Preliminary Study Of The Regulatory Mechanism Of Rna Interference. Cho Cells

With the discovery of the phenomenon of RNA interference (RNAi) and the wide application of RNAi technique, The regulatory small RNAs have become a focus of current research. Researchers have already identified a variety of classes of regulatory small RNAs in plants and animals, and are on the way to a full unco very of their physiological function and action mechanism. These 19-31 nt non-coding RNAs work in a sequence-specific manner to facilitate mRNA degradation, translational inhibition, heterochromatin formation and transposon control, thus leading to gene silencing. Regulatory small RNAs play important roles in the regulation of cell growth, development, differentiation and other important physiological processes. Nonetheless, there is still much to be elucidated in the pathway of small RNA regulation, as well as the regulation of regulatory small RNAs themselves. Hong et al. had reported an enhancement in mouse eri-1(meri-1) gene transcription and a consequential decrease in RNAi efficiency in the mammalian cells after treatment with E.coli expressed and enzyme digested siRNA (esiRNA). To better understand the physiological responses of the mammalian cells to high-dose esiRNA, we brought about a preliminary study on the transcriptional regulatory mechanism of meri-1 gene. We analyzed the secretion of interferon-Ⅰby the mammalian cells after liposomal transfection with high-dose esiRNA, and the effect of interferon-γon meri-1 gene promoter. We also preliminarily studied the relationship between RNAi and RIG-Ⅰ, a cytoplasmic receptor of RNA.In the first part of our work, by adopting green fluorescent protein (GFP) and Secretory Alkaline Phosphatase (SEAP) respectively as reporter genes, we analyzed the ability of different deletion mutants of meri-1 gene promoter to respond to high-dose esiRNA, thus identified the cis-acting elements on the meri-1 gene promoter which respond to high-dose esiRNA. The result suggested that there are two cis-elements on the proximal region of the meri-1 gene promoter that are important for the high-dose-esiRNA-induced enhancement of meri-1 gene transcription. These two cis-elements are situated from 47 to 77 nucleotides (5'-CCCGCCCA-3') and 30 to 47 nucleotides (5'-CGCTCCCGGAAGTAGGAG-3') upstream of the transcription initiation site.In the second part of our work, through cyto logical and biochemical approaches, we identified the trans-acting elements that can elevate meri-1 gene transcription level in response to high-dose esiRNA. The results of EMSA and Oligonucleotide Precipitation Assay showed that the proximal region of the meri-1 gene promoter can interact with Sp1 and Ets-1. The result of Oligonucleotide Precipitation Assay further established that only after exogenous siRNA induction can Sp1 associate with Ets-1, forming a complex that can bind to the proximal region of the oligonucleotides representing the meri-1 gene promoter. The expression of the reporter genes significantly decreased after RNAi knockdown of Sp1 and Ets-1 genes expression. The expression of the reporter genes also decreased after transfecting the cells with the dominant negative mutants of Spl and Ets-1 genes. All of these results are consistent with the assumption that high-dose esiRNA can activate Spl and Ets-1 to elevate meri-1 gene transcription level.In the third part of our work, we first studied the difference between the abilities to induct IFN-γsecretion by high-dose and low-dose esiRNA, and the ability of the proximal region of meri-1 gene promoter to respond to IFN-y. Then we analyzed the change in the ability of the proximal region of the meri-1 gene promoter to respond to high-dose esiRNA when RIG-I is overexpressed and downward-regulated, respectively. The results showed that high-dose esiRNA has got a higher ability to induct IFN-y secretion than low-dose esiRNA, and that the proximal region of the meri-1 promoter can respond to IFN-y. These results suggested a correlation between the enhancement of meri-1 gene transcription and the activation of immune system, both by high-dose esiRNA. Nonetheless, the RIG-I does not take part in this process. Oppositely, overexpression of RIG-I enhanced the ability of esiRNA to silence the expression of HBsAg, and knock-down of RIG-I undermined the ability of esiRNA to down-regulate the expression of HBsAg. Thus, we inferred that RIG-I may be related with certain steps in RNAi pathway.In conclusion, our work has provided necessary experimental data for the study of the regulation of RNAi, as well as important basic information for further study in RNAi application.