The Role Of Immune Regulatory Molecules PDCD4and TIPE2in Inflammation And Tumor

ObjectiveLung cancer is one of the most common malignant tumor and the leading cause of cancer death in the world. In pathologically, lung cancer can be divided into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). NSCLC accounts for more than80%of all kinds of lung cancers, including many histological types. The abnormal expression of many genes are involved in the development and progression of lung cancer. To explore new genes related to lung cancer and to investigate the role in cancer will be helpful for dignosis and therapy in furture.TIPE2, tumor necrosis factor-alpha-induced protein8(TNFAIP8)-like2(TNFAIP8L2), is a newly described negative immune regulator. It regulates both the innate immunity and the adaptive immunity by which it plays an important role in immune homeostasis. TIPE2preferentially expressed in immune organs and lymphatic tissue in mice. Our previous research has indicated that human TIPE2expressed in many non-immune cells, such as stratified squamous epithelial cells, seminiferous epithelial cells and some glandular epithelial cells, suggesting TIPE2may has other functions except for immune regulation.Previous research has found that TIPE2is able to bind to RGL specifically and then inhibit the activation of Ras signaling pathway, by which it suppresses the proliferation and migration of cells. So we wonder if TIPE2can serve as a new tumor suppressor. In our research from hepatocellular carcinoma (HCC), we found that there is a markedly low expression of TIPE2in cancer tissues of HCC compared with the adjacent non-tumor tissues. However, the fearture of TIPE2expresion in othter tumors is unclear. Meanwihile, the role of TIPE2in human tumor remians to be invastigated. Recent research reported that the murine TIPE2N-terminal lysine or arginine residues (Lys-15, Lys-16, and Lys-24) could combind with the C-terminus of Racl CAAX motif, by which it inhibits Rac membrane translocation, activation, and then down-stream Rac signaling. Rac is an important effector for cell movement, then we suppose if TIPE2is involved in the lung cancer and whether TIPE2could regulate the function of lung cancer cells by inhibiting Racl signal pathway.In present research, we explored the expression and function of TIPE2in NSCLC.Methods1. The expression of TIPE2mRNA in human lung cancer tissues and the adjacent tissues detected by Realtime PCR.Tissue specimens were collected from Qilu Hospital, including lung cancer tissues and the corresponding adjacent lung tissues.Total RNAs were extracted and were reversely transcripted to cDNA and then TIPE2expression was detected in by Realtime PCR with TIPE2specific primers.2. The expression of TIPE2protein in human lung cancer tissues and adjacent tissues detected by Western-blot.Proteins were extracted and were used in Western-blot to detect TIPE2expression using TIPE2-specific antibody in lung cancer tissues and adjacent tissues.3. TIPE2protein expression in lung cancer tissues and adjacent tissues by immunohistochemistry (IHC)The expression of TIPE2in human NSCLC tissue microarray were detected by IHC. We also observed the expression condition of TIPE2in lung cancer tissues with different pathological grading.4. Establishment of TIPE2overexpression and silence systemLung cancer cell lines A549and H1975were seeded onto24-well plate and then were transfected with recombinant TIPE2vector or mutant TIPE2vector or specific SiRNA for TIPE2. Tweenty-four hours after transfection, the cells were used for function research.5. Detection of cell proliferation by CCK8assayH1975and A549cells were seeded into96-well plate and their proliferation was detected using Thermo Scientific Microplate Reader. Growth curve was done based on the absorbance data.6. Colony forming assayH1975and A549cells were seeded into24-well plate and transfected with recombinant TIPE2vector or mutant TIPE2vector or specific SiRNA for TIPE2. Tweenty-four hours after transfection, cells were transfered into6-well plate in an appropriate density. After10days, the cells was fixed with methanol, then stained with1%crystal violet. The number of colony including more than50cells was counted under the microscope and colony-forming rate was calculated.7. The effect of TIPE2on migration and invasion capability of lung cancer cellsCell invasion was measured using Matrigel coated Transwell cell culture chambers (8μm pore size; Costar, Acton, MA), and cell migration was detected with transwell uncoated with Matrigel.8. Statistical analysis.Non-paired t test was used to analyse the difference of TIPE2expression between lung cancer tissues and adjacent tissues on both mRNA level and protein level. Spearman correlation test was used for the analysis of the expression of TIPE2and clinical parameters,Results1. TIPE2expression and clinical significance in lung cancer tissues and adjacent tissues.(1) There was no significant difference of TIPE2expression on mRNA level by Realtime PCR between lung cancer tissues and adjacent tissues(2) TIPE2expression by Western blot was markedly higher in lung cancer tissues than that of the adjacent tissues.(3) The IHC results from lung cancer micro-array showed that TPIE2expression in lung cancer was up-regulated compared to the corresponding adjacent non-tumor lung tissues. The absence or low TIPE2expression was detected in some cancer tissues with III pathological grade. There was no significant difference between TIPE2expression and sex, age, histopathological type, and tumor size.2. The effect of malignant behavior in lung cancer cells by over-expression of exogenous TIPE2gene.(1) CCK-8assay indicates that there was no difference between any periods, indicating that TIPE2did not affect the proliferation of lung cancer cells.(2) The over-expression of TIPE2markedly inhibited the colony forming ability of lung cancer cells both in A549and H1975cell lines.(3) Cells transfected with TIPE2plasmid had a significant decreased migration and invasion ability, indicating that TIPE2is able to inhibit the migration and invasion of lung cancer cells.3. The function of endogenous TIPE2in lung cancer.(1) Gene sequencing showed that no mutant happened to endogenous TIPE2in lung cancer cells.(2) Knockdown of endogenous TIPE2in lung cancer cells by using TIPE2specific siRNA could significantly increased the migration and invasion ability of lung cancer cells.4. TIPE2act as a tumor suppressor through targeting the Rac signal pathway(1) The inhibition of wild type TIPE2on the colony formation of lung cancer cells was reversed by mutation of TIPE2in Rac1-binding region(2) The mutant TIPE2in Rac1-binding region losed the ability of suppression to the migration of lung cancer cells.(3) The mutant TIPE2in Rac1-binding region losed the ability of suppression to invasion of lung cancer cells.Conclusions:1. High TIPE2expression was detected in lung cancer tissues compared with the adjacent tissues on protein level.2. TIEP2decreases the colony formation, migration and invasion of lung cancer cells through targeting the Rac1signal pathway.Originality and significance:1. We characterize the feature of TIPE2expression and function in lung cancer for the first time.2. TIPE2shows the tumor suppressor-like action in lung cancer, which will be a new potential target gene for therapy of lung cancer.Limitations:1. Because the limited number of cases in micro-array, and most of the cases were in grade II while cases in grade III were rare, an accurate relation of the expression of TIPE2and clinopathological features need to be further verification.2. As the limited research time, the sepcific Racl signal pathway through which TIPE2acts on lung cancer malignant behavior should be studied in the future. ObjectiveProgrammed cell death4, PDCD4, was firstly found in upregulating genes cell during cell apoptosis inmice in1995. PDCD4is an inhibitor of gene translation and a tumor suppressor gene which plays an important role in the suppression of multiple tumors. However, recent researches found that PDCD4is also related to the inflammational diseases, including experimental autoimmune encephalomyelitis (EAE), type I diabetes and LPS-induced endotoxic shock,sugesting PDCD4may affact function of immune cells.T cell and dendritic cell (DC) are both important effective cells in immune response. Here,we try to demonstrate whether PDCD4could affect the differentiation of both T cell and DCs.Methods1. The influence of PDCD4on the differentiation of T cell subsets:(1) Spleens and lymph nodes were separated from both Pdcd4-/-and wild type mice.(2) Flow cytometry was used to detect the proportion differences of CD8+T cells, CD4+T cells and Th1,Th2, Th17and CD25+Foxp3+Tregs in spleens and lymph nodes between Pdcd4-/-and wild type mice.2. The effect of PDCD4on the differentiation of bone marrow-derived dendritic cells (DC):(1) Bone marrow cells were separated from Pdcd4-/-and wild type mice, then cultured in complete RPMI1640medium supplemented with GM-CSF and IL-4,Half of medium was replaced at day3and5. At day7, the cells were collected as immature DC. To induce maturation, the immature DC were stimulated with LPS (1μg/ml) for additional24h..(2) To investigate the effects of PDCD4on DC differentiation, DC were collected. Then the percentage of CD11c positive cells and the relevant costimulatory molecules CD80, CD86and MHC-II on DC were detected by flow cytometry.(3) To explore whether PDCD4could impact the cytokine expression pattern of DC, the mature DC cultured supernatant was collected and used for the detection of TGF-β expression by ELISA and for IL-6, IL-10, MCP-1, IFN-γ, TNF and IL-12p70 expression by BD CBA Mouse Inflammation Kit.(4) Recent researches have demonstrated that some DC could secrete NO which acts as a negative regulator to T cell proliferation. To investigate whether the deficiency of PDCD4could affects NO production by DC, the concentration of NO in DC culture supernatant was detected by NO detection kit.(5) Both RT-PCR and Western blot were used to detect the expression of iNOS in the two kinds of DC from transcriptional and translational level.Results1. The influence of PDCD4on differentiation of T cell subsets:(1) The effect of PDCD4on CD4+and CD8+T cellsFlow cytometry was used to detecte the percentage of CD4+T cell and CD8+T cell, we found that the proportion of CD8+T cell in spleen and lymph nodes from Pdcd4-/-mice was significantly decreased compared to that from wild type mice. However, there was no significant difference on the proportion of CD4+T cell.(2) The effect of PDCD4on Thl、Th2and Th17subsetsThere was no significant differences on the proportion of Thl,Th2and Th17cells in spleen and lymph nodes that from the two kinds of mice.(3) The effect of PDCD4on CD4+CD25+Foxp3+regulatory T cell (Treg) differentiationThe proportion of CD4+CD25+Foxp3+Treg significantly increased in the Pdcd4-/-mice.2. The effect of PDCD4on the differentiation of bone marrow-derived dendritic cells:(1) PDCD4deficiency could not impact the induction of immature DC from bone marrow precursorsThe percentage of CDllc+DC from PDCD4deficient mice had no significant difference compared with wild type mice. Correspondingly, the level of CD80, CD86and MHC-II expression on PDCD4-deficient immature DC showed similar to the wild type DC. These results suggest that PDCD4could not impact the induction of immature DC.(2) PDCD4deficiency inhibited the expression of CD86on mature DC.Deficiency of PDCD4did not affect the levels of CD80and MHCII on mature DC. However,it significantly decreased the expression of CD86compared with C57BL/6DCs. These results implied that PDCD4may participate in the maturation of DC. (3) PDCD4deficiency had no impact on cytokines secreted by DCsMature DC cultured supernatant was collected and used for detection of TGF-P expression by ELISA and for IL-6, IL-10,MCP-1, IFN-y, TNF and IL-12p70expression by BD CBA Mouse Inflammation Kit. The results showed that although the concentration of TGF-β secreted by PDCD4deficient DC slightly increased compared with C57BL/6DC, it did not reach to statistical significance.In addition, the expression of other cytokines such as IL-6, IL-10, MCP-1,IFN-y, TNF and IL-12p70did not have significant difference between the two kinds of DC.These results indicate that PDCD4deficiency had no impact on cytokines produced by DC.(4) PDCD4deficiency could up-regulate NO secretion of DCsTo investigate whether the deficiency of PDCD4could affects NO production by DCs, the concentration of NO in DCs culture supernatant were detected by NO detection kit. Results showed that the concentration of NO from PDCD4deficient DCs was significantly higher than that from C57BL/6mice.(5) PDCD4could regulate the expression of iNOS in DCs on the transcriptional levelWe further detected the effect of PDCD4deficiency on the expression of iNOS at both mRNA level and protein level in DCs. Data shown that, iNOS expression on mRNA levels had no difference between the two kinds of DCs. However, iNOS expression in PDCD4-deficient DCs showed significantly increased on protein levels compared with C57BL/6DCs. These results indicated that the deficiency of PDCD4in DCs could increase the expression of iNOS on translational level.Conclusion1.PDCD4gene could influence the percentage of CD8+T and CD4+CD25+Foxp3+Treg in spleen and lymph nodes, but not CD4+T and its subsets Thl, Th2and Thl7cells, indicating that PDCD4could partially modulate the differentiation of T cell subsets.2. Although PDCD4has no influence on the induction of bone marrow derived DC, it affects CD86expression and NO secretion in mature DC These results indicate that PDCD4may influence the function of DC.Innovation and SignificanceWe proved that PDCD4affects the differentiation of immune cells including T cells and dendritic cells, which enriches the study of PDCD4function and also provides experimental and theory evidence for further research on PDCD4function in immune diseases. Limitations1. Due to technology restriction, this paper only takes gene-knockout mice as study objects, while we did not verify our results in PDCD4over-expression system.2. This study only limits to some phenomenon, However, its underlining mechanism needs to be further studied.