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Objective To investigate the effect of over-expressed macrophage migration inhibitory factor (MIF) on epithelial-mesenchymal transition (EMT) in human cervical carcinoma SiHa cells.Methods Recombinant eukaryotic expression plasmid liposome enhanced transfection of green fluorescent protein gene (pEGFP-N1)-MIF was constructed and then transfected into human cervical cancer SiHa cells.Experimental cells were classified into three groups (SiHa-pEGFP-N1-MIF,SiHa-pEGFP-N1,and SiHa).Western blot was used to detect the expression of MIF protein,and the expressions of EMT-related markers such as E-cadherin and vimentin in SiHa cells were determined before and after transfection.Results The eukaryotic expression vector pEGFP-N1-MIF significantly increased the expression of MIF protein in SiHa cells (P ＜ 0.05),and after overexpression of MIF gene in SiHa cells,the expression of E-cadherin protein in SiHa-pEGFP-N1-MIF group was significantly lower than that in control groups (P ＜0.05),while the expression of vimentin in SiHa-pEGFP-N1-MIF group was significantly higher than that in control groups (P ＜ 0.05).Conclusions Overexpression of MIF in cervical cancer SiHa cells can promote the EMT occurrence.
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Objective To explore the effect and molecular mechanism of N-myc downstream regulated gene 1 (NDRG1) on transforming growth factor-beta (TGF-β)-induced epithelial-mesenchymal transition (EMT) in human lung cancer cells.Methods Lung cancer A549 cells were transfected with NDRG1 overexpressed vector,and then treated with 5 μg/L TGF-β1.The abilities of invasion were detected by Transwell assay.The expressions of NDRG1 mRNA and protein were analyzed by teal-time reverse transcription polymerase chain reaction (RT-PCR) were examined with Western blot.The expressions of EMT-associated markers E-cadherin and Vimentin,and EMT-associated signaling molecules Snail,AKT and Smad were detected with Western blot.Results We found that TGF-β1 treatment could induce morphological alteration of A549 cells from epithelial morphology to mesenchymal morphology.TGF-β1 significantly increased the migration of A549 cells,and increased the expression of mesenchymal maker vimentin and decreased epithelial marker E-cadherin.More importantly,overexpression of NDRG1 significandy reversed the effects of TGF-β1 on A549 cells.Moreover,NDRG1 significandy decreased the levels of phospho-AKT,and suppressed the expression of EMT-related transcription factor Snail.Conclusions NDRG1 could reverse the effects of TGF-β1 on EMT in A549 cells,by which mechanism is related to reduction of the expressions of phospho-AKT and Snail.
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Objective To investigate the effect of methylguanidine and 1-methylhydantoin on cells cytotoxicity, apoptosis in human renal tubular epithelial cell line (HK-2). Methods Human PTEC cell line HK-2 was used in this study. HK-2 was cultured and divided into 3 groups: Norma1 control group (A), methylguanidine group(B) and 1-methylhydantoin group (C). The cell inhibitory rate of HK-2 was detected by MTT method. The cytotoxicity of methylguanidine to HK-2 was determined by NAG release test. Cell apoptosis was evaluated by using Hoechst stain and FACS with Annexin-V/PI. Results The OD value and NAG concentration of creatinine, methylguanidine and 1-methylhydantoin group were compared with normal control group. OD value decreased and NAG concentration significantly increased(0.188±0.011, 0.176±0.010 vs 0.545±0.021, F＝1557.74, P＜0.01; 20.488±0.473, 22.225±0.565 vs 5.125±0.198, F＝3848.22, P＜0.01). By Hoechst stain, pycnosis and apoptotic body could be found when HK-2 was cultivated in methylguanidine 1-methylhydantoin group. In methylguanidine, 1-methylhydantoin group apoptotic HK-2 apparently increased, compared with that in control group (18.23±1.1581, 20.22±1.1433 vs 2.473±0.321, F＝526.06, P＜0.01). Compared with group B, the OD value in group C decreased significantly (0.176±0.010 vs 0.188±0.011,t＝2.26, P＜0.05), NAG concentration increased significantly (22.225±0.565 vs 20.488±0.473,t＝-6.67, P＜0.01), and apoptotic rate in-creased significantly (20.22±1.1433 vs 18.23±1.1581,t＝-2.762, P＜0.05). Conclusions 1-methylhydantoin has more powerful cytotoxic effect to renal tubular epithelial cells than that of Methylguanidine.
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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.
