28 cell line co-transfected with si-ASNR+miR-519e-5p inhibitor through the Matrigel in the Transwell chamber had no difference compared with the Control group and the Vector group.13.The results of the Transwell cell invasion experiment showed that compared with the Control group and the si-NC group,the number of cells of the MKN45 cell line through the Transwell chamber matrigel was significantly reduced after the inhibition of ASNR(P<0.05),and the number of cells in the MKN45 cell line co-transfected with si-ASNR+miR-519e-5p inhibitor through the Matrigel in the Transwell chamber had no difference compared with the Control group and the si-NC group.Summary:1.There were binding sites between ASNR and miR-519e-5p,miR-519e-5p and FGFR2.2.The study found that ASNR acted as the ce RNA of miR-519e-5p through the influence on the ASNR/miR-519e-5p/FGFR2 pathway,thereby increasing the expression level of FGFR2 and its protein.3.ASNR/miR-519e-5p/FGFR2 axis promoted the invasion and migration ability of gastric cancer cells.Conclusions:1.The expression level of ASNR in gastric cancer tissues was obviously increased,and was markedly associated with tumor size,infiltrating depth,nodal status and TNM staging.ASNR expression was increased in HGC27,MKN45,AGS and MKN28 cells.2.Overexpression of ASNR could obviously enhance the migration and invasion ability of MKN28 cell line,which could be associated with the up-regulation of the expression levels of MMP2,MMP9 and ICAM1 and the down-regulation of the expression levels of TIMP1,TIMP2 and NM23.Inhibition of ASNR could significantly inhibit the proliferation,migration and invasion of MKN28 cells in vitro and in vivo,which might be related to the down-regulation of MMP2,MMP9 and ICAM1 expression levels and the up-regulation of TIMP1,TIMP2 and NM23 expression levels.3.Overexpression of ASNR could obviously reduce the E-cadherin expression in the MKN28 cell line,and the expression of N-cadherin,Snail,ZEB1,Twist,and Vimentin protein were significantly increased.Therefore,overexpression of ASNR might promote the occurrence of EMT in MKN28cells.Inhibiting ASNR could markedly increase the E-cadherin expression in the MKN45 cells and the expression of N-cadherin,Snail,ZEB1,Twist,and Vimentin protein were significantly reduced.Therefore,overexpression of ASNR might inhibit the occurrence of EMT in the MKN45 cell line.4.ASNR acted as the ce RNA of miR-519e-5p,which increased the expression level of FGFR2 and its protein,promoted the proliferation,migration and invasion of gastric cancer cells,thereby promoting the development of gastric cancer.