OPN Promotes Invasion And Migration Ability In Human Endometrial Cancer Cells Research

Endometrial cancer(EC) is the most common form of malignant influence of the female reproductive tract diseases, the incidence rate increased year by year. In recent years, with the rapid development of C hina’s economy, changing people’s lives and eating habits, irregular hormone replacement therapy and sex abuse and other factors, leading to a marked increase in the rate of endometrial cancer and tend to be young er, become a serious threat to our country health of the female reproductive tract malignant disease.OPN(osteopontin, OPN) is a combination with a variety of biological functions phosphorylated glycoprotein widely present in various human tissues and bod y fluids, and tumor growth, proliferation, invasion and metastasis are closely related,are considered to be a new tumor markers. Studies have shown that the function and role of osteopontin in gynecological tumors are more and more attention.Objective: To investigate the effect of human OPN recombinant protein(rhOPN) expression on the proliferation, migration and invasion of endometrial carcinoma HEC-1A cells. To detect OPN how to influence the MMP-2, E-cadherin, N- cad he r in, Vimentin expression and analysis related pathways molecules.Methods:(1) We use human endometrial carcinoma HEC-1A cells in vitro model, with different concentrations of rhOPN(0ng/ml, 100ng/ml, 200ng/ml, 300ng/ml, 400ng/ml) treated cells 24 h, using CCK-8 assay, Transwell assay and Wo und healing assay to detect proliferation,invasion and migration abilities of HEC-1A cells.(2) rhOPN protein was used to induce HEC-1A cells and detect the expression of MMP-2 by Western-blot. Using rhOPN concentration 300ng/ml, at different times(0h, 4h, 8h, 12 h, 24 h, 48h) induce HEC-1A cells, Westen-blot and Zymography assay detect MMP-2 protein expression and activity impact.(3) Using rhOPN concentration of 300ng/ml treated HEC-1A cells 24 h, detect the expression and location of E-cadherin, N-cadherin, Vimentin by Western-blot. Immunofluorescence results compared with the control group: E-cadherin, N-cadherin and Vimentin were located in the cell membrane.(4)Using rhOPN concentration of 300ng/ml treated HEC-1A cells 24 h, then add the relevant signaling pathways specific inhibitor U0126(MEK1/2 inhibitor), LY294002(PI3K inhibitor) respectively inhibit ERK and AKT activation detect MMP-2, E-cadherin, N-cadherin and Vimentin protein expression by Westen-blot.Results:(1) CCK-8 assay, Transwell assay and Wound healing assay showed the strongest proliferation, migration and invasion ability of HEC-1A cells at the rhOPN of 300(ng/ml)24h.(2) Both diffierent concentration and hours, MMP-2 was slightly changed from 0 to 400 and the strongest expression of MMP-2 at the point of 24 hours 300(ng/ml) rhOPN. At the best rhOPN conditions, the activity of MMP-2 was best by gelatin zymography.(3) The concentration of rhOPN 300(ng/ml)could increase the expression of N-cadherin, Vimentin and dcrease the expression of E-cadherin by Western-blot and Immunofluorescence staining.(4) In this condition rhOPN could active the AKT, ERK, when AKT and ERK were inhibited by LY294002(a specific inhibitor of PI3K/AKT phosphatidylinositol 3-kinase), U0126(a specific inhibitor of mitogen activated protein kinase kinase(MEK)/extracellular signal regulated kinase ERK), the expression of MMP-2, N-cadherin and Vimentin were decreased the expression of E-cadherin was increased.In meantime, the activity of MMP-2 was inhibited by Gelatin zymography.Conclusion:(1) OPN could influence the expression of MMP-2, N-cadherin, Vimentin and E-cadherin to participate in the process of proliferation, invasion and migration in HEC-1A cells.(2) OPN activate AKT and ERKsignaling pathways increase MMP-2, N-cadherin, Vimentin and decrease E-cadherin expression.