SuhRNAs Up-regulate The Expression Of Target Gene In Partly GRSF1-dependent Manner

[Objective]The mechanism of microRNAs(miRNA)induced post-transcriptional gene silencing has been proved and acceptable in molecular biology.It is well understood that the regulation network of microenviroment in vivo is complex and has mutability,as the saying ’a slight move in one part may affect the situation as a whole’,the occurence of biomolecules function disorder would cause deseases in the body.The well-studied mechanism of microRNAs mediated negative regulation prompted researchers to think whether there is an opposite way of small RNAs induced gene regulation.RNA activation(RNAa),in fact,is becoming a familiar topic.The mechanism of RNAa has been explored much during the past decade,and different types of research results have been achieved.The mechanisms of RNAa have different forms and differences between species,however,one thing for certain—the biomolecular which causes upregulation of gene expression do exist.Previous study of our lab has discovered a new mechanism of RNAa,which form similar to that of miRNAs mediated gene silencing.We found the incompeletly binding,which showing an exposed motif,miR-346 middle sequence motif "CCGCAU",between the miRNA(miR-346)and its target could result in up-regulated expression of gene.Next,we further found that GRSF1 was the key protein factor that mediated the gene upregulation via directly interacting with middle sequence motif "CCGCAU"of miR-346.This study will take the advantage of the previous work above to further explore the universality and specificity of the mechanism of GRSF1 induced mRNA translation in miR-346 "CCGCAU" motif-depentent manner.[Methods and results](1)We designed and constructed a 21nt siRNA and its corresponding SuhRNA which has the motif "CCCGCAU" in its middle seqeunce to target the EGFP gene.EGFP reporter assay,qRT-PCR and western blot assays were performed with HeLa cells co-expressed the siRNA(shR-EGFP)or SuhRNA(shR-U-EGFP)or their control(pSilencerNC)and pcDNA3/EGFP,not surprisingly,the results confirmed that shR-EGFP down-regulated the expression of EGFP whereas shR-U-EGFP up-regulated the expression of EGFP.Next,we designed and constructed the other two groups small RNAs which target the different region of EGFP mRNA to test the extent of influence of these three groups small RNAs by using EGFP reporter assay,qRT-PCR and western blot assays.Treated with all the three groups small RNAs respectively,the expression level of EGFP showed no significant differences between the groups.(2)EGFP reporter assay,qRT-PCR and western blot assays were performed to verify SuhRNAs mediated gene up-regulation in GRSF1 dependent manner.The results indicated that knockdown of GRSF1 led to failure of shR-U-EGFP induced up-regulate the expression of EGFP in HeLa cells.(3)We designed and constructed four mutants of the original motif "CCCGCAU"of SuhRNA by changing its bases as mutant 1-4(" CCCGCCG"/" CCCAUGC"/"AUCCCGC"/" CATATAT")and one mutant with different binding form as mutant 5.EGFP reporter assay,qRT-PCR and western blot assays were performed to detect whether the fuction of these five mutants would similar to the original SuhRNA.The result showed that all of the mutants had more or less positive influence on up-regulating the EGFP expression.Following experiments focus on whether the positive affection of these five mutants also depend on GRSF1,so we knockdown of GRSF1 to test the influence of these five mutants on EGFP by using EGFP reporter assay,qRT-PCR and western blot assays.The results suggested that all the mutants could be affected by GRSF1 except the mutant 4.[Conclusion]SuhRNA with the motif "CCCGCAU" mediated gene up-regulation in GRSF1 dependent manner is universality,but not specificity.