Study On The Mechanism Of FN1 MRNA 3’UTR,as The Core Of CeRNA Regulatory Network,Influencing The Invasion And Metastasis Function Of Gastric Cancer Through Multiple Targets

Background and objective: In China,the diagnosis rate of early gastric cancer(GC)is low and the prognosis of stage III/IV patients is poor.Therefore,in view of the current situation of GC in China,it is urgent to promote studies related to early diagnosis and precise treatment.There are many non-codingRNAs with unknown functions existing in the human genome,and exploring the mechanism of non-codingRNAs in tumor development can help to clarify the specific molecular mechanism of tumor formation.Among the various functions of non-codingRNAs,the mode of competing endogenousRNA(ceRNA)has been discussed by many researchers.Given its network regulation mode,exploring the core-acting molecules can help to effectively screen the targets of targeted therapy and improve the therapeutic effect.Fibronectin(FN)is a multifunctional protein found in the extracellular matrix(ECM).As a large glycoprotein in this family,FN1 plays various roles in tumor microenvironment.However,the mechanisms and functional differences of FN1 at theRNA and protein levels inside tumor cells,especially in GC cells,need to be further explored.Therefore,this study aims to clarify the prognostic impact of FN1 in GC at theRNA and protein levels and its correlation with clinicopathological characteristics.And we plan to investigate the effect of FN1 3’UTR on GC cells through in vitro and in vivo experiments,and to compare whether there are functional differences between theRNA and protein levels.The ceRNA regulatory network with FN1 3’UTR as the core was further constructed by sequencing technology and validated in experiments,and the target genes that play a central role in the network were screened.The study also delves into the specific molecular mechanisms underlying the effects of FN1 3’UTR and the molecular basis for the functional differences at theRNA and protein levels.Methods: In this study,we analyzed the expression levels of FN1 mRNA in GC and paracancerous tissues via the data of TCGA-STAD cohort.And we used the FN1 3’UTR expression data in 222 GC patients’ cancer tissues,which from the First Affiliated Hospital of China Medical University,obtained from in situ hybridization experiments and FN1 protein expression data in 102 GC patients’ cancer tissues,which from the First Affiliated Hospital of China Medical University,obtained from previous studies to analyze the correlation with the clinicopathological characteristics of GC;survival analyses were applied to explore the relationship of prognosis with FN1 expression at theRNA and protein levels in multiple cohorts.The expression levels of FN1 at theRNA and protein levels in GC cell lines were measured by Western Blot and qRT-PCR assays,and suitable cell lines were selected for subsequent experiments.The FN1 3’UTR overexpression cells and FN1 protein overexpression cells were constructed by plasmid transfection and evaluated by migration invasion assay,scratch assay and adhesion assay in vitro.The stable overexpression FN13’UTR cell line was constructed by lentivirus and were injected into the tail vein or abdomen of nude mice for assessment of cell invasion and metastasis in vivo.Up-regulated differentially expressed genes(DEGs)and highly enriched miRNAs after FN1 3’UTR overexpression were obtained by transcriptome sequencing and miRNA sequencing afterRNA pull-down.The functional and signaling pathway changes of cells after FN1 3’UTR overexpression were analyzed.The ceRNA regulatory network with FN1 3’UTR as the core was constructed,and the oncogenic target genes were screened to construct the oncogenic sub-network and core sub-network via the public databases.The dual-luciferase reporter gene assay,qRT-PCR and functional assays were used to validate the regulating relationship of FN1 3’UTR-miRNAs.The relationship of miRNAs-target genes in the core sub-network was verified by Western Blot and qRT-PCR assays,and the ceRNA mechanism of FN1 3’UTR-target genes was verified by RIP assays.We used Western Blot to validate the signaling pathway alterations generated after FN1 3’UTR overexpression in the sequencing analysis.The target genes were screened by bioinformatics analyses to serve as key target genes.The key target gene was further intervened to verify their roles and specific molecular mechanisms in the process of FN1 3’UTR affecting the phenotype of GC cells.Results: Analysis of transcriptome expression profiles and clinicopathological characteristics of TCGA-STAD cohort revealed that the expression level of FN1 mRNA was significantly higher in the cancer tissues of 375 patients than in the paracancerous tissues of 32 patients,and the expression level was significantly correlated with T stage and tumor differentiation degree.The expression levels of FN1 3’UTR in 222 clinical GC patients’ cancer tissues were analyzed by in situ hybridization experiment,and we found that FN1 3’UTR was highly expressed in 65.8% of patients,and the expression level was significantly correlated with T stage(P = 0.025)and N stage(P = 0.031).The expression data of FN1 protein from 102 clinical GC patients’ cancer tissues was obtained from the previous study of we published,and we revealed that the FN1 protein expression level was only correlated with T stage(P = 0.028).In the prognostic analysis,the postoperative survival of patients with high FN1 mRNA or 3’UTR expression was significantly worse than those with low expression in the TCGA-STAD cohort,the ACRG cohort(GSE62254dataset)and the 222-patient cohort(all P<0.05),and the FN1 3’UTR expression level was confirmed to be an independent risk factor for prognosis in the analysis of the 222-patient cohort.Whereas,no significant correlation between FN1 protein expression level and prognosis was found in the 102-patient cohort(P=0.807).The expression levels of FN1 3’UTR,FN1 mRNA and FN1 protein in SGC7901,MGC803,HGC27,AGS and MKN45 cell lines were detected by Western blot and qRTPCR assays,and HGC27 and AGS were selected as the main subjects for the lowest expression.The invasion and migration assays revealed that FN1 3’UTR overexpression significantly improved the invasion and migration abilities,scratch healing speed and adhesion ability of GC cells,and FN1 protein overexpression promoted these abilities to a lesser extent than FN1 3’UTR overexpression.After constructing GC cell lines stably overexpressing FN1 3’UTR,transcriptome sequencing of HGC27 after stably overexpressed FN1 3’UTR yielded 323 DEGs with 241 up-regulated and 82 downregulated genes.GSEA enrichment analysis of transcriptome sequencing data revealed that FN1 3’UTR also affects the epithelial-mesenchymal transition(EMT)-related signaling pathway,TGFβ signaling pathway and receptor interaction in ECM.Based on the results of sequencing data analyses,we further investigated the relationship between GC cells and mesothelial cells,and found that HMr SV5 had different abilities to adhesion and resistance on the invasion of GC cells after co-culture with FN1 3’UTR overexpression,FN1 protein overexpression and control cells,respectively.After co-cultured with FN1 3’UTR overexpressed or FN1 protein overexpressed GC cells,the barrier ability of HMr SV5 to GC cells was weakened compared to the control group,with HMr SV5 being the weakest after co-cultured with FN1 3’UTR overexpressed cells.And adhesion assays suggested that HMr SV5 after co-cultured with FN1 3 ’UTR overexpressed cells significantly enhanced the adhesion ability to GC cells and was stronger than HMr SV5 after co-cultured with FN1 protein overexpressed cells.We also found that the overexpression of FN1 3’UTR in HGC27 could promote the formation of metastatic nodules in lung in vivo after tail vein injection in nude mice,and were also found to be more easily implanted in the peritoneal mesentery after intraperitoneal injection.To further build the ceRNA regulatory network,RNA pull-down assay was performed on FN1 3’UTR stably overexpressed HGC27 and AGS cells,and 59 miRNAs with high enrichment on FN1 3’UTR were obtained by miRNA sequencing,and 16 miRNAs were obtained by rigorous screening.Target Scan 7.2 and DIANA Micro T-CDS databases were used to predict the target genes of 16 miRNAs,and the ceRNA regulatory network was constructed by combining the results of above analyses:FN1 3’UTR served as the core in the ceRNA regulatory network to regulate the mRNA expression of 66 target genes by highly enriching 16 miRNAs.Through the expression profile and prognostic data of the ACRG cohort,we obtained 14 genes that play a tumorpromoting role in the 66 target genes and constructed oncogenic sub-network and core subnetwork.In order to validate the relationship in the core sub-network of FN1 3’UTR with hsa-let-7i-5p,hsa-mi R-629-5p,hsa-mi R-423-5p,and hsa-mi R-296-3p,we applied qRTPCR,dual luciferase reporter gene and functional assays.After transfections of the above four miRNAs’ mimics in FN1 3’UTR overexpressed cells,respectively,we found the expression level of FN1 3’UTR was decreased and the tumor-promoting effect of FN13’UTR overexpression was restored.The dual luciferase reporter gene assay suggested the existence of direct binding sites for the above four miRNAs in the FN1 3’UTR region.Western blot,qRT-PCR assays and database prediction were used to clarify that let-7i-5p could down-regulate the expression of THBS1 and CPED1,and mi R-629-5p could down-regulate the expression of AMOTL2 in the core sub-network.We confirmed the positive correlation trend of expressions of THBS1,CPED1 and AMOTL2 with FN1 mRNA in both ACRG cohort and TCGA-STAD cohort,with THBS1 showing the highest correlation.And bioinformatics analyses also revealed that a high THBS1 expression in both ACRG cohort and TCGA-STAD cohort suggested poor prognosis.The RIP assay via Anti-Ago2 antibody confirmed the ceRNA regulatory mechanism that FN1 3’UTR regulates THBS1,CPED1 and AMOTL2 expression levels through competitive enrichment of miRNAs andRNA-induced silencing complexes.Based on the results of GSEA enrichment analysis,immunofluorescence staining,Western Blot and Elisa assays revealed that FN1 3’UTR overexpression significantly promoted the transformation of EMT(up-regulation of N-cadherin,Slug and Snail,down-regulation of E-cadherin,ZO1,β catenin and CLDN1),up-regulate the expression and exocytosis of MMP2/9 and TGFβ1/2.We screened THBS1 as a key factor in the ceRNA network and used si THBS1 to down-regulate the expression of THBS1 in FN1 3’UTR overexpressed cells.Functional assays evaluated that downregulation of THBS1 expression could restore the tumorpromoting effect,the EMT phenotypic changes and MMP2/9 expression levels after FN13’UTR overexpression.Immunofluorescence staining experiments revealed that the degree of EMT in the FN1 protein overexpressed cells was lower than that in the FN1 3’UTR overexpressed cells,and the expression level of THBS1 and the exocytosis level of TGFβ1/2 positively regulated by THBS1 were higher in the FN1 3’UTR overexpressed cells than in the FN1 protein overexpressed cells.Conclusions: 1.In GC,the postoperative survival of patients with high FN1 3’UTR expression was significantly worse than those with low expression,which can be used as an independent risk factor for prognosis assessment of GC,and expression level of FN13’UTR was correlated with pathological information related to invasive phenotype;2.FN13’UTR overexpression significantly increased the invasive and migration abilities of GC cells in vivo and in vitro assays,and we was also confirmed that FN1 3’UTR promoted the malignant phenotype of cells to a greater extent than FN1 protein in vitro assays;3.Based on the analysis of sequencing data,FN1 3’UTR regulates a large molecular network in GC through ceRNA mechanism,in which it has been demonstrated that FN1 3 ’UTR could upregulate the expression of THBS1,CPED1 and AMOTL2 through enrichment of hsa-let-7i-5p,hsa-mi R-629-5p,hsa-mi R-423-5p,hsa-mi R-296-3p;4.FN1 3 ’UTR may promote cell invasion and migration by promoting EMT,up-regulating the expression and exocytosis of TGFβ1/2 and MMP2/9 in GC;5.FN1 3’UTR—let-7i-5p—THBS1 axis was confirmed to be one of the core factors in this ceRNA network,and the regulatory relationship between FN1 3’UTR and THBS1 might be one of the main factors leading to the difference of FN1 3’UTR and FN1 protein in promoting cancer.