Study On The Promoting Angiogenesis Of Ovarian Carcinoma By Long Non-coding RNA TUG1 Via Upreglating The Expression Of LRG1

Background:The lethality rate of ovarian cancer(OC)is most highly in female carcinoma as the complicated pathogenesis,difficult decttion,diagnose and therapy in early stage.Study on pathogenesis of OC are benefit to analysis on pathogeny of tumor tissue and providing the basis for diagnose and therapy in clinic.Angiogenesis,the symbol of oncogenesis,can provide oxygen and nutriment for prolification,differentiation and metastasis of tumor.On the contrary,if the angiogenensis is inhibitated,the effect of described above would be opposite.The inhibitor of angiogenesis is first-line therapeutic drug for cancer.Study on angiogenesis of tumor is significant for diagnose and therapy.Long non-coding RNA(lncRNA)can mediate the biologic function via regulating the expression of RNA,DNA or protein.LncRNA TUG1 which is up regulated in many tumor tissues can mediate the prolification,differentiation and angiogenesis.This project is aim to study on the expression level of lncRNA TUG1 in ovarian tumor and analysis its regulatory mechanism on tumor via angiogenesis.Part Ⅰ The expression level of LncRNA TUG1 in ovarian cancer and its clinical meaningsObject:The measurement on expression level of IncRNA in tumor is the precondition and basis for the study on its regulatory on angiogenesis.The expression of lncRNA TUG1 in ovarial cancer can provide some valuable information for the oncogenesis and its clinical features.Whether the expression level of IncRNA TUG1 relate to the clinical meanings in ovarial tumor tissue is need to be clarified in this part.Material and Method:56 cases of patients with OC were recruited in this study.The tumor tissues and paracancerous normal tissues were both collected via surgery.The expression level of IncRNA TUG1 in tumor tissues and paracancerous normal tissues were measured by qRT-PCR after the extraction of total RNA.The relationship between the expression level of lncRNA TUG1 and clinical features of ovarian cancer patients were analysized by statistical software.Addionaly,the postoperative patients were tracking to determine the relationship between the expression of IncRNA TUG1 and prognosis.Results:The expression level of lncRNA TUG1 in ovarian tumor measured by qRT-PCR was significantly higher than in paracancerous normal tissue,which suggested that the expression of IncRNA TUG1 was up regulated in tumor tissue.In ovarial tumor tissue,the expression of IncRNA TUG1 related to the tumor size and N status of patient,while not with age,histological grade and clinical stage.The survival curve indicated high expression level of IncRNA TUG1 is adverse toprognosis.Conclusion:The expression level of lncRNA TUG1 was positive relative to the formation of ovarial cancer and its clinical meanings.The up regulated indicated the potential of neoplasia and poor prognosis.Part Ⅱ The effects of interference of LncRNA TUG1 on angiogenesis in ovarian canerObject:Angiogenesis in tumor tissue is the symbol of tumorigenesis.Study on the regulatory mechanism of angiogesis in ovarrial tumor tissue is the base for the therapy of ovarial cancer.As knowing the regulatory of IncRNA on tumor,the effect of expression of IncRNA TUG1 in ovarial cancer cell on angiogenesis was studied.Meanwhile,how did IncRNA TUG1 regulate the angiogenesis was analyized.Material and Method:5 strains of cells were screened by qRT-PCR via measuring the expression level of IncRNA TUG1.The screened SKOV3 and CA03 were used to study the effect of knock down of IncRNA TUG1 on proliferation,migration and angiogenesis of co-cultured HUVEC via RNA interference technology.The result of knock down was determined by qRT-PCR.HUVEC cells were treated with conditioned medium.Cell viability and clone formation assays were both used to determine the prolification ability of HUVEC cells which were culture with conditioned medium of the ovarian carcinoma cells.The wound healing and transwell assays were used to measure the metastasis ability of cells mentioned above.The effect of knocking down IncRNA TUG1 on angiogenesis was assessed by Matrigel based angiogenesis experiment in vitro.Result:LncRNA TUG1 in ovarial cancer cells were successed knocked down.Cell vibility and clone formation assay proved the knocked down of IncRNA TUG1 inhited the prolification of ovarian carcinoma cell.The results of wound healing and transwell assay indicated that the cell migration ability was also inhibited.In vitro matrigel based assay suggested that the angiogenesis can be inhibited by knock down of IncRNA TUG 1.Conclusion:The interference of IncRNA TUG1 can inhibit the profilication,metastasis,and agiogenesis of co-cultured HUVEC cell.The high expression of lncRNA TUG1 may promote the angiogenesis as its potential regulating effect.Part Ⅲ Study on the regulatory mechanism of LncRNA TUG1 on angiogenesis in ovarian cancerObject:Angiogenesis is affected by the angiogenic regulatory factor.Study on the expression level of angiogenic regulatory protein is necessary for the understanding of angiogenic mechanism.Exploring whether lncRNA TUG1 can affect the angiogenic regulatory protein and regulate angiogenesis via LRG1 was the key point.Material and Method:After interference of lncRNA TUG1,the expression level of LRG1 protein was measured by qRT-PCR,western blot and Elisa.The expression of LRG1 protein in ovarian tumor was also measured by qRT-PCR.The correlationship between lncRNA TUG1 and LRG1 protein were analyized by Spearman’ correlation coefficients.To analysis how does lncRNA TUG 1 affect on the angiogenesis,the expression of VEGF,Ang-1 and TGF-β were all measured by qRT-PCR and western blot.The group of shRNA TUG1 supplemented with LRG1 protein was set to determine whether LRG1 can promote the excretion of angiogenesis regulators(VEGF,Ang-1 and TGF-β).Result:After knock down of lncRNA TUG1,the expression of LRG1 protein measured by qRT-PCR,western blot and Elisa was down regulated.The analysis of spearman correlation suggested that the expression of lncRNA TUG1 in tumor tissue was positive correlation to the expression of LRG1 protein.The expression of VEGF,Ang-1 and TGF-β tested by qRT-PCR and western blot was down regulated after interference of lncRNA TUG1.However,VEGF,Ang-1 and TGF-β would be up regulated again when the LRG1 protein was supplemented in shRNA TUG1 group.Conclusion:After the knock down of lncRNA TUG1,the expression level of LRG1 protein,VEGF,Ang-1 and TGF-β were all down regulated.LncRNA TUG1 can mediate the angiogenesis via promoting the excretion of LRG1 protein.LRG1 protein can promote the agiogenesis via up regulating the expression of VEGF,Ang-1 and TGF-β.