| Regulatory T cells (Tregs) play an essential role in sustaining self-tolerance and immune homeostasis. The accumulation or deficiency in Tregs frequency will lead to different types of immune disorders. On the one hand, the accumulated Tregs increases susceptibility to cancer. On the other hand, the deficiency in Tregs were considered to be involved in the development of autoimmune diseases. Studies have shown that cellular apoptosis is involved in peripheral Tregs accumulation, but the related mechanism hasn’t been well explored. SENEX gene is a newly identified and apoptosis related gene, the potential role of SENEX gene expression involved in Tregs apoptosis remains largely unknown. In preliminary experiments, we found that SENEX gene expression up-regulated in peripheral Treg cells sorted from bladder cancer patients, while SENEX mRNA down-regulated in autoimmune diseases. Therefore, we hypothesized that "SENEX is a new gene which regulates Tregs cellular apoptosis". From hyperactive immune suppression (eg.Malignancies) to immunosuppressive deficiency (eg. autoimmune diseases), we assessed the vital role of SENEX gene in regulating Tregs apoptosis.First of all, we analyzed the peripheral Tregs frequency in patients with autoimmune diseases and bladder cancer, and assessed the clinical significance of Tregs expression. Secondly, we checked SENEX gene and related pro-apoptotic gene mRNA expression in autoimmune diseases and bladder cancer patients. The results showed that up-regulated SENEX expression may contribute to Tregs accumulation, while down-regulated SENEX gene was related to Tregs deficiency. These suggested that SENEX gene may regulate Tregs cellular apoptosis through a variety of ways. Finally, using FCM sorted CD4+CD25hi Tregs for primary culture, we found that silencing SENEX gene expression increased cellular apoptosis and pro-apoptotic gene expression in short-term cultured Tregs by RNA interference. In conclusion, oui study provides a new insight into understanding of peripheral Tregs apoptosis. This study includes the following three parts:Part I Detection and clinical significance of peripheral Tregs and related cytokines in patients with autoimmune diseases and bladder cancerObjective:To detect the proportion of regulatory T(Treg) cells, FoxP3gene mRNA levels and serum levels of cytokine TGF-β1, IL-10, IL-2, IL-17and TNF-a in peripheral blood of autoimmune diseases patients, aged bladder cancer patients and healthy volunteers. The correlation between Tregs frequency, FoxP3gene expression and clinical stage were also explored.Methods:23newly diagnosed autoimmune diseases patients,38newly diagnosed bladder cancer patients,34aged healthy volunteers and36young healthy volunteers were involved. Serum specimens were collected, then density gradient centrifugation were used for separation of peripheral blood mononuclear cell(PBMC). CD4+CD25+CD127Iow were used as molecular markers for Treg cells. Flow cytometry (FCM), QRT-PCR and ELISA were used for detecting the Tregs frequency, FoxP3mRNA expression and serum cytokine concentrations, respectively.Results:1. Tregs frequency significantly decreased in patients with autoimmune diseases:Compared with young healthy controls (5.09±0.37%) and aged healthy controls (7.14±0.41%), Tregs frequency in SLE (3.12±0.35%) and RA (2.98±0.31%) decreased significantly.2. Tregs frequency significantly increased in bladder cancer patients and aged healthy controls:Compared with young healthy controls (5.09±0.37%), the peripheral Tregs frequency in bladder cancer patients (10.67±0.58%) and aged healthy controls (7.14±0.41%) increased significantly. Tregs frequency in bladder cancer also increased as compared to aged healthy controls.3. FoxP3mRNA expression significantly decreased in patients with autoimmune diseases:Compared with young healthy controls (2-△△CT=0.0097±0.0039) and aged healthy controls (2-△△CT=0.0184±0.0059), FoxP3mRNA gene expression in SLE (2-△△CT=0.0063±0.0041) and RA (2-△△CT=0.0071±0.0048) patients decreased significantly.4. FoxP3mRNA expression significantly increased in bladder cancer patients: FoxP3mRNA expression in peripheral blood of bladder cancer patients (2-△△CT=0.0272±0.0081) was significantly higher than young healthy controls (2-△△CT=0.0097±0.0039) and aged healthy controls (2-△△CT=0.0184±0.0059)(p<0.05for each T test). There was no significant difference in FoxP3mRNA expression between aged healthy controls and young healthy controls (p=0.241).5. A series of cytokines secretion disorders in Rheumatoid Arthritis patients: Compared with both young and aged healthy controls, serum IL-10(14.89±1.07pg/ml) concentration in Rheumatoid Arthritis patients was significantly elevated. Compared with young healthy controls, serum IL-2(565.73±38.07pg/ml) and IL-17(18.86±4.72pg/ml) concentration were significantly elevated, whlie serum TGF-β1(6.19±2.08ng/ml) levels decreased significantly in Rheumatoid Arthritis patients. There was no significant difference in cytokine concentrations between SLE patients and healthy controls.6. A series of cytokines secretion disorders in bladder cancer patients:Compared with both young healthy controls and aged healthy controls, serum IL-10(13.03±2.16pg/ml), IL-17(18.29±3.45pg/ml), TGF-β1(16.22±3.35ng/ml) and TNF-α (25.18±5.74pg/ml) concentrations increased significantly in bladder cancer patients. Meanwhile, serum IL-2concentration significantly decreased in bladder cancer patients (165.29±26.45pg/ml) and aged healthy controls (196.69±32.38pg/ml) as compared to young healthy controls (409.24±90.81pg/ml). In addition, There was no significant difference of IL-2serum concentrations between bladder cancer patients and aged healthy controls (p=0.243).7. Tregs frequency and Foxp3mRNA expression were correlated with tumor stage in bladder cancer patients:The average Tregs frequency in21bladder cancer patients who were older than70years old (including70) was12.38%, that was significantly higher than patients who were younger than70years old (averaged Treg frequency8.96%)(P=0.017). Meanwhile, Tregs frequency was significantly correlated with tumor-infiltrating. Tregs frequency in patients with TisT1Ta stage (9.25%) was significantly lower than which in patients with T2-T4stage (12.13%)(p=0.006).FoxP3mRNA expression in21bladder cancer patients (2-△△CT=0.0368) who were older than70years old (including70),was significantly higher in patients younger than70years old (2-△△CT=0.0176)(P=0.024). FoxP3mRNA expression in patients with TisT1Ta stage (2-△△CT=0.0155) was significantly lower than patients with T2～T4stage (2-△△CT=0.0389)(p=0.043). In addition, FoxP3mRNA expression in patients with lymph node invasion (2-△△CT=0.0358) increased significantly as compared to patients without lymph node invasion (2-△△CT=0.0186).Conclusion:Tregs frequency and FoxP3mRNA expression significantly decreased in both SLE and RA patients. Tregs frequency and FoxP3mRNA expression increased in bladder cancer patients, while a series of cytokines secretion disorders was observed. Tregs frequency and FoxP3mRNA expression in bladder cancer patients were statistically correlated with tumor invasion. Peripheral CD4+CD25+CD127low Tregs play a vital role in maintaining immune homeostasis.On the one hand, a deficiency in Tregs will induce excessive immune response, and lead to autoimmune diseases; on the other hand, accumulated Tregs will excessively suppress the immune response, facilitate the tumor growth, and accelerated tumor metastasis in bladder cancer.Part Ⅱ The characteristics of SENEX gene and pro-apoptotic gene expression in peripheral blood of autoimmune diseases and bladder cancer patientsObjective:To detect SENEX gene and pro-apoptotic gene P53, P16, P21and Caspase-3expression in peripheral Tregs sorted from bladder cancer patients and healthy volunteers. To explore the potential correlation between SENEX gene mRNA expression, pro-apoptotic gene expression and cytokine concentration in Tregs. To detect SENEX gene and pro-apoptotic gene P53, P16, P21and Caspase-3expression in peripheral blood mononuclear cells (PBMC) sorted from autoimmune diseases patients and healthy volunteers.Methods:23newly diagnosed autoimmune diseases patients,16newly diagnosed bladder cancer patients,10aged healthy volunteers and12young healthy volunteers were involved. Serum specimens were collected, then density gradient centrifugation was used for separation of PBMCs. Flow cytometry was used for sorting peripheral CD4+CD25hiTregs/CD4+CD25-Teffs. QRT-PCR was used for detecting SENEX gene and pro-apoptotic gene P53, P16, P21and Caspase-3mRNA expression. Pearson correlation analysis was used for investigating the potential correlation between SENEX gene expression, pro-apoptotic gene mRNA levels and serum cytokine concentrations.Results:1. The purity test of FCM sorted CD4+CD25hiTregs:A total of16bladder cancer patients,12young healthy volunteers and10aged healthy volunteers were successfully obtained CD4+CD25hiTreg/CD4+CD25-Teff cell subsets, purity tested was checked as the CD4+CD25+CD127lowTregs frequency and the purity was above90%.2. The characteristics of SENEX gene expression in Tregs/Teffs:SENEX gene expression significantly increased in CD4+CD25hiTregs sorted from bladder cancer patients, as compared to Tregs from aged healthy controls and young healthy controls (P<0.05for each test). Meanwhile, SENEX gene expression in Tregs sorted from bladder cancer patients also significantly increased, as compared to Teffs sorted from bladder cancer patients. In aged healthy controls and young healthy controls, SENEX gene expression in Tregs was slightly higher than Teffs, but no statistical difference was calculated.3. The characteristics of pro-apoptotic gene expression in Tregs/Teffs sorted from bladder cancer patients:The pro-apoptotic gene P53, P16, P21and Caspase-3expression decreased significantly in Tregs sorted from bladder cancer patients, as compared to Teffs (P<0.05).4. SENEX gene expression was correlated with P53gene expression and serum concentration of TGF-(β1and IL-2in bladder cancer patients:SENEX expression was negatively correlated with P53mRNA expression (Pearson correlation coefficient=-0.405,p=0.012) and IL-2concentration (Pearson correlation coefficient=-0.362, p=0.026) in bladder cancer patients. Positive correlation between SENEX expression and TGF-β1concentration (Pearson correlation coefficient=0.507,p=0.001) was figured out by Pearson’s coefficient test.5. The characteristics of SENEX gene expression in PBMCs sorted from autoimmune disease patients and healthy controls:SENEX gene expression(2-△△CT=0.0354±0.0015) significantly decreased in PBMCs sorted from RA patients, as compared to PBMCs from both aged healthy controls and young healthy controls (P <0.05for each test). Meanwhile, SENEX gene expression in PBMCs sorted from SLE patients decreased slightly, as compared to PBMC sorted from aged controls and young healthy controls, but no statistical difference was calculated.6. The characteristics of pro-apoptotic gene expression in PBMCs sorted from autoimmune disease patients and healthy controls:Compared with healthy controls, the pro-apoptotic gene P53, and Caspase-3expression increased significantly in PBMCs sorted from RA patients. No statistical differences in pro-apoptotic gene expression were calculated between SLE patients and healthy controls.Conclusion:On one hand, SENEX gene expression was upregulated in Tregs sorted from bladder cancer patients, while pro-apoptotic gene P53, P16, P21and Caspase-3mRNA expression decreased significantly. Meanwhile, SENEX gene expression was significantly correlated with P53mRNA levels, serum TGF-β1and IL-2concentration. On the other hand, SENEX gene expression was downregulated in PBMCs of RA patients, while pro-apoptotic gene P53and Caspase-3expression increased in RA patients. SENEX gene may antagonize Tregs apoptosis and promote its accumulation by down-regulating pro-apoptotic gene P53gene expression in bladder cancer patients. Furthermore, upregulated SENEX gene may also increase the serum TGF-β1levels, and inhibit IL-2secretion in bladder cancer patients. The novel SENEX gene was involved in peripheral Tregs apoptosis.Part Ⅲ SENEX gene antagonistic peripheral CD4+CD25hiTreg cellular apoptosis in vitroObjective:To detect the spontaneous apoptosis of CD4+CD25hiTreg/CD4+CD25-Teff cells in aged bladder cancer patients when cells were primarily cultured for a short term. To observe the cellular apoptosis in Tregs/Teffs induced by oxidative stress. To research the cellular apoptosis and pro-apoptotic genes expression in Tregs/Teffs in response to SiRNA-SENEX transfection in bladder patients. To observe the cellular apoptosis in PBMCs in response to SENEX gene RNA interference in RA patients.Methods:Five newly diagnosed Rheumatoid Arthritis patients and five newly diagnosed bladder cancer patients were involved. Serum specimens were collected, then density gradient centrifugation was used for separation of PBMCs. Flow cytometry were used for sorting CD4+CD25hiTreg/CD4+CD25-Teff cells. During short-term primary culture, we used100μM H2O2as an inducer of oxidative stress damage. RNA interference was used for specificity inhibiting SENEX gene expression. QRT-PCR was used for detecting SENEX gene and pro-apoptotic genes P53, P16, P21and Caspase-3mRNA expression. Annexin V (+) were detected as the proportion of apoptotic cells by flow cytometry. By RNA interference, we specifically inhibited SENEX gene mRNA expression in PBMCs sorted from RA patients, and detected the PBMCs cellular apoptosis.Results:1. The spontaneous apoptosis of Tregs/Teffs in bladder cancer patients after primary cultured for24h:In the same culture conditions, after24h short-term cultured, spontaneous apoptosis of Tregs (Annexin V+cell ratio:3.53±0.17%) was significantly lower than Teffs (5.78±0.46%) in bladder cancer patients, The experiment results showed that Tregs from bladder cancer patients had the characteristics of apoptosis resistance. 2. Different doses of H2O2could induce diversed cellular apoptosis in Tregs/Teffs: Different doses of H2O2could induce cellular apoptosis in varying degrees in both Tregs and Teffs, the H2O2-induced apoptosis possesd a dose-response effection. When using25μM,50μM,100μM and150μM H2O2treatment for2h, instantly Treg cellular apoptosis rates were4.3±0.32%,4.9±0.48%,5.7±0.41%and9.7±0.95%, respectively. When using25μM,50μM,100μM and150μM H2O2treatment for2h, instantly Teff cellular apoptosis rates were5.1±0.53%,5.7±0.76%,6.8±0.75%and11.7±1.04%, respectively. When using the same dose of H2O2treatment, instantly Teff cellular apoptosis rates was higher than Tregs.3. Establishment of cellular model by SENEX gene RNA interference:After SENEX SiRNA transfection in Tregs/Teffs in vitro, real-time PCR test results suggest that using liposomes LipofectaininTM2000transfected SiRNA-SENEX, the SENEX mRNA inhibition rate was more than80%in both Tregs and Teffs.4. SENEX genes can antagonize Treg cellular apoptosis induced by H2O2:100μM H2O2treatment for2hours increased apoptotic cells in both Tregs (11.2±2.6%) and Teffs (13.1±4.3%), with no significant difference observed in H2O2induced apoptosis between Tregs and Teffs. Silencing SENEX gene expression with RNA interference for24h, follwed by H2O2treatment for2hours, increased apoptotic cells in Tregs (21.5±3.4%), but undergoing the same procedures, apoptotic cells wasn’t found to increase in Teffs (13.9±3.1%). No difference was observed in cellular apoptosis between Tregs and Teffs when cells were treated with SiRNA transfection alone.5. Silencing Tregs SENEX gene led to upregulation of P53, P16, P21and Caspase-3mRNA expression in response to stress:After SENEX gene was silenced down for24hours, followed by H2O2treatment for2hours, apoptosis regulatory genes Caspase-3, P53, P16and P21mRNA expression (2-△△CT=6705.73±124.07,253.08±16.01,154.58±35.12and5135.79±985.47, respectively) upregulated in CD4+CD25hi Tregs (p<0.05for each Mann-Whitney U Test).6. The spontaneous apoptosis of PBMCs in RA patients after primary cultured for24h:In the same culture conditions, after24h short-term cultured, spontaneous apoptosis of PBMCs (Annexin V+cell ratio:4.96±0.31%) was significantly higher than which in healthy controls (3.75±0.36%), the results showed that PBMCs from RA patients were more easily to be subjected cellular apoptosis.7. Different doses of Dexamethasone(Dxm) could induce diverse cellular apoptosis in PBMCs:Dxm-induced apoptosis possesd a dose-response manner. In RA patients, when using0×(5×10-2)mg/L,1×(5×10-2)mg/L,2×(5×10-2)mg/L,5×(5×10-2)mg/L,10×(5×10-2)mg/L and50×(5×10-2)mg/L Dxm treatment for24h, instantly PBMCs cellular apoptosis rates were19.0±4.18%,21.4±3.95%,22.9±4.99%,23.4±5.48%,23.9±5.17%,23.5±5.11%and23.8±5.32%, respectively. In healthy controls, when using0×(5×10-2)mg/L,1×(5×10-2)mg/L,2×(5×10-2)mg/L,5×(5×10-2)mg/L,10×(5×10-2)mg/L and50x(5×10-2)mg/L Dxm treatment for24h, instantly PBMCs apoptosis rates were16.3±3.76%,18.5±4.21%,19.3±5.07%,20.1±4.12%,19.9±4.02%,20.6±3.29%and20.2±4.28%, respectively. When using the same dose of Dxm treatment, instantly PBMCs cellular apoptosis rates was higher in RA patients.8. SENEX genes antagonize PBMCs apoptosis induced by Dexamethasone:5×10-2mg/L Dxm treatment for8hours increased PBMCs cellular apoptosis in both RA patients (13.5±2.86%) and healthy controls (5.2±1.04%). In the same condition, silencing SENEX gene expression by RNA interference, increased cellular apoptosis in both RA patients (13.5±2.86%) and healthy controls (10.6±2.95%).Conclusion:Cellular apoptosis were significantly decreased in Tregs sorted from bladder cancer patients, as compared to Teffs. Tregs were resistant to cellular apoptosis. When RNA interference was used for silencing SENEX gene expression, Tregs cellular apoptosis significantly increased in response to oxidative stress damage. Up-regulated SENEX gene expression antagonized Tregs apoptosis by down-regulating pro-apoptotic gene expression, promoted Tregs accumulation and tumorigenesis, and increased immune escape in bladder cancer patients. In addition, SiRNA-SENEX transfection increased PBMCs cellular apoptosis in RA patients, which proved that SENEX gene antagonized cellular apoptosis from the opposite side. SENEX gene should be expected as a potential target for immune intervention for bladder cancer, and could give us new ideas for prevention and treatment for cancer research. |
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