Regulation Of Epithelial Ovarian Carcinoma SKOV-3 Cell Migration And Chemosensitivity By YKL-40

Ovarian cancer is a common gynecological malignancy with the highest mortality rate.As the main pathological type,epithelial ovarian cancer(EOC)which has obscure symptoms is high malignant degree and easy to invade and metastasize.In those cases,there are about 70%patients occurring the invasion of the abdominal cavity organs or distant metastasis when making an accurate diagnose.At present,the main treatment of EOC is cytoreduction surgery and postoperative chemotherapy(platinum combined with paclitaxel)which can lead to the majority of patients get complete remission.But still existing 75%-80%patients with advanced stage will be recurred after repeated cycle of chemotherapy,and some patients will develop acquired drug-resistance as the sensitivity of platinum and other first-line chemotherapeutic 'drugs decreased.Due to the high invasion ability and the metastasis characteristics and the emergence of chemotherapy resistance,the mortality of epithelial ovarian cancer is high,which effecting the prognosis of patients and becoming a serious threat to women's health.Therefore,it is important to explore the molecular mechanism of the invasion,metastasis and reduced chemotherapy drugs'sensitivity of epithelial ovarian cancer.It is of great significance to understanding the molecular mechanism for improving the prognosis of patients.YKL-40,also known as chitinase 3-like 1(CHI3L1),is a secretory glycoprotein that highly expresses in breast cancer,lung cancer,glioma and other malignant tumors.YKL-40 is involved in the process of cell invasion and metastasis in lung cancer,cholangiocarcinoma and other tumor by interacting with hyaluronic acid,fibronectin,collagen and other extracellular matrix components.In addition,YKL-40 can mediate the TMZ's resistance of TMZ-R U87 cell line(temozolomide resistance cell line).However,it is still not clear that its role in the process of cell migration and chemotherapeutic sensitivity in epithelial ovarian cancer.In this study,we use the classical epithelial ovarian cancer cell line SKOV-3 to explore the effects of YKL-40 on cell migration ability and the sensitivity of platinum drug in vitro.The research is divided into three parts.Part One The expression of YKL-40 in ovarian cancer SKOV-3[Objective]To detect the expression of YKL-40 in ovarian cancer SKOV-3.[Methods]YKL-40 mRNA and protein(secreted protein and total protein)expression in ovarian cancer SKOV-3 cells were detected by RT-PCR,ELISA and Western Blot respectively.U87 cell line was used as positive control group.[Results]The expression of YKL-40 mRNA and protein was not detected in SKOV-3 cells.The agarose gel electrophoresis test showed the band of YKL-40 mRNA amplification products in U87 cells,and no amplification band in SKOV-3 cells;The protein secretion level of YKL-40 protein in U87 cells was up to(18.41 ± 1.69)ng/mL,and the clear YKL-40 protein band was found in Western Blot.The expression of YKL-40 secreted protein and intracellular total protein was not detected in SKOV-3 cells.[Conclusion]YKL-40 has no obvious expression in SKOV-3.Part Two The effect of YKL-40 on migration of epithelial ovarian cancer SKOV-3 cell[Objective]To investigate the effect of YKL-40 on epithelial ovarian cancer SKOV-3 cell migration in vitro and to explore the underlying mechanism.[Methods]According to the results of part one,construct the modified lentiviral vectors containing YKL-40 gene.Then SKOV-3 cells were transfected with this modified lentivirus to construct YKL-40 over-expression cell LV-SKOV-3 and YKL-40 over-expression was validated by qRT-PCR,ELISA,Western Blot.The cells transfected with empty vector were used as control(NC-SKOV-3);Transwell assay was used to evaluate the effect of YKL-40 on SKOV-3 cell migration.p38MAPK pathway was repressed by specific inhibitors and its effect on LV-SKOV-3 cell migration was further investigated.[Results]1.YKL-40 over-expression cell line LV-SKOV-3 and negative control cell line NC-SKOV-3 were establish.qRT-PCR,ELISA,Western Blot analyses further confirmed YKL-40 was significantly increased in LV-SKOV-3 cells on both mRNA and protein levels compared to NC-SKOV-3.The relative mRNA expression of LV-SKOV-3 was 9.89 times more than NC-SKOV-3 while the level of secreted protein reached to(32.35±1.38)ng/ml.2.After YKL-40 over-expression,the migration ability of LV-SKOV-3 was significantly enhanced.The averaged migration number of LV-SKOV-3 was(255:7±12.75)/view while NC-SKOV-3 was(119.25±13.25)/view.The migration ability of LV-SKOV-3 was increased 1.14 times compared with negative control cell NC-SKOV-3.3.Inhibition of p38MAPK pathway can partly decrease the LV-SKOV-3 migration ability.Transwell test showed that the migration ability of LV-SKOV-3 cellsin the inhibitor group was 63.30%lower than the control group.[Conclusion]YKL-40 significantly promotes the migration ability of SKOV-3 cells,which is partly mediated by p38MAPK pathway.Part Three The effect of YKL-40 on chemosensitivity of epithelial ovarian cancer SKOV-3 cell[Objective]To investigate the effect of YKL-40 on epithelial ovarian cancer SKOV-3 cell chemosensitivity in vitro and to explore the underlying mechanism.[Methods]YKL-40 over-expression cell LV-SKOV-3 and negative control cell NC-SKOV-3 were treated with cisplatin at different concentrations(1?g/ml,2.5?g/ml,5?g/ml,10?g/ml),CCK-8 assay was used to detect the cells' inhibition rate after 48 hours.The expression of NF-Kb in LV-SKOV-3 was detected by Western Blot assay.The specific inhibitor Bayl 1-7082 repressed the activity of NF-?B in LV-SKOV-3 cell and then detected the change of cells 'sensitivity to cisplatin.[Results]1.The result of Western Blot indicated that the level of NF-?B protein in LV-SKOV-3 was enhanced compared with negative control cell NC-SKOV-3.2.After inhibition of NF-kB,ELISA and Western Blot assays showed that YKL-40 expression of LV-SKOV-3 was decreased.The level of secreted YKL-40 protein was(25.40±3.30)ng/ml,which was 0.64 times less than DMSO control group.3.Being treated with different concentrations cisplatin,LV-SKOV-3 cell performed lower cell inhibition rate than NC-SKOV-3.This indicates that YKL-40 over expression inhibits the killing effect of cisplatin on SKOV-3 cells.4.After inhibition of NF-?B,the cell inhibitor rate of LV-SKOV-3 cell in inhibitor group was higher than DMSO control group,but still lower than NC-SKOV-3 cell group.It indicated that Inhibition of NF-?B can partly restore thekilling ability of cisplatin on SKOV-3.[Conclusion]1.NF-?B can regulate the expression of YKL-40 in epithelial ovarian carcinoma SKOV-3 cell.2.YKL-40 decrease the platinum drugs sensitivity of epithelial ovarian carcinoma SKOV-3 cell,which is partly mediated by transcription factor NF-?B.