| Background: Ovarian cancer is the most fatal of all gynecological reproductive tumors.One of the main reasons for the rising mortality of ovarian cancer is that it was found in advanced stage.We urgently need to find new regulatory factors to control the aggregation of ovarian cancer,which may be used as a target for targeted therapy in the treatment of ovarian cancer.Resistin,a macrophage derived cytokine,is associated with obesity and insulin resistance.In many human cancers,resistin levels are associated with high-grade cancers and / or relapse free survival,such as breast cancer,chondrosarcoma,colorectal cancer,endometrial cancer,gastroesophageal cancer,lung cancer,multiple myeloma,prostate cancer,and renal cancer.Resistin is not only associated with the survival of high-grade cancer patients,but also increases the possibility of its role in chemotherapy.High resistin levels are associated with reduced chemosensitivity in a variety of cancers.In addition,acquired resistance to chemotherapy is a major clinical challenge for patients with ovarian cancer.Resistin plays an important role in the growth,proliferation,angiogenesis,metastasis and drug resistance of different tumors.However,this role of resistin has never been evaluated and important research value in ovarian cancer.More and more relevant studies have proved that miRNAs play a crucial role in the proliferation,invasion and metastasis of tumor cells,and miRNAs play a key role in the regulation of EMT.At the same time,some miRNAs can affect the cancer cell stemness and drug resistance of cancer.Therefore,understanding the relationship between some miRNAs and the stemness and drug resistance of EMT and CSCs is conducive to basic research and clinical treatment.Objective: Based on the role of resistin in different cancers,especially in acquired drug resistance,we speculate that resistin may play a role in promoting cancer and drug resistance in ovarian cancer cells.In this study,we explored the role of resistin in the growth,clonality,invasion and cisplatin resistance of ovarian cancer cells,and elucidated the mechanism of these roles mediated by resistin,so as to provide the basis for the role and mechanism of resistin in cisplatin resistance of ovarian cancer cells.Methods: 1.A2780,SK-OV-3 and A2780 cis cells were treated with different doses of cisplatin for 72 h in the absence and presence of resistin(10 ng/mL and 25 ng/mL).MTT assay was used to observe the cell growth index and calculate the IC50 value of cisplatin resistance in different ovarian cancer cell lines.2.The secretion of VEGF and MMP2 in resistin treated A2780,A2780 cis and SK-OV-3 ovarian cancer cells were detected by ELISA.Western blotting and quantitative real time PCR were used to detect the protein expression and mRNA level of VEGF and MMP2.3.Two ovarian cancer cell lines A2780 and SK-OV-3 were treated with different doses of resistin(0,5,10,15,20 and 25 ng / mL).The cell growth and invasion were observed by MTT cell proliferation assay,colony formation assay and cell invasion assay.4.The levels of E-cadherin,vimentin,ZEB1,Sox2,Oct4 and Nanog mRNA and protein expression in A2780 and SK-OV-3 cells treated with 25 ng / mL resistin were detected by quantitative RT-PCR and Western blotting.5.A2780 and SK-OV-3 cells were transfected with pre-let-7a,pre-mir-200c and pre-mir-186.The levels of miRNAs(let-7a,mir-200c and mir-186)in A2780 and SK-OV-3 cells were detected by quantitative RT-PCR.6.A2780 was selected to establish SCID mouse tumor model and randomLy divided into two groups,after 3 weeks of tumor formation,the mice were treated with resistin or PBS.The expression levels of E-cadherin,vimentin,ZEB1,Sox2,Oct4 and Nanog were detected by quantitative RT-PCR.Results: 1.In the absence and presence of resistin(10 ng/mL and 25 ng/mL),the resistance of A2780,SK-OV-3 and A2780 cis cells to cisplatin was significantly increased in the lower dose resistin group(10 ng/mL),and further increased in the higher dose resistin group(25 ng/ mL),indicating that resistin resistance to cisplatin was dose-dependent.The IC50 value of resistin treated cells was significantly higher than that of the control group.2.25 ng / mL resistin increased the secretion of VEGF and MMP2 in A2780,A2780 cis and SK-OV-3 ovarian cancer cells,and A2780 cis cells secreted more VEGF and MMP2 than the parental A2780 cells.3.Resistin can significantly stimulate the growth of A2780 and SK-OV-3 ovarian cancer cells,and the cell growth increases with the increase of resistin dose and exposure time,indicating that the effect of resistin on ovarian cancer cells is dose-and time-dependent.4.Resistin reduces the expression of E-cadherin by ?2-folds(p < 0.01)in both A2780 and SK-OV-3 cells.This was accompanied by ?4-folds(p < 0.01)increase in vimentin and ?3-folds(p < 0.01)increase in ZEB1 in A2780 cells.and ?2-folds(p < 0.01)increase in vimentin and ZEB1 in SK-OV-3 cells.We also observed that resistin induces stemness in ovarian cancer cells.resistin can increase the mRNA transcription level of Sox2,Oct4 and Nanog.These results fully support the induction of EMT and stem cell charac teristics by resistin.5.We confirmed that miRNAs were involved in resistin induced cisplatin resistance: compared with control,the expression level of miRNAs(let-7a,mir-200c,mir-186)in 25 ng / mL resistin group was continuously down regulated by resistin,and A2780 and SK-OV-3 cells were transfected with pre-let-7a,pre-mir-200c,and pre-mir-186.The increase of pre miRNAs level destroyed resistin mediated invasion.6.Animal experiments showed that the levels of miRNAs,let-7a,mir-200c and mir-186,were down-regulated in tumors of resistin-treated mice compared with the control group.We observed induced EMT(E-cadherin decreased,vimentin/ZEB increased)and stemness(Sox2,Oct 4 and Nanog increased)in residual tumor.In addition,the tumor volume of mice treated with resistin increased significantly(P < 0.05).Conclusions: 1.After A2780 cells and A2780 Cis cells were treated with the same dose of resistin,the IC50 value of A2780 Cis cells was higher.At the same time,resistin could induce the increased expression of MMP2 and VEGF in cisplatin resistant cells,which suggested that resistin may lead to cisplatin resistance in ovarian cancer.2.Resistin can significantly stimulate the growth and invasion of ovarian cancer cells,and it has a dose-and time-dependent action,which may be one of the reasons for cisplatin resistance of ovarian cancer cells.3.Resistin can promote the decrease of E-cadherin and the up regulation of vimentin and ZEB1,and also can promote epithelial mesenchymal transition and lead to cisplatin resistance in ovarian cancer cells.Overexpression of let-7a,mir-200c and mir-186 significantly reversed resistin-mediated invasion and chemoresistance.4.Rsistin can promote the up-regulation expression of Sox2,Oct4 and Nanog in ovarian cancer cells,which shows the stemness of tumor and leads to cisplatin resistance.5.In vitro and in vivo experiments confirmed that overexpression of let-7a,miR-200c and miR-186 significantly reversed the growth,invasion,EMT and stemness of ovarian cancer mediated by resistin,and suggested that miRNAs could regulate cisplatin resistance of ovarian cancer through the above mechanisms. |
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