Primary Investigation The Role Of MEKK3 In Tumor Necrosis Factor-α-induced Interleukin-6 Production In Cancer Cels

Cancer is well known to hazard human health and life, and many cytokines are closely related with it.A large number of studies have shown that the cancer can autocrine or generate a large number of IL-6 by the proinflammatory factor (TNF-a,LPS etc.)stimulating, which is the incidence of cancer developmen,invasion and metastasis and is closely related to poor prognosis.On account of the literature and previous work, this study investigated the role of the mitogen-activated protein kinase MEKK3 in the IL-6 production of the cancer cells by TNF-a-induced to clarity the mechanism of the production of IL-6 by TNF-a-induced and find a possible role of therapeutic drug targets by using RNA interference, gene cloning, gene transfection, Westernblot, real-time fluorescence quantitative PCR, RT-PCR and ELISA techniques.Lung cancer is the most common and highly malignant tumors,and the first one which lead to death; breast cancer is not only the most common malignant tumor of women all over the world, but also one of the main cause of females death, therefore, the topic select lung cancer and breast cancer cells for study.In the first part of this study, the production of IL-6 in tne wild-type A549 lung cancer and breast cancer MDA-MB-231 cells after TNF-astimulating was detected by ELISA.The results showed that:after the stimulation of TNF-a, A549 and MDA-MB-231 cells can produce higher levels of IL6.0n this basis, the changes of phosphorylation and gene expression changes of MEKK3 were detectd in the A549 and MDA-MB-231 cells receiving TNF-a stimulated by using Western blot and RT-PCR, in order to determine whether is the TNF-a signaling pathway components.Western-blot results showed that:in the A549 accepting TNF-a stimulated, the phosphorylation of MEKK3 happened after 10min,30min peaked and 2h returned to normal; in the MDA-MB-231 accepting TNF-a stimulated, the phosphorylation of MEKK3 happened after 30min,1h peaked and 2h recoveryed normal.RT-PCR results showed that:compared with non-stimulated samples, after two kinds of cells receiving TNF-a stimulated 6h and 8h, the expression of MEKK3 mRNA were significantly increased.Western blot and RT-PCR results showed that: MEKK3 can be actived by TNF-a; MEKK3 is a molecule of the TNF-a signaling pathway, the changes about the phosphory-lation and its mRNA expression after accepting TNF-a stimulated is entirely similar to the phosphorylation and gene expression changes of other protein kinase reported in the literature due to accept cytokines stimulating.In the first part results of this study based, the second> third and fourth parts focused on exploring the role of the mitogen-activated protein kinase MEKK3 in the IL-6 production of the cancer cells by TNF-a-induced.In the second part of this study, four different siRNA template DNA sequence targeting in MEKK3 gene were designed by the application of Ambion's siRNA design software, synthesized the corresponding DNA fragments in vitro, cloned into the pSilencer 4.1-CMV hygro vector with its two restriction enzyme BamHI and HindlⅢcloning point to construct four targeted MEKK3 gene siRNA expression vector—pSilencer4.1-MEKK3 siRNA, then were transfected into A549 cells with Lipofectamine2000, Western-blot was carried out to analyze the suppression of MEKK3 in siRNA expression vectors. The results showed that these siRNA expression vector silenced,especially the suppression ratio in pSilencer4.1-MEKK3 siRNA2 expression vector was 85%.So transfecting it into A549 and MDA-MB-231, pSilencer4.1-MEKK3 siRNA2-harboring cell lines were acquired by hygromycin B selecting transfected cells, Real-time fluorescence quantitative PCR found that the suppression ratio of their MEKK3mRNA was more than 80%, so ensuring the follow-up study.In the third part of this study, The full length cDNA sequences of MEKK3 were obtained by RT-PCR., then cloned into pcDNA3.1/hygro (+) vector with KpnⅠand XhoⅠrestriction sites to construct pcDNA3.1/hygro (+)-MEKK3 eukaryotic expression vector, and transfected into A549 and MDA-MB-231 cells. Western-blot showed that the expression of MEKK3 protein were decreased in these two cells. pcDNA3.1/hygro(+)-MEKK3-harboring cell lines were acquired by hygromycin B selecting transfected cells. Real-time fluorescence quantitative PCR found that the expression level of their MEKK3 mRNA was increased comparing with empty vector stable clone.The results showed that pcDNA3.1/hygro (+)-MEKK3 eukaryotic expression vector was successfully constructed, and pcDNA3.1/hygro (+)-MEKK3-harboring cell lines were acquired, so ensuring the follow-up study.In the fourth part of this study, RT-PCR and ELISA were carried out to detect the expression of IL6 mRNA and the production of IL6 in the non-transfected A549 and MDA-MB-231 cells (wild type), down-expressed MEKK3 siRNA2/A549 and MDA-MB-231 stable cell clones, the MEKK3 gene in transiently transfected into down-expressed MEKK3 siRNA2/A549 and MEKK3 siRNA2/MDA-MB-231 cells which the expression of MEKK3 was restored by RT-PCR analying, A549 and MDA-MB-231 siRNA negative-control stable cell clone,over-expressed MEKK3 stable cell clone and pcDNA3.1 empty vector stable cell clone after TNF-a stimulating.The results showed that:the expression of IL6 mRNA and the production of IL6 in the down-expressed MEKK3 siRNA2/A549 and MDA-MB-231 stable cell clones were significantly lower than the wild strain or the negative control clone, the difference was significant (P<0.01); the two cell lines was restored the expression of MEKK3,their expression of IL6 mRNA and production of IL6 have largly improved, and were similar to the wild strains (P> 0.05), the production of IL6 in the over-expressed MEKK3 A549 and MDA-MB-231 stable cell clon were significantly higher.The preliminary results indicated that MEKK3 is closely related to the IL-6 production of the lung cancer and breast cancer cells by TNF-a-induced, and played an important role in regulating the IL-6 production of the lung cancer and breast cancer cells accepting TNF-a stimulated, the relevant mechanisms and downstream signaling pathways remains to be further studied.