The Mechanism Study Of Osteopontin (OPN) On Invasion And Metastasis In Tumor Cells Mediated By TLR2Signaling Pathway

Breast cancer is one of the cancers with high incidence and great harm in females, whichseriously affects physical and mental health of women. Invasion and metastasis of breast cancercells are the difficulty lie in the treatment, also is the main reason cause high mortality in breastcancer patients. Therefore, in-depth study on the mechanism of invasion and metastasis in breastcancer cells has great significance for the prevention and clinical treatment of breast cancer.Osteopontin (OPN), a secreted glycoprotein phosphorylation, can participate in manyphysiological and pathological processes, such as inflammation, immune response and cellularapoptosis, and also mediate tumor cells’ adhesion, invasion and metastasis by binding withspecific cell surface receptors. At present, the role of OPN in tumor cells and its mechanism isremain unknown, wich need for further research.TLR (Toll-like receptors) is a class of widely present in human body and is highlyconserved important pattern recognition receptors, which can recognize and combine with thepathogen-associated molecular pattern (PAMP), play an important role in natural immuneresponse. Recent studies have shown that a variety of tumor cells can express TLR,and had somerelevance with tumorigenesis, development, invasion and metastasis, but its mechanism isunclear.The prior research in our laboratory showd that toll-like receptor2and OPN expresseddifferently in human breast cancer cells MDA-MB-231(high metastatic breast cancer cell line)and MCF-7(low metastatic breast cancer cell line). The expression of TLR2and OPN inMDA-MB-231was higher than that in MCF-7. Research also showed that TLR2and OPN couldpromote the invasion and metastasis of breast cancer cells separately, so whether OPN havemany relationships with the effect of TLR2on the invasion and metastasis of breast cancer cells?And what is the mechanism that the interaction through? To solve these questions, we selectedbreast cancer cells MDA-MB-231as the object, and used Pam3CSK4(TLR1-TLR2heterodimeractivator) and LTA (TLR2activator) to activate TLR2signaling pathway to study the expressionchange of OPN, then further study the mechanism of TLR2and OPN in tumor invasion andmetastasis, which provided a theoretical basis for the clinical treatment of breast cancer. In this study, MTT, cell wound healing, Transwell cell Matrigel invasion assay, soft agarcolony formation assay and other means were used to test the abilities of breast cancer cellsMDA-MB-231in proliferation, migration, invasion and anchor growth after the stimulation ofPam3CSK4and LTA. The results showed that the stimulation of Pam3CSK4and LTA canpromote proliferation, migration,and invasion abilities of breast cancer cells.Real-time quantitative PCR and ELISA were used to test the expression of TLR2and OPNin breast cancer MDA-MB-231cells after the stimulation of Pam3CSK4and LTA. The resultsshowed that Pam3CSK4and LTA stimulation could notablely increase the expression of TLR2,and both stimulation could notablely increase the expression of OPN. The results of ELISA testsshowed that Pam3CSK4and LTA stimulation up-regulated the expression of iOPN and sOPNprotein.Use tumor metastasis PCR Array to analyse the expression of84genes，which associatedwith tumor metastasis. The results showed that Pam3CSK4and LTA stimulation inMDA-MB-231cells affected the expression of tumor metastasis associated genes.In summary, this experiment preliminary studied the mechanism of osteopontin (OPN) oninvasion and metastasis in tumor cells mediated by TLR2signaling pathway, and through tumormetastasis PCR array detected the different expression in tumor metastasis-related genes. All ofthese provided new ideas for the study on the mechanism of cancer cells’ invasion and metastasis,and also provided basic data for the clinical detection and treatment of breast cancer.