Expressional Regulation Mechanism Of Tumor Metastasis-related Factor OPN

Breast cancer is a malignant tumor which usually occurs in the breast epithelial tissue. It isthe most common malignant tumor in females. It severely affects physical and mental healthand even threats lives of women. Therefore the researches on prevention and treatment of breastcancer are increasingly valued, and prevention of malignant invasion and metastasis of cancercells is thought to be an effective treatment against breast cancer.Inflammation has been considered as one of the most important factors in cancerprogression. Local chronic inflammation is easy to induce tumorigenesis through manymechanisms such as inhibition of DNA damage and apoptosis, and it can recruit immune cells,leading to immune cell infiltration, and as a consequence, forming a tumor microenvironmentto promote the development of tumor. In recent years, many studies have found that toll-likereceptors expressed stably in a wide variety of tumor cell. TLRs can launch all sorts ofinflammation through a variety of mechanisms, and TLRs may participate in the genesis anddevelopment of tumor.Osteopontin (OPN) is a secretory phosphorylation glucoprotein that binds to themetastasis-associated cell surface receptors αvβ3-containing integrin as well as CD44,mediating malignant cell attachment, migration and invasion. But the upstream of molecularsignaling pathways to initiat OPN expression in tumor cells are still not clear and waiting forfurther researches.Activation of TLRs signaling has been identified as an early molecular event, contributingto genesis and progression of cancer. Therefore it comes to a question that whether TLRssignaling affect metastatic capability of advanced cancer cells by mediating the expression ofOPN. And what is the specific signal mechanism? In this regard, we used two kinds of humanbreast cancer cell line MDA-MB-231and MCF-7to detect the TLRs and OPN expression inthem. First we detected the mRNA expression of TLRs and OPN in two human breast cancercells by real-time quantitative PCR technology. The results showed that TLR4have high levelsof expression and OPN have stable expression in both of the two breast cancer cell lines. But theexpression of TLR4and OPN in MDA-MB-231cells are significantly higher than that in MCF-7 cells, and MDA-MB-231is a high metastatic breat cancer cell line. Therefore, we chooseTLR4and MDA-MB-231cell line as the research object, and use LPS as the TLR4agonist, tostudy the effect of TLR4signals on OPN expression and tumor metastasis abilities.By using real-time quantitative PCR and ELISA test, we found that the activation of TLR4signaling pathways have improved the OPN expression and other tumor metastasis-relatedcytokines in MDA-MB-231cell. Then we used MTT assay, in vitro scratch assays, Transwellinvasion assay and soft-agar colonization assay to respectively test the cell proliferation,metastasis, invasion, and anchor-independent growth abilities of in vitro tumor under LPSstimulation. The results showed that LPS stimulation can improve the metastasis and invasionabilities of MDA-MB-231cells. Then we used OPN specific neutralizing antibody to block thesecreted OPN, and it reversed the enhancement of metastasis-related abilities by LPS stimulation.It suggested that OPN played an important role in the enhancement of metastasis-related abilitiesby LPS stimulation. Thus we demonstrated that TLR4signals promoted the metastasis of breastcancer cells through upregulating the OPN expression.Finally, in order to preliminarily explore the mechanism of TLR4signals affecting theOPN regulation, we used LY294002, a specific inhibitors of PI3K, to suppresses the activityof the downstream kinase of TLR4signaling pathways, and detected its effect on OPNexpression and metastasis-related abilities of MDA-MB-231cells. We found that thesuppression of PI3K reversed the upregulation of OPN expression and enhancement ofmetastasis-related abilities by LPS stimulation. This suggested that PI3K involved in the TLR4signal mediated upregulation of OPN expression and enhancement of metastasis-relatedabilities.Taken together, this study found that the upregulation of OPN expression by TLR4signalshave a significant influence on the malignization of breast cancer cells, and its possiblemolecular mechanism was studied preliminarily. Exploring the relationship between TLRsignaling pathway and OPN expression provides new clues for the researches of OPNregulation mechanism. And it may provide new possibilities for the prevention and treatment ofbreast cancer.