The Effects And Mechanism Of SUSD2 And CCNG1 In Ovarian Cancer

Ovarian cancer is an important malignant disease threatening women's health.Because the ovary is located in the deep pelvic cavity,ovarian cancer often occur without typical symptoms and signs in the early stage.When symptoms occur,ovarian cancer are always diagnosed as advanced.Ovarian cancer has become the most malignant tumors in the mortality among gynecological malignant tumors.Though the 5-year cause-specific survival for serous carcinoma was improved by the development of cytoreductive surgery and platinum combined with paclitaxel chemotherapy,there is still no substantial progress in the clinical diagnosis and treatment of ovarian cancer This is mainly due to the lack of clinically useful early diagnostic tools and long-lasting therapeutic regimens,and the root causes reside in limited understanding for the molecular mechanism of ovarian cancer early diagnosis?occurrence?development and prognosis.Therefore,the key and difficult point of ovarian cancer research still reside in predicting the prognosis of ovarian cancer,looking for early screening methods and indicating new therapeutic targets.There are four main histological subtypes of ovarian cancer:serous,endometrioid,mucinous and clear cell types.High serous ovarian cancer(HGSOC)accounts for about 70%of all ovarian cancers,which owns the characteritics of easily peritoneal metastasis and high mortality,and it is the most common and malignant subtype of epithelial ovarian cancer.This study mainly focused on HGSOC.Notch3 is an important member of Notch family which could regulate cell development and differentiation.Recent evidences suggest Notch3 is associated with the occurrence and development of multiple tumors,such as lung cancer,breast cancer and ovarian cancer.In 2011,the genome and expression profile analysis of ovarian cancer by TCGA consortium revealed that Notch3 exists gene amplification and alterations in Ovarian Cancer.In addition,amplification and overexpression of Notch3 gene was detected in 66%serous ovarian cancer,and the overexpression of Notch3 was associated with poor prognosis of serous ovarian cancer.However,the potential molecular mechanisms involved in Notch3 pathway have not yet been fully understood?SUSD2(sushi domain containing 2)is an type ? membrane protein and often expressed in bone marrow mesenchymal stem cells,perivascular smooth muscle cells,and many cancer cell lines.Several papers demonstrated that over-expression of SUSD2 inhibited clonogenicity,anchorage independent growth,migration,and invasion in tumors such as liver cancer,RCC and lung cancer.However,in breast cancer,SUSD2 had a significantly high expression level and could increase the metastasis of breast cancer cells and induce T cell apoptosis;and SUSD2 also participates in the malignant behavior of cancer cells in gastric cancer.Thus it can be seen that SUSD2 might have a complex function in different cancers.In addition,it has been suggested that SUSD2 may be a potential downstream gene of Notch3.Therefore we hypothesize that SUSD2 is associated with the metastasis?chemotherapy resistance and then prognosis in serous ovarian cancer,which will provide an new idea for the treatment of ovarian cancer.Cyclin G1(CCNG1)is a subtype of cyclin G family protein that has a positive and negative regulator of cell growth.Although the function of CCNG1 remains unclear,it is known that CCNG1 is abnormally expressed in many types of malignant tumors,such as EOC,hepatocellular carcinoma and lung cancer.In addition,the role of CCNG1 in tumor cell proliferation and apoptosis has also been reported.It has been reported that Notch3 can regulate the expression of CCNG1 in hepatocellular carcinoma cells.Therefore,we suspect that there may be a relationship between CCNG1 and Notch3 in ovarian cancer cells,which could be involved in the occurrence and development of ovarian cancer.Based on the previous results,we will also discussed the effect of CCNG1 on malignant behavior of ovarian cancer.This study mainly aims to clarifing the relationship between SUSD2?CCNG1 and Notch3 in ovarian cancer,exploring their role in the occurrence and development of ovarian cancer,and studing their specific mechanisms.The main contents of the research are as the following:Part ?:The expression and clinical significance of SUSD2 and CCNG1 in ovarian cancer.Part ?:The effects and mechanism of SUSD2 in ovarian cancer cells metastasis and chemotherapy resistance.Part ?:The underlying effects and mechanism of CCNG1 in ovarian cancer.PART ?:The expression and clinical significance of SUSD2 and CCNG1 in serous ovarian cancerBackground and Object:Ovarian cancer,especially serous ovarian cancer,with the high mortality and poor prognosis,is a malignant tumor endangering women's health all over the world.The reason for its poor prognosis lies in the lack of early effective molecular markers and therapeutic targets in clinic.Notch3 has been proved to be crucial for the growth and the drug resistance of ovarian cancer.Therefore,it isexpected to improve the prognosis of ovarian cancer by studying the downstream regulatory genes of Notch3.SUSD2(sushi domain containing 2),which is an 822 amino-acid type ? membrane protein,plays an important role in the occurrence and development of various tumors,but its role and relative mechanism in ovarian cancer is unclear yet.CCNG1 is a cyclin,which can promote the proliferation of ovarian cancer cells,but its role in metastasis and chemoresistance of ovarian cancer cells is still unclear.In this study,we first clarified the relationship between SUSD2,CCNG1 and Notch3,and then examined the protein expression of SUSD2?CCNG1 in cancer tissue versus the epithelium from fimbriae of uterine tube(FTE)and analyzed the relationship based on their expression level with clinical information,and then discover the role of SUSD2?CCNG1 in ovarian cancer.Material and Methods:Construct the ovarian cancer cell lines with the Notch3 gene overexpressed or knocked down,western blot(WB)and RT-q PCR were used to detect regulatory effect of Notch 3 on SUSD2 and CCNG1.Obtain the ovarian cancer specimens from primary ovarian cancer patients and the normal FTE tissues were used as normal controls.Immunohistochemistry(IHC)?WB and RT-q PCR were used to detect the differet expression of SUSD2?CCNG1 between the two groups.According to immunohistochemical staining,ovarian cancer patients were divided into high-expression group and low-expression group.The clinical information of ovarian cancer patients was summarized and the relationship between the expression of SUSD2?CCNG1 and prognosis was analyzed.Results:Overexpression of Notch3 in ovarian cancer cell lines could up-regulate the expression of SUSD2 and CCNG1,while the expression of SUSD2 and CCNG1 decreased after interfering with Notch3 expression.The expression of SUSD2 and CCNG1 in HGSOC was significantly higher than that in normal control.IHC showed that the expression of SUSD2 in ovarian cancer was significantly higher than that in normal control;RT-q PCR?WB and IHC showed that the expression of CCNG1 in ovarian cancer was significantly higher than that in normal control.Survival analysis showed that the prognosis of patients with high SUSD2 expression and CCNG1 was significantly worse than patients with low SUSD2 expression.The 2-year survival rate and 2-year progression-free survival rate of ovarian cancer patients with high SUSD2 expression was lower than patients with low SUSD2 expression.In addition,the high SUSD2 expression was positively correlated with omental metastasis of ovarian cancer,1ymph node metastasis and chemotherapy resistance.The overall survival(OS)and progression-free survival(PFS)of patients with both SUSD2 and CCNG1 overexpression were significantly shorter than those of patients with both SUSD2 and CCNG1 underexpression.Conclusions:Notch3 can regulate the expression of SUSD2 and CCNG1 in ovarian cancer cell lines.SUSD2 and CCNG1 are highly expressed in ovarian cancer,and the high expression of SUSD2 and high expression of CCNG1 are associated with the adverse prognosis of ovarian cancer.Combined analysis of SUSD2 and CCNG1 expression will better predict the prognosis of ovarian cancerPART ?:The effects and mechanism of SUSD2 in ovarian cancer cells metastasisand chemotherapy resistanceBackground and Object:ovarian cacer owns a high mortality rate and poor prognosis,mainly because of the strong invasive ability of cancer cells and the prone to early metastasis.Ovarian cancer often has extensive pelvic and omental metastasis,which makes it impossible to completely resect the tumors.The results of part I showed that the high expression of SUSD2 was positively correlated with ovarian cancer metastasis,suggesting that SUSD2 was involved in the metastasis of ovarian cancer cells.In addition,chemotherapy resistance is also an important factor for the poor prognosis of ovarian cancer.It is not clear whether SUSD2 plays a role in the chemoresistance of serous ovarian cancer.From the results of part I,we can see that the high expression of SUSD2 is associated with cisplatin resistance,suggesting that SUSD2 is involved in cisplatin resistance in ovarian cancer cells.Therefore,the aim of this study is to verify the effect of SUSD2 on the metastasis and chemoresistance of ovarian cancer cells,and to explore its molecular mechanism.Material and Methods:Construct the ovarian cancer cell lines with the SUSD2 gene overexpressed or knocked down by using the lentivirus packaging system.The number of adherent cells passing through the transwell chamber was counted by Transwell experiment,and then the metastasis ability of cells in SUSD2 overexpression group,SUSD2 interference group and corresponding control group was compared.WB assay was used to detect the expression of EMT(Epithelial-mesenchymal transition)-related proteins and cell surface adhesion protein EpCAM.Rescue assay was used to detect the effect of changes in EpCAM on the relationship between SUSD2 and EMT.In addition,in vivo nude mice metastasis assay,ovarian cells were injected into the lateral tail vein to verify the effect of SUSD2 on lung metastasis of ovarian cancer cells.After stimulating ovarian cancer cells with cisplatin at a certain concentration,the expression of SUSD2 in ovarian cancer cells was detected by WB.MTT assay was used to detect the effect of changing the expression of SUSD2 on the chemosensitivity of ovarian cancer cells to cisplatin.WB and RT-PCR was used to detect the.expression of autophagy-related molecules after changing SUSD2 expression.After GFP-RPF-LC3 adenovirus infected SKOV3 cells,the effect of SUSD2 on autophagy was observed under fluorescence microscope.After inhibiting autophagy by chloroquine,the effect of overexpression of SUSD2 cell line on cisplatin sensitivity was detected.Results:Transwell experiment showed that SUSD2 could change the invasive and migratory ability of ovarian cancer cells:the invasive and migratory ability of ovarian cancer cells increased significantly after up-regulating the expression of SUSD2;and the invasive and migratory ability of ovarian cancer cells decreased significantly after down-regulating the expression of SUSD2.Westen-blot analysis showed that SUSD2 could regulate the occurrence of epithelial-mesenchymal transition(EMT),which was mainly manifested by the down-regulation of E-CAD and up-regulation of N-CAD and SLUG after up-regulating the expression of SUSD2,while after down-regulating the expression of SUSD2,these factors showed opposite changes.After interference with SUSD2,the expression of EpCAM decreased following with down-regulating SUSD2,while over-expression of SUSD2 could up-regulate the expression of EpCAM in ovarian cancer cells.Rescue assay showed that down-regulation of EpCAM could weaken the up-regulation of E-CAD and enhanced invasiveness of ovarian cancer cells induced by SUSD2 overexpression.In vivo experiments showed that interfering with SUSD2 could inhibit lung metastasis of ovarian cancer cells in nude mice.WB showed that after cisplatin stimulated ovarian cancer cells,SUSD2 expression increased with the prolongation of cisplatin treatment time.MTT showed that after up-regulating the expression of SUSD2,the sensitivity of ovarian cancer cells to cisplatin decreased and the half lethal dose(IC50)increased significantly;after knocking down the expression of SUSD2,the sensitivity of ovarian cancer cells to cisplatin increased,and IC50 was significantly lower than that of normal control group.By detecting autophagy-related molecules,we found that autophagy could be induced by overexpression of SUSD2,while inhibited by knocking down the expression of SUSD2.An increased number of autophagosomes and autophagic lysosomes was observed after SUSD2 overexpression under fluorescence microscope.After ovarian cancer cells was inhibited autophagy by chloroquine,the increased cisplatin resistance of cells with SUSD2-overexpression was weakenedConclusions:Overexpression of SUSD2 in ovarian cancer cells can regulate the changes of EMT-related molecules through the up-regulation of EpCAM,thus inducing EMT in ovarian cancer cells and ultimately promoting the metastasis ability of ovarian cancer cells.Furthermore,SUSD2 can promote cisplatin resistance of ovarian cancer cells by inducing cell autophagy.PART ?:The effects and mechanism of CCNG1 in ovarian cancerBackground and Object:As the standard treatment for ovarian cancer,cytoreductive surgery combined with cisplatin and paclitaxel chemotherapy have improved the 5-year survival rate of ovarian cancer patients,but its mortality rate is still the highest in gynecological malignant tumors because of the predominance of aggressive high-grade serous carcinomas and generation of chemoresistance.Notch3 plays an important role in the metastasis and drug resistance in ovarian cancer.The results of the part ?suggested that Notch3 can regulate the expression of CCNG1,the effect and mechanism-of CCNG1 in ovarian cancer cell metastasis and chemoresistance remains unclear.In addition,the results of the part ? showed that the high expression of CCNG1 was associated with the poor prognosis of ovarian cancer,suggesting that SUSD2 wasinvolved in the metastasis and cisplatin resistance of ovarian cancer cells.This study will further explore the effect of CCNG1 on metastasis and chemotherapeutic resistance of ovarian cancer cells.Material and Methods:Construct the ovarian cancer cell lines with the CCNGI gene overexpressed or knocked down by using the lentivirus packaging system.Subsequently,cell function experiments were carried out to study a series of biological functions of ovarian cancer cells by altering the expression of CCNGI,including:the number of adherent cells passing through the transwell chamber was counted by Transwell experiment,and then the metastasis ability of cells in CCNGI overexpression group,CCNGI interference group and corresponding control group was compared;WB assay was used to detect the expression of EMT-related proteins;in vivo nude mice metastasis assay,ovarian cells were injected into the lateral tail vein to verify the effect of CCNG1 on lung metastasis of ovarian cancer cells.After stimulating ovarian cancer cells with cisplatin at a certain concentration,the expression of CCNG1 in ovarian cancer cells was detected by WB.MTT assay was used to detect the effect of changing the expression of CCNG1 on the chemosensitivity of ovarian cancer cells to cisplatin.Results:Transwell experiment showed that CCNG1 could change the metastatic capacity of ovarian cancer cells:after over-expression of CCNG1,the invasive and migratory ability of ovarian cancer cells increased significantly;and the invasive and migratory ability of ovarian cancer cells decreased after interfering CCNG1 expression.Westen-blot analysis showed that CCNG1 could regulate the EMT-related molecules,which was mainly manifested by up-regulation of N-CAD?SNAIL and SLUG after over-expression of CCNG1,while after down-regulating the expression of CCNG1,these factors showed opposite changes.In vivo experiments showed that interfering CCNG1 could inhibit lung metastasis of ovarian cancer cells in nude mice.WB showed that after cisplatin stimulated ovarian cancer cells,CCNG1 expression increased with the prolongation of cisplatin treatment time.MTT showed that after interfering CCNG1 expression,the sensitivity of ovarian cancer cells to cisplatin increased,and IC50 was significantly lower than that of normal control group.Conclusions:CCNG1 can affect the changes of EMT-related molecules in ovarian cancer cells,and then promote ovarian cell metastasis.In addition,CCNG1 can promote the resistance of ovarian cancer cells to cisplatin chemotherapy.