| Cervical cancer is a primary cancer of the uterine cervix in females and has a high incidence and mortality. The mortality of cervical cancer is decreasing in advanced countries with well healthcare systems, but the incidence is still high in developing countries and the disease is the second most common female cancer worldwide, with an estimated global incidence of 470,000 new cases and approximately 233,000 deaths per year. Radical hysterectomy with pelvic lymphadenectomy and definitive radiotherapy (external pelvic beam irradiation plus brachytherapy) yield similar disease-free survival and survival rates in early cervical cancer whereas concurrent chemo-radiotherapy is the primary treatment for locally advanced disease with poor treatment effection which is the leading cause of death in patients with malignant tumor.Focal adhesion that is the binding sites of cells and cells or cell and extracellular matrix have changed, which may be one of mechanisms of tumor tissues with local invasion and distant metastasis.Focal Adhesion Kinase (FAK) is a non-receptor tyrosine kinase which plays a significant role in integrin mediated signal transduction pathway, involving in survival, motility, invasion, metastasis, angiogenesis, lymphangiogenesis, cancer stem cell functions, tumor microenvironment and epithelial to mesenchymal transition (EMT). However, the role and mechanism of FAK in cervical cancer is still not clear.MicroRNAs (miRNAs) are endogenous, small noncoding single-stranded RNAs that regulate body’s almost all important physiological process including growth, development, differentiation, metabolism and death. miRNAs can act as either oncogenes or suppressor genes by interacting with mRNAs 3’untranslated region(3’UTR) and inducing either translation suppression or degradation of mRNA at the post-transcriptional level, and might represent promising biomarkers for human cancer. Emerging evidence suggests that miR-7 as a tumor suppressor might be involved in the cell proliferation and migration of many human cancers including cervical carcinoma, and bioinformatics analysis predicts that FAK 3’UTR and miR-7 have conservative binding binding sites in a variety of biological species, then whether miR-7 can target FAK affecting cell proliferation and migration in cervical cancer deserves further study.Latest research has pointed out that circular RNA (circular RNA, circRNA) as competitive endogenous RNA (ceRNA) can competitively combine with the miRNA recognition element(MRE) of miRNA mRNA 3’UTR region, to regulate expression of mRNA and protein of miRNA-target genes by blocking the degradation or restraint of the target mRNA. Studies have shown that antisense to the cerebellar degeneration-related proteinl transcript (CDRlas, also termed ciRS-7), which harbors more than 70 conventional miR-7 binding sites, can reduce miR-7 activity and increased levels of miR-7-targeted transcripts in mouse neural tissues. However, what is the relationship between ciRS-7 and miR-7 and whether ciRS-7 can regulate miR-7-targeted transcripts by competitively combing with the miR-7 in cervical cancer have not reported so far.Shikonin, a natural naphthoquinone pigment isolated from Lithospermum erythrorhizon, its chemical structure is naphthoquinone compounds, has been reported play an important role in anti-inflammatory and anti-tumor. Studies have reported that Shikonin can inhibit cell proliferation, invasion and migration of a wide variety of tumor. However, the study about the role and mechanisms underlying the effect of Shikonin on cell proliferation, invasion and migration of cervical cancer are not anderstood.In the present study, we choose to detect human cervical cancer cells to study the expression and significance of FAK in cervical squamous cancer, and to study whether ciRS-7 and miR-7 can infect cell proliferation, invasion and migration in cervical cancer by targeting FAK, and to study whether shikonin can suppress cell proliferation, invasion and migration by inhibitting phosphorylation of FAK in cervical cancer. The research will be described in four parts as follows:Part I The expression and clinical significance of the FAK in cervical cancerObjective:To study the expression of focal adhesion kinase (FAK) in normal cervical tissues and cervical cancer tissues, and to explore the relationship between FAK and occurrence and development, invasion and metastasis of cervical squamous carcinomas.Methods:The mRNA and protein expression of FAK were detected in 47 cases of cervical cancer tissues and 35 cases of normal cervical tissues which were paraffin samples and fresh frozen Biopsy specimen by immunohistochemical SP and qRT-PCR.Results:1,The expression of the FAK is higher in cervical cancer than normal cervical tissue, meaning the two groups are significant difference (P< 0.05).2,FAK expression is increasing in FIGO stage Ⅲ-Ⅳ than stage Ⅰ-Ⅱ of cervical cancer, meaning there is a significant difference (P< 0.05).3,The expression of the FAK is decreasing in cervical cancer infiltration depth< 1/2 organization than infiltration depth> 1/2 organization, meaning there is a significant difference (P< 0.05).4,The FAK expression is obviously increasing in cervical cancer tissue with lymph node metastasis of cervical cancer than cancer tissue without lymph node, with significant differences between the two groups (P< 0.05).Conclusions:The expression of FAK increasing gradually along with the progression of the cervical cancer, which may be involved in the occurrence, invasion and metastasis process of cervical cancer, may be one of the high-risk biomarkers used as a judgment index from early cervical cancer to advanced cancer, providing a new predictive molecular target for diagnosis and prognosis of cervical cancer.Part II The mechanism of miR-7 inhibit the cell proliferation, invasion and migration of cervical cancer through FAK pathwayObjective:To Study the effection of miR-7 on the cell proliferation, invasion and migration of cervical cancer, and to explore whether its mechanism is related to miR-7 targets gene FAK.Methods:Constructed miR-7-MSCV-PIG plasmid, and detected the cell proliferation of cervical cancer Hela and C33A cells transfected with miR-7 by FCM. Detected the cell invasion and migration of cervical cancer Hela and C33A cells transfected with miR-7 by transwell. Constructed FAK-PGL3 plasmid following predicting that FAK is the potential target of miR-7 by bioinformatics, and then detected that whether FAK can be derectly regulated by miR-7 through Dual Luciferase Reporter Gene methods in HER293 cells. Detected the expression of FAK mRNA and protein in cervical cancer Hela and C33A cells transfected with miR-7 by qRT-PCR and Western blotting.Results:1,The expression of miR-7 obviously increased in cervical cancer Hela and C33A cells transfected with miR-7-MSCV-PIG plasmid than MSCV-PIG plasmid(negative control) (P< 0.05).2,The GFP fluorescence which reacting the cell proliferation weakened gradually in cervical cancer Hela and C33A cells transfected with miR-7(P< 0.05).3,The invasion and migration of cells were suppressed in cervical cancer Hela and C33A cells transfected with miR-7(P< 0.05).4,FAK 3’UTR have conservative binding sites with miR-7 in a variety of biological cells including human beings by bioinformatics, and then verified that miR-7 can be negatively regulated through biding with FAK 3’UTR by Dual Luciferase Reporter Gene methods in HER293 cells.5, The expression of FAK mRNA and protein is obviously decreased in cervical cancer Hela and C33A cells transfected with miR-7.Conclusions:miR-7 inhibited the cell proliferation, invasion and migration of cervical cancer through targeting FAK, to clarify that FAK and miR-7 were involved in the occurrence, invasion and metastasis process of cervical cancer, which may be new molecular targets for interventing the prognosis and treatment of cervical cancer.Part Ⅲ The mechanism of ciRS-7 regulate FAK expression through competitivly combining with miR-7 to promote the cell proliferation, invasion and migration of cervical cancerObjective:To Study the relationship of ciRS-7 and miR-7 in cervical cancer tissues, and to explore the effection of ciRS-7 on the cell proliferation, invasion and migration of cervical cancer, and to explore whether its mechanism is related with regulating FAK through competitivly combining with miR-7.Methods:Detected the expression of miR-7 and ciRS-7 in cervical cancer tissues and normal cervical tissues, and analyze the relationship of them. Detected the expression of ciRS-7 and miR-7 in cervical cancer Hela cells transfected with miR-7 and ciRS-7 by qRT-PCR. Detected the cell proliferation in cervical cancer Hela and C33A cells transfected with ciRS-7 by trypan blue cell count. Detected the cell invasion and migration of cervical cancer Hela and C33A cells transfected with ciRS-7 by transwell. Detected the expression of FAK mRNA and protein in cervical cancer Hela and C33A cells transfected with ciRS-7 by qRT-PCR and Western blotting.Results:!,The expression of miR-7 is obviously higher in cervical cancer tissues than in normal cervical tissues, and the expression of ciRS-7 is obviously lower in cervical cancer tissues than in normal cervical tissues (P<0.05), and there is significantly negative relationship between the expression of miR-7 and ciRS-7 (r=-0.644, P<0.05).2,The expression of ciRS-7 and miR-7 decreased obviously in cervical cancer Hela cella.3,ciRS-7 promoted the cell proliferation, invasion and migration of cervical cancer and upregulated the expression of FAK mRNA and protein in cervical cancer Hela and C33A cells transfected with ciRS-7.Conclusions:There is significantly negative relationship between the expression of miR-7 and ciRS-7 in cervical cancer tissues and cells. ciRS-7 promoted the cell proliferation, invasion and migration of cervical cancer by upregulating FAK through competitivly combining with miR-7. Molecular targeted therapy for the RNA regulation network is expected to afford a new direction for diagnosis and treatment for cervical cancer. Part IV The mechanism of Shikonin inhibit the cellProliferation, Invasion and Migration in Cervical Cancer Hela Cells through FAK pathway Objective:To investigate the effect of shikonin on proliferation, invasion and migration of human cervical cancer Hela cells and to explore the possible mechanism. Methods:The human cervical cancer Hela cells were cultivated and divided into control group and shikonin group (concentration respectively 4,8,16,32,64 umol·L-1). The effect of shikonin on cell viability, apoptosis, invasion and migration of cells cultivated after 24 h were studied by MTT assay, flow cytometric, transwell assay, respectively. When cells cultivated after 24 h, the expression of FAK protein, the level of phosphorylated FAK, and mRNA were assessed by western blot and quantitative real-time PCR analysis.Results:Shikonin inhibited proliferation and induced apoptosis of Hela cells in a dose-dependent manner. When treated with low-toxic doses of shikonin (4,8,16 μmol·L-1), cell invasion and migration were markedly suppressed. Furthermore, shikonin significantly down-regulated the expression of FAK and the level of phosphorylated FAK. In addition, shikonin could increase the expression of FAK mRNA.Conclusions:Shikonin inhibits proliferation and invasion of cervical cancer Hela cells by inhibiting phosphorylation of FAK.In summary, the high expression of FAK is one of the mechanisms for development and metastasis of cervical cancer. FAK is the potential targets of miR-7. miR-7 inhibit the cell proliferation, invasion and migration of cervical cancer through targeting FAK. ciRS-7 promot the cell proliferation, invasion and migration of cervical cancer by upregulating FAK through competitivly combining with miR-7. Further study shows that traditional Chinese medicine monomer shikonin can inhibit cell proliferation and invasion of cervical cancer Hela cells by inhibiting phosphorylation of FAK. In this study, we investigate the effection of FAK and its related RNA regulation network on cell proliferation, invasion and migration of cervical cancer and afford new Molecular target for diagnosis and treatment of cervical cancer, trying to treat cervical cancer by combining traditional Chinese and western medicine. |
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