Silencing Human Rb2/p130 with shRNA

We designed and validated by real-time PCR, western blot and immunofluorescence, two new shRNAs directed against RB2/p130 and we set up the protocol to efficiently target this gene in a human lymphoblastoid cell line, by both transient and stable transfection. The clones, that stably express short hairpins directed against Rb2/p130, will be useful to further investigate the role of this Rb family member in lymphoid cells both in physiological and pathological conditions. pRb2/p130 is a member of the Rb tumor suppressor family; this family is also composed of pRb/p105 and pRBL1/p107 [6,8,9]. Due to the dominant role of pRb/p105 in the family and to some overlapping functions, the roles of pRb2/p130 and RBL1/p107 were underestimated for a long time. It should be noted, however, that although they show partially overlapping functions, the three members are differentially regulated during cell cycle and interact with different E2F factors, which are the best-characterized retinoblastoma proteins targets. Through E2Fs, RBs regulate transcription and cell cycle transition [4]. In particular pRb2/p130 is abundant during the G0 phase in non-proliferating cells, and its expression is altered in a large number of human cancers [1–3,7]. pRb2/p130 interacts with the repressing E2F4-5 and this interaction is responsible for the repression of cell cycle regulatory genes, such as cdc25, cdc2, E2F1-3 and RBL1/p107 [4,6,8,9]. When cells enter the cell cycle, pRb2/p130 becomes phosphorylated and this event leads to dissociation of pRb2/p130 from its E2F partner and to loss of transcriptional repression. We chose RNAi to knockdown RB2/p130, with the purpose of fully elucidating its cellular functions in lymphoid cells. Unfortunately, no validated shRNA existed to target human Rb2/p130 to date, so we designed 10 oligos using web available software and cloned oligos in pSilencer4.1CMV vector. We show here by realtime PCR, immunofluorescence and western blot that oligo 4 and oligo 9 are effective in silencing, thus supporting their use for research purpose. To design shRNA we used GenScript software (sequences are shown in Fig. 1a); these sequences were then adapted to pSilencer4.1CMV-Puro vector, using the siRNA converter by Ambion. After cloning shDNA 4 and 9 into pSilencer4.1CMV-Puro vector, we transiently transfected cells to screen our sequences for efficient silencing. Cells were collected 24 and 31 hours after transfection, to further proceed to RNA and protein extraction. In Fig. 1b, we show by Real-time PCR that sequences 4 and 9 showed a strong reduction in Rb2/130 mRNA. To confirm that the downregulation at the mRNA level correlated with a corresponding protein decrease, we performed western blot analysis. In Fig. 1c, we show that pRb2/p130 levels in scrambled transfected cells were unchanged with respect to the control, whereas sequence 4 and sequence 9 are effective in silencing, showing a reduction respectively of 55% and 78% in the optimal condition. We then proceeded to create stable clones expressing shRNA against Rb2/p130. Cells nucleofected with pSilencer4.1CMV-shRb2 4, pSilencer4.1CMV-shRb2 9 or pSilencer4.1CMV-scrambled were selected using puromycin. RB2/p130 expression was then checked at the RNA and the protein levels by Real-time PCR (Fig. 1d), Western blot (Fig. 1e) and Immunofluorescence (Fig. 1f). Our results confirm transient transfection data. Sequence 4 is more efficient than sequence 9, but both are able to silence RB2/p130. Moreover, as we can detect by immunofluorescence, in sequence 9 most of pRb2/p130 signal is localized within the cytoplasm, where the protein is supposed to be inactive. Our results show clearly that sh4 and sh9 are effective in silencing RB2/p130, both in transient and stable transfection assays. Moreover, despite of the rapid transgene expression obtained by nucleofection, our analysis demonstrates that silencing is still efficient 31 hours after transfection. We also found that the two shRNAs showed different silencing dynamics during this time; in fact sh9 is more efficient 31 hours after transfection while sh4 shows higher efficiency at 24 hours.