The expression of IFN-γ and IL-4 on T lymphocytes that infiltrate in nasal polyps

AIM:To investigate the expression of Th1-typed cytokine IFN-γ and Th2-typed cytokine IL-4 on T lymphocytes that infiltrate in nasal polyps for searching the pathogenesis of nasal polyps. METHODS:Nasal polyps tissue samples and peripheral blood were obtained from 21 patients. Normal human inferior turbinate mucosa and peripheral blood were obtained as well. Flow cytometry was adopted to detect the expression of IFN-γ and IL-4 of T lymphocytes. RESULTS: Th cytokines were rarely detected in inferior turbinate from normal human. Nasal polyps tissue consisted of abundant T lymphocytes. The expression of IL-4 and IFN-γ increased in peripheral blood from patients compared with normal human (P<0.05). The expression of IL-4 increased but the expression of IFN-γ decreased in nasal polyps compared with that of peripheral blood from the same patient (P＜0.05). CONCLUSION:There were generous of T lymphocytes infiltrating in nasal polyps. There was abnormal immune status in the local nasal mucosa from the patients, and the predomination of Th cytokine secretion changed compared with peripheral blood from the same patients, which resulted in the change of microenvironment of nasal mucosa and possibly close related to the formation of nasal polyps.     

Effects of IL-6 and AG490 on growth of diffuse large B-cell lymphoma and Burkitt lymphoma transplanted into nude mice

AIM: To observe the effects of interleukin-6 (IL-6) and AG490 on diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL) transplanted into nude mice, and to explore the effects of STAT3 activation on growth of these kinds of lymphoma in nude mice and its related mechanisms. METHODS: The nude mouse models with DLBCL and BL were established by transplantation with OCI-LY8 cells and Raji cells, respectively, and were divided into 3 groups:control group, IL-6 group and AG490 group. The body weight of mice and tumor size were measured. Western blot and immunohistochemical staining were used to detect the protein levels of p-STAT3, survivin and vascular endothelial growth factor (VEGF), and real-time PCR was used to detect the mRNA expression of survivin and VEGF. RESULTS: The tumorigenic rate of 2 kinds of tumor cell lines in nude mice was 83.3% (25/30) totally. The tumorigenicity of OCI-LY8 cells (66.7%, 10/15) was significantly lower than that of Raji cells (100%, 15/15) (P<0.05). The tumor size and body weight on days 9 and 10 in IL-6 group increased as compared with the control group, and the total difference value of tumor size between day 1 and day 10 in IL-6 group was obviously larger than that in control group (P<0.05). The positive protein of p-STAT3 was found in the nucleus, while the positive expression of survivin and VEGF was found in the cytoplasm. As compared with control group, the expression of survivin and VEGF was significantly increased (P<0.05), while the protein level of p-STAT3 was not significantly increased in IL-6 group of DLBCL. The protein levels of p-STAT3 and VEGF were significantly decreased (P<0.05), while the expression of survivin did not significantly decreased in AG490 group of DLBCL. The p-STAT3 and VEGF levels significantly increased (P<0.05) in IL-6 group of BL, while the levels of 3 kinds of proteins significantly deceased (P<0.05) in AG490 group of BL, as compared with control group. No statistical difference of mRNA expression of survivin and VEGF among IL-6, AG490 and control groups was observed. CONCLUSION: IL-6 and AG490 affect the growth of DLBCL and BL through activation of STAT3 pathway. The activated STAT3 participates in pathogenesis and progress of DLBCL and BL by up-regulating the expression of survivin and VEGF.     

Clinical significance of STMN1 expression in cervical cancer and effect of inhibition of its expression on viability and apoptosis of cervical cancer SiHa cells

AIM: To investigate the clinical significance of stathmin 1 (STMN1) expression in cervical cancer and the influence of its expression on the viability and apoptosis of cervical cancer cells. METHODS: Western blot was used to detect the protein expression of STMN1 in cervical cancer tissues, and the relationship between the expression and clinical characteristics of cervical cancer was analyzed. STMN1-siRNA was transfected into cervical squamous-cell carcinoma SiHa cells. The protein levels of STMN1, STAT3, p-STAT3 and survivin were determined by Western blot after transfection for 48 h. The cell viability was measured by MTT assay. The apoptosis was analyzed by flow cytometry. DCFH-DA probe was used to detect the level of reactive oxygen species (ROS). RESULTS: The protein expression of STMN1 in cervical cancer tissues was significantly higher than that in paracancerous tissues (P<0.01). The STMN1 protein expression level was not correlated with age and histological types of cervical cancer patients, but was related to clinical stage, histological differentiation and lymph node metastasis (P<0.01). Transfection with STMN1-siRNA significantly reduced the expression of STMN1 in SiHa cells. Compared with control group, the cell viability in STMN1-siRNA group was significantly decreased, the apoptotic rate and ROS content were increased, and the protein levels of p-STAT3 and survivin were down-regulated (P<0.01). However, no significant difference of the STAT3 protein level was observed between STMN1-siRNA group and control group. CONCLUSION: STMN1 is highly expressed in cervical cancer, and its expression is related to clinical stage, histological differentiation and lymph node metastasis. Inhibition of STMN1 expression reduces the viability and promotes apoptosis of cancer cells by down-regulating STAT3 signaling pathway.     

Effect of IQGAP1 siRNA on biological characteristics of glioma cells by regulating STAT3 signaling pathway

AIM: To investigate the effect of IQGAP1 gene expression knock-down on invasion, migration and immunosuppression of glioma cells and its mechanism. METHODS: Human glioma U251 cells were randomly divided into blank group, negative control group and si-IQGAP1 group. AG490, an inhibitor of STAT3 signaling pathway, was used to treat the cells for 48 h. The cell viability was measured by MTT assay. The protein levels of IQGAP1, vascular endothelial growth factor (VEGF), transforming growth factor-β1 (TGF-β1), STAT3 and p-STAT3 were determined by Western blot. The cell invasion and migration abilities were detected by Transwell assays. RESULTS: The protein expression of IQGAP1 in si-IQGAP1-1 group and si-IQGAP1-2 group was significantly lower than that in blank group (P<0.05). Compared with blank group, the viability, the invasion ability and the migration ability of the cells in si-IQGAP1 group and AG490 group were significantly decreased, while the protein levels of VEGF, TGF-β1 and p-STAT3 were significantly decreased (P<0.05). Compared with AG490 group, the cell viability, invasion ability and migration ability in AG490+si-IQGAP1 group were significantly decreased, and the protein levels of VEGF and TGF-β1 were significantly decreased (P<0.05). CONCLUSION: Silencing of IQGAP1 gene expression reduces the invasion and migration abilities of glioma cells and decreases the protein expression of cellular immunosuppression molecules VEGF and TGF-β1, which is related to down-regulation of STAT3 signaling pathway.     

Effect of high mobility group box protein 1 on expression of STAT/SOCS/cyclin D1 in rat class fibroblast-like synovial cells

AIM: To investigate the regulatory effect of high mobility group box protein 1 (HMGB1) on STAT/SOCS signal transduction in RSC-364 synoviocytes. METHODS: RSC-364 cells induced by 10 μg/L human recombinant HMGB1 were collected at 6 h, 12 h or 24 h after treatment, respectively. The cells without any treatment were also collected at same time points for normal control. The mRNA expression of STAT 1/3 was analyzed by RT-PCR. The phospho-STAT 1/3 (p-STAT 1/3) and STAT 1/3 proteins were detected by Western blotting. The flow cytometric analysis (FCM) was applied to detect the protein expression of p-STAT 1/3, SOCS 1/3 and cyclin D1. RESULTS: HMGB1 at concentration of 10 μg/L significantly up-regulated the mRNA and protein levels of STAT1 expression, as well as cyclin D1 protein, and markedly increased the protein expression of p-STAT1 (P<0.01). Compared to control group, no significant difference of STAT3 and p-STAT3 expression was observed. HMGB1 elevated the protein expression of SOCS1, while the protein level of SOCS3 was only up-regulated at 6 h. CONCLUSION: HMGB1 may activate the process of STAT1 signal transduction and up-regulate the gene expression of cyclin D1, suggesting the role of HMGB1 in promoting the proliferation of synoviocytes.     

Effect of Qingyi decoction on p-STAT3 expression in pancreas of mice with severe acute pancreatitis induced by L-arginine

AIM: To explore the role of STAT3 signaling pathway in acute pancreatitis induced by L-arginine, and the mechanisms of Qingyi decoction(QYD) treatment on severe acute pancreatitis (SAP). METHODS: The Kunming mice were randomly divided into 3 groups (n=10): control group, SAP group and QYD treatment group (SAP+QYD). The mice in SAP group and SAP+QYD group were intraperitoneally injected with 20% L-arginine (3 g/kg, bid). The mice in SAP+QYD group were also administered intragastrically with QYD(10 mL/kg) 30 min after the second injection of 20% L-arginine and twice a day for the following 2 days. The mice were anesthetized and sacrificed 72 h after SAP induction. The activity of amylase was measured in serum, the relative pancreatic weight was assayed, and the activity of myeloperoxidase (MPO) was analyzed to evaluate the neutrophil infiltration in lung tissues. The morphology of pancreas and lung was observed. The protein of pancreas was extracted to detect the expression of p-STAT3 by Western blotting. The mRNA expression of monocyte chemoattractant protein-1(MCP-1) was determined by real-time PCR. RESULTS: Compared with control group at 72 h, L-arginine induced SAP with increased serum amylase activity, pancreatic wet weight ratio and MPO activity in lung tissues (P<0.05). In SAP+QYD group, the activity of amylase, pancreatic wet weight ratio and MPO levels were significantly lower than those in SAP group (P<0.01). Compared with control group at 72 h, the pancreas and lung were obvious injured, the protein level of p-STAT3 and mRNA expression of MCP-1 in pancreas tissues increased significantly in SAP group. Compared with SAP group, the pathologic damage of the pancreas and lung tissues, the protein level of p-STAT3 and mRNA expression of MCP-1 in pancreas were significantly reduced in SAP+QYD group. CONCLUSION: The expression of p-STAT3 in pancreas increases in the mice with SAP induced by L-arginine. The activation of STAT3 may take part in the development of SAP. Inhibition of STAT3 activation in pancreas is one of the mechanisms of QYD treatment for SAP.     

Growth inhibition of colorectal cancer HCT116 cells by lincRNA-p21 through STAT3 signaling pathway

AIM: To study the effect of long intergenic non-coding RNA-p21 (lincRNA-p21) on the growth inhibition of colorectal cancer HCT116 cells via STAT3 signaling pathway. METHODS: The human colorectal cancer cell line HCT116 was used to construct the cells with over-expression of lincRNA-p21 by transfection of pcDNA-lincRNA-p21, and negative control cells were also set up. After transfection, the expression level of lincRNA-p21 was detected by RT-qPCR. The cell viability and proliferation were examined by MTT assay and plate colony formation assay, respectively. The protein levels of STAT3 and phosphorylated STAT3 (p-STAT3) were determined by Western blot. After STAT3 signaling pathway activator SD19 was used to treat the colorectal cancer HCT116 cells with over-expression of lincRNA-p21, Western blot was used to detect the protein levels of STAT3 and p-STAT3, MTT assay was used to measure the viability of the cells, and flow cytometry analysis was used to determine the cell apoptosis. RESULTS: Compared with control group and pcDNA group, the expression of lincRNA-p21 in pcDNA-lincRNA-p21 group was significantly up-regulated, the cell proliferation was inhibited, and the protein levels of STAT3 and p-STAT3 were significantly decreased (P<0.05). After treatment with STAT3 activator SD19, the protein levels of STAT3 and p-STAT3 in pcDNA-lincRNA-p21+SD19 group were higher than those in pcDNA-lincRNA-p21 group, the cell viability was increased, and the apoptotic rate was decreased significantly (P<0.05). CONCLUSION: Over-expression of lincRNA-p21 inhibits the growth of colorectal cancer HCT116 cells. STAT3 signaling pathway activator abolishes the growth inhibitory effect of lincRNA-p21 over-expression. lincRNA-p21 inhibits the growth of colorectal cancer cells by inhibiting the activation of STAT3 signaling.     

Effects of sodium valproate on RPMI8226 and U266 cell proliferation and IL-6/JAK/STAT signaling pathway

AIM: To investigate the effects of sodium valproate (VPA) on the proliferation of multiple myeloma cell lines RPMI8226 and U266 and the regulation of IL-6/JAK/STAT signaling pathway. METHODS: The cells were treated with different concentrations of VPA for 12 h and 24 h. The growth of RPMI8226 cells and U266 cells was detected by MTT assay. Apoptotic rates and cell cycle were analyzed by flow cytometry. The mRNA expression of STAT3, STAT5 and STAT target genes Bcl-xL, Mcl-1, c-Myc, CCND1 and VEGF was measured by RT-PCR. Western blotting analysis was used to determine the total proteins and protein phosphorylation levels of JAK2 and STAT5. RESULTS: VPA inhibited the growth and induced the apoptosis of RPMI8226 cells and U266 cells in a concentration- and time-dependent manner. The levels of IL-6 in the culture supernatants of RPMI8226 cells and U266 cells treated with VPA were significantly higher than that in negative control group. VPA down-regulated the mRNA expression of STAT3, STAT5, Bcl-xL, Mcl-1, c-Myc, CCND1 and VEGF. After treated with VPA, the protein levels of p-JAK2, JAK2, p-STAT5 and STAT5 in RPMI8226 cells and U266 cells were significantly lower than those in control group. CONCLUSION: VPA inhibits the proliferation of PRMI8226 cells and U266 cells in vitro. The modulation of IL-6/JAK/STAT signaling pathway may be involved in its potential mechanisms.     

Effect of captopril on AGS nude mouse model of gastric cancer

AIM: To observe the effect of captopril on the genesis and development of gastric cancer, and to explore its clinical treatment feasibility for gastric cancer. METHODS: The human gastric cancer cell line AGS was used to establish a tumor model in nude mice, and the model mice were randomly divided into 3 groups: positive control (5-fluorouracil) group, normal control (saline) group and experimental (captopril) group. After intraperitoneal injection or intragastric administration of the drugs, the tumor growth curve was determined, and the tumor tissues were also sampled to detect the expression of Ki-67, STAT3, Bax and Bcl-2 by real-time quantitative PCR and immunohistochemistry. The apoptosis was detected by TUNEL+DAPI staining. RESULTS: The tumor growth curve showed that the tumor model in the nude mice was successfully established. The tumor volumes among groups showed significantly different after 14 d growth. The increase in the tumor volume in normal control group was significantly faster than that in the other two groups, and that in positive control group was the slowest. The expression of Bax in captopril group increased, and the expression of STAT3, Ki-67 and Bcl-2 was reduced as compared with normal control group and positive control group. Compared with normal control group, the apoptotic rate increased significantly, and the protein expression of p-STAT3 and STAT3 decreased obviously in positive control group and captopril group. CONCLUSION: With better feasibility, angiotensin-converting enzyme inhibitor captopril has a significant effect on treating gastric cancer in the AGS nude mouse model by regulating the expression of STAT3, Bax, Bcl-2 and Ki-67 to accelerate the apoptosis of cancer cells, thus inhibiting tumor growth.     

Roles of PI3K/Akt and JAK2/STAT3 signaling pathways in protection of SO2 against limb ischemia/reperfusion-induced acute lung injury in rats

AIM: To investigate the role of PI3K/Akt and JAK2/STAT3 pathways in the protection of sulfur dioxide (SO₂) against limb ischemia/reperfusion (I/R)-induced acute lung injury (ALI) in rats. METHODS: ALI was induced by limb I/R in the SD rats. Na₂SO₃(0.54 mmol/kg, ip)/NaHSO₃ (0.18 mmol/kg, ip) as SO₂ donor was injected at 20 min before reperfusion. The inhibitors of JAK2/STAT3 and PI3K/Akt pathways, Stattic (3 mg/kg, iv) and LY294002(40 mg/kg, iv), respectively, were injected at 1 h before reperfusion. Peripheral blood and lung tissues were collected for determining the contents of the cytokines, the protein levels of the molecules related to the signaling pathways, apoptosis and histopathologic changes by ELISA, TUNEL and Western blot. RESULTS: Compared with control group, the content of MDA, the activity of MPO, lung coefficient, apoptotic index, cytokine expression, and the protein levels of p-Akt and p-STAT3 in I/R group all increased significantly, and administration of Na₂SO₃/NaHSO₃ attenuated the damage in the lung. Besides, the results of Western blot showed that the rat lung tissues expressed p-STAT3 protein and p-Akt protein. After I/R, the protein levels of p-STAT3 and p-Akt were increased. After using Na₂SO₃/NaHSO₃, p-Akt was increased, but p-STAT3 was decreased (P<0.05). CONCLUSION: Both JAK2/STAT3 and PI3K/Akt pathways are likely involved in the protective effect of SO₂ against limb I/R-induced ALI in rats. The activation of JAK2/STAT3 signaling pathway increases I/R injury. Reversely, the activation of PI3K/Akt signaling pathway reduces I/R injury. Besides, JAK2/STAT3 and PI3K/Akt signaling pathways may have crosstalk during I/R-induced ALI and JAK2/STAT3 pathway may have an impact on the P13K/Akt pathway.     

Effect of HDAC1 silencing on apoptosis of squamous cell carcinoma of skin via regulating STAT3 signaling pathway

AIM: To investigate the effect of histone deacetylase 1 (HDAC1) silencing on apoptosis of squamous cell carcinoma of skin. METHODS: Skin squamous cell carcinoma A431 cells were transfected with HDAC1 small interfering RNA (HDAC1 siRNA) or small interfering RNA negative control (siRNA NC). The expression levels of HDAC1 in transfected cells were detected by RT-PCR and Western blot. The cell viability was measured by MTT assay, and the apoptosis was analyzed by flow cytometry. The protein levels of STAT3, p-STAT3 and cleaved caspase-3 were determined by Western blot. The inhibitor of STAT3 signaling pathway was used to treat the A431 cells transfected with HDAC1 siRNA. The cell viability was detected by MTT assay, the apoptosis was analyzed by flow cytometry, and the protein levels of STAT3, p-STAT3 and cleaved caspase-3 were determined by Western blot. RESULTS: HDAC1 siRNA inhibited the expression of HDAC1 at mRNA and protein levels in the A431 cells. After interfering with the expression of HDAC1, the cell viability and the protein level of p-STAT3 in the cells decreased, while the apoptotic rate and the protein level of cleaved caspase-3 in the cells were increased. After treatment with the inhibitor of STAT3 pathway, the viability of A431 cells transfected with siRNA and the protein level of p-STAT3 decreased, while the apoptotic rate and the protein le-vel of cleaved caspase-3 in the cells were increased. CONCLUSION: Interference with HDAC1 expression may regulate the STAT3 signaling pathway to inhibit the viability of skin squamous cell carcinoma cells, thus promoting the apoptosis of squamous cell carcinoma of skin.