The Molecular Mechanism Of Brazilin Inhibiting The Growth Of T24 Cells Through C-Fos And GADD45?

Bladder cancer is the common malignant tumor in male,with the characteristics of high incidence and postoperative recurrence.Su Funing lotion developed by ShanXi tumor hospital can effectively decreased the recurrence of bladder cancer with perfusion after operation.Brazilin,the effective ingredient of Su Funing lotion had showed the powerful ability on inhibiting the growth of bladder cancer T24 cells,with simulating the high expression level of c-Fos and GADD45?.Over-expression c-Fos and GADD45? could also inhibit the activity of T24 cells,respectively,indicating these two genes were the potential target genes for Brazilin inhibiting the growth of T24 cells.In this paper,we will further detect the effect of c-Fos and GADD45? on T24 cells through co-expression and co-interference experiments and explore the signal pathways of Brazilin inhibiting T24 cells mediated by c-Fos and GADD45? through the comparative transcriptomics of Brazilin and target gene c-Fos or GADD45?.Firstly,we established the fluorescent reporter vector system based on the GFP fused target protein to screen the positive siRNA.We found the interference efficiency of siRNA-274 with fluorescent reporter vector system p-c-Fos-EGFP-N1 to c-Fos was 64%,the interference efficiency of siRNA-638 with fluorescent reporter vector system p-GADD45?-EGFP-N1 to GADD45? was 67%.The obtained siRNA fragment could be used to interference the expression of the target genes in the following experiments.Secondly,we used the efficient siRNA for c-Fos and GADD45? to test the gene function in the process of Brazilin inhibiting T24 cells.siRNA-c-Fos interference in T24 cells could rescue the cell viability for 37.9% caused by Brazilin,while interference of GADD45? also rescued the death of T24 cells induced by Brazilin for 45.7%.Both c-Fos and GADD45? absence reduced the cell death of bladder cancer caused by Brazilin,while both c-Fos and GADD45? over-expressed lead to the cell death of bladder cancer,indicating that these two genes were the potential target genes of Brazilin inhibiting the growth of T24 cells.Thirdly,we found that co-transfected c-Fos and GADD45? into T24 cells resulted in the cell viability significantly decreased compared with T24 cells transfected c-Fos and GADD45? singly.Meanwhile,co-interference of c-Fos and GADD45? thorough siRNA technology could rescue the cell death more obviously caused by Brazilin compared with the group of transfected with siRNA-c-Fos or siRNA-GADD45? individually.The expression of c-Fos and GADD45? were not influenced with each other in mRNA level both in over-expression and interference group in T24 cells,exhibiting that Brazilin inhibiting the growth of T24 cells through the target gene c-Fos and GADD45?,independently.Finally,we further investigated the signal pathways of Brazilin inhibiting T24 cells through potential target genes c-Fos and GADD45? by comparative transcriptomics.There were 289 and 352 significant changed genes found in c-Fos over-expressed T24 cells and GADD45? over-expressed T24 cells through RNA-Seq technology and analysis.Compared the significant changed genes in target gene over-expressed group and Brazilin treatment.We continued to sort out 9 common genes in Group c-Fos/Brazilin and 8 common genes in Group GADD45?/Brazilin,respectively.However,we could not find common genes between group c-Fos/Brazilin and GADD45?/Brazilin,which showed that Brazilin inhibited the growth of T24 cells through the c-Fos and GADD45? mediated pathway independently.The research of c-Fos and GADD45? could help to confirm the target genes and pathways in the process of Brazilin inhibiting T24 bladder cancer cells,and also provide new molecular targets for developing anti-cancer drugs.