Interventive Effects Of STC1 Gene On Biological Changes Of Human Ovarian Cancer Stem Cells

Since the early diagnosis of ovarian cancer is difficult,it is difficult for the patients to achieve early treatment.In this case,ovarian cancer is one of the most lethal carcinomas in females.Since many scholars have carried on the systematic research on the occurrence and development process of ovarian cancer,and have made great progress,but the comprehensive treatment effect of ovarian cancer is far from satisfactory.Stanniocalcin is a glycoprotein hormone involved in calcium/phosphate(Pi)homeostasis.STC1 gene was found in a wide range of human tissues and organs,and involved in a variety of physiological and pathological process.In recent years,more and more studies have found that STC1 is closely related to the tumor occurrence and development.we have confirmed that STC1 gene has a significant impact on human ovarian cancer on the biological behavior and chemotherapy resistance.Previous studies reported that tumor tissue contained a small fraction of cells that exhibited the stem-like properties,which called cancer stem cells(CSCs).CSCs are characterized by their self-renewing capability,high tumorigenicity,and resistance to chemotherapy and radiotherapy.In this study,we used the human ovarian cancer SKOV3 and HEY cells lines as the research objects.The ovarian cancer stem cells were enriched by serum-free medium culture method,and the related characteristics of stem cells.In this case,we can laid the foundation to further study of ovarian cancer stem cell and the related mechanisms.Research purposes: 1.Obtain ovarian cancer cells spheroids by suspension culture method and verify whether STC1 gene is a basic feature of ovarian cancer stem cells 2.After silencing of STC1 gene of the cells,we explore the effects of STC1 gene on the characteristics of ovarian cancer stem cells.Research methods: 1.The ovarian cancer cells were cultured in the serum-free medium.The cell viability was observed under the condition of cell suspension growth.The expression levels of STC1 gene in different growth periods were detected by Western blotting and the RT-PCR methods 2.Both the ovarian cancer cells which the STC1 gene was knocked down and the control group cells were cultured in serum-free medium.The growth of the two groups was observed and the cells spheroids were collected.3.MTT assay was used to detect the effects of STC1 gene on the proliferation of ovarian CSCs.4.Using colony formation test explored the variation of two groups’ clonogenic capacity.5.Using scratching test and Transwell experiments explored the variation of two groups’ scavenging capacity.6.The expression levels of stem cell markers and EMT-related proteins in ovarian cancer stem cells were detected by Western blotting and RT-PCR.7.The tumorigenic ability of ovarian cancer stem cells was detected by transplanted the cells into nude mice.8.Drug-sensitivity tests were used to measure the sensitivity of these two groups of stem cells to carboplatin and paclitaxel.Research results 1.The adherent ovarian cancer cells were inoculated in serum-free medium for suspension culture.A large number of cells died within 24 hours.The viable cells formed the cell spheres.2.After cultured a few days later,the spheres volume gradually increased larger,the number of cells increased,and more cells were contained in every sphere.3.By Western blotting and RT-PCR,the expression level of STC1 gene was increased.4.Knockdown of STC1 expression resulted in the size of spheroids dramatically reduced in comparison with control spheroids.5.The proliferation ability of STC1-silencing cells was significantly lower compared with control group.6.The results of Transwell invasion assay showed that the invasive ability of ovarian cancer stem cells was significantly attenuated by STC1 gene.We found that the migration ability of ovarian cancer stem cells significantly reduced after silencing STC1 gene was also.7.The expression of stem cell markers was decreased by Western blotting and RT-PCR.The expression of E-cadherin in STC1 silencing cells was increased,while N-cadherin,Vimentin,Snail-1,Snail-2 expression level was significantly decreased.8.STC1-silencing cells were more sensitive to the treatment of carboplatin and paclitaxel 9.Tumorigenesis experiments in nude mice showed that the tumorigenicity of STC1-silencing cells was significantly lower than that of control cells.Analysis conclusions 1.The STC1 over-expressed ovarian cancer stem cells cultured in serum-free medium are capable of forming cells spheres,which is a basic characteristic of cancer stem cells.2.After silencing STC1 gene,the cancer stem cells biological behavior of the cells spheres were significantly inhibited.