| Backgroud and objective Ovarian cancer(OC)is the most lethal of malignant gynecological cancers,frequently,attributable to the pathogenesis is still unclear,late diagnosis and responsiveness to chemotherapy.Therefore,the prognosis is very poor.Epithelial ovarian cancer(EOC)accounts for more than 90% of all OCs.Micro RNA(mi RNA)are approximately 22-nucleotide noncoding RNAs.Recent studies have shown that mi RNA is closely related to cancer progression.In many human cancers were found the abnormal expression of mi RNA,the previous researches were focused on apoptosis,cell cycle,angiogenesis,epithelial mesenchymal transition(EMT)and resistance to chemotherapy.Recently,it was discovered the expression of mi RNA from circulate in the body fluids of healthy and diseased patients,suggesting that they may serve as a novel diagnostic marker.It could help cancer patients to do early diagnosis and treatment.Let-7a as a member of the let-7 family,which is one of the first mi RNA was found.It is down regulated in a variety of malignant tumors,such as breast cancer,prostate cancer and pancreatic cancer,and it is considered as a tumor suppressor gene.HMGA2(high-mobility group AT-hook 2)and cell cycle regulatory factor D1(Cyclin D1,CCND1)as target genes of let-7a have been demonstrated.Let-7a negatively control genes expression by binding to the 3’ untranslated region(UTR)of target m RNAs.HMGA2 and Cyclin D1 were related with the occurrence of tumor,histological type,degree of differentiation,the invasiveness and prognosis of the patients.However,there is no research of let-7a,HMGA2 and Cyclin D1 in EOC.The study is to detect the expression of let-7a from plasma with EOC,then predict the possibility of let-7a as a diagnostic marker of EOC.To study the expression of let-7a,HMGA2 and Cyclin D1 in EOC tissues,and to explore the role in the development of EOC.Materials and Methods 1.Study object 48 cases of EOC patients as malignant group were recruited from September 2014 to May 2016 in the Third Affiliated Hospital of Zhengzhou University.21 cases of benign epithelial ovarian neoplasms patients as benign group.Plasma were collected before operation,neoplasma tissues were collected during operation.23 cases of healthy subjects as normal healthy group were recruited at the same period and same hospital.26 cases of normal ovarian tissues as normal group.All people gave informed consent to use excess specimens for research.The protocols employed in this study and the use of human tissues was approved by the Ethics Committee of the Third Affiliated Hospital of Zhengzhou University.2.Methods(1)Use q RT-PCR to investigate the plasma levels of let-7a in the malignant group,benign group and normal healthy group.Malignant group,benign group and normal group were investigated the expression of let-7a,HMGA2 and Cyclin D1 m RNA using q RT-PCR.(2)Detection of HMGA2 and Cyclin D1 protein expression and location by immunohistochemistry.3.Statistical analysis All the data were analysed using the Statistical Package for the Socail Sciences version 21.0 softwore.Data were presented as mean±SD.The Student’s t test was used for comparisons of two independent groups.One-way ANOVA or non– parametric Kruskal-Wallis test were used by multiple quantitative data.χ2 test and Fisher exact probability method were used to categorical variables.Use Pearson test to analysis correlation.Significant level α=0.05.The comparision between two groups,significant level α′=α/comparision times=0.0167.Result 1.Analysis of let-7a in plasma The relative quantity of let-7a in plasma with normal,benign and malignant group were(2.046±0.734),(2.046±0.734)and(1.329±0.610).Compared the plasma let-7a levels in the three groups,malignant group was lower than the normal group and benign group,the difference was statistically significant(p<0.01).Levels of benign group was lower than the normal controls,the difference had no statistically significant(p>0.05).The area under ROC curve was 0.826(95%:0.728~0.923,p<0.001).When Youden index was 1.975,the sensitivity was 91.7% and the specificity was 65.2%.2.The expression of let-7a,HMGA2 and Cyclin D1 m RNA in different tissues The expression of let-7a,HMGA2 and Cyclin D1 m RNA in three groups were significantly different.The expression of let-7a in normal group,benign group and malignant group were(5.045 ±4.435),(4.178±2.856)and(2.144±2.048).Compared the let-7a levels in the three groups,malignant group was lower than the normal group and benign group,the difference was statistically significant(p<0.01).Levels of benign group was lower than the normal controls,the difference had no statistically significant(p>0.05).The expression of HMGA2 m RNA in normal group,benign group and malignant group were(1.040±1.107),(1.516±1.088)and(6.667±5.139).Compared the HMGA2 levels in the three groups,malignant group was higher than the normal group and benign group,the difference was statistically significant(p<0.01).Levels of benign group was higher than the normal controls,the difference had no statistically significant(p>0.05).The expression of Cyclin D1 m RNA in normal group,benign group and malignant group were(1.138±0.838),(1.384±1.207)and(3.355±2.358).Compared the Cyclin D1 levels in the three groups,malignant group was higher than the normal group and benign group,the difference was statistically significant(p<0.01).Levels of benign group was higher than the normal controls,the difference had no statistically significant(p>0.05).3.Correlation between let-7a and clinicopathologic geatures The expression of let-7a in malignant group tissues with FIGO I-II and III-IV were(2.920±2.352)and(1.679±1.720),the level of expression of let-7a in patients with advanced-stage was lower than that of patients with early-stage disease,the difference was statistically significant(p<0.05).The expression of let-7a in tumour tissues of EOC patients with no metastasis and with lymph nodal metastasis were(3.029±2.207)and(1.564±1.740),the level of expression of let-7a with lymph nodal metastasis was significantly lower than that of EOC patients with no metastasis(p<0.05).4.The correlation analysis of let-7a,HMGA2 and Cyclin D1 m RNA The Pearson correlation analysis was used.The level of let-7a was significantly negatively correlated with HMGA2 m RNA(r=-0.729,p<0.05),and was significantly negatively correlated with Cyclin D1 m RNA(r=-0.735,p<0.05).The expression of let-7a between plasma and tissues had no correlation(r= 0.163,p =0.270).5.The expression of HMGA2 and Cyclin D1 protein and location The positive expression rate of protein with HMGA2 in normal group,benign group and malignant group were 26.9%(7/26),47.6%(10/21)and 85.4%(41/48).Compared the HMGA2 protein in the three groups,malignant group was higher than the normal group and benign group,the difference was statistically significant(p<0.01).Levels of benign group was higher than the normal controls,the difference had no statistically significant(p>0.05).The positive expression rate of protein with Cyclin D1 in normal group,benign group and malignant group were 30.8%(8/26),52.4%(11/21)and 81.3%(39/48).Compared the Cyclin D1 protein in the three groups,malignant group was higher than the normal group and benign group,the difference was statistically significant(p<0.01).Levels of benign group was higher than the normal controls,the difference had no statistically significant(p>0.05).HMGA2 and Cyclin D1 were all located in the cell nucleus.6.Correlation between HMGA2,Cyclin D1 protein and clinicopathologic geatures The positive expression rate of HMGA2 protein in malignant group tissues with FIGO I-II and III-IV were 66.7%(12/18)and 96.7%(29/30),the positive expression of HMGA2 in patients with advanced-stage was higher than that of patients with early-stage disease,the difference was statistically significant(p<0.05).The positive expression of HMGA2 protein in tumour tissues with no metastasis and with lymph nodal metastasis were 68.4%(13/19)and 96.6%(28/29),the positive expression of HMGA2 with lymph nodal metastasis was significantly higher than that of EOC patients with no metastasis(p<0.05).The positive expression rate of Cyclin D1 protein in malignant group tissues with FIGO I-II and III-IV were 61.1%(11/18)and 93.3%(28/30),the positive expression of Cyclin D1 in patients with advanced-stage was higher than that of patients with early-stage disease,the difference was statistically significant(p<0.05).The positive expression of Cyclin D1 protein in tumour tissues with no metastasis and with lymph nodal metastasis were 63.2%(12/19)and 93.1%(27/29),the positive expression of Cyclin D1 with lymph nodal metastasis was significantly higher than that of EOC patients with no metastasis(p<0.05).Conclusion 1.Let-7a was significantly down-regulated in plasma with epithelial ovarian cancer patients.The ROC curve analyses showed that the detection of plasma let-7a had certain accuracy in the diagnosis of EOC.It may be as a potential diagnostic tool.2.The expression of let-7a was significantly down-regulated in tissues with epithelial ovarian cancer patients,HMGA2 and Cyclin D1 were significantly upregulated in EOC tissues,they may be involved in the occurrence and development of EOC. |
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