Rab1A knockdown inhibits malignant biological behaviors of breast carcinoma cells by inhibiting phosphorylation of AKT

AIM: To investigate the role of Rab1A gene in the malignant biological behaviors of breast carcinoma cells. METHODS: The expression levels of Rab1A in breast carcinoma tissues and normal adjacent tissues, and the basic expression level of Rab1A in different breast carcinoma cell lines were measured by Western blot. Small interfering RNA (siRNA) targeting Rab1A was designed, synthetized and transfected into the breast carcinoma MDA-MB-231 cells. After validation of efficiency of Rab1A gene expression knock-down, the malignant biological behaviors of the MDA-MB-231 cells were measured by CCK-8 assay, wound healing assay, Transwell assay and flow cytometry. The protein levels were determined by Western blot. RESULTS: Rab1A was expressed in normal breast tissue and cells at low level, and at high level in the cancer tissues and cancer cells (P<0.05). Compare with control group, after knock-down of Rab1A expression, the viability of MDA-MB-231 cells was significantly inhibited (P<005), the abilities of migration and invasion were reduced (P<0.05), the apoptosis was decreased (P<0.05), the percentage of G²/M phase was increased, the protein levels of p53, Bax, cleaved caspase-3 and PTEN were significantly increased (P<0.05), and the protein levels of Bcl-2, cyclin D1, cyclin B1, matrix metalloproteinase 2 (MMP2), p-AKT and mTOR were significantly decreased (P<0.05). CONCLUSION: Rab1A modulates the breast carcinoma cell viability, inhibits the migration and invasion abilities, induces G² arrest and effectively regulates the cell growth-, cell cycle-and apoptosis-related proteins. Knock-down of Rab1A expression inhibits the evolution and development of breast cancer by inhibiting the phosphorylation of AKT pathway, and Rab1A may function as a potential target in breast carcinoma treatment.     

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.     

Effects of BCL6B on proliferation and migration of human colorectal carcinoma LoVo cells and its potential mechanism

AIM: To detect the endogenous expression of B-cell leukemia/lymphoma 6 member B (BCL6B) in FHC and LoVo cells, and to investigate the effects of BCL6B on proliferation and migration of LoVo cells for further exploring the underlying mechanism. METHODS: The endogenous expression of BCL6B in the FHC and LoVo cells was detected by RT-PCR and Western blot. The methods of MTT assay, colony formation assay, wound healing assay and Transwell chamber experiment were employed to examine the biological functions of BCL6B in the LoVo cells. The mRNA and protein levels of BCL6B, cyclin D1 and matrix metalloproteinase-9 (MMP-9) were determined by RT-PCR and Western blot, respectively. The level of phosphorylated protein kinase B (p-AKT) was detected by Western blot. RESULTS: BCL6B expression was notably repressed in the LoVo cells as compared with the FHC cells, which were significantly increased by transfection with pcDNA3.1-BCL6B. The abilities of proliferation and migration of the LoVo cells at 72 h were inhibited by 28.33%(P<0.01) and 36.11%(P<0.05) in BCL6B group. The mRNA levels of cyclin D1 and MMP-9 in the cells of BCL6B group were decreased by 39.90%(P<0.01) and 77.36% (P<0.05), and the protein levels of cyclin D1, MMP-9 and p-AKT were reduced by 44.00%(P<0.05), 47.06%(P<0.01) and 32.88% (P<0.05), respectively. CONCLUSION: BCL6B inhibits proliferation and migration of the LoVo cells, and the PI3K/AKT signaling pathway is involved in this process.     

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.     

Effect of lentinan on apoptosis and PI3K/AKT signaling pathway in leukemic HL-60 cells in vitro

AIM:To study of the regulatory effect of lentinan on human leukemic HL-60 cell apoptosis and its effect on PI3K/AKT signaling pathway in HL-60 cells in vitro.METHODS:Lentinan at concentrations of 0 mg/L, 15 mg/L, 30 mg/L and 45 mg/L was applied to HL-60 cells cultured to the logarithmic phase in vitro, and the inhibitory effect of lentinan on the viability of HL-60 cells was measured by MTT assay after 24 h, 48 h and 72 h. The apoptosis induced by lentinan was analyzed by flow cytometry. The protein levels of cleaved PARP, cleaved caspase-9, cleaved caspase-3, cleaved caspase-8, cytochrome C, PI3K, AKT and p-AKT were determined by Western blot. After treatment with PI3K inhibitor LY294002 at 5 mg/L for 72 h, the apoptosis of HL-60 cells was analyzed by flow cytometry. RESULTS:The viability of HL-60 cells was inhibited after treatment with lentinan at concentrations of 15 mg/L, 30 mg/L and 45 mg/L for 24 h, 48 h and 72 h in concentration-dependent and time-dependent manners (P<0.05). The apoptosis of HL-60 cells was promoted after treatment with lentinan (15 mg/L, 30 mg/L and 45 mg/L) for 72 h in a concentration-dependent manner (P<0.05). The protein levels of cleaved PARP, cleaved caspase-9, cleaved caspase-3 and cytoplasmic cytochrome C in the HL-60 cells induced by 30 mg/L lentinan were increased significantly with the increase in the treatment time (P<0.05), but caspase-8 did not show any change. The protein levels of PI3K, AKT and p-AKT were decreased obviously with the increase in the lentinan concentration (P<0.05). Treatment of HL-60 cells with LY294002, a PI3K pathway inhibitor, produced apoptosis-inducing effect similar to lentinan (P<0.05). CONCLUSION:Lentinan induces HL-60 cell apoptosis by inhibiting PI3K/AKT signaling pathway.     

Curcumin inhibits migration and invasion of lung cancer PC-9 cells via down-regulation of nectin-4 expression

AIM: To investigate the effects of curcumin on the abilities of migration and invasion in the lung cancer PC-9 cells, and to observe the relationship between curcumin and nectin-4 expression.METHODS: The viability, migration and invasion of lung cancer PC-9 cells treated with curcumin or transfected with siNectin-4 were measured by MTT assay, wound healing test and Transwell assay, respectively. The protein levels of nectin-4, p-AKT and AKT in the PC-9 cells treated with curcumin or transfected with siNectin-4 were detected by Western blot.RESULTS: Curcumin inhibited the viability of PC-9 cells. The wound healing rates and the numbers of the transmembrane cells in curcumin 10 μmol/L and 20 μmol/L groups were decreased compared with control group without curcumin treatment. The expression level of nectin-4 was reduced after curcumin treatment for 24 h. The viability of the PC-9 cells was significantly inhibited after transfected with siNectin-4 for 48 h or 72 h (P<0.01), and the wound healing rates was decreased in siNectin-4 group compared with NC group (P<0.01). The numbers of the transmembrane cells in siNectin-4 group was significantly reduced (P<0.01). Curcumin and knockdown of nectin-4 suppressed the activation of AKT pathway in PC-9 cells. In siNectin-4+curcumin group, the cell viability reduced compared with curcumin group, and wound healing rates, cell invasive ability and AKT phosphorylation levels were decreased.CONCLUSION: Curcumin inhibits migration and invasion of the lung cancer PC-9 cells via down-regulation of nectin-4 expression and inhibition of AKT pathway.     

siRNA targeting TRIM29 gene induces apoptosis of nasopharyngeal carcinoma cells by inhibiting PI3K/AKT signaling pathway

AIM:To investigate the effect of TRIM29 gene expression silencing on the apoptosis and PI3K/AKT signaling pathway in human nasopharyngeal carcinoma 5-8F cells. METHODS:The 5-8F cells were divided into blank group, negative control (NC) group (transfected negative control siRNA) and si-TRIM29 group (transfected TRIM29 specific siRNA). The viability of the 5-8F cells transfected with si-TRIM29 for 0~96 h was measured by CCK-8 assay. The apoptotic rate and the protein levels of TRIM29, cleaved caspase-3, cleaved caspase-9, Bcl-2, Bax, t-AKT and p-AKT in the 5-8F cells transfected with si-TRIM29 for 48 h were determined by flow cytometry and Western blot, respectively. PI3K/AKT signal specific inhibitor LY294002 at 10 μmol/L and si-TRIM29 alone or in combination were treated with the 5-8F cells, and the cells were divided into blank group, LY294002 group and LY294002+si-TRIM29 group. The apoptotic rates in the 3 groups were detected by flow cytometry. RESULTS:The protein expression of TRIM29 in the 5-8F cells transfected with TRIM29 siRNA was significantly lower than that in blank group (P<0.05). Compared with blank group, the cell viability was significantly decreased, the apoptotic rate was significantly increased, the protein levels of cleaved caspase-3, cleaved caspase-9 and Bax were significantly increased, and the protein levels of Bcl-2 and p-AKT were significantly decreased in si-TRIM29 group (P<0.05). The apoptotic rate in LY294002 group was higher than that in blank group, while that in LY294002+si-TRIM29 group was even higher than that in LY294002 group (P<0.05). CONCLUSION:Silencing of TRIM29 gene expression induces apoptosis of nasopharyngeal carcinoma 5-8F cells by inhibiting PI3K/AKT signaling pathway.     

Effect of arctigenin on apoptosis of human nasopharyngeal carcinoma cell line CNE-1

AIM: To investigate the effect of arctigenin on the apoptosis of human nasopharyngeal carcinoma cell line CNE-1 and its potential mechanism. METHODS: The inhibition of cell viability was analyzed by CCK-8 assay. The activity of caspase-3 and caspase-9 was analyzed by caspase-3 and caspase-9 activity kit. Apoptotic cell percentage was evaluated by Annexin V-PI staining. The expression of PI3K/AKT/XIAP signal pathway-related molecules at mRNA and protein levels was analyzed by real-time PCR and Western blot. RESULTS: Arctigenin inhibited the cell activity in a dose- and time-dependent manner after treatment with arctigenin at concentrations of 10, 20, 40 and 80 μmol/L for 24 h, 48 h and 72 h (P<0.01). Arctigenin also increased the activity of caspase-3 and caspase-9 and the apoptotic rate (P<0.05), and down-regulated the mRNA and protein expression of PI3K/AKT/XIAP signal pathway-related molecules (P<0.05).CONCLUSION: Arctigenin induces the apoptosis of CNE-1 cells through PI3K/AKT/XIAP signal pathway.     

Muscarinic receptor 3 agonist promotes epithelial-meschymal transition in human lung cancer A549 cells by activating PI3K/AKT signaling pathway

AIM: To investigate the possible signaling pathway that promotes epithelial-mesenchymal transition (EMT) of the lung cancer A549 cells stimulated with muscarinic receptor 3 (M3R) agonist carbachol. METHODS: The lung cancer cells A549 were treated with 400 μmol/L carbachol. The morphological changes of the cells were observed under inverted phase contrast microscope. The migration and invasion abilites were measured by Wound healing and Transwell assays. qPCR was used to detect the mRNA level of vimentin and E-cadherin. The protein levels of p-AKT, vimentin and E-cadherin were determined by Western blot. RESULTS: After treatment with carbachol, the A549 cells showed loss of the close connection and the cell morphology was transformed from irregular polygon to spindle-like cells. The results of Wound healing and Transwell assays showed that the migration and invasion abilites of the A549 cells were enhanced. Carbachol increased the vimentin expression and decreased the E-cadherin expression at mRNA and protein level (P<0.05). The phosphorylation of AKT in the A549 cells was up-regulated (P<0.05). These changes was inhibited by M3R antagonist 4-DAMP. CONCLUSION: Carbachol promotes EMT in the human lung cancer A549 cells by activating PI3K/AKT signaling pathway.     

FGFR2-IIIb D3 extracellular domain inhibits lipid synthesis in keratinocytes

AIM:To investigate the inhibitory effect of extracellular domain of fibroblast growth factor receptor 2 (FGFR2)-Ⅲb D3 on lipid synthesis in HaCaT keratinocyteas. METHODS:The bioactivity of FGFR2-Ⅲb D3 extracellular domain was detected by isothermal titration calorimetry (ITC) and CCK-8 assay. The inhibitory effect of FGFR2-Ⅲb D3 extracellular domain on lipid synthesis in HaCaT cells was analyzed by real-time PCR, Western blot, flow cytometry and oil red O staining. RESULTS:The results of ITC, real-time PCR and CCK-8 assay showed that FGFR2-Ⅲb was highly expressed in HaCaT cells, and FGFR2-Ⅲb D3 extracellular domain specifically bound to FGF7 to inhibit HaCaT cell viability. The results of real-time PCR and Western blot showed that FGF7 significantly up-regulated the mRNA expression of sterol regulatory element binding protein-1c (SREBP-1c), fatty acid synthase (FAS), acetyl-CoA synthase (ACS), stearoyl-CoA desaturase (SCD) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), and the protein levels of p-FGFR, p-AKT and SREBP-1 in HaCaT cells, and these effects were inhibited after the treatment with FGFR2-Ⅲb D3 extracellular domain and AKT inhibitor, which was significantly different from the FGF7 group. and The results of oil red O staining and flow cytometry showed that the lipid content of HaCaT cells was increased after FGF7 induction. After treatment with FGFR2-Ⅲb D3 extracellular domain and AKT inhibitor, the lipid content was decreased (P<0.01). CONCLUSION:FGFR2-Ⅲb D3 extracellular domain inhibits the autophosphorylation of FGFR2-Ⅲb by its competitive binding to FGF7, and then blocks downstream PI3K-AKT signaling pathway, thus resulting in the down-regulation of SREBP-1c and downstream lipid synthases, and inhibiting lipid synthesis in keratinocytes.     

Quercitrin promotes apoptosis of gastric cancer cell line SGC7901 by inhibiting PI3K/AKT signaling pathway

AIM: To investigate whether quercitrin induces apoptosis of gastric cancer cell line SGC7901 by inhibition of PI3K/AKT signaling pathway. METHODS: The human gastric cancer SGC7901 cells were selected as the research object. The cytotoxicity of quercitrin was detected by MTT assay, and IC₅₀ value of quercitrin was calculated. The SGC7901 cells were divided into control group, quercitrin group (incubated with 200 μmol/L quercitrin), insulin-like growth factor-1 (IGF-1) group (incubated with 100 μg/L IGF-1) and quercitrin+IGF-1 group (incubated with 200 μmol/L quercitrin and 100 μg/L IGF-1). After 48 h, the apoptosis of SGC7901 cells was analyzed by flow cytometry, and the protein levels of cleaved caspase-3, p-AKT (Ser473), AKT, p-PI3K (Tyr508) and PI3K were determined by Western blot. RESULTS: The viability of SGC7901 cells was significantly decreased as the concentration of quercitrin increased, starting at 100 μmol/L (P<0.05). The IC₅₀ value of quercitrin for 48 h was 275.40 μmol/L. After treatment with 200 μmol/L quercitrin for 48 h, the apoptosis rate and the protein level of cleaved caspase-3 in quercitrin group were significantly increased (P<0.05), and the phosphorylated levels of AKT and PI3K were significantly decreased compared with control group (P<0.05). Treatment with quercitrin and IGF-1 inhibited the effect of quercitrin on SGC7901 cells compared with quercitrin group. CONCLUSION: Quercitrin may induce apoptosis of gastric cancer cell line SGC7901 by inhibiting the activation of PI3K/AKT signaling pathway.     

Over-expression of SIRT1 gene inhibits high glucose-stimulated H9c2 cardiomyocyte apoptosis and ROS level through PI3K/AKT signaling pathway

AIM: To investigate the effects of silent information regulator 1 (SIRT1) over-expression on the apoptosis and the level of reactive oxygen species (ROS) in high glucose-induced H9c2 cardiomyocytes. METHODS: H9c2 cardiomyocytes were transfected with empty plasmid (pcDNA3.1-NC) and SIRT1 over-expression plasmid (pcDNA3.1-SIRT1), and then stimulated by high glucose. The H9c2 cells were divided into control group, high glucose group, high glucose + pcDNA3.1-NC group and high glucose + pcDNA3.1-SIRT1 group. The expression of SIRT1 at mRNA and protein levels in each group was determined by qPCR and Western blot. The viability of the cells was measured by MTT assay. The apoptotic rate was analyzed by flow cytometry. The protein levels of phosphatidylinositol 3-kinase (PI3K), phosphorylated PI3K, AKT and phosphorylated AKT were examined by Western blot. RESULTS: SIRT1 was significantly decreased in high glucose-induced H9c2 cardiomyocytes, the cell viability was significantly decreased compared with control group, while the ROS levels and apoptotic rate were increased, and the phosphorylated PI3K and AKT protein levels were down-regulated (P<0.05). Over-expression of SIRT1 significantly promoted the viability of H9c2 cardiomyocytes induced by high glucose, decreased the ROS levels and apoptotic rate, and up-regulated phosphorylated PI3K and AKT protein levels (P<0.05). CONCLUSION: SIRT1 over-expression reverses the decrease in the viability of high glucose-stimulated H9c2 cardiomyocytes, and the increases in apoptotic rate and oxidative stress by regulating PI3K/AKT signaling pathway.     

miRNA-126 inhibits proliferation,migration and invasion of human lung cancer A549 cells via EGFR/AKT/mTOR pathway

AIM: To investigate the effects of microRNA(miRNA)-126 on the proliferation, migration and invasion of human lung cancer cell lines, and to explore its mechanism. METHODS: The A549 cells were transfected with miRNA-126 agomir by Lipofectamine 2000. The expression of miRNA-126 was detected by real-time PCR. The cell activity was detected by MTT assay. The number of viable A549 cells was counted by the method of Trypan blue exclusion. The cell colony-forming capability was determined by cell colony formation test. The cell migration and invasion abilities were assayed by wound healing and Transwell methods, respectively. The protein levels of p-EGFR, EGFR, p-AKT, AKT, p-mTOR and mTOR were determined by Western blot. RESULTS: The expression level of miRNA-126 was significantly increased in the A549 cells compared with negative control(NC) group and control group(P<0.01). The proliferation of A549 cells was decreased extremely after transfected with the miRNA-126 agomir(P<0.01), so did the result of the cell colony-formation test. The migration and invasion abilities of the lung cancer cells were also significantly inhibited. The protein levels of p-EGFR, p-AKT and p-mTOR were significantly down-regulated compared with NC group and control group(P<0.01). CONCLUSION: Over-expression of miRNA-126 significantly inhibits the proliferation, migration and invasion ability of human lung cancer A549 cells by down-regulation of EGFR/AKT/mTOR pathway.     

Paeonol affects viability and migration ability of hepatocellular carcinoma cells by up-regulating miR-424-3p and inhibiting PI3K/AKT signaling pathway

AIM To investigate the effect of paeonol on the viability and migration ability of hepatocellular carcinoma cells and its molecular mechanism. METHODS Human hepatocellular carcinoma Hep3B cells was treated with paeonol at different concentrations (50, 100, 200 and 400 mg/L). The cell viability was measured by CCK-8 assay to determine the optimal drug concentration. The Hep3B cells were divided into normal control (NC) group, paeonol group, miR-NC group, miR-424-3p group, paeonol+anti-miR-NC and paeonol+anti-miR-424-3p group. The expression level of miR-424-3p was detected by RT-qPCR. The migration ability was detected by Transwell assay. The protein levels of cyclin D1, matrix metalloproteinase 2 (MMP2), MMP9 and PI3K/AKT signaling pathway-related molecules were determined by Western blot. RESULTS Paeonol intervention inhibited the viability of Hep3B cells in a concentration-dependent manner (P<0.05). The concentration of paeonol at 200 mg/L was selected for the following study. Paeonol intervention inhibited the protein expression of MMP2 and MMP9 in the Hep3B cells, and inhibited the migration ability of the Hep3B cells. Paeonol intervention promoted the expression of miR-424-3p in the Hep3B cells (P<0.05). Over-expression of miR-424-3p inhibited the expression of cyclin D1, MMP2 and MMP9 in the Hep3B cells and inhibited cell viability and migration ability (P<0.05). Inhibition of miR-424-3p reversed the effect of paeonol on the viability and migration ability of the Hep3B cells (P<0.05). Paeonol inhibited phosphorylation levels of PI3K and AKT in the Hep3B cells and inhibited the activation of PI3K/AKT signaling pathway (P<0.05). Inhibition of miR-424-3p reversed the effect of paeonol on PI3K/AKT signaling pathway in the Hep3B cells (P<0.05). CONCLUSION Paeonol inhibits the viability and migration ability of hepatocellular carcinoma cells by up-regulating miR-424-3p and inhibiting PI3K/AKT signaling pathway.     

Effect of KLF6 gene on apoptosis of THP-1 cell-derived macrophages

AIM: To investigate the effects of Kruppel-like factor 6 (KLF6) over-expression on the viability, apoptosis, reactive oxygen species (ROS) level and AKT signaling pathway of THP-1 cell-derived macrophages. METHODS: Human monocyte cell line THP-1 was induced to differentiate into macrophages by phorbol myristate acetate (PMA), and the macrophages were randomly divided into pcDNA3.1 group, oxidized low-density lipoprotein (ox-LDL) group, ox-LDL+pcDNA3.1 group and ox-LDL+pcDNA3.1-KLF6 group. pcDNA3.1 was transfected according to Lipofectamine^TM 2000 Kit. The cell viability, apoptotic rate and ROS level were detected by MTT assay, flow cytometry with Annexin V-FITC/PI double staining and H₂DCF-DA probing, respectively. The protein levels of Bcl-2, Bax and p-AKT were determined by Western blot. RESULTS: After pcDNA3.1-KLF6 was transfected into the macrophages, the expression of KLF6 was increased significantly (P<0.05). ox-LDL significantly inhibited the viability of the macrophages, induced apoptosis and ROS production, up-regulated the protein expression of Bax, and down-regulated the protein levels of Bcl-2 and p-AKT (P<0.05). Over-expression of KLF6 significantly reduced the effects of ox-LDL on cell viability, apoptosis, ROS level and the protein levels of Bcl-2, Bax and p-AKT (P<0.05). CONCLUSION: KLF6 significantly reduces the apoptosis of THP-1 cell-derived macrophages induced by ox-LDL, which may be related to the reduction of ROS level and activation of AKT 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.     

MicroRNA-497 inhibits viability,invasion and migration of thyroid papillary cancer cells

AIM:To investigate the underlying mechanisms of mircoRNA-497 (miR-497) inhibiting the viability, migration and invasion of papillary thyroid cancer (PTC) cells. METHODS:TargetScan 6.0 was used to predict the potential targets of miR-497. The target gene was confirmed by luciferase reporter assay, RT-qPCR and Western blot. The expression levels of miR-497 and its target gene in the PTC tissues were detected by RT-qPCR. Gene transfection, MTT assay, and cell migration and invasion assays were used to investigate the effects of miR-497 and its target gene on PTC cell viability, migration and invasion. RESULTS:AKT3 was demonstrated to be the direct target gene of miR-497. In addition, AKT3 expression was higher in the PTC tissues than that in normal tissues (P<0.05) and negatively correlated with miR-497 expression (r=-0.573 7, P<0.01). Furthermore, down-regulation of AKT3 also suppressed cell viability, migration and invasion of PTC, which played similar roles of miR-497 over-expression in PTC cells. CONCLUSION:miR-497 inhibits the viability, migration and invasion of PTC cells by directly targeting AKT3.     

Effect of HC-1119 on biological behaviors of triple-negative breast cancer BT549 cells

AIM: To explore the effect of new artificially synthesized androgen receptor (AR) antagonist HC-1119 on the biological function of triple-negative breast cancer (TNBC) BT549 cells and the molecular mechanism. METHODS: The AR expression was assessed in different human breast cancer cell lines MDA-MB-231, T47D, MCF-7, SKBR3 and BT549 by Western blot. The TNBC BT549 cells with AR positive expression were treated with HC-1119. The cell viability was measured by CCK-8 assay. The apoptosis rate and cell cycle distribution were analyzed by flow cytometry. The migration and invasion abilities were detected by Transwell assay in vitro. The protein expression of E-cadherin, vimentin and P21 was determined by Western blot. RESULTS: AR was positively expressed in BT549 cells. HC-1119 inhibited the cell viability in a time-and dose-dependent manner (P<0.05), increased the percentage of apoptotic cells and the percentage of S-phase cells significantly, repressed the migration and invasion abilities (P<0.05), and decreased P21 expression at protein level (P<0.01). No influence on the expression of E-cadherin and vimentin in the BT549 cells was observed. CONCLUSION: AR antagonist HC-1119 decreases the viability, migration ability and invasion ability, enhances the apoptosis, and arrests the cell cycle distribution of TNBC BT549 cells. HC-1119 represses the viability of BT549 cells by down-regulating P21 expression, while the process of epithelial-mesenchymal transition is not involved in the inhibition of cell migration.     

Effects of axitinib on biological behavior of adrenocortical carcinoma cell line SW-13

AIM: To investigate the effects of axitinib on the biological behavior of adrenocortical carcinoma cell line SW-13. METHODS: CCK-8 assay was used to measured the viability of SW-13 cells treated with axitinib at different concentrations. The cell cycle distribution was analyzed by flow cytometry. The apoptotic rate was also analyzed by flow cytometry with Annexin V/PI double staining. Wound healing experiment and Transwell invasion assay were used to observe cell migration and invasion abilities,respectively. The protein levels of vascular endothelial growth factor receptor 2(VEGFR2), extracellular regulated protein kinases 1/2 (ERK1/2) and p-ERK1/2 were determined by Western blot. RESULTS: After treated with axitinib, the viability of SW-13 cells was significantly inhibited, the cell cycle was blocked in G₂/M phase, and the apoptosis rate was increased. The migration and invasion abilities of SW-13 cells were markedly inhibited by axitinib (P<0.01). The protein levels of VEGFR2 and p-ERK1/2 in the SW-13 cells were significantly decreased with axitinib treatment (P<0.01). CONCLUSION: Axitinib inhibits the viability, blocks the cell cycle, promotes cell apoptosis, and inhibits the migration and invasion abilities of SW-13 cells. The mechanism may be related to inhibition of VEGFR2 expression and reduction of ERK1/2 phosphorylation.