Preliminary Investigation The Role Of MEKK3 In LPS Induced Interleukin-6 Production In Cancer Cells

With science development, clinical treatments including neoadjuvant radiother- apy,chemotherapy,biotherapy,and gene therapy,as well as traditional medicine has made amazing progress in the treatment of cancer,but on the whole,at present most of the effects of cancer treatment and prognosis remains poor,with 5-year survival rate is still very low.Occurrence of malignant tumors and development is the result of a number of factors,long-term chronic inflammation of stomach,liver, lung,colorectal,prostate cancer,breast cancer,ovarian cancer,and other important factors while cytokines play an important role in this process.A large number of studies have shown that the cancer cells can autocrine or generate a large number of IL-6 by the proinflammatory factor (TNF-α,LPS etc.)stimulating,which is the incidence of cancer development,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 LPS-induced using RNA interference,gene transfection,Western-blot, RT-PCR and ELISA techniques.Ovarian cancer,lung cancer is highly malignant tumors,and gastric cancer and breast cancer is most common in China or a malignant tumor in women.We first applied LPS to stimulate these four different tumor cells,and then to detect the production of IL-6 levels.It is found that four kinds of cells could produce different concentrations of IL-6,but ovarian cancer cell SKOV3 and gastric adenocarcinoma cell AGS generated higher concentration,therefore,we selected ovarian cancer cell SKOV3 and gastric adenocarcinoma cell AGS for study.In the first part of this study,the production of IL-6 in the wild-type AGS gastric adenocarcinomas cell and SKOV3 ovarian cancer cell post-LPS stimulating was detected by ELISA.The results showed that post-stimulation with LPS, AGS and SKOV3 cells can produce higher levels of IL-6.In order to determine whether MEKK3 is the LPS signaling pathway component,the changes of gene expression of MEKK3 were detectd in the AGS and SKOV3 cells in response to LPS stimulating by using RT-PCR. The results of RT-PCR showed that compared with non-stimulated samples,the expression of MEKK3 mRNA were significantly increased after the two kinds of cells had been stimulated with LPS.RT-PCR results showed that MEKK3 can be actived by LPS.Therefore,MEKK3 is a component of the LPS signaling pathway in these two different cancer cells,the changes about MEKK3 mRNA expression in response to LPS stimulating is entirely similar to gene expression changes of other protein kinase reported in the literature due to accept cytokines stimulating.Based on the first part results of this study,the second and third parts focused on exploring the role of MEKK3 in the IL-6 production of the cancer cells by LPS-induced.In the second part of this study,three different siRNA template DNA sequence targeting in MEKK3 gene and three different siRNA template DNA sequence targeting in MEKK2 gene were designed by the application of Genscript siRNA design software,synthesized the corresponding DNA fragments in vitro,cloned into the pRNAT-H1.1/Adeno shuttle vector with its two restriction enzyme MluI and XhoI cloning point to construct three targeted MEKK3 and MEKK2 gene siRNA expression vector:AD-MEKK3siRNA and AD-MEKK2siRNA, respectively, then transformed into Escherichia coli BJ5183 carrying backbone plasmid pADEasy-1 to obtain adenovirus plasmid through homologous recombination.The adenovirus plasmid was transfected into 293A cells to form adenovirus particle.The successfully packaged virus was upscaled through 3 to 5 circles and then its titer was determined. The successfully transfected was confirmed by GFP expression.Western blot was carried out to analyze the suppression of MEKK3 in AGS and SKOV3 cells that had been transduced with the AD-MEKK3 siRNA.The results showed that the best siRNA expression vector could silence MEKK3 expression with a suppression ratio which was up to 89%.While the best siRNA expression vector could silenced MEKK2 expression with a suppression ratio which was up to 75%.In the third part of this study, RT-PCR and ELISA were carried out to detect the expression of IL-6 mRNA and the production of IL-6 in the non-transfected AGS and SKOV3 cells (wild type),down-expressed MEKK3 siRNA3/AGS and SKOV3 cell, the MEKK3 gene transiently transfected into down-expressed MEKK3 siRNA3/AGS and MEKK3 siRNA3/SKOV3 cells which the expression of MEKK3 was restored by Western blot analying, and AGS and SKOV3 siRNA negative-control cells post-LPS stimulating.The results showed that the expression of IL-6 mRNA and the production of IL-6 in the down-expressed MEKK3 siRNA3/AGS and SKOV3 cell were significantly lower than the wild strain or the negative control clone, the difference was significant (P<0.01);After the two cell lines had been restored the expression of MEKK3, their expression of IL-6 mRNA and production of IL-6 were largly improved, and were similar to that of wild strains (P>0.05).The preliminary results indicated that MEKK3 is closely related to the IL-6 production of the gastric adenocarcinoma cancer and ovarian cancer cells by LPS-induced, and played an important role in regulating the IL-6 production of the gastric adenocarcinoma cancer and ovarian cancer cells in response to LPS stimulating.The relevant mechanisms and downstream signaling pathways remains to be further studied.