Silencing of Mouse RBBP6 Using Interference RNA Implicates It in Apoptosis and the Cell Cycle

Abstract

Retinoblastoma-binding protein 6 (RBBP6) is one of
the few proteins that interacts with both tumour suppressor
proteins p53 and Rb and by implication plays an important role
in cell regulation. Previous studies carried out to monitor the role
of RBBP6 targeted mouse spliced variants (P2P-R and PACT) of
the gene. These studies showed that over-expression of the P2P-R
protein promote camptothecin-induced apoptosis, whilst
knocking out the PACT domain using homologous recombination
promoted apoptosis. In this study, the role of RBBP6 in
camptothecin induced apoptosis was investigated by knocking
down the whole gene by targeting the 5’ DWNN domain of
RBBP6 using RNA interference (RNAi). Two distinct small
interference RNA (siRNA) oligonucleotides designated DWNN-A
and DWNN-B targeting different regions of the gene were
designed and two stable siRNA-expressing cell lines were
established, namely RU6A and GU6B expressing DWNN-A and
DWNN-B respectively. Qualitative fluorescent microscopy and
quantitative Real-Time polymerase chain reaction (qRT-PCR)
analysis were used to evaluate the silencing effect on RBBP6
expression in the NIH 3T3 parent cell line as compared to the
stable cell lines RU6A and GU6B. The DWNN-A oligonucleotide
effect appeared to be more potent than that exerted by DWNN-B.
The stable cell lines proved to be significantly more resistant to
apoptosis induced by camptothecin compared to the parental
NIH 3T3 cell line using several apoptosis assays. Camptothecin
induced apoptosis was restored to that observed in the NIH 3T3
cell line after the re-introduction of the full-length RBBP6 cDNA
(DWNN-200). Furthermore, the results suggest that Apoptosis
mediated through RBBP6 is dependent on p53 expression and
possibly follows an intrinsic pathway as shown by the Bax/Bcl-2
ratio. Bcl-2 was higher than Bax in RU6A as compared to NIH
3T3 where Bax was higher compared to Bcl-2. Growth of the
siRNA expressing cell lines RU6A and GU6B were also shown to
be restricted in the G1 phase of the cell cycle implicating the
RBBP6 gene in cell cycle progression. In conclusion the role of
RBBP6 was established in camptothecin induced apoptosis and
the cell cycle although the exact mechanisms have not been fully
elucidated.