Specific Growth Suppression Of Wild-Type P53 Tumor Cells By Dna-Modified Sirna Sequences Targeting Mdm2

Background: Murine double minute 2 (MDM2) is an oncoprotein that inactivates p53 tumor suppressor protein by ubiquitination and subsequent proteasomal degradation. MDM2 is overexpressed in 10%-20% of human cancers, most of which carry wild-type (wt) p53. Synthetic siRNAs silence gene expression by RNA interference. However, siRNAs also suppress the expression of unintended targets containing complementary sequences to as short as 6 bases of the seed region of siRNA (off-target effects). Such nonspecific effects can be avoided by nucleotide modifications, including DNA replacement in the seed region. Aim: We conducted this study to select canonical and DNA-modified siRNAs possessing high silencing activity for MDM2 with less nonspecific effects. In addition, we examined the validity of the MDM2-p53 interaction as a target for RNAi cancer therapy. Methods: Twenty-four siRNA sequences targeting MDM2 were newly selected using the siDifect software. In total, 33 siRNAs, including 9 previously reported sequences, were chemically synthesized. Effective siRNAs were converted to double-stranded RNA-DNA chimera (dsRDC), which had a DNA replacement in the 5′ region of the guide strand (nucleotide 1-6 from the 5′ end) and its complementary sequence of the passenger strand, including the adjacent 2-b overhang. Three cell lines, SJSA-1 osteosarcoma (high MDM2 expression, wt p53), NUGC3 gastric cancer (mutant p53), and Kato-III gastric cancer (mutant p53), were used. MDM2 protein and mRNA levels were examined using Western blotting and real-time RT-PCR, respectively. Cell viability was measured using WST-8 assay. siRNA and dsRDC were transfected using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer9s instructions. Results: Among the 33 siRNAs examined, 7 siRNAs (2 previously reported and 5 newly designed) at 1 nM suppressed MDM2 mRNA expression and cell viability of SJSA-1 cells by 72%-86% and 51%-75%, respectively. These effective siRNAs were converted to dsRDC-modified form. Two of them demonstrated MDM2 knockdown and growth suppression of wt p53 tumor cells as efficiently as the unmodified forms. Further, in comparison with the unmodified forms, the modified forms showed less nonspecific growth suppression of cells carrying mutant p53. These 2 dsRDC-modified siRNAs were both newly selected sequences. Conclusions: Our 2 newly selected dsRDC-modified siRNAs had high knockdown activity and less nonspecific cytotoxicity. Thus, they represent potent therapeutic agents against cancers carrying wt p53 with MDM2 overexpression. Citation Format: Mitsuaki Hirose, Kenji Yamato, Rie Saito, Takunori Ueno, Sachiko Hirai, Hideo Suzuki, Shinji Endo, Ichinosuke Hyodo. Specific growth suppression of wild-type p53 tumor cells by DNA-modified siRNA sequences targeting MDM2. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2603. doi:10.1158/1538-7445.AM2014-2603