Effect Of Human Adipose Derived Stem Cells On Ovarian Cancer SK-OV-3 Cells And Its Mechanism

Objective: Ovarian cancer is one of the most common malignant tumors in gynecology,with the highest mortality rate among female reproductive system tumors.A lot of patients were diagnosed at the advanced stage,51% of patients were in FIGO III,29%were in FIGO IV,and the 5-year survival rate for patients at stage III and IV was only27%.Tumor metastasis is the key factor to affect the survival rate of patients with ovarian cancer.The growth of cancer cells depend on the tumor microenvironment.Studies have found that cancer cells can recruit mesenchymal stem cell(MSC)into the tumor microenvironment to play a role.Adipose derived stem cell(ADSC)is an important member of the mesenchymal stem cell family.ADSC has been shown to participate in the malignant process of most cancer cells,such as breast cancer,lung cancer,osteosarcoma,glioblastoma,prostate cancer,etc.It has been reported that ADSC activated the Wnt signaling pathway and enhanced the metastatic capacity of colon cancer cells through a paracrine manner.In this study,we extracted human ADSC to make conditioned medium(CM)to culture ovarian cancer SK-OV-3 cells,simulating its role in the tumor microenvironment.We observed and studied changes in tumor characteristics,and explored the role and underlying mechanism of ADSC in the development of ovarian cancer which aimed to provide new directions for the treatment of ovarian cancer.Methods: 1.The isolation,cultivation,and identification of ADSC.(1)Using collagenase digestion to extract ADSC from omental adipose tissue,isolated and cultured them;(2)Using flow cytometry to identify primary cell surface markers and adipogenic and osteogenic differentiation in vitro;(3)Making ADSC conditioned medium(ADSC-CM)to culture SK-OV-3 cells and a blank medium is set as a control group.2.To study the effect of ADSC on the proliferation and apoptosis of ovarian cancer SK-OV-3 cells,(1)detecting cell proliferation by CCK-8 assay;(2)detecting cell cycle by PI staining;(3)detecting cell apoptosis by Annexin-V/PI double staining;(4)detecting the expression of apoptosis related proteins by Western Blot.3.To study the effect of ADSC on the migration and invasion of ovarian cancer SK-OV-3 cells,(1)detecting the migration of cells through a Wound healing assay and a Transwell migration assay;(2)detecting the invasion of cells by Transwell invasion assay;(3)detecting the expression of EMT related markers by RT-PCR and Western Blot,respectively.4.To study the potential mechanism of the role of ADSC on ovarian cancer SK-OV-3 cells,(1)detecting the expression of SALL4 and Wnt/β-catenin signal pathway markers by Western Blot;(2)silencing the expression of SALL4 in SK-OV-3 cells which treated with ADSC-CM by si RNA;(3)re-detecting the biological behaviors of SK-OV-3 cells after using SALL4-si RNA and XAV-939(the inhibitor of Wnt/β-catenin signaling pathway);(4)detecting the relationship between SALL4 and β-catenin through a double-labeling immunofluorescence staining assay.Results: 1.Primary ADSC exhibited a fibroblastoid morphotype and cell surface expressed the antigens CD105,CD73,and CD90(≥95% positive)and the absence of hematopoietic lineage markers CD45,CD34,CD11 b,CD19,and HLA-DR(≤2%positive).Additionally,it presented the expected capacity to differentiate into osteogenic and adipogenic lineages in vitro.2.The cell numbers of S and G2-M increased,the apoptosis rate decreased,and the expression of apoptosis inhibitory protein Bcl2 was up-regulated while the expression of apoptosis promoting protein Bax was down-regulated in SK-OV-3 cells treated with ADSC-CM.3.The migration and invasion capacities of SK-OV-3 cells treated with ADSC-CM were enhanced and the expression of epithelial marker E-cadherin was down-regulated while the expression of mesenchymal markers vimentin and N-cadherin was up-regulated.4.The biological behaviors of SK-OV-3 cells was inhibited after using SALL4-si RNA and XAV-939.SALL4 and β-catenin co-localized in the nucleus and cytoplasm.Conclusion: 1.ADSC was successfully extracted from human omental adipose tissue by collagenase digestion.2.ADSC promotes the progression of ovarian cancer by activating the SALL4-mediated Wnt/β-catenin signaling pathway.