A Study On The Expression Of CKS2 In Uterine Leiomyosarcoma And Its Influence On Tumor Biological Behaviors

BackgroundUterine leiomyosarcoma(ULMS)is a very malignant kind of tumor and accounts for 1.5%of uterine corpus malignancies and 40%of uterine sarcoma.It is characterised by early metastasis,poor prognosis and high rate of recurrence.Cyclin—dependent kinase subunit 2(CKS2)is a small protein which prevails among eukaryotes and it is involved in the cell cycle regulation as a part of CDK.Recently,plenty of reports had indicated that CKS2 was up-regulated in many types of tumors and the overexpression of CKS2 was connected with tumor genesis and development,however,to the best of my knowledge,there are few studies about the CKS2 expression in ULMS or its effect on ULMS.This study explores the CKS2 expression status in ULMS and the relationship between CKS2 expression and clinicopathological parameters.This study also tries to detect the effect of CKS2 on ULMS cells.Part I The expression of CKS2 in ULMS and its clinical significanceMethods1.Specimens including 38 cases with ULMS and 38 cases with leiomyoma during 2005 to 2015 were collected at Qi Lu Hospital of Shandong University and Shandong Provincial Hospital.2.The expression of CKS2 was detected by immunohistochemistry in experimental group and control group respectively and the results were scored according to the Formwitz standard.3.According to the score,the difference of CKS2 expression in ULMS and leiomyoma was measured;the ULMS specimens were divided into two groups—high expression group(?4)and low expression group(<4).The connection between CKS2 expression and clinicopathological parameters was analyzed by statistical methods.Results1.The expression of CKS2 was mainly localized in nucleus in uterin leiomyosarcoma and leiomyoma.The up-regulated expression of CKS2 was detected in 63.2%(24/38)of ULMS tissues,whereas only 18.4%(7/38)of leiomyoma tissues showed high CKS2 expression.Compared with leiomyoma,ULMS has up-regulated expression of CKS2(p=0.000).2.Statistical results showed that high CKS2 expression was associated with larger tumor size(p=0.014)and lower PR expression(p=0.048),whereas the correlation between CKS2 expression and other clinicopathologic features,such as age,FIGO stage or ER was not found.3.Kaplan-Meier analysis suggested that patients in the group with high expression of CKS2 had a significantly poorer prognosis than those in the group with low expression(Log Rank Chi-square=4.735?p<0.05).Multivariate analysis revealed that CKS2 expression(Risk ratio,RR=3.124,p<0.05)and FIGO stage(Risk ratio,RR=2.087,p=0.001)were significant risk factors for overall survival.Conclusion1.The expression of CKS2 was mainly localized in the nucleus.2.Compared with the expression in leiomyoma,the expression of CKS2 was up-regulated in ULMS,High CKS2 expression was associated with larger tumor size and lower PR expression.CKS2 expression and FIGO stage were significant risk factors in overall survival for patients with ULMS.Part ? The effect of CKS2 on cell biological behavior in leiomyosarcoma and its mechanismMethods1.Small interfere RNA(siRNA)targeting CKS2 was transfected into ULMS cell lines(SK-UT-1 and SK-UT-1B)by Lipofectamine 2000 to interfere the expression of CKS2.2.After transfection,the whole RNA and protein were extracted from cells,and the expression of CKS2 mRNA and protein were examined through real-time qPCR and western blot respectively to examine the interference efficiency.3.To determine the effect of CKS2 silencing on proliferation of ULMS cell lines,proliferation rates of tumour cells were measured using a cell counting kit(CCK-8)and growth curve of those cells was drawn thereafter.4.To determine the effect of CKS2 silencing on migration and invasion of ULMS cell lines:the migration and invasion assays were carried out using the transwell model.5.The colony formation ability of si-Ctrl and si-CKS2 ULMS cells were evaluated by the plate clone formation assay.6.To determine the effect of CKS2 silencing on cell cycle of ULMS cell lines:the effects of CKS2 on cell cycle progression were assessed using propidium iodide staining and subsequently analyzed by flow cytometry.7.To determine the effect of CKS2 silencing on cell cycle of ULMS cell lines:the apoptosis rates of si-Ctrl and si-CKS2 cells were determined with AnnexinV-FITC/PI double-staining assay and subsequently analyzed by flow cytometry.8.To determine the effect of CKS2 silencing on the expression of protein:the change of protein expression was measured by western blot.Results1.The level of CKS2 mRNA and protein was significantly down-regulated after the transfection of si-CKS2.2.SK-UT-1 and SK-UT-1B cells showed less proliferation ability after the thansfection at 48h and 24h respectively.3.Si-CKS2 could significantly inhibit the migration and invasion of SK-UT-1 and SK-UT-1B cells(p<0.01,p<0.01).4.Compared with the control group,si-CKS2 SK-UT-1 and SK-UT-1B cells significantly formed less foci(p<0.01,p<0.01).5.Silencing of CKS2 resulted in cell cycle arrest at the G1/S transition in SK-UT-1 and SK-UT-1B cells.6.The difference in apoptosis rate between si-CKS2 cells(8.2%)and si-Ctrl cells(5.9%)did not reach significance(p>0.05).7.The expression of cyclin A2 was downregulated after the knockdown of CKS2,but there is no change on the expression of claudinl,p-AKT,p-ERK,p-P38 and Bax.Conclusion1.CKS2 was expressed both in SK-UT-1 and SK-UT-1B cell lines.2.While CKS2 may contribute to the G1/S transition,proliferation,migration,invasion and clone formation in vitro of ULMS cell lines,it may have little effect on ULMS cells apoptosis.CKS2 can exerts its effect via cyclin A2.