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<p><b>OBJECTIVE</b>The main goal is to investigate the role of P120-catenin (P120ctn) in cadherin switching, as well as migration and invasion, of oral squamous cell cancer (OSCC) cells.</p><p><b>METHODS</b>The plasmid pGFP-V-RS-P120ctn shRNA was used to transfect TSCCA cells and significantly reduce the expression of P120ctn in these cells. Real-time fluorescent quantitative polymerase chain reaction and Western blot were conducted to determine the mRNA and protein expression levels of P120ctn, E-cadherin (E-cad), and N-cadherin (N-cad). By contrast, the Transwell cell invasion and cell migration assay was used to determine the invasion and migration capacities before and after the transfection.</p><p><b>RESULTS</b>After the plasmid pGFP-V-RS-P120ctn shRNA was transfected into the TSCCA cells, we found that as the P120ctn expression significantly decreased, E-cad mRNA and protein expression decreased significantly. Moreover, N-cad mRNA and protein expression increased significantly (P<0.05). Lastly, the cell migration and invasion capacities were augmented significantly (P<0.05).</p><p><b>CONCLUSIONS</b>In OSCC cells, P120ctn may be involved in cadherin switching and promote metastasis and invasion.</p>
Subject(s)
Humans , Cadherins , Carcinoma, Squamous Cell , Catenins , Cell Line, Tumor , Cell Movement , Mouth Neoplasms , Neoplasm Invasiveness , Neoplasm Metastasis , TransfectionABSTRACT
Objective:To study the effects of activating protein kinase C (PKC)on oral squamous-cell cancer(OSCC) cell epithelial-mesenchymal transition(EMT).Methods:Plasmid pCMV6-AC-GFP-P120ctn was used to transfect HN12 cells to make P120-catenin (120ctn) overexpress and thereafter PKC activator PMA (phorbol 12-myristate 13-acetate) was added to the culture.Real-time fluorescent quantitative PCR and Western blot were adopted to test mRNA and protein expression of PKC,P120ctn,E-cadherin(E-cad),N-cadherin(N-cad) and Vimentin(Vim).Transwell cell invasion and cell migration assay were used to test the invasion and migration capacity before and after the activation.Results:When PKC was activated by PMA,the expression of P120ctn and E-cad were decreased,the cell morphology changed,nRNA and protein expression of mesenchymal marker protein N-cad and Vim increased significantly.Meanwhile,the migration and invasion capacity of the tumor cells increased significantly(P ＜ 0.05).Conclusion:In OSCC cells,PKC may be involved in promoting EMT and the metastasis and invasion by adjusting P120ctn/E-cad expression and cell adhesion.
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Objective To investigate the role and possible mechanism of P120 catenin involving in the hemodynamic changes by inducing vascular endothelial cells injury through an in vitro experiment. Methods The hemodynamic environment under the different hemodynamic conditions at the vascular bifurcations was simulated through a T-shaped flow chamber system designed by ourselves. The human umbilical vein endothelial cells (HUVEC)cultured in vitro were stimulated and used the HUVEC cells of the small interfering RNA (SiRNA)after P120ctn gene fragments being knocked out. After loading flow rate of 250 and 500 ml/ min respectively and acting on for 12 h,the HUVEC morphology,growth pattern,and expression of P120ctn and other proteins were observed. Results (1)Normal HUVEC:500 ml/ min was loaded for 12 h,the cells were fused excessively at the impinging point,the cell gaps became narrowed,the cell density decreased and the morphology was elongated in the high wall shear stress (WSS)and wall shear stress gradient (WSSG)regions. A part of cells migrated downstreamly,and their arrangement direction was consistent with the direction of impinging flow. Compared with the unloaded impinging flow field,after the 2 kinds of impinging flows being loaded for 12 h,the expression levels of P120ctn,vascular endothelial calcium (VEC),Kaiso,α-catenin,and other proteins were decreased. The expression level of matrix metalloproteinase 2 (MMP-2)was increased. There were significant differences (all P < 0. 05). (2)HUVEC after P120ctn being knocked out:Under the impact of the impinging flow,the cell growth time was reduced to 60 min. 250 ml/ min being loaded for 60 min,the impinging point and its surrounding cells still maintained the polygon,but some cells in the high WSS and high WSSG regions began to move downstreamly and aggregated,the cell arrangement mode partly arranged along with the direction of the flow;500 ml / min being loaded for 60 min,the cell density in the high WSS and high WSSG regions were decreased significantly and the morphology was elongated. A large number of cells migrated downstreamly and aggregated. The arrangement mode was parallel and consistent with the direction of the impinging flow. Compared with the unloaded impinging flow field,after the 2 kinds of velocities being loaded for 60 min,the expression levels of VEC,Kaiso,α-catenin proteins were decreased. The expression level of MMP-2 was increased,There were significant differences (all P < 0. 05) Conclusions The hemodynamic change may induce the changes in vascular endothelial cell morphology,growth pattern,and expression of P120ctn and other related proteins, leading to the decrease of vascular endothelial cell adhesion connection stability and the expression changes of related proinflammatory factors. The knockout of P120ctn may result in a further decrease of the vascular endothelial cell adhesion connection stability.
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Objective To investigate the expression of p 120-catenin in human bladder cell line BIU-87 and T24.And to explore the effect of down-regulation of p120-catenin on proliferation, invasion and adhesion of BIU-87 cell line. Methods The study was from Aug .2010 to May.2011.Real-time RT-PCR and Western blot were used to examine the expression of p 120-catenin in human bladder cell line BIU-87 and T24.There were three study groups:non-transfected group , the group transfected with p 120-catenin siRNA and the group transfected with control siRNA .p120-catenin siRNA was used to decrease the expression of p120-catenin.MTT assay and colony formation assay were used to study the BIU-87 cell growth and cell pro-liferation.The invasion of BIU-87 cells was measured by Transwell chamber assay .Cell adhesion artificial re-constituted basement membrane assay was used to examine the change of BIU-87 cell adhesion capacity . Results In both BIU-87 and T24 cells there were the expressions of p 120-catenin.But the expression in BIU-87 cells (0.11±0.39) was more than that in T24 cells (0.48±0.17).The decreased of p120-catenin ex-pression could enhance the proliferation (182.7±13.4%) and the invasiveness (217.5±15.9) and decrease the adhesion capacity (57.3±6.4%) of BIU-87 cells. Conclusions There is higher expression level of p120-catenin in lower-grade malignant bladder cancer cells .The down-regulation of p120-catenin promotes the bladder cancer proliferation and invasiveness of bladder carcinoma cells , and inhibited the bladder canc-er cell adhesion .
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Objective To investigate the relationship between endothelial damage and p120-catenin (p120-ctn) in a model of paraquat intoxication,and the modulatory effect of mangiferin on p120-ctn.Methods Human umbilical vein endothelial cells (HUVECs) were cultured in two compartment spreading apparatus in vitro.The endothelial cells were divided into three groups:control group (cultured in DMEM with 10％ fetal bovine serum),paraquat group (paraquat was added to the medium with final concentration of 0.05 μmol/L) and mangiferin group (cultured in medium with addition of paraquat for 30 minutes,then mangiferin was added in a final concentration of 20 μmol/L).The cellular permeability at 6,12,24,48,72 hours after culture in the three groups was measured.The expressions of p120-ctn 1A,p120-ctn 3A mRNA and p120-ctn protein were measured by reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot analysis.The distribution of p120-ctn protein was observed by immunofluorescence.Results Compared with control group,cellular permeability in paraquat and mangiferin groups were increased with prolongation of time,and peaked at 72 hours [(29.86 ± 3.98)％,(24.39 ± 2.79)％ vs.(11.71 ± 1.67)％,both P＜0.05].The cellular permeability was significantly lower in mangiferin group than that in paraquat group at different time points (all P＜0.05).At 6 hours after intoxication,the expressions of p120-ctn 1A,p 120-ctn 3A mRNA (gray value) and p 120-ctn protein (gray value) were significantly lower in paraquat group than those in control group (p120-ctn 1A mRNA:0.150 ± 0.024 vs.0.433 ± 0.024,p120-ctn 3A mRNA:0.316 ± 0.043 vs.0.701 ±0.020,p120-ctn protein:0.485 ±0.031 vs.0.763 ±0.038,all P＜0.01).The expressions of p120-ctn 1A,p120-ctn 3A mRNA and p120-ctn protein were significantly higher in mangiferin group than those in paraquat group from 6 hours on (p120-ctn 1A mRNA:0.281 ± 0.021 vs.0.150 ± 0.024,p120-ctn 3A mRNA:0.602 ± 0.042 vs.0.316 ± 0.043,p120-ctn protein:0.675 ± 0.031 vs.0.485 ± 0.031,all P＜0.01),and they were gradually increased with prolongation of time,and peaked at 72 hours (p120-ctn 1A mRNA:1.376 ±0.128 vs.0.150 ± 0.024,p120-ctn 3A mRNA:1.251 ± 0.059 vs.0.316 ± 0.043,p120-ctn protein:0.844 ± 0.050 vs.0.485 ± 0.031,all P＜ 0.01).Under upright fluorescence microscope,p120-ctn was mainly distributed in the cell membrane in control group,with a slight expression in cytoplasm,and no expression in the nuclei.With prolongation of time,p120-ctn expression in the cell membrane was gradually decreased in paraquat group,while it was increased in the cytoplasm and nuclei,with blurring of cell membrane and widening of cellular gap.p120-ctn expression was improved on the cell membrane in mangiferin group at corresponding time points,with decreased in expression in nuclei and cytoplasm.Conclusion The p120-ctn protein plays an important role in the enhancement of endothelial permeability in paraquat intoxication,and mangiferin may attenuate endothelial injury in paraquat intoxication possibly through modulation of p 120-ctn protein.
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Objective: To construct the recombinant lentiviral vector expressing specific shRNA of p120 catenin gene, and to identify the RNA interference efficiency by infecting PANC-1 cells. Methods: Three pairs of shRNA targeting p120ctn mRNA were designed, synthesized and ligated into enzyme-digested and linearized pGCSIL-GFP lentiviral vectors. The recombinant lentiviral vectors were co-transfected with the pGC-FU-p120ctn-3FLAG containing p120ctn gene into 293T cells after identification by PCR and sequencing analysis. Western blotting analysis was applied to select the most effective shRNA. Recombined lentivirus was packaged and the concentration of the virus titer was measured. Real-time PCR and Western blotting analysis were used to identify the interference efficiency after infection of the PANC-1 cells by recombined lentivirus. Results: PCR and DNA sequencing analysis confirmed that p120ctn-shRNA-LV was successfully constructed and the concentration of the virus titer was 3×109TU/ml. Real-time PCR showed that expression of p120ctn mRNA was decreased by 82.6% in PANC-1 cells infected with the recombined lentivirus (P<0.05). Western blotting analysis showed that the expression of p120ctn protein was also greatly deceased after infection. Conclusion: We have successfully constructed lentiviral vector expressing shRNA of p120 catenin gene, and this lays a foundation for studying the role of p120ctn in invasion and metastasis of pancreatic carcinoma.