Effects Of AGO3 Proteins On Cell Cycle Of HEK293 Cell Line And The Localization Of AGO3 Protein Complex

The Argonaute protein family was first identified in plant, as a growth regulator, and extensive reaserch has found that they are the core in gene-sliencing pathways. It is close related with the biogenesis and cell function of small non-coding RNAs (siRNA, miRNA, piRNA) , it can combine the small RNAs ,control protein synthesis or affect the stability of mRNA that RNA intrference technology(RNAi). Argonaute (AGO) is a highly conserved and it include many protein members, at the same time, the different Ago proteins have different biological functions.Because knock down of Ago3 can not cleavage degrade target mRNA and can not inhibit the human protein expression, furthermore, it can not obtain a stable line expression Ago3 in human cell by transfection Ago3 plasmids.Therefore, the function and features of Ago3 are not yet clear in the human cells. To use the RNA intrference technology and the methods of transfection exogenous Ago3 plasmids for control the upward and downward of Ago3 in HEK293 cells, and then study the effects of Argonaute3 Proteins on Cell Cycle of HEK293 Cell line and preliminary exploration of Ago3 protein. This article first introduces the RNAi interference technology and then detailed explanation the Argonaute protein family and its function.HEK293,MCF,Hela cells were transfected with si-ago3 and c-flag-ago3 using Calcium phosphate and Lipofectamine 2000 ,culture 24h, fluorescence microscope observe the effect of transfection,select the best transfection conditions;RT-PCR detect the mRNA transcription level of ago3 gene; Western blotting detect the expression level of flag-ago3 plasmid in 293 cell; MTT method draw the growth curve of si-ago3 plasmid are transfected into HEK293 cell24h, 48h, 72h; After Tranfect plasmid 48h, determine the effects of Argonaute 3 Proteins on Cell Cycle of HEK293 Cell line by flow cytometry; Finally Immunofluorescence target Ago3 protein in the HEK293 cell.Exogenous Ago3 gene can stable express in HEK cells using Lipofectamine 2000. In HEK293,low-expression of Ago3 protein can inhibit cell normal growth and block G0/G1,on the contrary, over-expressin of Ago3 protein can promote G0/G1 phase.This results shows a centain amount of Ago3 protein is necessary for the growth of normal HEK293 cell,low-expression or over-expressin all can affect Cell Cycle of HEK293 Cell line.Then detect the transcription level of P161INK4a and cyclinD1 gene that regulate G1/S change in cell of Ago3 protein of low-expression and over-pression. The result showed that the different expression levels of Ago3 protein directly impact on the expressin level of cyclinD1 in cell.but,the expression level of p16 gene no significant changes were found in results.Therefore ,the regulatory mechanism is still not clear. Finally, Ago3 protein was targeted in the cytoplasm, indicating the protein complex of small RNA bind to Ago3 mediated post-transcription gene silencing in the hunman cells. This airticle laid a function for further studying the effect of Ago3 protein on cell cycle and for further exploring the specific function of Ago3 protein complex in the RNA interference technology provide more effective conditions.