Study On The Role And Mechanism Of CircRNA CircFN1 In The Occurrence And Development Of Gastric Cancer | Posted on:2022-08-12 | Degree:Master | Type:Thesis | Country:China | Candidate:N Lin | Full Text:PDF | GTID:2544306335970089 | Subject:Surgery | Abstract/Summary: | PDF Full Text Request | Objective:Gastric cancer(GC)remains one of the most malignant tumors worldwide.Globocan 2020 statistics show that among the eight tumor types with the highest morbidity and mortality,GC global morbidity and mortality rank sixth and fourth,respectively.Circular RNAs(circRNAs)play an important role in regulating gene expression,but the specific mechanism of circRNAs in GC remains very limited.Chip analysis of human GC tissue and carcinoma-adjacent tissue and circRNA expression profiles showed that,in the cancerous tissue,713 circRNAs,including hsacirc0058132,had significantly higher or lower expression by>2 times.Via the circBase database,we found that hsacirc0058132 was located on chromosome 2q35;inferring from the host gene FN1(fibronectin 1,protein fiber connection),we termed it circFN1 Based on the early research results,we selected circFNl as the object of further study,and explored its biological function and mechanism of action to provide a theoretical basis for targeted therapy of GC.Methods:The GC cell lines SGC-7901 and BGC-823 were infected by lentivirus plasmid to construct the lentiviral vector,and a stable GC cell line(sh-circFN1)and negative control(sh-NC)were constructed.The role of circFN1 in GC cell proliferation was verified with the Cell Counting Kit-8 assay.The role of circFN1 in GC cell migration and invasion was detected with the Transwell assay.The effect of circFN1 on xenograft tumor growth was evaluated with a nude mouse subcutaneous tumor model.Further,we established a nude mouse lung metastasis model to observe the effect of circFN1 on GC cell lung metastasis.For identifying the downstream target genes,the target genes were first predicted with bioinformatics,then circRNA pulldown was combined with RT-qPCR,and verified with the dual luciferase assay,functional complement assay,and western blotting to reveal the specific molecular mechanism of circFN1 involvement in GC occurrence and development.Based on sh-circFN1 and sh-NC,miRNA and mRNA expression profiles were detected using high-throughput sequencing,and the expression profiles of differentially expressed miRNAs and mRNAs were analyzed with bioassay to construct miRNA-mRNA pairs significantly related with GC.Results:The in vitro cell function testing showed that,compared with the sh-NC group,the sh-circFN1 group had significantly inhibited GC cell invasive and migration ability.The in vivo tumor transplantation showed that,compared with the sh-NC group,the sh-FN1 group had significantly reduced subcutaneous GC cell tumor-forming ability and significantly inhibited tumor-forming ability of lung metastases.Bioinformatics analysis,circRNA pulldown,dual luciferase assay,functional complement assay,and western blotting confirmed that circFNl could directly bind to miR-515-5p in the GC cells.The possible downstream target gene of miR-515-5p was MARK4.High-throughput sequencing yielded a total of 2390 significantly differentially expressed mRNAs:1130 were upregulated and 1260 were downregulated.A total of 483 miRNAs were significantly differentially expressed:220 were upregulated and 263 were downregulated.Conclusion:Inhibiting circFN1 can significantly inhibit GC cell invasion and migration.circFNl acts as an miR-515-5p sponge,which may relieve the inhibition of miR-515-5p of the downstream target gene MARK4,subsequently upregulating MARK4 expression and ultimately promoting GC cell invasion and migration.The differential miRNA and mRNA expression profiles in the GC cells were obtained.A total of 2390 significantly different mRNAs and 483 significantly different miRNAs were screened.Based on the differentially expressed miRNAs and mRNAs,a total of 2220 relationship pairs were formed between the top five upregulated miRNAs and mRNAs,where the miRNA with the most association pairs was hsa-miR-29c-3p.A total of 1549 miRNA-mRNA pairs were formed between the bottom five downregulated miRNAs and mRNAs,where the miRNA with the most correlations was hsa-miR-193a-3p. | Keywords/Search Tags: | CircFN1, Proliferation, Migration and Invasion, Sequencing, Gastric Cancer | PDF Full Text Request | Related items |
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