Foxp3Expression In A549Cells Regulated By TLR4Through NF-kB

Foxp3(Forkhead box protein3) has been identified as a master regulator of thedevelopment and function of Tregs（CD4~+CD25~+regulatory T cells）. Recent papers reportthat Foxp3is not only expressed in Tregs but also in tumor cells, moreover, the expression ofFoxp3in tumor cells is associated with tumor escape. However, it is unclear how theexpression of Foxp3is regulated in tumor cells. Many reports show that Foxp3is closelyconnected with NF-κB（Nuclear factor-kappa B）. Studies found that the NF-κB signallingpathway was involved in the LPS/IL-2induced upregulation of Foxp3expression. NF-κBcan also promote the transcription of Foxp3.TLR4(Toll-like receptor4) is one member of Toll-like receptors that are mainlyexpressed in immune cells and considered to be an important link between innate andadaptive immunity, NF-κB is the key factor of TLR4signaling pathway. Recent studies havefound that TLR4is also expressed in many tumors. Studies found that lung cancer cellsexpressing TLR4were functionally associated with anti-apoptotic activity through activationof NF-κB by LPS. In many cancers, NF-κB is persistently activated, which contributes totumorigenesis and cancer therapy resistance. Studies demonstrated that LPS, a TLR4ligand,promoted tumor invasion through the NF-κB pathway in breast cancer and colorectal cancercells.Our previous studies found that the expression of Foxp3and TLR4were expressed innon small-cell lung cancer (NSCLC), and both expression was positively correlated;Moreover, our studies indicated that TLR4signaling pathway may be involved in theregulation of Foxp3in Lewis lung caner(LLC) cells, but the accurate mechanism is stillunclear.Based on the above background, we proposed the following questions:1. Whether TLR4and Foxp3are expressed in A549cells?2. If so, what are the relations between TLR4andFoxp3?3. Could TLR4regulate the expression of Foxp3in A549cells through NF-κB?So, Our research include three parts and the results as follows:1. TLR4and Foxp3are expressed in A549cellsFirstly, in order to investigate whether TLR4and Foxp3are expressed in A549cells, the expression of TLR4and Foxp3in A549cells was examined by RT-PCR and flow cytometry(FCM). The results show that both TLR4and Foxp3can be expressed in A549cells.2. Activation of TLR4promotes the expression of Foxp3In order to investigate if TLR4signaling pathway is involved in the regulation of theexpression of Foxp3, LPS was used as TLR4ligand to activate TLR4signaling pathway,after LPS stimulating the mRNA and protein expression of Foxp3were detected in A549cells by RT-PCR, RT-qPCR and FCM. The result showed that the expression of Foxp3mRNA and protein in LPS stimulation groups were significantly increased compared withcontrol group. To further verify that the TLR4signalling pathway is involved in theregulation of the expression of Foxp3, PMB (Polymyxin B) was used to block the TLR4signalling pathway, and the expression of Foxp3mRNA and protein were examined byRT-PCR, RT-qPCR and FCM. The results showed that the expression of Foxp3mRNA andprotein were significantly decreased with PMB treatment compared with the control LPSstimulation group. These results indicate that the expression of Foxp3can be regulated byTLR4signalling pathway in A549cells.3. NF-κB is involved in the expression of Foxp3induced by the TLR4signallingpathwayTo explore if NF-κB is involved with regulation of Foxp3by the TLR4signallingpathway, the expression of p65protein after LPS stimulation was detected by westernblotting. The results showed that the nuclear p65in LPS stimulated group was significantlyhigher than the control group. To investigate if the expression of Foxp3is regulated by theTLR4signalling pathway through NF-κB, PDTC was used to inhibit NF-κB and then theexpression of Foxp3mRNA and protein were examined by RT-PCR, RT-qPCR and FCM.The results showed that the expression of Foxp3mRNA and protein were significantlydecreased upon PDTC treatment compared with the control LPS stimulation group. Theseresults suggest that NF-κB is involved in the regulation of Foxp3expression.From the studies of above, the conclusions are:1. Both TLR4and Foxp3are expressed in A549cells.2. Foxp3expression can be regulated by TLR4signaling pathway through NF-κB inA549cells.This study indicates that Foxp3expression can be regulated by TLR4signaling pathwaythrough NF-κB in A549cells which might provide some clue for understanding theregulating mechanisms of Foxp3in tumor cells and may also provide new insight on clinical immunotherapy for lung cancers.