The Effect Of Foxp3 On The Biological Behaviour Of Lung Adenocarcinoa And Its Regulation By TLR4

Lung cancer is one of the highest morbidity and mortality of malignant tumors, which is one of the biggest threats to people health and life. It has been reported that the incidence and mortality of lung cancer were significantly increased in many countries.According to histological classification, lung cancer is divided into two major categories, small cell lung cancer(SCLC) and non-small cell lung cancer(NSCLC).NSCLC includes squamous carcinoma, large cell carcinoma and adenocarcinoma.While lung adenocarcinoma shows an earlier average age of onset than other types of lung cancer, and they usually have no obvious clinical symptoms at the early stage,most of these patients are already in advanced stage when they got their diagnosis.Although the growth of lung adenocarcinoma is slower than other types of lung cancer, it is usually metastasis at early stage, and the 5-year survival rate is still low.Therefore to study the molecular mechanism of the tumorigenesis and development for lung adenocarcinoma may help to provide the potential therapeutic targets of lung adenocarcinoma.Foxp3(Forkhead box P3) is a member of forkhead/winged-helix transcription factor family. It has been identified as a master regulator of the development and immunosuppressive function of CD4+CD25+ Tregs(Regulatory T cells). In 2007,Hinz et al was first found that Foxp3 expressed in pancreatic carcinoma, subsequent studies showed that Foxp3 also expressed in prostate cancer, breast cancer, ovarian cancer, gastric cancer and other malignant tumors, and influenced the progress and prognosis of cancers. However, these studies reported that Foxp3 appeared to be a multifaceted factor with seemingly opposite functions in tumor progression. Our previous study revealed that Foxp3 was expressed in NSCLC and was positively co-related with lymph node metastasis and TNM stage. However, the functions and molecular mechanisms of Foxp3 in lung adenocarcinoma still remain unclear.TLR4, one of the pattern recognition receptors, is a member of Toll-like receptors family and plays a role in innate and acquired immunity. It was reported that TLR4 was overexpressed in a variety of tumor tissues and cells, such as colorectal cancer,gastric cancer and ovarian cancer and so on. In the tumor microenvironment, the TLR4 signaling pathway could be activated to upregulate Foxp3 expression in Tregs and thereby enhance the immunosuppressive function of Tregs. Besides inflammatory cytokines release would be increased to promote tumor immune escape. Meanwhile, it has been reported that TLR4 activation could cause histone methylation changes at multiple sites. Nonetheless, it is still unknown the effects of the changes of histone methylation in the process of TLR4 participate in tumor immune escape. Our previous study revealed that TLR4 signaling pathway activation could induce Foxp3 expression in the human lung adenocarcinoma cell line, but the related epigenetics mechanism is still unclear.Based on the backgrounds, we proposed the following questions: what is the role and the related mechanisms of Foxp3 in the biological behavior of lung adenocarcinoma?Whether and how histone methylation changes participate in the regulation of Foxp3 expression by TLR4 in tumor immune escape? In order to solve our questions, we study the effect of Foxp3 on the biological behaviour of lung adenocarcinoa and its regulation by TLR4 with human lung adenocarcinoma tissue array and human lung adenocarcinoma cell line A549 as the research objects.I. The effect of Foxp3 on the biological behavior of lung adenocarcinoa.Our previous study revealed that Foxp3 was expressed in NSCLC and was positively co-related lymph node metastasis and TNM stage. Although the function of Foxp3 in lung cancer remains unexplored, the available data suggested that Foxp3 might function as a carcinogenic gene in lung cancer. In order to investigate the role of Foxp3 in lung adenocarcinoa and possible mechanisms, the lung adenocarcinoma array and human lung adenocarcinoma cell line A549 were used as the research objects. The study includes three aspects:1. The expression of Foxp3 in lung adenocarcinoa tissues.To investigate Foxp3 expression in lung adenocarcinoma specimens, a tissue array containing 40 lung adenocarcinoma samples and their corresponding noncancerous lung tissues was generated to determine Foxp3 expression by immunohistochemical staining. The data showed that(1) The expression of Foxp3 was increased in cancerous tissues, and Foxp3 protein was primarily localized in the cytoplasm and the minority localized in nucleus of the tumor cells. No obvious staining was observed in noncancerous lung tissues, it only had a low expression of Foxp3 and expressed in the cytoplasm/nucleus.(2) The positive rate of Foxp3 in tumor cells was 40.0%, while in the normal cells was 5.0%, the comparison between the two groups were statistically significant(P = 0.000). These results revealed that remarkable Foxp3 overexpression in lung adenocarcinoma and the location of Foxp3 was different between the tumor cells and normal cells.2. The effect of Foxp3 on migtarion, invasion and proliferation in lung adenocarcinoma cells.(1) Identification of silenced Foxp3 using si RNA.The Foxp3 gene was silenced in A549 cells using si RNA. The transfection efficiency was detected by fluorescence microscope and flow cytometry. The results showed that the transfection efficiency was 98.7%. The silencing efficiency was detected by RT-q PCR and Western Blot. The results showed that the expression of Foxp3 could be inhibited specifically by the two si RNAs that can be used for following experiments.(2) The effect of Foxp3 silencing on migration and invasion in A549 cells.The effect of Foxp3 silencing on migration in A549 cells were detected by wound healing and transwell migration assay. The results showed that the migration was impeded significantly after silencing Foxp3(P < 0.05). The effect of Foxp3 silencing on invasion in A549 cells were detected by matrigel invasion assay. The result showed that the invasion ability decreased significantly after silencing Foxp3(P < 0.05).These data indicated that Foxp3 enhanced lung adenocarcinoma cells migration and invasion.(3) The effect of Foxp3 silencing on proliferation in A549 cells.The effect of Foxp3 silencing on proliferation in A549 cells were detected by CCK8 assay. The result showed that the proliferation was inhibited significantly after silencing Foxp3(P < 0.05). The data indicated that Foxp3 enhanced lung adenocarcinoma cells proliferation.3. The mechanisms of Foxp3 effect on proliferation of lung adenocarcinoma cells.(1) The effect of Foxp3 silencing on cell cycle in A549 cells.To elucidate the mechanisms by which Foxp3 promotes A549 cell proliferation, the cell cycle was analyzed by PI staining assay. The result showed G1 cell cycle was arrest after Foxp3 silencing in A549 cells. The result suggested a proliferation-promoting role of Foxp3 in lung adenocarcinoma cells via accelerated G1/S transition.(2) The effect of Foxp3 silencing on related cell cycle checkpoint genes in A549 cells.The expression changes of associated G1/S checkpoint genes(CCND1, CCND2,CCND3, CCNE1, CCNE2, CDK2, CDK4 and CDK6) were examined by RT-q PCR and Western Blot after Foxp3 silencing in A549 cells. The results showed that the expression of CCND1 was obviously decreased after silencing Foxp3(P < 0.05).These data indicated that Foxp3 increased the expression of CCND1 genes, which promoted G1/S transition in lung adenocarcinoma cells.(3) The mechanisms of CCND1 regulated by Foxp3.To thoroughly investigate the role of Foxp3 in the expression of CCND1, the subcellular localization of Foxp3 and CCND1 were detected by immunofluorescence assay. The result showed that Foxp3 and CCND1 were colocaliaztion in A549 cells.Then we generated a construct in which luciferase expression was driven by regulatory regions of CCND1. The result showed that Foxp3 silencing significantly suppressed luciferase activity driven by the CCND1 promoter(P < 0.05). These results elucidated that Foxp3 regulated CCND1 transcription by binding its promoter directly.(4) The correlation assay among Foxp3 and CCND1 expression in lung adenocarcinoma tumor tissues.The expression of CCND1 in the lung adenocarcinoma tumor tissues and their corresponding noncancerous lung tissues(n = 40) were detected by immunohistochemical staining. The result showed that the CCND1 expression positively correlated with Foxp3 expression(R = 0.408, P = 0.001). While, there was no expression of CCND1 in the noncancerous lung tissues. It illustrated that a positive correlation between Foxp3 and CCND1 in lung adenocarcinoma tissues.From the results, we can conclude that the Foxp3 gene was over-expression in lung adenocarcinoma tissues. Functionally, Foxp3 could promote lung adenocarcinoma cells migration and invasion. In addition, Foxp3 could positively regulate the G1/S checkpoint gene CCND1 in both lung adenocarcinoma cells and clinical tissues. Our study has demonstrated an oncogenic role of Foxp3 in lung adenocarcinoma.II. The study of Foxp3 regulated by TLR4 and related epigenetics mechanisms.Our previous studies have shown that the TLR4 level was positively correlated with Foxp3 expression in human non-small cell lung tumor tissues. Additionally, TLR4 signaling pathway activation could induce Foxp3 expression increased in the human lung adenocarcinoma cell line A549. Nonetheless, it is still unknown whether histone methylation changes during TLR4 activation or specific molecular mechanisms of TLR4 participate in the regulation of Foxp3 expression in lung cancer cells. In order to analyze the epigenetics mechanisms of Foxp3 regulation by TLR4, the human lung adenocarcinoma cell line A549 was used as the research object. The study includes four aspects:1. The effects of TLR4 activation on the Foxp3 expression and inhibitory cytokines secretion.To observe the effects of TLR4 activation on Foxp3 expression and the relevant inhibitory cytokines(TGF-β1, IL-35, IL-10 and HMOX1) secretion in A549 cells, we first tested the expression of Foxp3 and the secretion changes of these inhibitory cytokines after LPS-induced activation of the TLR4 signaling pathway in A549 cells by RT-q PCR, ELISA and flow cytometry. The results showed that the expression of Foxp3 and the secretion of inhibitory cytokines(IL-10 expression not detected) were increased at both the m RNA and protein level after LPS activation of the TLR4 signaling pathway in A549 cells(P < 0.05). Further, TLR4 was activated with LPS for48 h after transfection with Foxp3 si RNA1/2 or NC si RNA, and then the secretion of inhibitory cytokines was detected by RT-q PCR and ELISA. The results showed that TGF-β1, IL-35, and HMOX1 expression levels were significantly reduced at both the m RNA and protein levels after Foxp3 silencing in A549 cells(P < 0.05). These data suggested that Foxp3 indeed might play a role in tumor immune escape by secreting inhibitory cytokines in A549 cells. Moreover, this process was regulated by TLR4.2. The change of relevant KDMs and KMTs after TLR4 activation.To observe the histone methylation changes participate in the regulation of Foxp3 expression by TLR4, the expressions of relevant KDMs and KMTs(KDM4C,KDM5 C, KDM3 A, KMT2 A, KMT2 D, KMT2 E, EZH1 and EZH2) after TLR4 activation were examined by RT-PCR and Western Blot. The results showed that KDM3 A was increased most significantly after TLR4 activation(P < 0.05) and maintained at a constant level. This finding suggested that the H3K9me1/2demethylase KDM3 A was associated with TLR4 activation. KDM3 A likely participated in TLR4-mediated transcriptional regulation of Foxp3.3. The effects of KDM3 A silencing on TLR4- induced Foxp3 expression and the inhibitory cytokines secretion.To examine the role of KDM3 A in the TLR4-mediated regulation of Foxp3, the KDM3 A gene was silenced in A549 cells using si RNA. Then the expression of Foxp3 and its downstream inhibitory cytokines secretion were detected by RT-q PCR, flow cytomertry and ELISA. The results showed that Foxp3 expression was significantly reduced after TLR4 activation when KDM3 A was silenced(P < 0.05). Meanwhile, the inhibitory cytokines secretion were significantly reduced(P < 0.05). These findings indicated that KDM3 A played a positive regulatory role during TLR4 regulation of Foxp3 and inhibitory cytokines secretion in A549 cells.4. The epigenetics mechanisms of Foxp3 regulation by TLR4.To further study the role of KDM3 A in the TLR4-mediated regulation of Foxp3 transcription, immunofluorescence and dual-luciferase reporter assay system were performed. The co-location of KDM3 A and Foxp3 was observed with immunofluorescence. Further, KDM3 A silencing suppressed luciferase activity driven by the Foxp3 promoter significantly(P < 0.05). The results elucidated that KDM3A could regulate Foxp3 expression directly.From the results, we can conclude that TLR4 activation could promote the expression of the H3K9me1/2 demethylase KDM3 A. KDM3 A bound directly to the Foxp3 promoter and promoted Foxp3 transcription, thereby inducing the secretion of Foxp3-related downstream inhibitory cytokines(TGF-β1, IL-35, and HMOX1),ultimately facilitating the immune escape of lung adenocarcinoma.In conclusion, in this research the role of Foxp3 in the development of lung adenocarcinoma were studied from both the effects of Foxp3 on lung adenocarcinoma biological behaviour and the epigenetic mechanisms of the regulation on Foxp3 expression by TLR4. The study might not only demonstrate the molecular mechanisms for tumorigenesis and development but also offer a novel therapeutic target for the precision treatment of lung adenocarcinoma.