The Study Of Effect And Mechanism Of ΔNP63 On Invasion In TCCB 5637 Cells

Objective: To explore the effects ofΔNp63 gene on invasion and migration in TCCB cells; to study the expression ofΔNp63 and claudin-1 genes in 5637 cells, to investigates the invasion and migration molecular mechanism ofΔNp63 and claudin-1 genes in 5637 cell.Methods: Amplified, extracted and identified theΔNp63-shRNA plasmids, Transfected 5637 bladder cancer cells; screened the 5637 cells by G418 stable express proteins ofΔNp63; The protein expression level ofΔNp63 was analyzed by Western-blot assays; The TCCB 5637 cells were divided into control group (without transfection), negative plasmid group (with transfection of shRNA negative) and the interference plasmid (with transfection of effective shRNA), analyzed the impact of 5637cells whenΔNp63 down-expression with invasion and migration assay, Homogeneous and Heterogeneous adhesion experiment; to explore theΔNp63 protein and claudin-1 protein by Laser scanning confocal microscope and western blot assay in 5637 cells treated byΔNp63-shRNA,ΔNp63-shRNA negative and vector; explore the mRNA expression level ofΔNp63 and claudin-1 with real-time PCR in 5637 cells that been transferred byΔNp63-shRNA, ΔNp63-shRNA negative and vector; to investigates the relevance and the potential molecular mechanism ofΔNp63 and claudin-1 genes in 5637 cells.Result: Plasmid sequence analysis proved thatΔNp63-shRNA plasmid sequences meet the requirements, compared with vector group and negative plasmid group,ΔNp63 protein expression decreased significantly in sh-RNA plasmid group(p<0.05);the cells of homogeneity adhesion was 2012.75±9.54 in sh-RNA plasmid group, but the cells of homogeneity adhesion was 1025.50±17.48 and 1020.25±20.25in vector group and negative plasmid group, compared with vector group and negative plasmid group that increased(p<0.05);the A value of heterogeneity adhesion was 0.295±0.008 in sh-RNA plasmid group, the A value of heterogeneity adhesion was 0.460±0.177and0.442±0.071 in vector group and negative plasmid group, that decreased compared with vector group and negative plasmid group(p<0.05);the number of invasion was 9.5±2.0 in sh-RNA plasmid group, but in vector group and negative plasmid group the number of invasion was 18.2±1.3and16.0±2.6,that decreased compared with vector group and negative plasmid group(p<0.05); the migration was not changed in 12h(p>0.05), the number of migration was 18.60±0.61,20.00±0.27 and 19.50±0.51 in different groups; but after 24h, the number of migration was 29.17±1.72 in sh-RNA plasmid group, but in vector group and negative plasmid group the number of migration was53.83±1.60 and 51.50±1.87 , that decreased compared with vector group and negative plasmid group(p<0.05); confocal laser scanning microscope showedΔNp63 and claudin-1 both appeared in 5637 cells,ΔNp63 mainly in the nucleus region and claudin-1 expression in the cell membrane;InΔNp63-shRNA groups, the fluorescence ofΔNp63 and claudin-1 down-expression; The expression ofΔNp63 and claudin-1 about protein and mRNA was obviously decreased in the interference plasmid group, Statistics analysis shows the interference plasmid group has significant difference compared with other groups, the change of claudin-1 was positively correlated withΔNp63.Conclusion: Obtain theΔNp63-shRNA plasmid, and theΔNp63-shRNA plasmid effectively transfer the 5637 cells; the down-expression ofΔNp63 gene could suppressed the invasion and migration in 5637 cells; and theΔNp63-shRNA plasmid effectively reduced the mRNA and protein expression ofΔNp63 in 5637 cells;the mRNA and protein change of claudin-1 in different groups was positive correlation withΔNp63 gene,ΔNp63 gene may regulated the invasion and metastasis of TCCB through changed the expression of claudin-1 .