Devlopment Of Arrayed RNAi Screening Approach And Its Application In The Study Of PrP Function

Prions are unprecedented infectious pathogens that cause a group of fatalneurodegenerative diseases in human and animals by an entirely novel mechanism. It hasbeen suggested that these disorders were caused by the conformation conversion of PrP^C toPrP^Sc. The highly conserved glycophosphatidylinosital (GPI) anchored- sialoglycoproteinPrP^C, was encoded by the Prnp gene and mainly expressed in the brain. The loss offunction of PrP might be associated with apoptosis in prion diseases, while the normalfunction of PrP is still poorly understood.To gain insights into the function of PrP, some groups of transgenic and knock-out micehas been developed, but the results were not satisfactory. Numerous partners of PrP havealso been identified by the method of yeast two hybridization, but the cellular data of theseinteraction seem being not enough. For a better understanding of this problem, it isobviously significant to explore and to develop more effective techniques to look into thefunction of PrP.Over the past decade, RNA interference (RNAi) has emerged as a new method fornocking down or silencing the gene expression. Recently, the high-throughput RNAi screening has been appearing to be an attractive and powerful tool in research oflarge-scale functional genomics.This paper reports our novel approach in the development of an economic andeffective arrayed-RNAi screening technique and its application in the study of the role ofPrP in STS-induced apoptosis.1. Establishment of the arrayed-RNAi screening methodFirst of all, we established a RNAi collection, which consists of 59 RNAi plasmidtargeting human apoptosis related genes as well as 6 PrP-RNAi candidates. At the sametime, we selected a highly effective, non-viral, non-liposomal cationic polymer,polyethylenimine (PEI), aiming to establish a reverse transfecting method. The results ofour experiments showed that the transfecting efficiency of PEI was rather high, 55ï¼…,being similar to that of liposome (x²=0.439, P＞0.05) cells, and the PEI reversetransfecting efficiency was even much higher than that of routine (x²=8.598, P＜0.05)ones in HEK293 cells. What was being remarkable was that during the reverse transfection,many tedious procedures such as, rinsing the cell with and antibiotic free medium beforetransfecting, changing the medium after transfecting etc., all could well be avoided; thewhole process could be completed just in one day rather than two days in routine method.After reverse transfected, cells containing PrP-RNAi were selected to be the PrP-RNAicandidate plasmid, PR6, that could alter STS-induced apoptosis and was, therefore,identified to be an effective PrP-RNAi plasmid by RT-PCR and Western blot respectively.This was again much simple than routine RNAi screening method.2. Screening the regulators of STS-induced apoptosisIn order to find the targets of PrP in the pathway, we screened the regulators ofSTS-induced apoptosis pathway. 22 genes including PrP, P53, Cyc, Bax, CDK6, CDK 7,E2F4 have been identified to be the involved in regulation of STS-induced apoptosis.Among these regulators, PrP enhanced STS-induced apoptosis in HEK293 cells and itsrelated effects could be abolished by the Bax-RNAi.Our data suggested that the present method is effective for studying apoptosis related gene function and seems to be potential to perform high-throughput RNAi screenings.These results also provided clues for further study of PrP regulation in STS-inducedapoptosis.3. Validating the results of RNAi screening: PrP decreased/enhanced STS-inducedapoptosis in Neuron2A and HEK293 cellsFor assessing the stringency, we transfected the Neuron2A and HEK293 cells withPrP-RNAi and PrP expression plasmid respectively to verify the results of arrayed RNAiscreening. In this regard, we tested the susceptibility of different cells to STS-inducedapoptosis. Result showed that the PrP-RNAi enhanced STS-induced apoptosis and PrPoverexpression reduced it in Neuron2A cells. On the contrary, PrP-RNAi decreasedSTS-induced apoptosis and PrP overexpression increased it in HEK293 cells. The effectsof PrP-RNAi and overexpression on cell susceptibility with STS treatment appeared to bealtering the cytotoxicity, DNA fragmentation, Bax and Cleaved-Caspase3 levels. Theseresults further proved that PrP regulates STS-induced apoptosis in different cells throughBax inspide of a contraty effect.In conclusion, we have developed an effective and economic arrayed-RNAi screeningmethods and successfully employed it in PrP function research. Use this approach, we findthat PrP played a key role in STS-induced apoptosis through Bax. Our data alsodemonstrated that the present method has the potential to facilitate genome-scale RNAisceening, in general, and in prion research, in particular.