MiRNA-4664-3p Inhibits Cell Proliferation,migration And Invasion By Targeting YAP1 In Gastric Cancer

BackgroundAs a common malignant tumor of the digestive system,gastric cancer(GC)has made great progress in its diagnosis and treatment,and the survival rate of early gastric cancer has been significantly improved.However,most of the patients are in the advanced stage when they are diagnosed,it is difficult to cure and the mortality rate is high.The occurrence and development of gastric cancer involves multi-factor and multi-step biological changes.Biological processes such as excessive cell proliferation,invasion and metastasis are the main causes of death in patients with gastric cancer.Studies have shown that 95%of gastric cancer cases are stomach adenocarcinoma(STAD).Due to the lack of specific early diagnostic markers and the high histological heterogeneity of STAD,pathological staging is not a good predictor of prognosis.Therefore,searching for markers for clinical diagnosis and prognostic assessment of gastric cancer and clarifying effective molecular targets have become a research hotspot in the current field.MicroRNA(miRNA)have been proven to inhibit translation or accelerate degradation by acting on the 3’UTR of their target genes,and regulate most tumor-related biological processes,including cell proliferation,apoptosis,differentiation,and metastasis.A large number of studies have shown that miRNAs,that participate in the occurrence and development of tumors,are abnormally expressed in gastric cancer and other tumors,indicating that miRNAs might be potential biomarkers for cancer.Studies have shown that miRNAs are abnormally expressed in GC,affecting the proliferation,invasion and metastasis.Therefore,to clarify the role of miRNA in GC is very important for the diagnosis and treatment.ObjectiveTo investigate the regulation mechanism of miRNA-4664-3p in gastric cancer.Method1.Relevant clinical case data of gastric cancer patients and miRNA data were download from TCGA database.The R language was used to analyze the data,and single-factor and multi-factor Cox regression analysis was used to screen the differentially expressed miRNAs in the cancer tissues of GC patients.Kaplan-Meier curve was used to perform survival analysis.Based on the TCGA database,the relationships between the miR-4664 level in GC tissues and the grade of the tumor and lymph node metastasis were determined.2.Real-time quantitative PCR were used to detect the expression of miR-4664-3p.Tissues were Gastric cancer and cell lines were four gastric cancer cells including AGS,SGC7901,MKN28,MKN45 and normal gastric cells(GES-1).Based on the Gastric cancer tissues,the relationships between the miR-4664 level in GC tissues and the grade of the tumor and lymph node metastasis were determined.Quantitative PCR were used to detect the overexpression and knockdown efficiency of designed miR-4664-3p overexpression and knockdown plasmids.CCK-8 experiment,scratch healing experiment and transwell experiment were used to study the role of miR-4664-3p in the process of GC cell proliferation,migration and invasion.3.Online tools such asmiRDB(http://www.mirdb.org/miRDB/),TargetScan(http://www.targetscan.org/),mirDIP(http://ophid.utoronto.ca/mirDIP/index.jsp)were used to predict the target genes and molecular pathway of differentially expressed miRNAs.TargetScan online prediction was used to predict the possible target binding site of YAP 1 for miR-4664-3p.Between the GC and normal gastric tissues,the difference in mRNA and protein expression levels of YAP1 were detected by quantitative PCR and Western blot,as well as normal gastric cell line and different gastric cancer cell lines.The dual luciferase reporter gene system was used to reveal the targeting relationship between miR-4664-3p and YAP1.Quantitative PCR was used to detect the expression changes of miR-4664-3p after silencing YAP1 by transfecting the YAP1 liposomes with siRNA and the expression changes of miR-4664-3p after up-regulating YAP1.Quantitative PCR and Western blot were used to detect the effect of up-regulation and down-regulation of miR-4664-3p on YAP1.CCK-8 experiment,scratch healing experiment and Transwell experiment were used to study the effects on the gastric cancer cells of overexpression of miR-4664-3p and YAP1,which were proliferation,migration and invasion.4.Nude mice were used to reveal the effects of miR-4664-3p and YAP 1 on tumor development.Nude mice were injected with constructed miR-4664-3p and YAP1 overexpression stable strains,measure the size of the tumor,calculate the change in tumor volume,and measure the weight of the tumor after the mice are sacrificed.Result1.Based on the results of the TCGA database analysis,7 miRNAs were related to the survival of GC patients,including hsa-miR-4664,hsa-miR-548ba,hsa-miR-520c,hsa-miR-515-1,hsa-miR-519b,hsa-miR-520g,hsa-miR-523.Multivariate regression analysis showed that miR-4664 was an independent risk factor for patient survival.The K-M survival curve showed that miR-4664 was more highly expressed in long-term survival gastric cancer patients,suggesting that the expression level of miR-4664 is positively correlated with the long-term prognosis of gastric cancer patients.In TCGA database,the expression level of miR-4664 in GC tissue was significantly lower than that in the corresponding adjacent tissues,and its expression level was related to the grade of the tumor and lymph node metastasis.Further verification in clinical tissues found that the relative expression level of miR-4664-3p in the cancer tissues of GC patients was significantly lower than that in the adjacent tissues,and the expression level of miR-4664-3p was significantly related to the grade of the tumor and lymph node metastasis,consistent with TCGA database verification results.Compared with normal gastric tissue cells GES-1,the expression level of miR-4664-3p in gastric cancer cell lines AGS,SGC7901,MKN45,and MKN28 cells was significantly reduced.2.The GC cell lines AGS with relatively high expression of miR-4664-3p and The GC cell lines SGC7901 with relatively low expression of miR-4664-3p were selected for follow-up experiments to reveal the effect of miR-4664-3p on the malignant biological behavior of GC cells.Transfection of miR-4664-3p inhibitor in AGS cells inhibited the expression of miR-4664-3p and promoted cell proliferation,migration and invasion;transfection of miR-4664-3p mimics in SGC7901 cells increased the expression of miR-4664-3p and inhibited cell proliferation,migration and invasion.3.In order to explore the potential molecular mechanism of miR-4664-3p in GC,we predicted the target genes of miR-4664-3p through miRDB,TargetScan and mirDIP databases,and found that miR-464-3p might directly target on YAP1 and the possible binding site is YAP1 3’UTR region sequence.YAP1 mRNA and protein levels in cancer tissues of GC patients were significantly higher than those in adjacent tissues,and YAP1 mRNA and protein levels in gastric cancer cell lines(AGS,SGC7901,MKN45,MKN28)were significantly higher than those in normal gastric cell line GES-1.There was a significantly negative correlation between the expression of YAP1 and the expression of miR-4664-3p in gastric cancer tissues and adjacent tissues.The luciferase reporter gene showed that in AGS cells,cells transfected with miR-4664-3p inhibitor and YAP1-WT had a significantly higher luciferase activity compared with the negative control,but no significance was found in luciferase activity in cells transfected with miR-4664-3p inhibitor and YAP1-MUT.In SGC7901 cells,miR-4664-3p mimics and YAP1-WT transfected cells had a significant decrease in luciferase activity compared with the negative control,but the luciferase activity of the cells transfected with miR-4664-3p mimics and YAP1-MUT did not change significantly,indicating that miR-4664-3p could directly bind to the 3’UTR region of YAP1.There was no significant difference in the expression of miR-4664-3p after silencing and up-regulating YAP1,suggesting that YAP1 could not regulate miR-4664-3p.Compared with cells transfected with negative control,YAP1 mRNA and protein levels were significantly increased in AGS cells transfected with miR-4664-3p inhibitor.Compared with cells transfected with negative control,The mRNA and protein levels of YAP 1 were significantly reduced in SGC7901 cells transfected with miR-4664-3p mimics.4.To verify whether miR-4664-3p functioned through YAP1,SGC7901 cells were co-transfected with miR-4664-3p mimics and pcDNA3.1/YAP1 plasmids.The results showed that the overexpression of YAP 1 could weaken the inhibitory effect of miR-4664-3p mimics,which could weaken the effects such as the proliferation,invasion and migration on gastric cancer cells.The up-regulation of miR-4664-3p led to the decrease of YAP1 expression,the up-regulation of E-cadherin,and down-regulation of Ki67 and N-cadherin.Compared with the group transfected with miR-4664-3p mimics only,YAP1 expression was up-regulated,with significantly down-regulation of E-cadherin protein level and up-regulation of Ki67 and N-cadherin in cells co-transfected with miR-4664-3p mimics/YAP1 plasmids.The above indicators did not change significantly in cells co-transfected with empty plasmid and miR-4664-3p mimics,indicating that miR-4664-3p targeted on YAP1 to regulate the expression of the key protein of gastric cancer cells,and then regulate the gastric cancer cells on the proliferation,migration and invasion.5.In experiment of tumor formation in nude mice,the results showed that the volume and weight of tumors in the miR-4664-3p agomir group were significantly lower than those in the miR-NC group,the volume and weight of tumors in the miR-4664-3p agomir/YAP1 group were significantly higher than those in the miR-4664-3p agomir group,but had no significant difference with those in miR-4664-3p NC group,indicating that the overexpression of YAP 1 weakened the inhibitory effect of miR-4664-3p agomir on tumor volume and weight.Conclusion1.Based on the bioinformatics analysis results of the TCGA database,miR-4664,as one of the miRNAs specifically expressed by GC,was down-regulated in GC cancer tissues and was an independent risk factor related to survival.the expression level of miR-4664 is positively correlated with the long-term prognosis of gastric cancer patients.The expression level of miR-4664-3p in gastric cancer tissues and cell lines AGS,SGC7901,MKN45,and MKN28 was significantly reduced by molecular biology experiment,and the expression level of miR-4664-3p was significantly related to the grade of the tumor and lymph node metastasis.2.Down regulation of miR-4664-3p promoted cell proliferation,migration and invasion;up regulation of miR-4664-3p inhibited cell proliferation,migration and invasion,suggesting its potential anti-cancer ability.3.The bioinformatics analysis results predicted miR-464-3p might directly target on YAP1 by binding in the 3’-UTR region of YAP1.YAP1 mRNA and protein levels are significantly higher in gastric cancer tissues and cell lines.There is a significantly negative correlation between the expression of YAP 1 and the expression of miR-4664-3p in gastric cancer tissues and adjacent tissues.The dual luciferase experiment showed the direct targeting relationship with them.YAP1 could not regulate miR-4664-3p.Overexpression of miR-4664-3p could significantly down-regulate YAP1 and inhibition of miR-4664-3p promoted YAP1 expression significantly,The above results suggest that miR-4664-3p plays a role in down-regulating the expression level of YAP 1.4.1n vitro experiments,results showed that miR-4664-3p/YAP1 overexpression can block the inhibitory effect of miR-4664-3p overexpression on the proliferation,migration and invasion of gastric cancer cells.In vivo experiments,results showed that miR-4664-3p inhibits tumor growth by down-regulating YAP1 expression.In summary,miR-4664-3p acted as a tumor suppressor gene in gastric cancer by targeting YAP1.With the deepening of research,miR-4664-3p is expected to become a new target for gastric cancer prevention and treatment in the future.