Mechanism And Clinical Significance Of Ku80 Involved In Lung Cancer Growth Through Regulating COX-2 Expression

Objective: Cyclooxygenase 2(COX-2) overexpression in tissue injury, inflammation or malignant cells, that is closely related to state COX-2 may be associated with inflammation, tumorgenesis. A large number of studies show that, COX-2 were highly expressed in lung cancer, esophageal cancer, gastric cancer, breast cancer, colon cancer, hepatocellular carcinoma, which has great significance in drug resistance and prognosis of tumor. PGE2 as a COX-2 product, stimulates blood vessel growth, suppresses the immune function, has the important meaning to regulate a variety of cellular proliferation, tumor growth signaling pathway etc.. Previous research indicates that, the overexpression of COX-2 can promote cell proliferation by weakening the anti proliferation effect of TGF- beta, and promote the anti-apoptotic protein Bcl-2 and inhibit the expression of apoptosis-protein Bax to inhibite apoptosis. COX-2 as the Bcl-2 upstream regulatory protein, can also activate PI3 K to upregulate Mcl-1 and inhibit the apoptosis of tumor cell. Others think the inhibition of COX-2 on the apoptosis signaling of tumor is induced by p53 gene mutant, or COX-2 mediates Fas protein weakending apoptosis signaling. The inhibition of COX-2 upregulates the expression of caspase proteins. In addition, COX-2 can reduce the activity of E-cadherin to promote cell invasion and metastasis. And there are correlation between COX-2 and MMP9. Inhibition of COX-2 expression downregulates the expression of MMP9 family proteins. Some research found that, PGE2 upregulate CD44,which is important in tumor migration and tumor stemness.In angiogenesis aspect, COX-2 induce VEGF and TGFbeta, which can promote endothelial cell migration and tube formation. COX-2 knockdown significantly downregulates VEGF and inhibit gastric cancer cell growth. Some studies suggest that COX-2 upregulate Akt expression, thereby promoting angiogenesis. At present, COX-2 inhibitor indomethacin, Celecoxib can significantly inhibit the growth of tumor and angiogenesis, induce apoptosis of tumor cells, but alsocan enhance the radiosensitivity of chemotherapeutic and drug cytotoxicity to tumor. However, drugs treated with COX-2 inhibitors are often easy to produce the high toxicity. In recent years, with the development of tumor molecular biology, tumor immunology, it makes the gene diagnosis, tumor targeted therapy, gene therapy has become possible. The knockdown or overexpression of some transcription factors to inhibite COX-2 expression. At present, NF-κB, AP-1, TFIIB, transcriptional coactivator of p300/CBP have been found to transcription factors of COX-2, makes the COX-2 targeted therapy entered a stage of deepening. Complexity of transcription regulation, COX-2 also exist some other regulatory factors, the purpose of this paper is to find the new COX-2 regulation factor, study the mechanisms of COX-2 transcriptional regulation, as well as the significance of tumor growth.Methods:(1) Using streptavidin-agarose pulldown and proteomics assay to discover and validate some new COX-2 promoter binding protein in lung cancer cell lines.(2) Gene transduction and si RNAme technology to overexpress or knockdown COX-2 promoter specific binding protein, further to effect the activitity of COX-2 promoter in lung cancer cells. And study the mechanism of COX-2 binding protein regulates cell proliferation, transformation and some other biological function in vitro and in vivo.(3) Using RT-PCR, Western blot, immunohistochemistry to detect the expression of COX-2 and COX-2 binding protein in human tumor cells and normal cells and patient tissues, and combined with the cell characteristics and clinical data analyse the specific expression of COX-2 binding protein in lung cancer and the value of clinical diagnosis, predict prognosis, recurrence and therapeutic target.(4) Constructing animal model to validate the effect of Ku80 on tumor growth and COX-2 expression.(5) Using immunoprecipitation assay to test the interaction of Ku80 and the transcriptional co activator of CBP, and its effect on the expression of COX-2.Results:(1) Using streptavidin-agarose pulldown and proteomics assay, we identified and validated Ku80 as a new binding protein of the COX-2 gene promoter, and verify the combination of Ku80 and COX-2 promoter by Ch IP assay.(2) Overexpression orknockdown of Ku80 can enhance or inhibit activity of COX-2 promoter and the expression of COX-2. Knockdown of Ku80 can inhibit cell proliferation and migration.(3) Using Western blot, immunohistochemistry to find the expression correlation between COX-2 and Ku80 in lung cancer cells and lung cancer patients, and Ku80 is of great significance on the prognosis and metastasis of lung cancer.(4)In vivo, knockdown of Ku80 could inhibit the growth of tumor and COX-2 expression.(5) Using immunoprecipitation, we found that Ku80 can interact with CBP, and acetylated by CBP to effect the transcription of COX-2.Conclusions: Successfully pulldown and identificate the new COX-2 binding protein by streptavidin-agarose pulldown and proteomics assay and test Ku80 regulate COX-2 expression. There are correlation between Ku80 and COX-2 expression through clinical datas analysis and Ku80 is of great significance on the metastasis and prognosis of lung cancer.