| Ovarian cancer is one of the most common malignant tumors in female reproductive organs,ranking third only in cervical cancer and uterine carcinoma.Due to the development of the embryo of the ovary,the anatomical and endocrine functions are more complex,the early symptoms are not typical,and the tissue type and benign or malignancy of the ovarian tumor are difficult before operation.There is no early detection way of ovarian cancer,and about 75%of patiants were diagnosed advanced stage.Ovarian cancer is the most deadly tumor in gynecological tumors.Surgical is the most impotant treatment of ovarian cancer and also combined with chemotherapy after operation.Although 20%-25%of patients with ovarian cancer in early stage after surgery,the survival of 5 years period is more than 90%,there are still 25%-40%patients with high-risk factors to carry out chemotherapy postoperation,advanced ovarian cancer after the satisfied total stage tumor reductive surgery,the standards chemotherapy method is paclitaxel plus carboplatin,complete response rate was 50%,but advanced ovarian cancer patiants median progression-free surial still no more than 2 years.The results of the treatment of ovarian cancer are not satisfactory,so many scholars have devoted to studying the pathogenesis of ovarian cancer,improving the efficacy of first-line chemotherapy and choosing new chemotherapy drugs.The development of tumor related to tumor cells,mesenchymal cells,immune inflammatory cells and tumor blood vessels,the function of tumor growth,development,metastasis and invasion are not only related to the tumor cell's genetic mutations,also related to the tumor stromal microenvironment.The relationship between inflammation and tumor is mainly studied in two aspects.1)The function of inflammatory cytokine interleukin-17 and signal transduction mechanism.2)function of Nod like receptor(pathogen and cell stress recognition receptor)and signal transduction activation mechanism.Il-17 is a characteristic cytokine secreted by TH17 cells,with six IL-17 family members,IL-17a(IL-17),IL-17b,IL-17c,IL-17d,IL-17e(also known as IL-25)and IL-17f.Il-17f has the highest homology(approximately 50%)with IL-17,and its coding genes are located in the same segment of the chromosome 6p12.CD4+IL-17+ cells to produce IL-17 and IFN-gamma can also be induced by tumor cells synergy Th1 type CXC chemokine ligand(CXC chemokine ligand,CXCL)9 and the generation of CXCL 10,which can mobilize effect T cells into the tumor microenvironment.CD4 + IL-17 + cells to produce IL-17 and IFN-? can also be induced by tumor cells synergy Th1 type CXC chemokine ligand(CXC chemokine ligand,CXCL)9 and the generation of CXCL10,which can mobilize effect T cells into the tumor microenvironment.In epithelial ovarian cancer research found that Treg cells in the tumor microenvironment(Regulatory T cells:Regulatory cells)can be achieved by IL-2 into CD4+ IL-17+ cells,thus reduce Treg cell-mediated immune inhibition.Recent studies have shown that blocking the PI3K pathway can raise IL-17 signals by increasing IL-17RA.Il-17 activates Erk through the SEFIR-TILL structure of IL-17RA,resulting in phosphorylation of the C/EBPb Thr188,and the phosphorylation of Thr 188 is also required for the IL-17 to mediate GSK3b phosphorylation C/EBPb by the end structure of IL-17RA.The two successive phosphorylation events resulted in the loss of C/EBPb and the negative regulation of IL-17.Il-17 plays a role in different types of tumor tissues.Some scholars analyzed the colon tumor microenvironment in the distribution of all kinds of T cell subgroup and its correlation with prognosis of patients,the results showed that CD4+IL-17+ cells in patients with high expression of prognosis is poorer,and Th1 cells the prognosis of patients with high expression had better prognosis.In the study of hepatocellular carcinoma model,the prognosis of Il-17+cells in tumor tissues is negatively correlated with the prognosis of liver cancer patients.Metastasis of ovarian cancer cells leads to recurrence of ovarian cancer and poor prognosis.Peritoneal metastasis is the most common transmission path,and stage ? is associated with 5 year survival rate of 32%to 47%.The study on the mechanism of metastasis of ovarian cancer and its desire to find a way to improve the prognosis of ovarian cancer patients.MicroRNAs(miRNAs)are small,non-coding RNAs of 19-25 nucleotides that post-transcriptionally regulate gene expression.The sequence at the 5'end of the mature miRNA,called the "seed region",bind sits complementary sequence within the 3 untranslated regions(UTR)of the target mRNA.MiRNA works by regulating target genes.miRNA can play the role of cancer gene or tumor suppressor gene in tumor,and miRNA,such as let-7d,mir-127,mir-15a,mir-34a,and mir-34b,in ovarian cancer,can be used to promote ovarian cancer.Researchers detecte miRNAs expression level in ovarian cancer tissues and blood circulation to study the cancer or tumor suppressor role,and can according to the miRNA expression in different tissue to study specific miRNA regulation genes,downregulated gene by the miRNA upregulation and study the gene function lack or obtain phenotype.miRNA may be used as a new biological marker for disease diagnosis in the future,and the analog may be a targeted drug to provide new treatments for malignant tumor therapy.miRNA plays a regulatory role in various biological processes,such as regulating cell cycle,differentiation,increment,apoptosis and angiogenesis.Epithelial mesenchymal EMT(EMT)is an important way to promote the metastasis of epithelial cells,which is characterized by down-regulation of epithelial markers E-cadherin,N-cadherin,ZEB1,ZEB2 and Vimentin.miR-200 proved to directly regulate E-cadherin via targeting of its transcriptional repressors ZEB1/2,and a double-negative feedback loop between ZEB1/2,and the miR-200 family regulating EMT and promoting metastasis was demonstrated.In another study,epithelial ovarian cancer cells transfected with EMT promotion factor HMGA2 reduced the levels of miRNA-15a,miRNA-18a,miRNA-22 and miRNA-29b and the expression of the let-7 family members increased significantly.The target gene and miRNA can be regulated each other,and can be transformed between epithelium and interstitium.Recently,miRNAs proved to be involved in normal into myofibroblast cell into a cancer related cells,through miRNA-31 and lower the miRNA-214,and chemotaxis factor CCL5 are identified as target genes of miRNA-214,this suggests that the tumor microenvironment plays an important role in regulation.Other important cellular processes such as hypoxia and angiogenesis are regulated by miRNAs in ovarian cancer HIF-1 alpha and HIF-2 alpha of the 3-UTR of miRNA-199a,the expression of miRNA-199a decreased in the hypoxia environment,causing HIF to rise.Members of miRNA-200 can inhibit angiogenesis by inhibiting the target gene CXCL1 and IL-8,reducing the formation of malignant tumors.This give us the prompt,ovarian cancer risk factors and the process is not a single way,using the ovarian cancer gene and the relationship between miRNA and micrornas oncogene and tumor microenvironment cytokines and inflammatory factor relationship to downregulate metastatsis of ovarian cancer.Part 1 Expression of inflammatory cytokines in ovarian epithelial cancer and their correlationObjective:By detecting the expression of IL-17 in epithelial ovarian tumor,the relationship between its clinical characteristics and the diagnosis and treatment of ovarian cancer and its prognosis were investigated.Experimental methods:Fifty cases of postoperative tissue specimens of ovarian malignant epithelial neoplasms received from January 2013 to December 2015 were collected from the department of gynecology of qilu hospital of shandong university.All the patients were diagnosed with malignant epithelial neoplasms and 10 benign ovarian epithelial tumor tissues were used as control.Immunohistochemical detection of inflammatory cytokine IL-17A,,IL-17D,epithelial markers E-cadherin and interstitial markers Vimentin in ovarian malignant epithelial tumors,ovarian benign epithelial tumors and their expression in normal ovarian epithelial tissue.The correlation between cytokines and epithelial-mesenchymal markers was analyzed.Experimental results:1.Upon testing,it was found that IL-17A was located in cytoplasm and turned brownish yellow after staining.Through detection,it was found that IL-17D was located in the cytomembrane and cytoplasm and turned light yellow after staining.2.The immunohistochemical results showed that 50 cases of malignant ovarian epithelial neoplasm were divided into IL-17A positive group and IL-17D positive group.There was no statistically significant difference in the expression of IL-17A,IL-17D in pathological types.There was no statistically significant difference in expression rates between IL-17A and IL-17D in no ascites,less than 500ml and more than 500ml of ascites.IL-17A and IL-17D showed high positive rate of expression in Vimentin and high positive rate,while E-cadherin expressed in low positive rate.Conclusion:1.Compared with normal ovarian tissue and benign epithelial ovarian tumor tissue,the expression of IL-17A,IL-17D and Vimentin was positive in epithelial ovarian cancer.The expression of E-cadherin in epithelial ovarian cancer was low,and the positive expression rate was high in benign ovarian tumors and normal ovarian tissue.2.High clinical stage,low differentiation epithelial ovarian cancer IL-17A,IL-17D and Vimentin was high expressed and E-cadherin expressed low positive rate.There was no significant difference in the positive expression rate in the amount of ascites and the types of epithelial ovarian cancer.3.The results suggest that inflammatory cytokines and interstitial markers may promote the aggressive metastasis of ovarian cancer.Part 2 The expression of mirna-544a in ovarian cancer and its effect on the biological behavior of ovarian cancerObjective:Through Rt-PCR we detected the level of microRNA-544a in normal ovarian epithelial tissues and epithelial ovarian cancer tissues,the miRNA-544a mRNA level expressed in normal ovarian epithelial cells and ovarian cancer cell lines,investigate its correlation with clinical features,explore its relationship with ovarian cancer metastasis and invasiveness.Respectively transefected with the miRNA-544a mimics in ovarian cancer cell lines,detected the miRNA-544a increase ovarian cancer cell migration,and found the relatively mRNA and protein expression level of E-cadherin(CDH1)and Vimentin,to determine if miRNA-544a promote the development of ovarian tumours.Experimental methods:A total of 36 cases of ovarian malignant epithelial neoplasms were collected from the department of Gynecology of Qilu hospital of shandong university from November 2015 to November 2016.All of them were diagnosed as malignant epithelial neoplasms,13 cases of normal ovarian epithelial tissues.RT-PCR to detect the miRNA-544a expression level in normal epithelium ovarian tissues and epithelial ovarian cancer tissues,epithelial markers E-cadherin and interstitial markers Vimentin in ovarian malignant epithelial tumors and normal ovarian epithelial tissue.The protein expression of E-cadherin and Vimentin was detected by Western blot of 3 patients with ovarian cancer tissue and normal.ovarian tissue respectively.By transfected miRNA-544a mimics to upregulat miRNA-544a in ovarian cancer cells.The effection of transfection was detected by mRNA level according to RT-PCR.The invasion and migration ability of ovarian cancer cells after upregulated miRNA-544A was detected in Transewell small room invasion.RT-PCR was used to detect the relative expression of CDH1 and Vimentin mRNA levels in the epithelial ovarian cancer cells and the protein levels of E-cadherin and Vimentin in ovarian cancer cells by Western blot.Experimental results:1 miRNA-544a mRNA levels were significantly increased in the ovarian malignant epithelial tumor tissue,while in the normal ovarian epithelial tissue was low expressed.2 miRNA-544a mRNA was lowly expressed in the normal ovarian epithelial cell HOSepiC,while epithelial ovarian cancer cells A2780,HO8910 and SKOV3 were highly expressed.3.In the western blot experiments:we random detected 3 cases of ovarian cancer tissues and 3 normal ovarian tissues.And detected E-cadherin protein level expressed in cancerous tissues were lower than normal ovarian tissues,and Vimentin protein in the cancer tissues is higher than that in normal ovarian tissues.4.Transwell small room experiment found that upregulated miRNA-544a induced ovarian cancer cells have the ability to promote migration and invasion.Conclusion1.Compared with normal ovarian tissues,miRNA-544a mRNA and Vimentin mRNA levels were highly expressed in epithelial ovarian cancer tissues.The mRNA and protein level of E-cadherin were downregulated in epithelial ovarian cancer.2.The mRNA and protein level of miRNA-544a and Vimentin in ovarian cancer cells were highly expressed,while E-cadherin in normal ovarian epithelial cells were highly expressed.RT-PCR detected a down-regulated mRNA level in the epithelial ovarian cancer cell CDH1 after transfected with miRNA-544a mimics and increased the mRNA level of Vimentin.3.In the immune imprinting experiment,the expression of the protein level of E-cadherin in ovarian cancer cells was decreased after miRNA-544a was upregulated,and the expression of Vimentin protein increased.4.The results suggest that miRNA-544a may promote the development of ovarian cancer by EMT.Conclusion:1.miRNA-544a has the ability to promote the migration and invasion of ovarian cancer cells2.In ovarian cancer cells,the level of mRNA of the epithelial ovarian cancer cells CDH1 was down-regulated and the mRNA level of Vimentin was up-regulated.3 The expression level of Vimenrin protein was elevated and the protein level of E-cadherin decreased by over expressed miRNA-544a?4.The results suggest that miRNA-544a may promote the development of ovarian cancer in the development of ovarian cancer by reducing the epithelial marker E-cadherin,and the elevated interstitial marker Vimenrin promotes EMT. |
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