Jagged1 regulates STAT3 signaling pathway and affects growth and apoptosis of multiple myeloma cells

AIM:To investigate the effect of Jagged1 on the growth and apoptosis of multiple myeloma cells. METHODS:Transfection with small interfering RNA targeting Jagged1 and negative control was carried out in multiple myeloma cell line U266, and the mRNA and protein levels of Jagged1 in the cells were determined by RT-qPCR and Wes-tern blot. The cells without transfection were used as blank control. Trypan blue staining was used to draw the cell growth curve. The cell viability was measured by MTT assay. The apoptosis was analyzed by flow cytometry. The protein levels of STAT3, p-STAT3 and Bax in the cells were determined by Western blot. STAT3 signaling pathway inhibitor AG490 was used to detect the activation level of STAT3 signaling in the cells. RESULTS:Compared with the U266 cells without transfection, the expression of Jagged1 at mRNA and protein levels decreased in the U266 cells transfeced with small interfering RNA targeting Jagged1 (P<0.05). However, the expression of Jagged1 at mRNA and protein levels did not change in the U266 cells transfected with small interfering RNA negative control. Knockdown of Jagged1 expression decreased the cell viability, increased the apoptotic rate, increased Bax levels, and decreased the protein level of p-STAT3 in the U266 cells (P<0.05). AG490 treatment decreased the protein level of p-STAT3, blocked the activation of STAT3 signaling pathway, promoted the cell apoptosis induced by Jagged1 knockdown, and inhibited the viability of the U266 cells. CONCLUSION:Knock-down of Jagged1 expression promotes the apoptosis of multiple myeloma cells by inhibiting STAT3 signaling pathway, thus suppressing cell growth.     

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.     

Procaine regulates CXCR7 and affects AKT/STAT3 signaling pathway to inhibit viability,migration and invasion of bladder cancer cells

AIM To investigate the effects of procaine (PCA) and CXC chemokine receptor 7 (CXCR7) on the viability, migration and invasion of bladder cancer cells and its potential mechanism. METHODS Human bladder cancer RT4 cells were treated with PCA at different concentrations， and were divided into PBS group (without PCA treatment), PCA group (treated with 4 mmol/L PCA), siRNA negative control (si-Con) group (transfected with si-Con), CX?CR7 siRNA (si-CXCR7) group (transfected with si-CXCR7), PCA+pcDNA group (treated with 4 mmol/L PCA and transfected with pcDNA) and PCA+pcDNA-CXCR7 group (treated with 4 mmol/L PCA and transfected with pcDNA-CX?CR7). The siRNA and pcDNA were transfected into the RT4 cells by liposome method. The mRNA expression of CX?CR7 in the RT4 cells was detected by RT-qPCR. The cell viability was measured by CCK-8 assay. The invasion and migration abilities of the cells were detected by Transwell assays. The protein levels of CXCR7, AKT, STAT3, p-AKT and p-STAT3 were determined by Western blot . RESULTS Compared with PBS group, the viability, migration ability and invasion ability of the RT4 cells treated with PCA at different concentrations were significantly decreased (P<0.05), and the expression of CXCR7 at mRNA and protein levels in PCA group was also significantly decreased (P<0.05). Compared with si-Con group, the expression of CXCR7 at mRNA and protein levels in si-CXCR7 group was significantly decreased, and the viability, migration ability and invasion ability of the cells were also significantly decreased (P<0.05). Compared with PCA+pcDNA group, the expression of CXCR7 at mRNA and protein levels in PCA+pcDNA-CXCR7 group was significantly increased, and the viability, migration ability and invasion ability of the cells were also significantly increased (P<0.05). Compared with PBS group, the protein levels of p-AKT and p-STAT3 in PCA group were significantly decreased(P<0.05). Compared with PCA+pcDNA group, the protein levels of p-AKT and p-STAT3 in PCA+pcDNA-CX?CR7 group were significantly increased (P<0.05). No significant difference in the protein levels of AKT and STAT3 among the groups was observed. CONCLUSION Treatment with PCA inhibits the viability, migration and invasion of bladder cancer cells by inhibiting the expression of CXCR7. Over-expression of CXCR7 reverses this effect of PCA. Its mechanism may be related to AKT/STAT3 signaling pathway.     

Effect of AG490 on expression of VEGF and HIF-1α in HEL cells

AIM: To investigate the effect of AG490 on the expression of VEGF and HIF-1α, and the capacity of invasion in human erythroleukemia (HEL) cells. METHODS: The HEL cells were treated with AG490 at different concentrations. The cell viability was detected by CCK-8 assay. The apoptosis was detected by Hoechst staining. The apoptosis and the cell cycle were analyzed by flow cytometry. The capacity of migration was evaluated by Transwell assay. The mRNA expression level of JAK2 was measured by RT-PCR. The protein levels of p-JAK2, VEGF and HIF-1α were determined by Western blot. RESULTS: The HEL cell viabilities were 88%, 75%, 48%, 10% and 0.12% after treated with AG490 at 20, 40, 60, 80 and 100 μmol/L for 48 h, respectively. The results of Hoechst staining showed that brilliant blue cells in 80 μmol/L AG490 group was significantly increased compared with control group for 48 h. The apoptosis rate of 80 μmol/L AG490 group was significantly increased compared with control group at 48 h after AG490 treatment. The number of membrane-permeating HEL cells in 20 μmol/L AG490 group at 24 h after AG490 treatment was significantly lower than that in control group (P<0.05). The mRNA level of JAK2 decreased in a concentration-dependent manner after the HEL cells were treated with different concentrations of AG490 for 48 h. The protein levels of p-JAK2, VEGF and HIF-1α were lower in AG490 treatment groups than those in control group (P<0.05). CONCLUSION: AG490 inhibits the expression of VEGF and HIF-1α in HEL cells by inhibiting JAK2 pathway.     

Niflumic acid inhibits the proliferation,migration and invasion of glioma U87 cells

AIM To investigate the effect of niflumic acid （NFA） on human glioma U87 cells and to clarify the potential mechanism. METHODS The U87 cells were cultured in vitro and divided into blank control group， and 50， 100 and 200 μmol/L NFA groups. MTT assay was performed to determine the viability of cells in various groups. Migration and invasion abilities were measured by real-time cell analysis （RTCA）. RESULTS The results of MTT assay showed that compared with blank control group， the viability of U87 cells was increased after treatment with NFA for 12 h （P<0.05 or P<0.01）， while the viability was significantly decreased after treatment with NFA for 24 and 48 h （P<0.05 or P<0.01） in a concentration-dependent manner. The results of RTCA showed that compared with control group， the cell migration and invasion abilities were inhibited in 100 and 200 μmol/L NFA groups （P<0.05 or P<0.01） and the inhibitory effects were more obvious in 200 μmol/L NFA group （P<0.01）. CONCLUSION NFA inhibits the viability， migration and invasion of human glioma U87 cells.     

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.     

IQGAP1 is overexpressed and interacts with ERK in human myeloma cells

AIM:To explore the role of IQ motif-containing GTPase-activating protein 1 (IQGAP1) in the cell proliferation of multiple myeloma and its mechanism. METHODS:RT-PCR and Western blotting were performed to detect the expression of IQGAP1 in human myeloma cell lines RPMI8226 and U266. shRNA-expressing plasmids were used to transfect into RPMI8226 cells to knock down the expression of IQGAP1. MTT assay was used to examine the proliferation of RPMI8226-shIQGAP1 (clone 1) cells, RPMI8226-shRNA negative control cells and un-transfected RPMI8226 cells with or without VEGF/IL-6 treatment. The protein levels of p-ERK1/2, ERK1/2, AKT, p-AKT, STAT3 and p-STAT3 in RPMI8226-shIQGAP1 (clone 1) cells, RPMI8226-shRNA negative control cells and un-transfected RPMI8226 cells were measured by Western blotting. The interaction between IQGAP1 and ERK was determined by the method of co-immunoprecipitation. RESULTS:IQGAP1 was overexpressed in human myeloma cell lines RPMI8226 and U266 as compared with normal lymphocytes. Transfection with shRNA targeting to IQGAP1 decreased the expression levels of IQGAP1 in RPMI8226 cells. The proliferation of RPMI8226 cells decreased when IQGAP1 was knocked down by shRNA with or without VEGF/IL-6 treatment. IQGAP1 affected the proliferation of RPMI8226 cells by regulation of MAP kinase (ERK1/2) pathway. The level of p-ERK1/2 in RPMI8226-shIQGAP1 (clone 1) cells decreased by 70.2% as compared with RPMI8226-shRNA negative control cells and un-transfected RPMI8226 cells. The interaction between IQGAP1 and ERK in RPMI8226 cells was observed. CONCLUSION: IQGAP1 plays an important role in the cell proliferation of multiple myeloma via MAP kinase (ERK) pathway.     

Effect of NEU3 on biological behaviors of human prostate cancer DU145 cells

AIM:To explore the effects of neuraminidase 3 (NEU3) on the viability, invasion and apoptosis of human prostate cancer DU145 cells and the molecular mechanism. METHODS:The human prostate cancer DU145 cells were divided into blank control group and treatment group. The cells in treatment group were treated with either neuraminidase inhibitor DANA, or NEU3 small interfering RNA (siRNA) to knock down the expression of NEU3. The cell viability was measured by CCK-8 assay. The cell invasion ability was detected by Transwell assay. The effects of the treatments on the mRNA level of Bcl-2 were detected by qPCR. The effects of the treatments on the protein levels of matrix metalloproteinase 2 (MMP2) and apoptotic inhibitory protein Bcl-2 were determined by Western blot. Apoptosis of the cells was analyzed by flow cytometry. RESULTS:The protein level of NEU3 and the apoptotic rate in DANA group were not significantly different from those in blank control group. The viability of DANA-treated DU145 cells was increased, and the invasion ability, MMP2 protein level, and Bcl-2 mRNA and protein levels were all decreased in these cells, compared with blank control group. On the other hand, the levels of NEU3 protein and Bcl-2 mRNA and protein in NEU3 siRNA group were significantly decreased compared with blank control group, while the viability and apoptotic rate of the cells with NEU3 siRNA transfection were increased (P<0.05). However, the protein expression of MMP2 and the invasion ability of the cells were not significantly changed after NEU3 siRNA treatment. CONCLUSION:The inhibition of NEU3 in enzyme activity and expression decreases the viability, and enhances the apoptosis of human prostate cancer DU145 cells. However, it has no obvious effect on the invasion ability of DU145 cells.     

Effect of DEC1 gene silencing on viability,invasion and migration of human breast cancer MDA-MB-231 cells under hypoxia

AIM: To investigate the effect of differentiated embryonic chondrocyte gene 1 (DEC1) expression silencing on viability, invasion and migration of human breast cancer MDA-MB-231 cells and its possible mechanism under hypoxia. METHODS: The expression of DEC1 was detected by RT-qPCR and Western blot in breast cancer MDA-MB-231 cells under normoxia and hypoxia. MDA-MB-231 cells were transfected with the siRNA targeting DEC1 and the protein levels of DEC1, Smad3 and phosphorylated Smad3 (p-Smad3) were examined under hypoxia. Subsequently, the changes in the viability, invasion and migration abilities of MDA-MB-231 cells were analyzed by CCK-8 assay, Transwell experiment and Scratch test, respectively. RESULTS: The expression of DEC1 in MDA-MB-231 cells under hypoxia was higher than that in the MDA-MB-231 cells under normoxia condition at both mRNA and protein levels (P<0.05). The viability, invasion and migration abilities of MDA-MB-231 cells in siRNA-DEC1 group were decreased significantly as compared with control group (P<0.01). Besides, the protein level of p-Smad3 in the MDA-MB-231 cells in siRNA-DEC1 group was lower than that in negative control group under hypoxia condition (P<0.05). CONCLUSION: Down-regulated DEC1 expression significantly decreases the viability, invasion and migration abilities of breast cancer MDA-MB-231 cells by blocking the TGF-β/Smad3 signaling pathway under hypoxia condition.     

Effects of CENP-W down-regulation on human glioma U87 cells

AIM: To study the effect of centromere protein W (CENP-W) down-regulation on human glioma U87 cells.METHODS: Small interfering RNA (siRNA) was used to inhibit the expression of CENP-W in the U87 cells. The interference effect of siRNA was evaluated by RT-qPCR and Western blot. The proliferation of the cells was analyzed by MTT assay, BrdU staining and colony formation experiment. Transwell chamber assay was used to detect the invasion ability of the cells. The cell migration ability was measured by a scratch test. The changes of the cell cycle distribution and apoptosis were analyzed by flow cytometry.RESULTS: The results of MTT assay, colony formation experiment and BrdU staining showed that the cell proliferation and colony formation abilities in experimental group were significantly lower than those in control group and negative control group. The results of Transwell and scratch experiments showed that the migration and invasion abilities in experimental group were weaker than those in blank control group and negative control group. The results of flow cytometry analysis showed that the cell cycle distribution in experimental group was arrested in G₀/G₁ phase. The percentage of apoptotic cells in experimental group was higher than that in control group (P<0.05).CONCLUSION: Down-regulation of CENP-W expression inhibits the proliferation, migration and invasion of human glioma cells and promotes the apoptosis of the cells, suggesting that CENP-W may be a potential target of gene therapy for human glioma.     

Expression of miRNA-22 in ovarian cancer and effect of miRNA-22 over-expression on SKOV-3 ovarian cancer cell proliferation,migration and invasion

AIM: To examine the expression of miRNA-22 in the ovarian tissues and the effect of miRNA-22 over-expression on the proliferation, migration and invasion in SKOV-3 cells. METHODS: The expression levels of miRNA-22 in different ovarian tissues and SKOV-3 cells were determined by qPCR. miRNA-22 was over-expressed by transfection of miRNA-22 mimic. The cell viability was examined by CCK-8 assay. The cell migration was measured by wound healing test. The cell invasion was analyzed by Transwell assay. The protein expression levels of VEGF and P53 were determined by Western blot. RESULTS: Compared with the normal ovarian tissue, the expression level of miRNA-22 was remarkably decreased in the ovarian tumor tissues. After transfection with miRNA-22 mimic, the expression level of miRNA-22 in the SKOV-3 cells was significantly increased, while the cell viability, migration and invasion were obviously decreased. Moreover, the protein expression of VEGF and P53 was dramatically inhibited after over-expression of miRNA-22. CONCLUSION: The decreased miRNA-22 expression may be correlated with the development of ovarian can-cer. Over-expression of miRNA-22 decreases the cell viability, migration and invasion by reducing the protein expression of VEGF and P53.     

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.