The Impact Of SND1 Protein On Chemosensitivity Of Ovarian Cancer Cell

Objectives: Ovarian cancer is the most lethal gynecological cancer,which seriously threaten the health of women.The incidence rate of ovarian cancer occupied the third only after cervical cancer and uterine body cancer,however,its mortality rate is the highest among all kinds of gynecological tumors.The main reasons include two aspects: On the one hand,due to the early onset of abdominal metastasis,most of the findings are in the late stage at the time of diagnosis;On the other hand,in spite of high response rates to primary therapy,most patients eventually relapse developing drug-resistant disease.SND1 is a multifunctional protein and express in almost all eukaryotes.Recent studies show that SND1 play a crucial role in the development of many kinds of tumors.SND1 proteins are over-expressed and can promote the proliferation,invasion and migration of cancer cells.Our previous works show that SND1 repress the expression of E-cadherin(CDH1),an epithelial marker,by up-regulating the transcriptional factor SLUG and thus promote epithelial–mesenchymal transition(EMT).Some evidence suggested that EMT has been indicated to be involved in resistance to chemotherapeutic agents in cancer treatment.The purpose of our project is to explore the role of SND1 in the development and chemosensitivity of ovarian cancer and its potential mechanism involved.Our study might be beneficial to early diagnosis of ovarian cancer and overcome ovarian cancer chemotherapy resistance.Methods: The report is composed of following three parts: Part I,In order to observe the effect of SND1 protein on apoptosis,Western blot was applied to detect the expression of marker proteins relate to apoptosis in ovarian cancer cells with stable over-expressed and knockdown of SND1 protein.Part II,To find out the association between SND1 protein and chemotherapeutic susceptibility,Real-time PCR and Western blot were applied to measure the level of mRNA and protein of apoptosis-related markers after using cisplatin,a chemotherapy agent.The proliferation ability of cells was detect by MTT and clone formation test.Cell flow cytometry was used to explore the impact of SND1 on and apoptosis in ovarian cancer cells with stable over-expressed and knockdown of SND1 protein.Part III,After analyzing the results of gene expression profiles in ovarian cancer cells with knockdown of SND1 protein,some genes associated with apoptosis and chemotherapeutic susceptibility were selected to be identified by Real-time PCR.Real-time PCR and Western blot were applied to measure the level of mRNA and protein of related genes to explore the potential mechanism of SND1 protein reduced the resistance to chemotherapeutic agent cisplatin in ovarian cancer cells.Results: Part I: The expression of cleaved caspase3,cleaved PARP and Bax,which are markers associated with apoptosis,are significantly higher in ovarian cancer cells with overexpressed SND1 and lower in SND1 knockdown cells than that of control groups.Part II:(1)The expression of apoptosis-related marker proteins in ovarian cancer cells increased in both SND1 overexpression and knockdown group when treated with cisplatin(upon exposure to cisplatin);When given same concentration of cisplatin,the expression of apoptosis-related markers decrease in SND1 overexpression and increase in SND1 knockdown cells.(2)Cell flow cytometry assay show that enhanced apoptosis occurred in both SND1 overexpression and knockdown ovarian cancer cells upon exposure to cisplatin.Given same concentration of cisplatin,there were reduced apoptosis in SND1 overexpression and increased apoptosis in SND1 knockdown group.(3)MTT assay show that the alteration of IC50 is positively correlated with the expression of SND1 protein in ovarian cancer cells treated with cisplatin.(4)Clone formation test show the same trends as MTT assay without cisplatin.However,comparing with no cisplatin the clone numbers were significant decreased both in SND1 overexpression and knockdown ovarian cancer cells upon exposure to cisplatin.All data above indicate that SND1 repress the chemotherapeutic susceptibility of ovarian cancer cells.Part III: After analyzing the results of gene expression profiles in stable SND1 knockdown transfectant ovarian cancer cells,several genes related to chemotherapeutic susceptibility and apoptosis were screened and verified by Real-time PCR.We found that mRNA level of GAS6 was positively correlated with the expression of SND1 protein in ovarian cancer cells treated with cisplatin.Given the same concentration of cisplatin,the expression of GAS6 and p-AKT protein altered positively with the expression of SND1 protein.Results above suggest that SND1 may have impact on the sensitivity of ovarian cancer cells to cisplatin through regulation of GAS6 and PI3K/AKT signaling pathways.Conclusion: Our study indicates that SND1 repress the apoptosis of ovarian cancer cells with treatment of cisplatin.SND1 knockdown enhance the chemotherapeutic susceptibility of ovarian cancer cells to cisplatin and its molecular mechanism involved in the GAS6 and PI3K/AKT signaling pathways.