Effects Of Inflammation Associated Factors On The Migration Ability Of MCF-7 Cells And The Underlying Mechamisms

Tumor is one of the major lethal causes threatening the health, which is the No1 killer some developed countries. For women, breast cancer is predominant in female cancers. The microenvironment of the tumor plays an important role in facilitating cancer progression and activating dormant cancer cells. One feature of many solid tumors is the influx of inflammatory cells, such as tumor infiltrating lymphocytes (TILs) and macrophages. These inflammatory cells are assumed to have an important function in the stages of cancer development such as cell transformation, angiogenesis, and metastasis. Indeed, focal macrophage infiltration has been linked to increased angiogenesis in human breast and colorectal cancer. Yet, the functional significance of cytokines produced in situ by inflammation and tumor cells is still unclear. Another feature of many tumors is the hypoxia of the tumor cells and the resulting production of reactive oxygen species (ROS).It has been reported that either ROS or nuclear factor kappa B (NFκB) could facilitate the epithelial-mesenchymal transition (EMT) in certain cell types and tumor necrosis factorα(TNF-α), one of the major factors released from the inflammatory cells, could cause NFκB activation and ROS production. It has been reported that TNF-αcan promote EMT in certain cell types, but the precise mechanism is still unclear. Based on the importance of the EMT in carcinoma progression and the prevalence of TNF-αand hypoxia in the presence of tumors, the present study was designed to determine whether TNF-αand ROS are capable of facilitating EMT and to understand the mechanisms involved.The continued expression and functional activity of E-cadherin are required for cells to remain tightly associated in the epithelium. In the absence of E-cadherin, many other cell adhesion and cell junction proteins expressed in epithelial cells are unable to support intercellular adhesion. The central role of E-cadherin in epithelia is demonstrated by the fact that loss of either its expression or function results in the dissolution of the epithelial architecture and the acquisition of a mesenchymal phenotype. This process, referred to as the epithelial–mesenchymal transition (EMT), occurs within the context of development and tumor progression. Snail functions as a key regulator in the induction of a phenotypic change called epithelial to mesenchymal transition (EMT). Aberrant expression of Snail prevails in the onset and development of tumor.Thus, our study aimed to elucidate that whether TNF-αand ROS could induce EMT in MCF-7 cells. Further, we observed the effects of TNF-αand ROS on the expression of Snail and subsequent E-cadherin. The underlying mechanism was also discussed. The migrating cell number in TNF-αtreated group is about 2–fold of that of the control group. Accordingly, the expression of E-cadherin was decreased and the expression of vimentin was increased upon TNF-αtreatment. These results showed that TNF-αcan promote epithelial- mesenchymal transition (EMT) of MCF-7 cells. Further, we found that the expression of Snail, an important transcription factor in EMT was increased in this process, which is inhibited by NFκB inhibitor Aspirin while does not affected by ROS scavenger NAC. Consistently, specific inhibition of NFκB by mutant IκBαalso blocked the TNF-αinduced upregulation of Snail promoter activity. Thus the activation of nuclear factor kappa B (NFκB), which causes an increase in the expression of the transcription factor Snail is essential in the TNF-αinduced EMT. Reactive oxygen species (ROS) caused by TNF-αseemed to play a minor role in the TNF-α-induced EMT of MCF-7 cells, though ROS per se can promote EMT. These findings suggest that different mechanisms might be responsible for TNF-α- and ROS-induced EMT, indicating the need for different strategies for the prevention of tumor metastasis induced by different stimuli. Accordingly, our results found that Aspirin and NAC can block the EMT induced by TNF and H2O2 respectively.In view of the above data and data from others, we found that ROS is potent inducer of Snail and consequently EMT. EMT can be triggered by different signaling molecules, such as by epidermal growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor beta (TGFβ), hepatocyte growth factor (HGF), WNTs and Notch. In agreement with the involvement of Snail in all studied processes of EMT, these signalling molecules have been shown to induce Snail genes in different cellular contexts. Previously, ROS were found to facilitate the EMT in certain cell types. In addition, ROS is supposed to be produced in these processes. Thus, it is reasonable to deduce that ROS functions importantly in these EMT process. From the above data, we can see NFκB does not play a major role, which prompt us to switch to posttranscriptional regulation of Snail.In addition to transcriptional events, gene expression programs are strongly influenced by posttranscriptional regulatory processes, such as those controlling mRNA turnover and translation. Although the mechanisms determining mRNA turnover are poorly understood, they are generally believed to involve RNA-binding proteins recognizing specific RNA sequences. There has been growing interest in a particular pathway which regulates mRNA stability, and is mediated by AU-rich elements (AREs), usually found in the 3` untranslated region (UTR) of short-lived mRNAs. HuR binds target mRNA subsets bearing AREs through its RNA recognition motifs and has been shown to regulate the expression of many target mRNAs, including those that encode c-fos, vascular endothelial growth factor, tumor necrosis factor alpha (TNF-α), b-catenin, c-myc, cyclooxygenase 2, myogenin, MyoD, several cyclins, granulocyte- macrophage colony-stimulating factor, several interleukins, p21, p27, p53, and hsp70. Interestingly, Snail mRNA bears multiple AREs in the 3`UTR. What's more, H2O2 enhance the cytoplasmic localization of HuR, suggesting the potential role of HuR in the regulation of Snail under the treatment of H2O2.First,MCF-7 cells were transfected with synthetic siRNA against HuR or the control siRNA. Knockdown efficiency was detected by the Western blotting 36 h after transfection. 70% of the endogenous HuR was knocked- down. Cells 36 h after transfection with either the control or the siRNA against HuR were treated with 50μM H2O2 for 12 h before harvested for RNA extraction. Decreased mRNA level of Snail was seen in the si-HuR group, suggesting a role of HuR in the process. Consistent changes of Snail at protein level were also observed. As a common role of HuR in the regulation of the mRNA stability of target genes, we asked whether HuR increased the expression of Snail in the same way. To test the possibility, we observed the half-life of Snail in the HuR knocked down cells and the control cells. Actinomycin D was added 12 h after H2O2 treatment to block the transcription. Shorter half-life of Snail was found in si-HuR group, pointing out that HuR increased the stability of Snail.Since HuR usually functions through the ARE located in the 3`UTR of target genes, we analyzed the 3`UTR of the Snail mRNA. The UTR region is highly AU rich and there are three typical AREs. We sought to directly test whether HuR was involved in the regulation of 30UTR of Snail through the supposed AREs. Both the UTR containing the AREs and the deletion without the AREs were cloned to pGL3-Control vector and transfected into si-HuR and the control MCF-7 cells. The construction bearing the full length of the UTR had lower activity compared with the deletion in MCF-7 cells without H2O2 treatment and HuR knockdown, suggesting the AREs functions as an element for instability. In addition, only the construct with AREs were responsive to H2O2 (about 2-fold under the treatment of H2O2) in MCF-7 cells without HuR knockdown, suggesting that both AREs and HuR are necessary for H2O2 induced Snail upregulation. In addition, in si-HuR MCF-7 cells, even the construction containing AREs rarely responded to H2O2. Together these results suggested that H2O2 increased the expression of Snail by the interaction of ARE and HuR.Subcellular re-distribution of HuR after H2O2 treatment HuR is predominantly localized in the nucleus while shuttled to the cytoplasm under stress conditions to function as an mRNA stabilizer or a translational regulator. To assess whether H2O2 treatment caused any changes in relative distribution of HuR, subcellular fraction were studied by western blot in untreated or H2O2-treated cultures. HuR were predominantly nuclear when without treatment, as reported earlier. By 6 h of H2O2 treatment, HuR were found to enrich in the cytoplasm. In addition, no obvious changes of the total level of HuR were found. Together these results pointing out that H2O2 increased Snail expression at least partially through the posttranscrip- tional regulation by enriching HuR in the cytoplasm. Of note, our study can not rule out the importance of transcriptional regulation. In fact, Snail was rarely expressed in cells without H2O2 treatment in MCF-7 cells. Thus, we can draw the conclusion that combination of transcriptional and post- transcriptional regulation contributes to the upregulation of Snail under the treatment of H2O2 in MCF-7 cells.At last, we observed the effects of knockdown of HuR on the migration ability induced by H2O2. To this end, first we observed the expression of E-cadherin both at mRNA level and protein level under H2O2 induction either in MCF-7 cells with or without HuR knockdown. H2O2 treatment reduced the transcription of E-cadherin in MCF-7 cells without HuR knockdown (about 4-fold). And this repression effect was only partially rather than totally eliminated in HuR knockdown MCF-7 cells (about 2-fold downregulation induced by H2O2 treatment), suggesting that HuR only functions partially in the induction of Snail and the consequent inhibition of E-cadherin. In addition, HuR knockdown itself seemed to have no effect on the expression of E-cadherin. Consistent with the mRNA data, the protein level of E-cadherin displayed similar changes. Then we observed the migrating ability in si-HuR cells and the control cells. As expected, decreased migrating ability was found in the si-HuR cells under the treatment of H2O2. Thus, besides the anti-apoptotic role of HuR, here we suggest a role of HuR in the migrating process.In summary, our study here has investigated the effects of TNFαand H2O2 in the migration of MCF-7 cells. It is important to note that NFκB mainly mediate the upreguation of Snail by TNFαwhile HuR mediate partially the increased expression of Snail in the process of H2O2 treated MCF-7 cells.