Discovery Of Novel Cell Differentiation-enhancing SiRNA As And Cell Proliferation-promoting Genes By Random SiRNA Library Based Combinatorial Screening

RNA interference (RNAi) is a sequence-specific posttranscriptional gene silencing process mediated by double-stranded RNA. It is an efficient genetic approach for silencing target genes. Small interfering RNA (siRNA) is a class of 21 nt-23 nt double-stranded RNAs, which is the mediator of RNAi. RNA interference technology is the gene blocking technique using principles of RNAi to artificially inhibit specific gene expression. Because of its specificity, efficiency, and easy implementing, RNAi immediately attracted the attention of researchers of gene function, and is successfully applied to more species in gene function studies. Its application fields include different classes of model organisms, from the lower organisms, such as C. elegans, Drosophila to mammals. RNAi technology has been used not only to study the function of individual gene, but also to perform the RNAi-based genome-wide functional analysis for mammalian cells, which promotes the construction of RNAi library.There are three types of RNAi libraries used for mammalian. The first is RNAi library targeting known genes. The second is semi-random RNAi library converted from cDNA library. The third is random RNAi library. We have constructed a random siRNA library, in which a siRNA coding sequence of 19bp was cloned into a siRNA expression vector with convergent pol III promoters.18bp of nucleotides in the siRNA coding sequence were chosen randomly during oligonucleotide synthesis, so that the siRNA library encodes all permutations of siRNA theoretically. The fully randomized library we constructed can be used in all cell types and all organisms and avoids siRNA sequence bias. Another obvious advantage of our library is that it is most suitable for studying the function of low expression gene. Screening with random siRNA library is a potent tool for genome-wide functional studies in post-genomics era.We used the random siRNA library in a screening for siRNA that can affect the cell differentiation of P19 cells. The cells were transfected with the siRNA library. The positive phenotype cells were selected by the method of selective conditions. Then the coding sequences of siRNAs were retrieved from the cells selected. In order to enriching the positive phenotype cells, the same screening was repeated once again. Then we tested the clones separately. We obtained the four siRNAs that can affect the differentiation of P19 cells, C-2-10, C-2-29, C-2-49 and C-2-87. Through further verification and identification, repetition maximum siRNAs, C-2-10 and C-2-29 can affect the differentiation of P19 cells, and differentiated cells were neural progenitor cells. C-2-49 and C-2-87 siRNAs also can affect the differentiation of P19 cells, and most differentiated cells were neural progenitor cells, which can further differentiate into mature neurons and glials cells. Minority of differentiated cells were neurons. We also tested the function of siRNAs, C-2-49 and C-2-87 in the E14/T mouse embryonic stem cell lines. C-2-49 and C-2-87 siRNAs can affect the differentiation of the E14/T ESC, and the differentiated cells were neural progenitor cells. We obtained siRNAs that can affect the differentiation of P19 cells through the screening of random siRNA library.In this study, we used the random siRNA library in a screening for siRNA that can affect the cell differentiation of P19 cells. And we obtained four siRNAs that can affect the differentiation of P19 cells. We hope that we can provide new data to cell differentiation.We also discovered some novel cell proliferation-enhancing genes by random siRNA library based combinatorial screening. Three genes, Txndc5?Tmed2 and Nov were verified to be able to enhance MC3T3-E1 cell proliferation when over-expressed through methods of MTS and cell cycle distribution. G1-S-phase transition of cells stably transfected with three genes plasmids were found to be increased. We preliminarily studied mechanism of Txndc5 and Nov in the fields of cell proliferation. By detection of some cyclins, we found two genes, Txndc5 and Nov able to regulate the transcription of cyclin A to increase expression of cyclin A, in order to enhancing MC3T3-E1 cell proliferation. MC3T3-E1 cell accelerated proliferation in the effect of?-Estradiol, and the expression of Txndc5 was increased. It is shown that the expression of Txndc5 is also increased in the physiological condition.We hope that we can discover some genes'novel functions and provide some new data to cell proliferation.In summary, we used the random siRNA library for phenotype-driven screening using cell differentiation model, and multiple siRNAs that affect the cell differentiation of P19 cells and some genes that significantly enhance cell growth were identified. This study provides new data for mechanism studies of cell differentiation and cell proliferation. Screening with random siRNA library introduces alternative potent strategy for functional studies in post-genomics era.