Moreover, BRCA2 expression in mice subcutaneous xenograft showed consistency with the expression with p53BER2 (Fig. reverse nutlin-3-induced G1-arrest and senescence in TP53-WT cell lines. What is more, the knockdown of p53BER2 showed resistance to nutlin-3 treatment in vivo. Additionally, we found BRCA2 could be regulated by p53BER2 in vitro and vivo; further experiment showed p53BER2 could induce cell-cycle arrest and DNA repair by mediating BRCA2. In summary, the Rabbit Polyclonal to CHST10 p53-associated enhancer RNA-p53BER2 mediates the cell cycle and senescence of p53 in TP53-WT renal cancer cells. (%)0.7640.382?55452025? 55392217Gender, (%)0.2230.637?Male592930?Female251312T stage, (%)4.4020.111?T1a421824?T1b?+?T2291415?T313103Fuhrman grade, (%)9.9730.007?G130822?G2432617?G3?+?G41183 Open in a separate window renal cell carcinoma, cycle threshold, Fuhrman nuclear grade. P53BER2 could be specifically expressed in TP53-WT renal cancer cell lines Since p53BER2 is an enhancer RNA mediated by wild-type p53, we wonder whether p53BER2 is mediated in wild-type p53 in renal cancer cells. First, as shown in Fig. ?Fig.1A,1A, we could find that p53BER2 expression in TP53 mutant cells (786-O) is the lowest, consistent with our conjecture. To further understand the relationship between p53BER2 and wild-type p53 protein, we used the p53 protein activator nutlin3a to treat p53 wild-type and TP53 mutant kidney cell lines. Western Blot showed that nutlin3 was effective in inducing p53 expression and its target- p21 in Caki-1, but not in the 786-O cell line (Fig. ?(Fig.2A).2A). qPCR results indicated that nutlin3a could induce p53BER2 in p53 wild-type cells (OSRC-2, ACHN, CAKI-1), but did not induce TP53 mutant cell expression (Fig. ?(Fig.2B).2B). Also, we included p21 and PUMA as the positive control, and the results showed the expression of p21 and PUMA were upregulated with nutlin-3 treatment (Fig. ?(Fig.2A2A and Supplementary Fig. 1C, D). But the PAPPA expression, the previous target of p53BER222, did not change with nutlin-3 treatment, which might be due to different biological mechanisms in different tissue and cells (Supplementary Fig. 1E). To further explore the relationship between p53 and p53BER2, we got the pLX313-TP53-WT and pLX313-TP53-P278A, then we used the corresponding lentivirus and empty vector (EV) lentivirus to infect H1299, which were found as a p53-null cell26. Then we used WB and qPCR to test the expression of p53 and p53BER2; the results showed that overexpression of p53 induced an obviously increased expression of AZD9898 p53BER2 in WT-TP53 H1299 cells, but not in the AZD9898 TP53-P278A H1299 (Fig. 2C, D). Then we used si-P53 to transfect H1299-wt-TP53; qPCR results revealed that downregulation of p53 could decrease the expression level of p53BER2 (Supplementary Fig. 2A, B). p53BER2 reporter could detect the expression of p53BER2 in TP53-wt RCC cell lines It has been reported in the literature that wild-type p53 can bind to p53BER2 to enhance promoter expression. Using this principle, we designed the p53BER2 reporter to further investigate whether P53 initiates the promoter by direct binding to p53BER2. First, we combined the p53BER2-specific sequence with the minimal promoter to form a p53 promoter that specifically recognizes the wild-type p53 protein. Further, we used the GAL4-UAS system to enhance promoter efficiency and use dual to report the AZD9898 gene, and finally form the p53BER2 reporter (Fig. ?(Fig.2E).2E). Since 293T and HK2 have a basic expression of wt-p5327,28 and p53, 786-O express a relatively low level of mut-p53 protein. Here we transfected the p53 reporter and control reporters into the HK2, 293T, and 786-O cell lines, respectively,.