The Function And Molecular Mechanisms Of Hsa?circ?0003204 In The Growth And Metastasis Of Human Cervical Cancer

Background and ObjectiveCervical cancer(CC)is the most common gynecological malignancy in women.It is still the main cause of death due to cancer in women in developing countries,and the metastasis and recurrence of CC are still the difficulties in the treatment of cervical carcinoma.In recent years,a large number of studies have shown that circRNAs are involved in the regulation of tumorigenesis and progression through the regulation of related signaling pathways by acting as a sponge of miRNA,but there are still few reports on the mechanisms of.cervical cancer.We selected targeted circular RNA through high-throughput sequencing methods,investigated its impact on the biological behavior of cervical cancer cells.The mechanisms of circular RNA in CC were explored through the dual luciferase reporter gene experiment,WB experiment,PCR amplification,nude mouse tumorigenicity assay.Circular RNA could regulated the growth?development in cervical cancer.This study offered a new method and theoretical basis for the diagnosis?treatment?prevention and treatment of recurrence for cervical cancer.MethodChapter 1:Differential expressed circRNAs were screened in cervical cancer tissues by high-throughput sequencing and verifying their expression in tissues and cell lines.hsa_circ₀003204 was verified and selected for further study.The relationships between hsa_circ₀003204 and the clinical and pathological characteristics of cervical cancer were explored.Chapter 2:In vitro experiments,hsa_circ₀003204 was downregulated by siRNA in cell transfection,upregualted by overexpressed plasmids of CC cell lines.After the expression of hsa_circ₀003204 determined by-Real time quantitative PCR(qRT-PCR),cell proliferation ability?cell cycle and apoptosis?migration and invasion ability were verified by MTS?flow cytometry?Transwell experiment.The mechanisms of hsa_circ₀003204 in the proliferation,apoptosis,migration and invasion of cervical cancer cells were explored.Chapter 3:MAPK signaling pathway was selected by GO enrichment assay and KEGG Pathway enrichment analysis.hsa-mir-7-5p and PAK1 was screened by bioinformatics prediction.Double luciferase reporter gene assay,Western Blot experiments and real-time PCR were used to confirm that hsa_circ₀003204 could regulate MAPK pathway by acted as sponge of hsa-mir-7-5p.Chapter4:Cervical cancer cell suspension transfected by overexpressed lentiviral vector system were injected subcutaneously in nude mouse tumorigenicity assay.The differences in subcutaneous tumor size,weight and protein expression levels of related signaling pathways between the overexpression group and the control group were performed to verify the function of hsa_circ₀003204 in the regulation to MAPK signaling pathway and to verify its role in the growth of cervical cancer cells.ResultChapter 1:There were differential expressions of circRNAs in cervical cancer tissues and adjacent normal tissues.And hsa_circ₀003204 was a circRNA and was significantly up-regulated in cervical cancer tissues and cervical cancer cell lines.The expression level of hsa_circ₀003204 were significantly up-regulated in cervical cancer patients with vascular invasion?lymph node metastasis.Chapter 2:In vitro,the down-regulation of hsa_circ₀003204 could inhibit the growth of cervical cancer cells,inhibit their migration and invasion,promote their apoptosis.The up-regulation of hsa_circ₀003204 could promote the growth of cervical cancer cells,reduce their apoptosis,and increase the ability of invasion and migration.Chapter 3:Hsa_circ₀003204 can up-regulate PAK1 and activate MAPK pathway by regulating hsa-miR-7-5p.Chapter 4:The overexpression of hsa_circ₀003204 can activate the MAPK pathway and promote the growth of cervical cancer cells in vivo.ConclusionHsa_circ₀003204 can up-regulate PAK1 and activate MAPK pathway by binding to hsa-miR-7-5p,thus promoting the growth of cervical cancer.