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Objective To observe the effect of miR-369-3p on vascular endothelial growth factor C (VEGFC) gene in bladder cancer cells EJ and 5637 and observe its effect on cell proliferation and apoptosis.Methods Bladder cancer cell lines EJ and 5637 were transfected with miR-NC (control group) or transfected with miR-369-3p (experimental group).The target gene of miR-369-3p was predicted by Targets can target gene prediction software.Fluorescence real-time quantitative polymerase chain reaction (qPCR) was used to detect the changes of VEGFC at mRNA level.The changes of VEGFC,p-Raf-1 and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) at the protein level were detected by Western blotting.The effect of miR-369-3p on the proliferation of bladder cancer cells was detected by cell counting kit (CCK-8) and clone formation experiment.The effect of miR-369-3p on apoptosis was detected by flow cytometry.Results After transfection with miR-369-3p,the expression of VEGFC at mRNA and protein levels was significantly decreased (P ＜ 0.05),the expression of p-Raf-1 and p-ERK1/2 protein was significantly decreased,the cell proliferation ability was significantly decreased (P ＜ 0.05),the number of clones formed was significantly decreased (P ＜ 0.05) and the apoptosis was significantly increased (P ＜ 0.01).Conclusions miR-369-3p can inhibit the proliferation and promote the apoptosis of bladder cancer cells by interfering with the expression of VEGFC gene,which may provide a new target for the biological therapy of bladder cancer.
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Objective To investigate the inhibitory effect of Withaferin A ( WFA) on the growth of orthotopic xenograft tumor of hepatocellular carcinoma in nude mice and the mechanism of its antitumoral effect. Methods For in vivo model, anti-tumor efficacy of Withaferin A was evaluated in nude mice mod-els of human liver cancer orthotopic xenograft. The nude mice were randomly divided into model group, Sunitinib group,and Withaferin A groups [6, 3 mg/(kg·d)]. All mice were given intraperitoneal injec-tion for 14 days. Tumor volume and tumor weight were observed. Antiangiogenic effects were assessed in vi-vo by the tumor inhibition rate and microvessel density. Quantitative polymerase chain reaction ( QPCR) as-say was used to detect the mRNA expression of vascular endothelial growth factor ( VEGF) , basic fibroblast growth factor (bFGF), angiopoietin-2 (Ang-2), vascular endothelial growth factor receptor 2 (VEGFR2) from tumor tissues. For in vitro experiments, the cell count kit 8 ( CCK8 ) assay was used to detect the effect of Withaferin A on HepG2 cells proliferation. QPCR assay and enzyme-linked immunosorbent assay ( ELISA) were used to detect the mRNA expression of VEGF. Results Compared to the model group, the high-dose Withaferin A group and the Sunitinib group had a significantly lower tumor weight (P<0. 05). The tumor inhibition rate was 42. 69％ in the high-dose Withaferin A group, 20. 22％ in the low-dose With-aferin A group, and 49. 43％ in the Sunitinib group. The growth of HepG2 cells was significantly inhibited by different concentrations of Withaferin A,and the 50％ concentration of inhibition ( IC50 ) of Withaferin A were (2. 64 ± 0. 18)μmol/L at 24 h. Withaferin A (6,3 μmol/L) could inhibit the protein and mRNA ex-pression of VEGF ( P<0. 05 ) . Conclusions Withaferin A significantly reduces the growth of orthotopic xenograft tumor of hepatocellular carcinoma in nude mice via antiangiogenic effect. Downregulation of the protein and mRNA expression of VEGF by WFA may be one mechanism of its anti-liver cancer effect.
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Objective To explore the effect of proliferation and apoptosis,and research its mechanism associated with RAS/extracellular signal regulated kinase/mitogen-activated protein kinase (RAS/ ERK/MAPK) in Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutation acute lymphocytic leukemia cell line MV4-11 cells treated by triptolide (TP).Methods MV4-11 cells were respectively treated by triptolide with diverse concentrations and different times.The proliferation inhibition rate was measured by methyl thiazolyl tetrazolium,the apoptosis rate was detected by flow cytometry,the mRNA expressions of FLT3,RAS,ERK,forkhead box protein M1 (FOXM1),and c-Myc were analyzed by realtime fluorescent quantitative polymerase chain reaction (PCR).Results The proliferation inhibition rate markedly increased in a dose-time dependent manner after MV4-11 cells were treated by TP.After cells were treated with 10,and 20 nmol/L TP,respectively,the apoptosis rates at 48 h were (17.30 ± 0.56) ％,(35.77 ±0.55)％,and those at 72 h were (49.83 ±0.45)％,(68.90 ±0.75)％ correspondingly.PCR data showed that the mRNA level of FLT3 mRNA decreased obviously,and that of RAS,ERK,FOXM1,and c-Myc also decreased.Conclusions Triptolide could significantly inhibit the MV4-11 cells proliferation and induce cell apoptosis,and its mechanism might be through inhibiting the expression of related genes on RAS/ERK/MAPK signaling pathway.
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Objective To investigate the effect of MicroRNA-9 (miR-9) on cell proliferation and collagen synthesis in hepatic stellate cells (HSCs),and to explore the potential mechanism.Methods HSC-T6 cells were cultured and transfected with miR-9 mimics with lipofectamine 2000.After incubation 48 h,the cells were collected and total proteins and RNAs were extracted.The expression of miR-9 was detected by quantitative real time polymerase chain reaction (qRT-PCR).The protein expression of type Ⅰ collagen and type Ⅲ collagen were measured by Western blot.The methyl thiazolyl tetrazolium (MTT) method was used to asses the proliferation of HSC-T6 cells.The expression of transforming growth factor-β1 receptor 2 (TGFBR2) was detected by qRT-PCR and Western blot.Results Compared to the control group,miR-9 expression in HSCs was increased in the miR-9 mimics group (P ＜ 0.05),type Ⅰ and type Ⅲ collagen protein expression was reduced by (44 ± 2) ％ and (50 ± 3) ％ (P ＜ 0.01),respectively.The proliferation activity of HSCs was decreased by (48 ± 4)％ (P ＜ 0.05).The expression of TGFBR2 was inhibited in the miR-9 mimics group.Conclusions Upregulation of miR-9 plays a role on suppressing cell proliferation and collagen synthesis in HSCs.This process might be mediated by downregulation of TGFBR2.
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Objective To investigate the influence of cyclooxygenase-2 (COX-2) knock-down on cell proliferation and apoptosis in gastric cancer cell line SGC7901.Methods The mRNA levels of COX-2 in SGC7901 and gastric epithelium cells (GES) were determined by quantitative real-time polymerase chain reaction (qRT-PCR) assays.The influence of COX-2 on the proliferation and apoptosis of cell line SGC7901 was evaluated with methyl thiazolyl tetrazolium (MTT) and fluorescence-activated cell sorter (FACS) assay.Results qRT-PCR assay indicated that the mRNA level of COX-2 in SGC7901 was significantly higher than that in GES (P ＜ 0.01).MTT and FACS assays showed that the proliferation was reversed by COX-2 knock-down in SGC7901 cells.Up-regulated Bax and down-regulated Bcl-2 can inhibit the expression of COX-2.Conclusions COX-2 could contribute to the proliferation of gastric cancer cell line SGC7901.
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Objective To investigate the impact of triptolide (TP) on proliferation and apoptosis of imatanib resistant CML cell (K562/G01) and its regulating effect on Wnt/β3-catenin signal pathway.Methods A series of 10,20,40,and 80 nmol/L of triptolide were used in CML cells K562/G01 for 12,24,and 48 hours.The cell proliferation was detected with methyl thiazolyl tetrazolium (MTT) test.The apoptosis was assessed with flow cytometry (FCM).The mRNA expressions of breakpoint cluster region-c-abl (BCR-ABL),β-catenin and its down-stream targets Lef-1,and cyclinD1 were analyzed with real-time quantitative polymerase chain reaction (RT-PCR),respectively.Results Triptolide significantly inhibited K562/G01 cell growth ability and induced apoptosis in a dose-dependent manner.Mter being treated with 20,40 nmol/L TP for 24 hours,the cell growth inhibition rates were (22.62 ± 1.33) ％,and (51.41 ±1.39) ％,respectively.The late apuptosis rates were (6.91 ± 0.14) ％,and (7.64 ± 0.47) ％,respectively.Meanwhile,PCR data showed that the mRNA levels of BCR-ABL were decreased,compared to the control group,the mRNA levels of β-catenin,Lef-1,and cyclinD1 were also decreased obviously after treatment.Conclusions Our data indicated that the triptolide could inhibit the proliferation and induce the apoptosis of K562/G01,and the mechanism might be related to the blockade of Wnt/β-catenin signal pathway.
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Objective To investigate the inhibitory effect of aqueous extract of Taxus chinensis.vat (AETC) combining Cisplatin (DDP) on vitro cultured human lung carcinoma A549 cells,and the effects on resistance genes.Methods The A549 cells were divided into different concentrations of DDP groups,different concentrations of AETC groups,and blank group,and drug effect of 48 h with the method of cell counting kit-8 (CCK-8) and the effect on cell survival were detected.Based on the above results,then A549 cells were divided into DDP combining different concentrations of AETC groups,DDP group,blank control group,and drug effect of 48 h with the method of CCK-8 and the effect on cells survival were detected.The gene expressions of adenosine triphosphate (ATP)-binding cassette subfamily B member 1 trans-porter (ABCB1),ABCG2,and ABCC1 were examined by polymerase chain reaction (RT-PCR).Results Cisplatin 12 μg/ml (DDP),DDP + ATEC 400 μg/ml,DDP + ATEC 800 μg/ml,DDP + ATEC 1 200 μg/ml,DDP + ATEC 1 600 μg/ml,A549 cell inhibition rate of each group was 44.36％,69.61％,74.73％,80.10％,and 74.73％,respectively;Different concentrations of AETC combining DDP could decrease the resistance related gene ABCC1,ABCB1 expressions,and correlated to the dose.AETC combining DDP showed no effects on ABCG2 gene expression.Conclusions AETC combining DDP could inhibit the growth of A549 cells,and decrease the resistance-related gene ABCC1,ABCB1 expressions.
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Objective To investigate the effect of miRNA-200b-specific inhibitor on hepatic stellate cells(HSCs) activation,proliferation,and extracellular matrix production.Methods The miRNA-200b-specific inhibitors were designed,synthesized,and transfected into HSCs with lipofectamine 2000.The supernatant and HSCs were collected after incubation for 48 h.The expression of miR-200b was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR).The expression ofα-smooth muscle actin (oα-SMA) protein in HSCs was detected by Western blotting.The cell proliferation was assayed by methyl thiazolyl tetrazolium (MTT) method.Contents of type Ⅲ procollagen and hyaluronic acid in supernatant were determined by radioimmunoassay.Results Compared to the control group,miRNA-200b expression was decreased in the miRNA-200b inhibitor group by 82％ (P ＜ 0.01),α-SMA protein expression was reduced in the miRNA-200b inhibitor group by (19 ± 3) ％ (P ＜ 0.05),and the activity of HSCs proliferation was reduced by(33 ± 5)％ (P ＜0.01),and the contents of type Ⅲ procollagen and hyaluronic acid in supernatant were reduced in miRNA-200b inhibitor group by (35 ± 4)％ and (31 ± 2)％,respectively(P ＜0.01).Conclusions The miRNA-200b-specific inhibitor could significantly reduce the expression of miRNA-200b,and inhibit HSC proliferation,activation,and extracellular matrix production.
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Objective To investigate the effect of miR-218 on cell proliferation and cell cycle in the gastric cancers (GC).Methods SGC-7901 and MGC-803 cells were transfected with miR-218 mimics.Meanwhile,SGC-7901 and MGC-803 cells were transfected with control mimics (Scramble mimics) as negative control.Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to detect the miR-218 expression in these transfected cells.The effect of miR-218 overexpression on cell proliferation was evaluated with direct cell counting and MTS [3-(4,5-dimethylthiazol-2-yl)-5 (3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium inner salt] assay.Moreover,the effect of miR-218 overexpression on cell cycle was examined by fluorescence activated cell sorting (FACS).Results miR-218 expression was remarkably induced in miR-218-transfected cells,miR-218 overexpression led to a significant decrease in cell proliferation rate compared to control cells (P ＜ 0.05).miR-218 overexpression induced GC G1 arrest.Conclusions miR-218 can suppress GC cell proliferation and block cell cycle progression.It maybe play an important role in tumorigenesis and development of GC.
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Objective To investigate the effect of astragalus polysaccharides (APS) on the growth and proliferation of hepatocellular carcinoma cells and its effect on adenosine monophosphate activated pro-tein kinase (AMPK) activity.Methods Hepatocellular carcinoma HepG2 cells cultured for 12,24,and 48 hours were treated with 200,300,and 400 mg/L concentration of astragalus polysaccharides.The cell inhibition rate was detected with methyl thiazolyl tetrazolium (MTT),and apoptosis was observed under the fluorescence microscope.Western blot method was used to measure the expression of total AMPK,phosphorylated AMPK (p-AMPK),and phosphorylate mammalian target of rapamycin (p-mTOR) protein expressions.Results Astragalus polysaccharides of each concentration significantly inhibited the proliferation of human hepatoma HepG2 cells (P ＜ 0.01),and the effect of 300 mg/L concentration astragalus polysaccharides was more significant than that of the 200 mg/L concentration (P ＜0.01);while inhibitory effect of 400 and 300 mg/L Astragalus polysaccharides on the proliferation of human hepatoma cell line HepG2 was not significant difference.We found that Astragalus polysaccharides of each concentration could promote the apoptosis of HepG2 cells,and the effect of 300 mg/L Astragalus polysaccharides was more significant.However,astragalus polysaccharide of 400 mg/L concentration could promote the apoptosis no more than the 300 mg/L concentration,which was observed by fluorescent microscope.Western blot results showed that astragalus polysaccharides could increase the expression of p-AMPK (P ＜ 0.05),and inhibit its downstream protein expressions of p-mTOR (P ＜ 0.05).The proliferation effect of astragalus polysaccharides was weakened after accession of AMPK antagonist compound C on hepatocellular carcinoma cells.Conclusions APS can inhibit the growth and proliferation of hepatocarcinoma cells,and its mechanism is related to the AMPK-mTOR pathway.
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Objective To investigate the effect of down-regulation of transient receptor potential melastatin 8 (TRPM8) on the proliferation and apoptosis of human bladder transitional cell carcinoma cells T24.Methods shRNA targeting TRPM8 was designed and synthesized, and then transfected into the T24 cells via lipofectamine 2000 mediation.The proliferation and apoptosis of T24 cells were detected with methyl thiazolyl tetrazolium (MTT) assay and flow cytometry.Expression of extracellular regulated kinase (ERK), cyclin D1, and Bcl-2 were detected with Western blot.Results TRPM8-targeted shRNA downregulated TRPM8 expression of T24 cells.MTT assay showed a significant acceleration of the proliferation of shRNA interference group compared to blank and control groups (P ＜0.01).Compared to control group, cell apoptosis rate was significantly higher in shRNA interference group (P ＜ 0.01).In addition, the expressions of PI3K, cyclin D1, and Bcl-2 were decreased in shRNA interference group.Conclusions Down-regulation of TRPM8 can induce inhibition of proliferation and promotion of cell apoptosis in human bladder transitional cell carcinoma cells T24 via regulating PI3K.It might be regarded as a novel target for clinical diagnosis and gene therapy of bladder cancer.
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Objective To investigate the effect of As2 O3 in combination with cinobufacini on proliferation and apoptosis of the K562 cells and provide theoretical basis for clinical application.Methods Cell proliferation was assayed by analyzing the growth and viability of the cells.Apoptosis was assayed by performing cell morphology,Annexin-V/PI staining,DNA-PI staining,and DNA gel electrophoresis.Results After exposure to As2O3 and cinobufacini,the growth of K562 cells was inhibited and the viability of K562 cells was decreased. After treated with 1.0μmol/L As2O3,0.125μg/ml cinobufacini,0.25μg/ml cinobufacini,1.0μmol/L As2O3 + 0.125 μg/ml cinobufacini,1.0μmol/L As2O3 + 0.25μg/ml cinobufacini for 24 and 48 hours,the proliferation inhibition rate were(24 ± 1.3)%,(21 ± 1.5)%,(38 ± 3.1)%,(57 ±2.7)%,(66 ±3.3)% and(49 ±2.9)%,(48 ±2.7)%,(61 ±2.1)%,(77 ±3.8)%,(82 ±4.2)%,the apoptosis rate of K562 cells were(4.8 ± 0.5)%,(5.6 ± 0.7)%,(9.8 ± 0.6)%,(11.9 ± 1.2)%,(15.2±1.5)% and(11.0 ±0.9)%,(12.9 ±1.1)%,(18.4 ±1.5)%,(21.0 ±2.0)%,(28.0 ±1.9)%.The percentage of apoptotic cells was a time- and dose-dependent manner.Typical DNA ladder was shown by DNA gel electrophoresis.Conclusions As2O3 combined with cinobufacini inhibited the proliferation of K562 cells and induced apoptosis of the K562 cells,the combination of the two drugs had better effect.
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Objective To explore the different concentrations of glycyrrhizin on hepatic stellate cell proliferation and its regulation mechanism.MethodsHepatic stellate cell proliferation rate of three different concentrations of glycyrrhizin was detected by MTT assay.The expression of cyclin E was detected by immunocytochemistry.ResultsMTT assay was used to detect the cell proliferation inhabitation in different concentration groups,from control group 0.8 mg/L glycyrrhizin group to 1.2 mg/L glycyrrhizin group and 1.6 mg/L glycyrrhizin group.The cell proliferation inhibited rates were 0,2.73%,14.75 % and 25.96%.The difference was significant (P＜0.05).The numbers of Cyclin E positive cells in different concentrations of glycyrrhizin group were significantly different compared with control group,and it was concentration dependently decreased (P＜0.01).ConclusionsGlycyrrhizin concentration dependently inhibited rat hepatic stellate cells proliferation and it could reduce the expression of cyclin E and inhibit the proliferation of rat hepatic stellate cells.
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Objective To investigate the influence of PPARγ excitomotor RSG and ATRA on gastric cancer SGC7901 cell line proliferation in vitro and its potential mechanism study.Methods Human gastric cancer SGC7901 cell line was cultured in vitro.Experiment samples were divided to blank group,10μmol/L ATRA group, 12.5μmol/L RSG group, 25μmol/L RSG group, 10μmol/L ATRA + 25μmol/L RSG group.Proliferation inhibitory effect was determined by MTI assay.Flow cytometry was used to detect cell cycle, H.E stain was used to observed micrography alteration.Expression of PPARγ protein in gastric cancer cells were measured by immunohistochemistry.PPARγ mRNA in gastric cancer cells were measured by RT-PCR.Results ATAR at concentration 10μmol/L, RSG at 12.5 μmol/L and RSG at 25 μmol/L could inhibit the proliferation of SGC7901 cells in a dose-and time-dependent, and when both agents were combined for 72h, growth inhibition ratio was (29.73 ± 0.69) %.Flow cytometry analysis revealed a cell cycle arrest at G1 and S phase, and when both agents combined, S% was (12.87 ± 0.35 )%, cell micrography tended to be normal when both agents combined.Up-regulation of PPARγ protein and PPARγ mRNA expressions were also observed, those effects were enhanced when both agents combined, and grey scale ratio was 0.646.Conclusion The ATRA and RSG could significantly induced growth inhibition of human gastric cancer SGC7901 cell, which may be associated with cell cycle arrest and inducing differentiation, activation of PPARγ protein and PPARγ mRNA expression.Synergistic effect could be caused by the combined use of the two agents.
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Objective To investigate the effect of proteasome inhibitor MG132 on the proliferation of human lens epithelial cells SRA01/04. Methods The SRA01/04 cells were treated with MG132 by different concentrations (0, 0. 1, 0. 5, 1. 0, 2. 5, 5.0, 10. 0μmol/L) for 36 hours. The cell viability in all groups was determined using methylthiazoltetrazolium (MTT) test. The effect of MG132 on the apoptosis and regulation of cell cycle about SRA01/04 cells were detected by flow cytometry (FCM). The SRA01/04 cells treated with MG132 were observed after Annexin V/FITC-PI staining by fluorescence microscope. Results The inhibitory effect of MG132 on SRA01/04 cells proliferation was enhanced with the increase of MG132 concentration. The 50% inhibiting concentration ( IC50 ) of MG132 was 2. 50μmol/L after SRA01/04 cells were treated with MG132 for 36 hours. The apoptosis index of the cells treated by MG132 at 2. 5μmol/L and 5 μmol/L for 36 hours was 6. 55 ± 0. 35% and 13.75 ± 3.18%, and 0. 75 ± 0. 21% for 5.0μmol/L for 36 hours in control group. After cells were treated with MG132 for 48h, the percentages of cells at G0/G1 phase were (42. 57 ± 0. 64) %, (73.42 ± 3.10) %, ( 80. 95 ± 3.83 ) % 0, 2. 5,5.0 μmol/Lgroups respectively, and those at S phase were (49. 44±1.36)%, ( 17. 40 ± 1.50)%, ( 19. 57 ± 1.29)%.Annexin V/FITC-PI staining was used, and MG132 was found to result to apoptosis. Conclusions MG132 could inhibit the proliferation of SRA01/04 cells by the effect of inducing apoptosis and regulation of cell cycle. The proteasome inhibitor-might play a key role in the prevention of posterior capsular opacification.