The Mechanism Of MiR-330-5P Targeting ITGA5 To Promote Gastric Cancer Proliferation,invasion And Migration

Part 1 Screening of differential gene ITGA5 in gastric cancer tissues and corresponding adjacent normal tissues based on high-throughputRNA-Sequence and bioinformatics methods.Objective: To screen the differentially expressed integrin family(Integrins)genes in gastric cancer tissues and corresponding normal tissues adjacent to cancer,and to explore the possible biological processes and signal pathways affected by the differential genes.Methods: Using Illumina HiSeqTM 2500 high-throughput sequencing technique,10 pairs of gastric cancer tissues(group T)and corresponding adjacent normal tissues(group N)were deeply sequenced by mRNA,and the differential genes were analyzed and screened.Bioinformatics methods were used to analyze the biological processes and signal pathways that differentially expressed genes may participate in,and cluster-Profiler was used for functional enrichment analysis.The differentially expressed genes were verified by GEPIA platform software and public database of Sun Yat-sen University Network on starbase platform,and the biological processes and signal pathways involved in them were analyzed by Gene Set Enrichment Analysis(GSEA)enrichment.Results: 1.2625 differential mRNA,were identified in N group and T group,including 2307 up-regulated genes and 318 down-regulated genes.Among them,ITG(A1,A5,A7,A10,AX,B1,B3,B5,B8)were significantly differentially expressed in(ITGs),and the highest multiple of difference in Fold Chang was ITGA5.2.GEPIA platform software and public database of Sun Yat-sen University Network on starbase platform showed that the expression level of ITGA5 in gastric cancer tissues was higher than that in adjacent normal tissues,the expression of ITGA5 gene was related to the prognostic information of gastric cancer patients,the prognosis of patients with high expression of ITGA5 was poor,and the prognosis of patients with low expression of ITGA5 was better.3.based on the high-throughput sequencing data,ITGA5 gene was enriched by Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG),and further GSEA enrichment analysis.It was found that the signal pathways involved in ITGA5 were mainly concentrated in Focal adhesion,ECM-receptor interaction and PI3K-AKT related pathways,and mainly involved in biological processes such as protein connection,cell adhesion,adhesion plaque,cell matrix receptor interaction and so on.Conclusion: The differential gene ITGA5,is mainly involved in the biological processes such as protein connection,cell adhesion,adhesion plaque and cell matrix receptor interaction,and may be involved in Focal adhesion,ECM-receptor interaction,PI3K-AKT and other signal pathways,but it needs further experimental verification.Part 2 Study on the expression of ITGA5 gene in gastric cancer tissues and cells and its correlation with clinicopathological features of patientsObjective: To detect the expression of integrin α5 in gastric cancer tissues and gastric cancer cells,and to analyze the correlation between integrin α5 and clinicopathological features.Methods: 1.Tissue samples and clinical data of patients with gastric cancer were collected,totalRNA and total protein were extracted,and tissue wax blocks were made.2.The expression of ITGA5 in gastric cancer and paracancerous normal tissues was detected by Quantitative Real-time PCR(qRT-PCR),Western Blot,immunofluorescence(IF)and immunohistochemistry(IHC).2.GES-1 and gastric cancer cell lines(AGS,MKN-28,and HGC-27)were collected,and totalRNA and total protein were extracted.qRT-PCR and Western Blot techniques were used to detect the expression of ITGA5 in cell lines.The measurement data were expressed by mean±standard deviation((?)±s).T-test was used for comparison between the two groups,and single factor analysis of variance(One-Way ANOVA)test was used for comparison between groups.Chi-square test was used for counting data.P<0.05 indicates that the difference is statistically significant.Results: 1.The results of Western blot and qRT-PCR detection in gastric cancer tissues and adjacent normal tissues showed that the protein expression level of ITGA5 in gastric cancer tissues was significantly higher than that in adjacent normal tissues,while the level of mRNA was consistent with the protein expression level,and there were significant differences.2.The results of immunohistochemistry showed that ITGA5 gene was significantly increased in tumor tissues and significantly correlated with tumor size,lymph node metastasis and TNM pathological stage(P<0.05),but not with age,sex and histological classification(P<0.05).3.The results of qRT-PCR detection showed that the expression level of ITGA5 in gastric cancer cell lines(AGS,MKN-28 and HGC-27)was significantly higher than that of GES-1(P<0.05).Western blot detection was consistent with that of qRT-PCR,and the expression level of ITGA5 in AGS and MKN-28 cells was relatively higher(P<0.05).These two cell lines were selected for follow-up experiments.Conclusion: 1.The expression of ITGA5 was significantly increased in gastric cancer tissues,but low in normal tissues adjacent to cancer.2.The expression of ITGA5 was significantly correlated with tumor size,lymph node metastasis and TNM stage,but not with sex,age and histological classification.3.The expression of ITGA5 was significantly increased in gastric cancer cell lines(AGS,MKN-28,and HGC-27).Part 3 Effect of ITGA5 gene on proliferation,apoptosis,invasion and migration of gastric cancer cells in vivo and in vitroObjective: To investigate the effect of ITGA5 gene on proliferation,apoptosis,invasion and migration of gastric cancer cells in vitro and in vivo.Methods: The plasmids sh-NC,sh-ITGA5-1,sh-ITGA5-2,sh-ITGA5-3 and OE-ITGA5 were synthesized by Guangzhou Funeng Biotechnology Co,Ltd,through transformation of competent cells,selection of monoclonal strains,expansion of culture,extraction of plasmids,transfection of MKN-28 and AGS gastric cancer cells with Lipofectamine 3000,screening of plasmids by qRT-PCR and Western blot,and selection of the ones with the best silencing efficiency for the next experiment.According to the selected plasmid,lentivirus was packaged with the third generation lentivirus packaging system,293 T tool cells and Lipofectamine 3000.After purification,gastric cancer cells were infected and screened with puromycin to construct gastric cancer cell lines stably transfected with puromycin.On this basis,the proliferation ability of gastric cancer cells was detected by CCK8,Edu and clone formation,and the apoptosis rate of gastric cancer cells was detected by flow cytometry.The migration ability of gastric cancer cells was detected by Transwell chamber test,and the invasive ability of gastric cancer cells was detected by smearing matrix glue in Transwell chamber.After MKN-28 and AGS cells were stably transfected with silenced and over-expressed ITGA5 gene,the tumor was implanted subcutaneously in nude mice.The volume and weight of the transplanted tumor were measured.The nude mice were scanned by living imager,and the expression of ITGA5 was detected by immunohistochemistry.All measurement data were expressed by mean±standard deviation((?)±s).T-test was used for comparison between the two groups,and one-way analysis of variance(One-Way ANOVA)test was used for comparison between groups.Chi-square test was used for counting data.P<0.05 indicates that the difference is statistically significant.Results: The results of qRT-PCR detection showed that the sh-ITGA5-1 was the best.Compared with the control group,the proliferation,migration and invasion of MKN-28 and AGS gastric cancer cells were significantly enhanced after overexpression of ITGA5 gene.After silencing ITGA5 gene,the proliferation,migration and invasion ability of MKN-28 and AGS gastric cancer cells were significantly decreased compared with the control group(P<0.05).However,there was no significant difference in the apoptosis rate of gastric cancer cells after overexpression and silencing of ITGA5 gene.In vivo experiments showed that overexpression of ITGA5 gene could accelerate the proliferation of transplanted tumor in nude mice,and after silencing ITGA5 gene,the growth of transplanted tumor in nude mice was slowed down(P<0.05).Conclusion: ITGA5 gene can promote the proliferation,invasion and migration of gastric cancer cells,but has no effect on apoptosis.Part 4 Screening and targeting of miRNA regulating the expression of ITGA5Objective: To screen the miRNA,that regulates the expression of ITGA5 and to explore the relationship between miRNA,and the targeted regulation of ITGA5 gene in gastric cancer cells.Methods: The miRNA of targeting ITGA5 predicted by online software such as Target Scan,miRDB,miRTar Base and mir DIP was further verified by qRT-PCR and Western blot.Bioinformatics was used to screen miRNA with negative correlation with ITGA5 and possible targeting relationship to carry out dual luciferase reporter gene experiment.ITGA5 3’-UTR wild-type(Wt)plasmid and mutant(Mut)plasmid were constructed and co-transfected with miRNA mimics mimic and ITGA5 Wt/Mut plasmid in 293 T cells by double luciferase reporter gene experiment,and the fluorescence intensity was detected.Through the ENCORI Pan-cancer Analysis Platform public database of Sun Yat-sen University,the correlation between ITGA5 and miRNA in gastric cancer tissue samples was analyzed.All measurement data were expressed by mean ±standard deviation((?)±s).T-test was used for comparison between the two groups,and one-way analysis of variance(One-Way ANOVA)test was used for comparison between groups.Chi-square test was used for counting data.P < 0.05 indicates that the difference is statistically significant.Results: Six stable and conservative miRNA(miR-26a-5p,miR-92a-3p,miR-148a-3p,miR-148b-3p,miR-330-5p and miR-152-3p)were selected from the four common online targeting prediction software.the results of qRT-PCR and Western blot showed that the expression of ITGA5 was significantly down-regulated after transfection of miR-330-5p mimics,and there was a negative correlation between miR-330-5p and ITGA5 expression.The results of double luciferase reporter gene assay showed that the relative fluorescence values of miR-330-5p mimics and ITGA5-Wt groups were significantly lower than those of miR-330-5p mimics and ITGA5-Wt groups.Compared with miR mimics-NC and ITGA5-Mut cotransfection groups,the relative fluorescence values of miR-330-5p mimics and ITGA5-Mut groups had no significant change(P>0.05).Conclusion: miR-330-5p may be the upstream regulatory molecule of ITGA5,which was verified by double luciferase report experiment.Part 5 Study on the effect of miR-330-5p-mediated ITGA5 on the proliferation,migration and invasion of gastric cancer cells and its regulatory mechanismObjective: To explore the effect of miR-330-5p on the proliferation,invasion and migration of gastric cancer cells mediated by ITGA5 and the regulatory mechanism of related signal pathways.Methods: After transfection of miR-330-5p mimics,miR-330-5p inhibitors,sh-ITGA5,OE-ITGA5;and cotransfection of miR-330-5p mimics and OE-ITGA5,miR-330-5p inhibitors and sh-ITGA5,the effects of miR-330-5p and ITGA5 on the proliferation of gastric cancer cells were detected by CCK8,Edu and colony formation assay,and the effects of miR-330-5p and ITGA5 on the invasion and migration of gastric cancer cells were detected by Transwell chamber.The protein expression levels of ITGA5,FAK,p-FAK,AKT and p-AKT in each group were detected by Western blot.All measurement data were expressed by mean±standard deviation((?)±s).T-test was used for comparison between the two groups,and single factor analysis of variance(One-Way ANOVA)test was used for comparison between multiple groups.Chi-square test was used for counting data.P<0.05 indicates that the difference is statistically significant.Results: In MKN-28 and AGS cells,compared with the control group,miR-330-5p mimics significantly inhibited the proliferation,invasion and migration of gastric cancer cells,OE-ITGA5 could significantly promote the proliferation,invasion and migration of gastric cancer cells(P< 0.05).After cotransfection of miR-330-5p mimics and OE-ITGA5,miR-330-5p mimics could significantly reverse the promoting effect of OE-ITGA5 on the proliferation,invasion and migration of gastric cancer cells.The results of Western blot detection showed that in MKN-28 and AGS cells,(1)after silencing ITGA5 gene,the expression of phosphorylated p-FAK and p-AKT was significantly down-regulated compared with Control group and negative control group,but there was no significant change in FAK and AKT.On the contrary,after overexpression of ITGA5 gene,the expression of phosphorylated p-FAK and p-AKT was significantly up-regulated compared with Control group and negative control group(P< 0.05),but there was no significant change in FAK and AKT.(2)compared with miR mimics NC group,the protein expression of ITGA5,p-FAK and p-AKT was significantly inhibited in miR-330-5p mimics group,while the expression of FAK and AKT protein had no significant change.The expression of ITGA5,p-FAK and p-AKT protein increased significantly in OE-ITGA5+miR mimics NC group,while the expression of FAK and AKT protein had no significant change.Compared with OE-ITGA5+miR mimics NC group,the expression of ITGA5,p-FAK and p-AKT protein in OE-ITGA5+miR-330-5p mimics group was significantly increased and significantly inhibited by miR-330-5p mimics(P < 0.05);FAK and AKT protein level had no significant change.(3)compared with miR inhibitors NC group,the expression of ITGA5,p-FAK and p-AKT protein in miR-330-5p inhibitors group was significantly increased,while the expression of FAK and AKT protein had no significant change.In sh-ITGA5+miR inhibitors NC group,the expression of ITGA5,p-FAK and p-AKT protein was significantly inhibited,while the expression of FAK and AKT protein had no significant change.Compared with sh-ITGA5+miR inhibitors NC group,the expression of p-FAK and p-AKT protein in sh-ITGA5+miR-3305 p inhibitors group was significantly decreased and significantly promoted by miR-330-5p inhibitors(P < 0.05),FAK and AKT protein level had no significant change.Conclusion: miR-330-5p mediates the effect of ITGA5 on the proliferation,invasion and migration of gastric cancer cells,and its mechanism may be related to the regulation of FAK/AKT signal pathway.