| Backgroud and objectiveOvarian cancer(OC)is a common malignant tumor in the female reproductive system and it’s morbidity is next only to the cervical cancer and endometrial cancer,while the mortality rate is the highest according to some statistics.Among them,the most common histologic type is epithelial ovarian cancer(EOC).It is so difficult to detect the cancer in the early stage due to the special anatomy of the ovaries which are located in the pelvic cavity that approximately 70%of the patients have already entered the advanced stage of ovarian cancer.Beyond that,drug resistance and relapse always lead to poor prognosis as well as high mortality rate,the 5-year survival rate of patients with adanced ovarian cancer is always less than 30%,and that of early ovarian cancer patients can often reach as much as 90%.At least 75%of the patients visiting have had lymph node metastasis or pelvic extraneous metasta-sis.Therefore,early diagnosis difficulty,metastasis,drug resistance and recurrence are the present situation of ovarian cancer which is also the difficult problems to be solved.By investigating the molecular pathogenesis of ovarian cancer,searching out the biomarker for this disease and developing the effective targeted agents will greatly improve the quality of treatment and offer help for estimating prognosis of ovarian cancer additionally.Embryonic stem cell associated transcripts 4(Nanog)is the key transcription fac-tor to maintain the self-proliferation and multi-differentiation potential of embryonic stem cells which belongs to NK-2 gene of the ANTP superfamily.Not only are plu-ripotent cells and germ cell tumors capable of expressing Nanog genes,but also leu-kemia and solid tumors.Its expression is up-regulated and operates as an onco-gene.FOXD3,one member of the forkhead box(FOX)family is essentials for mam-malian embryos’development.FOXD3 plays crucial roles in promoting the formation,migration and differentiation of neural crest as well as maintaining the pluripotency of embryonic stem cells.An experiment of transplanted tumor on nude mouse had di-rectly proved Foxd3’tumorigenicity.However,FOXD3 is mostly reported to be down-regulated in tumors,and is regarded as a tumor suppressor.Experiments showed that both Nanog and FOXD3 were closely related to tumors.They have both engaged in some tumors’occurrence and development,such as cell proliferation and differentiation,epithelial-mesenchymaltransition(EMT),metastasis,chemotherapy re-sistance,immune tolerance and tumor stem cell regulation.FOXD3 is an activation factor of Nanog.When ESCs’special transcription start regional upstream is rein-forced by FOXD3,Nanog’promoter is activated and it’expression increases.Also some study put forward that Nanog is the upstream gene of FOXD3 which is targeted by Nanog.At present,there are few researches on FOXD3 gene,and its expression mecha-nism is not clear.There is no study of FOXD3 in OC at present,and there are no re-ports of FOXD3 and Nanog in OC or other tumors at home and abroad.In this study,we explore the role of the two factors in EOC by investigating the expression of Nanog and FOXD3 in mRNA and protein level in epithelial ovarian tumors and nor-mal ovarian tissues.It may provide ideas and opportunities for the treatment of EOC.Materials and Methods1.Study objectA total of 91 cases of patients are recruited who were admitted and operated on in the Third Affiliated Hospital of Zhengzhou University from January 2015 to March2017.Among them,25 cases of normal ovarian patients as normal group,they were normal vertified by pathology,which obtained from the excised attachments owing to endometrial cancer,cervical cancer and other benign lesions.26 cases of benign epithelial ovarian neoplasms patients as benign group.40 cases of EOC pa-tients as malignant group.We obtained the informed consent of patients to use the specimens for research and got the permission from the Ethics Committee of the Third Affiliated Hospital of Zhengzhou University.The tissues were collected during surgical procedures.None of the patients re-ceived chemotherapy,hormonetherapy or immunotherapy before surgery.They had no history of radiation exposure or infectious disease.Metastatic carcinomas of other organs were not inculuded yet.All specimens were vertified by pathology after sur-gery.2.Methods(1)quantitative Reverse transcription-polymerase chain reaction was used to detectand compare the mRNA expression of Nanog and FOXD3 in the malignant group,benign group as well as normal group.(2)Immunohistochemical staining was employed in detecting and comparing the protein expression of Nanog and FOXD3 in these three groups.3.Statistical analysisUsing the Statistical Package for the Socail Sciences version 21.0 softwore to do data statistics and analysis.The measurement data that conforms to the normal dis-tribution were presented as mean±SD,Single factor analysis of variance was adopted and LSD-t test was used for comparisons of two independent groups.χ~2 test andχ~2test divided by R×C were used to categorical data.And Spearman correlation analysis was employed in analyzing the correlation of the two factors.Significant levelα=0.05.Result1.Expression of Nanog,FOXD3 mRNA in three groupsThe relative expression of Nanog mRNA in the normal group,benign group and malignant group were 0.827±0.243,1.009±0.747and3.266±1.413 successively.The expression of Nanog mRNA in malignant group was higher than that in benign group and normal group,the differences were both statistically significant(p<0.01).The dif-ference between benign group and the normal controls had been found no statistically significant however(p>0.05).The relative expression of FOXD3 mRNA in the normal group,benign group and malignant group were 0.571±0.266,0.976±0.369and1.457±0.496 successively.The expression of Nanog mRNA in malignant group was higher than that in benign group and normal group,and levels of benign group was higher than the normal controls,and the differences of expression were all found statistically significant(p<0.01).2.Expression of Nanog,FOXD3 protein in three groupsNanog and FOXD3 protein were both mainly expressed in the cytoplasm of ovarian epithelial tissues which appeared tan and presented a granular distribution.Nanog protein’positive expression rate in the normal group,benign group and malignant group were 26.9%(6/25),47.6%(9/26)and 80.0%(32/40)successively.The expression level of Nanog protein in malignant group was obviously higher than that in other two groups and the differences were both statistically signifi-cant(p<0.01).The difference of expression between the benign group and normal control showed no statistically significant however(p>0.05).FOXD3 protein’positive expression rate in the normal group,benign group and malignant group were 20.0%(5/25),65.0%(17/26)and 87.5%(35/40).The expression level of FOXD3 protein in the malignant group,benign group and normal group showed gradual downward,and the differences were all statistically signifi-cant(p<0.05).3.Correlation between Nanog,FOXD3 protein and clinicopathologic fea-turesThe protein’positive expression rate of Nanog in epithelial ovarian carcinoma tissues with FIGO I-II and III-IV were 62.5%(10/16)and 91.7%(22/24).The positive expression rate increased with the increase of pathological stage,and the difference between them was statistically significant(p<0.05).Then,the protein’positive ex-pression rate of Nanog in epithelial ovarian carcinoma tissues with moderate-well differentiation group and poorly differentiated group were 62.5%(10/16)and91.7%(22/24),the difference between them was statistically significant as well(p<0.05).As the level of differentiation decreased,the positive expression in-creased.Nonetheless,the expression level of Nanog protein was not correlated with the patients’age,tumors’histologic types and lymph node metastasis or not(p>0.05).The expression rate of FOXD3 protein in epithelial ovarian carcinoma tissues was not correlated with all of the clinicopathological features which included the patients’age,tumors’histologic types,pathological stage,degree of cell differentiation and lymph node metastasis or not(p>0.05).4.Correlation analysis between Nanog,FOXD3 protein in in epithelial ovarian carcinoma tissuesThrough the Spearman correlation analysis,expression of Nanog protein was positively related to that of FOXD3 expression in the epithelial ovarian carcinoma(r=0.652,p<0.001).Conclusion1.The expression of Nanog mRNA and FOXD3 mRNA in epithelial ovarian cancer tissues were both up-regulated.2.The expression of Nanog protein and FOXD3 protein in epithelial ovarian cancer tissues were both up-regulated.The expression level of Nanog protein was re-lated to the pathological stage and degree of cell differentiation.3.The correlation between Nanog protein and FOXD3 protein is positive in epi-thelial ovarian cancer tissues.Nanog and FOXD3 may all participate in the occurrence and development of epithelial ovarian cancer.The two factors are potential biology and prognostic markers and may become target molecules for the treatment and in-tervention of EOC. |
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