| Part I Overexpression of microRNA-205 predicts lymph node metastasis and indicates an unfavourable prognosis in endometrial cancerBackground:Endometrial cancer(EC)is one of the most common malignancies of female genital system worldwide.In Chinese women,there has been a tendency for this disease to increase recently.As an integral component of surgical staging system,lymphadenectomy for patients with EC remains controversial,particularly in clinical stage I disease including not only low-risk but also high-risk subgroups.In order to maximize the therapeutic effect of lymph node excision for high-risk patients who can potentially obtain survival benefits from it while minimize its reverse effects in low-risk group,preoperative risk stratification of lymph node metastasis are called for.Up-regulation of miR-205 in carcinoma of the endometrium has been consistently reported recently and was found to correlate with poor survival.However,whether miR-205 can be used as a predictive molecule for lymph node metastasis of EC patients is still unknown.Objective:In the current study,we aimed toexplore whether overexpression of miR-205 in curettage samples of EC could identify clinical stage I patientswho are at high risk for lymph node metastasisbefore surgery and validate the role of miR-205 as a prognostic marker in EC.Methods:Relative quantification detection of miR-205 in both curettage and hysterectomy specimens of patients with EC was performed by FQ-PCR(fluorescent quantitative polymerase chain reaction).Prediction of lymph node metastasis based on miR-205 expression in addition to tumor type and grade in curettage samples was done for all EC patients and patients with clinical stage I disease via AUC(area under curve)of ROC(receiver operating characteristic curve).Moreover,survival analysis was conducted.Results:1.We observed that miR-205 was significantly and consistently elevated in both curettage and hysterectomy samples of EC relative to normal controls.2.The increased expression of miR-205 in the curettage and hysterectomy samples was significantly correlated with pre-operative histological characteristics,including subtype and grade(both P<0.001).The upregulated expression of miR-205 in the curettage and hysterectomy samples of EC was also significantly correlated with the presence of lymph node metastasis(both P<0.001).Besides,a good concordance was found concerning the expression of miR-205 between pre-operative and post-operative detection.3.Furthermore,we documented that overexpression of miR-205 in curettagespecimens before surgery could predict lymph node metastasis with a high accuracy especially for patients with clinical stage I disease.4.miR-205was revealed again to be linked to poor prognosis in EC.ConclusionLymph node metastasis could be predicted by overexpression of miR-205with a high accuracy for clinical stage I disease.When combining miR-205 expression with preoperative histologic characteristics including tumor subtype and grade,the predictive value for lymph node metastasis could be further improved,indicating that overexpressed miR-205 in endometrial biopsies could provide additional and helpful information for reinforcing risk stratification before surgery.Furthermore,up-regulated miR-205 was found to be linked to poor prognosis in endometrial cancer.Part ? microRNA-205 plays a cancer-promoting role by targeting PHLPP2 in endometrial cancer cellsBackground:Incidence ofendometrial cancer(EC)is second to none among femalereproductive organ in the United States.In recent years,EC is more and more common in China due to the change of lifestyle.More than 75%of EC patients can be diagnosed at early stage thus having a favorable outcome.However,a small part of patients with advanced or aggressive EC can't obtain good prognosis because they will unavoidably suffer from relapse,metastasis and resistance for conventional treatment and the specific mechanisms under this phenomenon are less well understood.Therefore,novel and more effective molecules or means beneficial to improvingsurvival for these patients are needed.Targeted therapy appears to be this kind of promising therapeutic method.Our previous study have documented that elevated expression of miR-205 could predict lymph node metastasis with high accuracy and indicatedpoor prognosis in EC.The biological role of miR-205 in EC and the underlying mechanism are needed to be further explored.PHLPP2(pleckstrin homology domain leucine-rich repeat protein phosphatase 2)is a recently found tumor suppressor which can directly dephosphorylate the phosphorylated AKT and has been documented lost in many varieties of human cancer.However,whether PHLPP2 is in a loss-of-function state in EC and the specific mechanismof its downregulation are unclear.Objective:In this part,we intended to investigate the expression of miR-205 in human EC cells and human normal endometrial epithelia cells,then to explore the biological role of miR-205 in oncogenicity and invasion of EC cells,to reveal the relationship between miR-205 and PHLPP2 as well as the potentialmechanism,thus to further verify whether miR-205 can be a candidate target for therapy of EC.Methods:FQ-PCR(fluorescent quantitative polymerase chain reaction)was used to detect the relative expression of miR-205 innormal endometrial epithelia cells as well as three types of EC cells including Ishikawa,HEC-1-A and AN3CA.We then performed CCK-8(Cell Counting Kit-8)assay,colony formation assay and Transwell invasion assay,respectively,to determine proliferation and invasion ability of Ishikawa and HEC-1-A after transfection of miR-205 mimics or negative control;for AN3CA and HEC-1-A,after transfection of miR-205 inhibitor or negative control at the same time.Next,flow cytometry assay was done to investigate apoptosis rate of Ishikawaafter transfection of miR-205 mimics or negative control;for AN3CA,after transfection of miR-205 inhibitor or negative control as well.Additionally,dual-luciferase reporter assay and Western blotting were carried out to validate whether miR-205 directly interacted with PHLPP2 to fulfill its role in EC.Furthermore,IHC(immunohistochemistry)was done to investigate PHLPP2 expression in clinical specimens of EC patients,and Spearman correlation analysis was applied to assess the relationship between PHLPP2 and miR-205 expression in EC tissue.Results:1.FQ-PCR revealed that the differences of endogenous miR-205 expression between EC cells and normal endometrial epithelia cells were all statistically significant(P<0.01).More specifically,miR-205 expressionin Ishikawa,HEC-1-A and AN3CA were all statistically higher than in endometrial epithelia cells.2.Our data indicated that forcedoverexpression of miR-205 promotedproliferation as well as invasiveness in Ishikawa and HEC-1-A transfected with miR-205 mimics when compared with the same cells transfected with negativecontrol(P<0.01).3.we found that the proliferative and invasive potential ofAN3CA and HEC-1-A after transfection with miR-205 inhibitordecreased in comparison with the same cells after transfection with negative control(P<0.01).4.Flow cytometry assay revealed that transfection with miR-205 mimics decreased apoptosis rate of Ishikawa as compared with negative control.In contrast,transfection with miR-205 inhibitor increased apoptosis rate of AN3CA when compared with negative control.5.Using the miRBase and TargetScan database,PHLPP2 was predicted to possess putative binding sites in the 3'UTR of its mRNA which could be targeted by miR-205.Dual-luciferase reporter assay revealed that when compared with negative control,miR-205 mimics transfection resulted in a reduction of relative luciferase activities of PHLPP2 luciferase reporter containing wild type sequence in its 3'UTR;on the contrary,relative luciferase activities of PHLPP2 luciferase reporter containing wild type sequence in its 3'UTR was increased by transfection of miR-205 inhibitor to AN3CA in comparison to negative control.Nevertheless,the regulatory effects of miR-205 mimics and inhibitor on relative luciferase activities were completely abolished via introduction of PHLPP23'UTR that contained mutations in the binding sites.Collectively,our data in this partdemonstrated that miR-205 directly bound to 3'-UTR of PHLPP2.Through Western blotting,we found that protein level of PHLPP2 decreased after transfection of miR-205 mimics in Ishikawabut increased by transfectionwith miR-205 inhibitor in AN3CAas compared with negative control,indicating that miR-205 negatively modulated PHLPP2 at a posttranscriptional level.6.We observed that positive rate of IHC staining of PHLPP2 in EC tissue was statistically lower than in its normal counterpart(P<0.01).7.Using Spearman correlation analysis,an inverse correlation between PHLPP2 and miR-205 expression was found in clinical samples of EC(r=-0.823,P=0.0001).Conclusion:Endogenous miR-205 expression in EC cells Ishikawa,HEC-1-A and AN3CA was statistically higher than in normal endometrial epithelia cells.Overexpression of miR-205 promoted proliferation and invasiveness,but inhibited apoptosis in EC cells.MiR-205 exerted cancer-promoting effects by targeting PHLPP2 in EC.MiR-205 could be a target for EC targeted therapy. |
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