| Background and objective: Gastric cancer(GC),as a common malignant tumor seriously endangering human health,is the third leading cause of cancer death in the world.Gastric cancer is characterized by occult onset and inconspicuous early symptoms,and most patients have advanced stage of the disease when first diagnosed,accompanied by lymph nodes or distant organs metastasis,which has become a difficulty in the prevention and treatment of gastric cancer.Therefore,it is extremely urgent to further study the molecular mechanism of the occurrence,development,metastasis and drug resistance of gastric cancer through various research technologies and methods,and search for molecular markers and drug targets for early diagnosis.Recent studies have shown that non-coding RNAs are involved in the process of tumor metastasis and malignant transformation.MiR-4295 is a newly discovered microRNA molecule.Through bioinformatics analysis,we found that miR-4295 has binding sites with lncRNA HOTAIR.Therefore,our study started with non-coding RNAs to investigate the functions and mechanisms of microRNA(miR-4295)and long non-coding RNA(lncRNA HOTAIR)in the growth,proliferation,invasion and migration of gastric cancer.Part 1 Study on the effect of miR-4295 on the growth and invasion as well as migration of gastric cancer by PTEN/PI3K/AKT pathwayMethods: The expression of miR-4295 among gastric cancer cell lines SGC-7901,MGC-803,BGC-823,MKN-28 and human gastric mucosal epithelial cell line GES-1 as well as gastric cancer tissues(or normal gastric mucosa adjacent to cancer)were detected by qRT-PCR in this study.miR-4295 eukaryotic plasmid overexpression vector and its inhibitor vector were constructed.The constructed vector was transfected into gastric cancer cell lines SGC-7901 and MGC-803 by transient transfection,and the expression of miR-4295 in the transfected cell lines was detected by qRT-PCR.The effect of miR-4295 and its inhibitor and miR-4295 combined with AKT inhibitor on the proliferation activity of gastric cancer cells was detected by CCK-8 assay.Flow cytometry was used to detect the effect of miR-4295 combined with AKT inhibitor on the apoptosis rate of gastric cancer cells.The effect of miR-4295 and its inhibitor on the cloning ability of gastric cancer cells was detected by plate cloning.Scratch test was used to detect the effect of miR-4295 and its inhibitor on the migration ability of gastric cancer cells.Effect of miR-4295 and its inhibitor and miR-4295 combined with AKT inhibitor on invasion and migration of gastric cancer cells were detected by transwell assay.In combination with bioinformatics software,miRanda,TargetScan,Pictar2,RNAhybrid,miRDB,RNA22-HAS,Targetminer,EMBL-EBI,miRWalk,DIANA-microT,mirbridge,miRNAMap and PITA online prediction software were used to predict the target genes of miR-4295.DAVID and KOBAS were combined to conduct GO analysis and KEGG signaling pathway analysis on the target genes of miR-4295,so as to analyze the possible molecular mechanism of miR-4295 in the biological process of gastric cancer.Hub gene,which plays a key role in the protein-protein interaction(PPI),was screened out from the potentialtarget genes of miR-4295 by STRING.The correlation of miR-4295 targeting hub gene in gastric cancer and its survival analysis of gastric cancer patients were analyzed by TCGA database GEPIA.Real-time quantitative PCR and western blot were used to verify the expression levels of the target genes of miR-4295 and related genes in the PI3K/AKT signaling pathway.Finally,double luciferase reporter gene system experiment was used to further verify the target genes of miR-4295,so as to clarify the relationship between miR-4295 and its target gene in gastric cancer.Results:1.Detection of miR-4295 expression level in gastric cancer cells and tissues.QRT-PCR results showed that(the quantitative expression results were expressed as Means ± SD,and the values of SGC-7901,MGC-803,BGC-823 and MKN-28 as follows: 1.832 ± 0.120,2.421 ± 0.187,1.328 ±0.102,1.000 ± 0.040,respectively),compared with human gastric epithelial cell lines GES-1(0.962 ± 0.070),the expression level of miR-4295 in the gastric cancer cell lines SGC-7901,MGC-803,BGC-823 and MKN-28 were all increased,with the most significant increase in SGC-7901 and MGC-803.Among the 31 patients with gastric cancer,miR-4295 expression in gastric cancer tissues was significantly up-regulated by 80.6%(25/31)compared with the normal gastric mucosa tissues.The expression level of miR-4295 was significantly negative correlated with the tumor size and distal metastasis of gastric cancer(p <0.05),but was not significantly correlated with the age,gender,histological grade and lymph node metastasis of gastric cancer patients(p > 0.05).2.Construction of miR-4295 overexpression plasmid vector and inhibitor plasmid vector.The sequencing results showed that the recombinant miR-4295 and its inhibitor sequences were inserted correctly,indicating that the recombinant miR-4295 and its inhibitor plasmid vector was constructed successfully.The expression level of miR-4295 in the transfected gastric cancer cells was detected by qRT-PCR,and the results showed that miR-4295 expression level in the transfected gastric cancer cell lines SGC-7901 and MGC-803 was significantly up-regulated compared with that in the miR-NC transfection group(p < 0.05).Compared with the Anti-miR-NC transfection group,the expression level of miR-4295 in gastric cancer cell lines SGC-7901 and MGC-803 transfected with miR-4295 inhibitor was significantly down-regulated(p< 0.05).3.CCK-8 assay was used to detect the proliferation activity of gastric cancer cells after transfection.The results showed that compared with the miR-NC transfection group,the proliferation activity of gastric cancer cell lines SGC-7901 and MGC-803 transfected with miR-4295 was significantly improved(p < 0.05).Compared with the Anti-miR-NC transfection group,the proliferation activity of gastric cancer cell lines transfected with miR-4295 inhibitor was significantly reduced(p < 0.05).Compared with miR-4295,the proliferation activity of miR-4295 combined with AKT inhibitor group was significantly reduced(p < 0.05).4.The apoptosis rate of gastric cancer cells treated with miR-4295 combined with AKT inhibitor was detected by flow cytometry.The results showed that compared with the miR-NC transfection group,the apoptosis rate of gastric cancer cell lines SGC-7901 and MGC-803 transfected with miR-4295 were significantly reduced(p < 0.05).Compared with the miR-4295 transfection group,the apoptosis rate of gastric cancer cell lines SGC-7901 and MGC-803 were significantly increased with miR-4295 combined with AKT inhibitor(p < 0.05).5.The cloning ability of transfected gastric cancer cells cultured continuously for 15 days was tested by plate cloning formationexperiment.The results showed that,compared with the miR-NC transfection group,the number of clones of miR-4295 transfected gastric cancer cell lines SGC-7901 and MGC-803 was significantly improved(p< 0.05).Compared with the Anti-miR-NC transfection group,the number of clones of gastric cancer cell lines SGC-7901 and MGC-803 transfected with miR-4295 inhibitor was significantly reduced(p < 0.05).6.The impact of transfection on the migration of gastric cancer cells was detected by scratch test.The scratch test results showed that upregulation of miR-4295 expression in the gastric cancer cells SGC-7901 and MGC-803,the migration spacing of the miR-4295 group was small at 24 h compared with the miR-NC group(p < 0.05).Nevertheless,downregulation of miR-4295 expression in gastric cancer cells SGC-7901 and MGC-803,the migration distance of miR-4295 inhibitor group was large at 24 h compared with the Anti-miR-NC group,(p < 0.05).7.Transwell assay was used to detect the effects of transfection on the migration and invasion of gastric cancer cells.The results showed that compared with the miR-NC group,the number of cell migration and invasion in the miR-4295 group was significantly increased(p < 0.05).Nevertheless,downregulation of miR-4295 expression in gastric cancer cells SGC-7901 and MGC-803,the migration and invasion of miR-4295 inhibitor group was significantly reduced compared with the Anti-miR-NC group(p < 0.05).Compared with miR-4295,the migration and invasion of miR-4295 combined with AKT inhibitor group significantly reduced(p < 0.05).8.Potential functions and mechanisms of miR-4295.In the early stage,a total of 1,5353 candidate target genes of miR-4295 were screened by 14 predictive software.A total of 600 target genes of miR-4295 were selected by 7 biological software to predict positive results at the same time for subsequent GO and pathway analysis as well as molecular network analysis of target genes.The GO enrichment of function analysis showed that 24 signaling pathways were significantly enriched in biological processe(p < 0.01),12 signaling pathways were significantly enriched in cellular component(p< 0.01),12 signaling pathways were significantly enriched in molecular function(p < 0.01).KEGG signaling pathway analysis showed that miR-4295 target genes were significantly enriched in 23 signaling pathways(p < 0.01).MiR-4295 target gene protein interaction(PPI)network screened 15 hub genes,including CUL3,PPARG,UBE2D1,FMR1,SUV39H1,RAB5 A,AGO4,RNF2,PTEN,RUNX2,KMT2 A,ITPKB,ESR1,DICER1 and LDLR.MiR-4295 hub gene PTEN is significantly correlated with BCL2L11.Bioinformatics software was used to predict the site binding of miR-4295 and PTEN or BCL2L11 3'UTR,the results showed that PTEN3'UTR target site was located at its 412-418 bp and the sequence(5'-3')was UUGCACU.BCL2L11 3'UTR target site was located at its 3'UTR4093-4099 bp,and the sequence(5'-3')was UUGCACU.The sequencing results of the positive cloning of PTEN 3'UTR and its mutant double luciferase plasmid showed that the recombinant PTEN3'UTR and its mutant sequences were inserted correctly,indicating that the recombinant plasmids of psiCHECK2-PTEN-3'UTR and psiCHECK2-PTEN-mut-3'UTR were successfully constructed.The sequencing results of the recombinant BCL2L11 3'UTR and its mutant double luciferase plasmid showed that the recombinant BCL2L11 3'UTR and its mutant were inserted correctly,indicating that the recombinant GV272-BCL2L11-3'UTR and GV272-BCL2L11-mut-3'UTR were successfully constructed.The results of double luciferase activity showed that compared with the PTEN-3'UTR + miRNA-NC co-transfection group,the luciferase activity in the PTEN-3'UTR + miR-4295 co-transfection group was significantly reduced,with statistically significant difference,indicating that miR-4295 could bind to PTEN 3'UTR and inhibit its expression.There was no significant difference in luciferase activity between the PTEN-3'UTR +miRNA-NC co-transfection group and the PTEN mut-3'UTR +miR-4295 co-transfection group,showing that miR-4295 could not bind to the target mutated PTEN 3'UTR and could not inhibit its expression.Compared with the PTEN-3'UTR + miR-4295 co-transfection group,the luciferase activity of the PTEN mut-3'UTR + miR-4295 co-transfection group was significantly increased,which further indicated that miR-4295 could not bind to PTEN 3'UTR target site mutation,and thus could not inhibit the target gene expression.These results suggest that miR-4295 can directly bind to the target gene PTEN 3'UTR and inhibit its expression.Compared with the BCL2L11-3'UTR + miRNA-NC co-transfection group,the luciferase activity of the BCL2L11 3'UTR + miR-4295co-transfection group was significantly reduced,and statistically significant difference,showing that miR-4295 could bind BCL2L113'UTR and inhibit its expression.There was no significant difference in luciferase activity between the BCL2L11-3'UTR + miRNA-NC co-transfection group and the BCL2L11 mut-3'UTR + miR-4295co-transfection group,indicating that miR-4295 could not bind to the target mutated BCL2L11 3'UTR and could not inhibit its expression.Compared with the BCL2L11-3'UTR + miR-4295 co-transfection group,the luciferase activity of the BCL2L11 mut-3'UTR + miR-4295co-transfection group was significantly increased,which further indicated that miR-4295 could not bind to the BCL2L11 3'UTR target site mutation,XVIIIand thus could not inhibit the target gene expression.These results suggest that miR-4295 can directly bind to the target gene BCL2L113'UTR and inhibit its expression.qRT-PCR was used to detect the effect of miR-4295 on the expression levels of target genes PTEN,BCL2L11 and their related genes mRNA expression in gastric cancer cell lines SGC-7901 and MGC-803.The results showed that compared with the miR-NC transfection group,the mRNA levels of PTEN and BCL2L11 in the miR-4295 transfection group were significantly reduced(p < 0.05),while the mRNA expression of PI3 K,AKT and mTOR were not significantly different.However,the expression of miR-4295 in gastric cancer cells SGC-7901 and MGC-803 was down-regulated,and compared with the Anti-miR-NC transfection group,the mRNA levels of PTEN and BCL2L11 in the miR-4295 inhibitor group were significantly increased,while the expression levels of PI3 K,AKT and mTOR genes were not significantly different.Western blot was used to detect the effects of miR-4295 on the expression levels of target genes PTEN,BCL2L11 and their related proteins in gastric cancer cell lines SGC-7901 and MGC-803.The results showed that,compared with the miR-NC transfection group,the protein levels of PTEN and BCL2L11 in the miR-4295 transfection group were significantly reduced,while the protein expression levels of PI3 K,p-AKT and p-mTOR genes were significantly increased(p < 0.05).However,the expression of miR-4295 in gastric cancer cells SGC-7901 and MGC-803 was down-regulated,compared with the Anti-miR-NC transfection group,the protein levels of PTEN and BCL2L11 in the miR-4295 inhibitor group were significantly up-regulated,while the protein expression levels of PI3 K,p-AKT and p-mTOR genes were significantly reduced(p <0.05).Part 2 Study on expression and function of lncRNA HOTAIR in gastric cancerMethods: The expression of HOTAIR among gastric cancer cell lines SGC-7901,MGC-803,BGC-823,MKN-28 and human gastric epithelial cell line GES-1 were detected by qRT-PCR in this study.The expression level of HOTAIR in 408 cases of gastric cancer was analyzed by GEPIA(Gene Expression Profiling Interactive Analysis)based on TCGA and GTEx public database.The sequence of small interfering HOTAIR was chemically synthesized.The siHOTAIR was transfected into gastric cancer cell lines SGC-7901 and MGC-803 by instantaneous transfection,and the expression of HOTAIR were detected by qRT-PCR.The effect of siHOTAIR on the proliferation activity of gastric cancer cells was detected by CCK-8 assay.Scratch test was used to detect the effect of siHOTAIR on the migration ability of gastric cancer cells.Effects of siHOTAIR on invasion and migration of gastric cancer cells were detected by transwell assay.UCSC(Version 2009)and StarBase v2.0were used to predict the binding site between miR-4295 and HOTAIR.Finally,the site binding between miR-4295 and HOTAIR was further verified by the double luciferase reporter gene system experiment.Results:1.The expression levels of HORAIR in gastric cancer cell lines(SGC-7901,MGC-803,BGC-823 and MKN-28)were analyzed.The results of qRT-PCR showed that(quantitative expression results were expressed as Means ± SD,and SGC-7901,MGC-803,BGC-823 and MKN-28 as follows: 1.000 ± 0.1000,0.370 ± 0.040,0.280 ± 0.030,0.310± 0.040,respectively).Compared with GES-1(0.190 ± 0.070),the expression levels of HOTAIR in human gastric epithelial cell lines were all increased.The expression levels of SGC-7901 and MGC-803 were the most significant.GEPIA was used to analyze the expression of HOTAIRXXin gastric cancer tissues,which was significantly higher than that in normal gastric mucosal tissues.2.siHOTAIR and siNC were transiently transfected into gastric cancer cell lines SGC-7901 and MGC-803,respectively.After transfection for 48 h,the expression level of HOTAIR was detected by qRT-PCR.The results showed that compared with siNC transfected of the gastric cancer cell lines SGC-7901 and MGC-803,the expression levels of HOTAIR in the gastric cancer cell lines SGC-7901 and MGC-803 transfected with siHOTAIR were significantly down-regulated(p < 0.05).3.CCK-8 assay was used to detect the proliferation activity of gastric cancer cells after transfection.The results showed that compared with the gastric cancer cell lines SGC-7901 and MGC-803 in the siNC transfection group,the proliferation activity of the gastric cancer cell lines SGC-7901 and MGC-803 transfected by siHOTAIR was significantly reduced(p < 0.05).4.The impact of transfection on the migration of gastric cancer cells was detected by scratch test.The scratch test results showed that downregulation of HOTAIR expression in gastric cancer cells SGC-7901 and MGC-803,the migration spacing of cells in the siHOTAIR group was large at 24 h compared with siNC group(p < 0.05).5.Transwell assay was used to detect the effect of transfection on the migration and invasion of gastric cancer cell lines.The results showed that compared with the siNC group,the number of cell migration and invasion in the siHOTAIR group was significantly reduced(p < 0.05).6.The sequencing results of the positive cloning of HOTAIR and its mutant double luciferase plasmid showed that the recombinant HOTAIR and its mutant sequences were inserted correctly,indicating the successful construction of the psiCHECK2-HOTAIR and psiCHECK2-HOTAIR-mut recombinant plasmids.The results of double luciferase activity showed that compared with the HOTAIR + miR-NC co-transfection group,the luciferase activity of the HOTAIR + miR-4295 co-transfection group was significantly reduced,and the difference was statistically significant,indicating that miR-4295 could bind to HOTAIR.Compared with the HOTAIR mut + miR-NC co-transfection group and the HOTAIR mut + miR-4295 co-transfection group,there was no significant difference in luciferase activity between the two groups,indicating that miR-4295 could not bind to HOTAIR with a target mutation.These results suggest that miR-4295 can directly bind to HOTAIR.Conclusions:1.MiR-4295 and HOTAIR were highly expressed in gastric cancer cell lines SGC-7901,MGC-803,BGC-823,MKN-28 and gastric cancer tissues,and the expression level of miR-4295 was positively correlated with the tumor size and distal metastasis of gastric cancer.2.MiR-4295 can promote the growth,proliferation,invasion and migration of gastric cancer cells,while down-regulation of the expression of miR-4295 can inhibit the growth,proliferation,invasion and migration of gastric cancer cells.MiR-4295 combined with AKT inhibitor can reduce the proliferation,invasion and migration of gastric cancer cells and increase the apoptosis rate of gastric cancer cells.3.PTEN and BCL2L11 were direct target genes of miR-4295.Up-regulation of miR-4295 in gastric cancer cell lines SGC-7901 and MGC-803 could significantly increase the protein expression levels of PI3 K,p-AKT and p-mTOR,while down-regulation of miR-4295 in gastric cancer cell lines SGC-7901 and MGC-803,the protein expression levels of PI3 K,p-AKT and p-mTOR were significantly reduced.4.Down-regulation of HOTAIR could inhibit proliferation,invasion andmigration of gastric cancer cells,and found that miR-4295 had binding sites with HOTAIR. |
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