The Effect Study Of Osteopontin (OPN) On Metastasis And Invasion In Human Ovarian Cancer Cells

Ovarian cancer is one of a common tumor for the female, its incidence is second only todevelop womb cancer and cervical cancer. But ovarian cancer is the first place in all kinds ofgynecological tumors,which is a serious threat to women’s life health. Therefore, the preventionof ovarian cancer research is more and more attention, and prevention of malignant invasionand metastasis of cancer cells is an effective treatment against ovarian cancer.Osteopontin (OPN) is a kind of multi-functional extracellular matrix proteins, that binds tothe metastasis-associated cell surface receptorsαvβ3 and CD44, mediated malignant cellattachment, migration and invasion.So,osteopontin may become a new targets of malignanttumor treatment through the effect of osteopontin in cancer development andmetastasis,blocking OPN and its receptor signal transmission may be new biological basis forcancer therapy.This study were designed three OPN siRNA, which were OPN mRNA 255 sites, 483 sites,831 sites, and constructed the expression vector pGPU6 / GFP/Neo-shOPN, and set the negativecontrol pGPU6 / GFP/Neo-shNC. It found the cell transfection efficiency is 60% that use ofLipofectamine 2000 Reagent through the fluorescence microscope. Then we got sevenpositive clone lines through G418 culture solution.We found the OPN expression was reducedafter RNA interference through ELISA test, which found out the lowest monoclonal cell lines ofOPN expression. It is monoclonal cells pGPU6/GFP/Neo-OPN1-3 (M1-3) that the constructionof ectors cells is 255 sites. Applying a cell fluorescence immunohistochemical experimentshowed that red fluorescent abated after knockdown OPN.This result explained the express ofOPN reduced, which confirmed RNAi OPN success.We use cell phenotype experiments including MTT cell proliferation assay, soft agargrowth assay, wound healing assay and cell invasion assay to analyse 7 kinds of monoclonal celllines. The results showed that: contrasting the experimental group M1-3 (pGPU6 /GFP/Neo-OPN1-3) with the control group MC (untreated HO-8910 PM cells), the cellproliferation ability, the number of clones, the migration ability and the invasive ability reduced significantly. These results suggested that OPN knockdown reduced the cell proliferation,invasiveness and metastasis of the human ovarian cancer cell. Therefore, we screened M1-3(OPN knockdown HO-8910PM) cells to use for the following experiments.There is evidence showed that TLRs expressed on the surface of various tumor cells. Usingthe TLR4 specific agonists lipopolysaccharide (LPS) to stimulate tumor cells, we found that LPScan promote the invasiveness and metastasis of the tumor cells. As it is known that osteopontinalso can strengthen the invasion and metastasis of tumor, are there any connections betweenTLR4 signal and OPN expression?Based on these questions, the ovarian cancer cells of MC and M1-3 were pretreated withLPS of TLR4 specific agonists and detected the phenotypic changes in the control andexperimental group of the two ovarian cancer cells by the experiment of tumor cell cellproliferation assay, colony formation in soft agar, wound healing and transwell matrigelinfection assays. The results showed:（1）the experimental group of M1-3 Compared with thecontrol group MC, cell proliferation, metastasis and aggressive significant reduction of OPN,explained human ovarian cancer HO-8910 PM cell proliferation, metastasis and attack abilityby OPN knockdown. (2) The ovarian cancer cells of MC and M1-3 were pretreated with LPSfor 6h, and compared to the normal group (not add LPS) that cell proliferation, metastasis andattack ability represent increased, and MC cells showed significant difference. But compared toMC cells, the metastasis and invasion ability was significantly reduced in M1-3 cells, explainedOPN could influence of metastasis and invasion by LPS stimulated in the ovarian cancer cell ofHO-8910 PM of OPN knockdown. Thus we demonstrated LPS promoted HO-8910 PM cellmetastasis and invasion through enhanced expression of OPN.Then we detected expression of OPN through LPS stimulation to ELISA test and cellfluorescence immunohistochemical, the results indicated that: (1) Compared to MC cells,expression of OPN obviously reduced in the M1-3 cells, and significantly different. (2) Theovarian cancer cells of MC and M1-3 were pretreated with LPS for 6h that OPN expressionwas increased comparion of the normal group cells. In addition,we found expression of OPNwas significantly downregulation in M1-3 cells. Thus we demonstrated LPS promotedHO-8910 PM cell proliferation, metastasis and invasion through upregulation expression ofOPN. Therefore we are interested with whether TLRs signaling affect metastatic capability of advanced cancer cells by mediating the expression of OPN and investigate what the potentialcorrelation between TLRs and OPN.This study will be providing theoretical support to use atechnique of RNA interference the application in cancer therapy.