The Study Of MicroRNA Function In Central Nervous System And Absorption Mechanism Of Exogenous MiRNA In Mammals

MicroRNA(miRNA)are a class of small non-coding single-stranded RNA molecules with the length about 22 nucleotides,and can regulate its target gene expression at the post-transcriptional level by completely or partially complementary binding to mRNA 3' end untranslated region sequence,resulting in target mRNA degradation or translation inhibition.MiRNAs are highly conserved by evolution.MiRNAs do not have open reading frames and do not code for proteins,but nevertheless are functionally important in many kinds of important physiological and pathological process.Recent researches have found that miRNA expression is highly conserved?time-ordered and tissue specific.MiRNAs are abundant in the nervous system of vertebrates and play an important role in brain development and regulating plastically.Besides,miRNAs are closely related to the occurrence of diseases in the nervous system.Recent researchs have found that exogenous plant miRNAs could be primarily acquired orally,through food intake by mice.Those miRNAs could be transported into peripheral blood and tissues against digestion through gastrointestinal tract and play biological functions in the target organs.However,the underlying mechanism still remains unknown.In this thesis,we focus on the miRNA functions in regulating neuron apoptosis in the nervous system and the absorption mechanism of exogenous miRNAs by mammals.In the first section,we investigated the role of miRNA in regulating neuron apoptosis through targeting Apaf-1 in physiological and pathological process.We initially examined the expression of Apaf-1 protein and mRNA in the mouse cortex during brain development,and found that the Apaf-1 protein levels decreased as cortical development progressed.Similarly,Apaf-1 mRNA also decreased during brain development.Compared with the downregulation of the mRNA level,the decrease of Apaf-1 protein level was more dramatic,and this indicates that Apaf-1 gene expression might be regulated at the posttranscriptional level.Using computer-aided algorithms,we found that four(miR-23a,miR-23b,miR-27a,and miR-27b)of the miR-23-27-24 clusters had conserved putative binding sites for Apaf-1 mRNA on 3'-UTR.We also found that expression of the miR-23-27-24 clusters clearly increased from quantitative reverse transcription-PCR(qRT-PCR)experiments,in a manner that was inversely correlated with the pattern of Apaf-1 expression.A RNA dot blotting assay and in situ hybridization staining were employed to further corroborate the qRT-PCR results.We peformed luciferase assay and Western blot to comfirm that these miRNAs actually suppress Apaf-1 gene expression.We also found that hypoxia caused increased neuronal apoptosis,decreased miR-23b-27b expression and increased Apaf-1 protein expression.Overexpression of the miR-23b and miR-27b in primary cortical neurons could attenuate neuronal apoptosis through suppression of Apaf-1.We constructed neuronal-specific miR-23b-27b cluster transgenic mice to further confirm the roles of miR-23b and miR-27b in neuronal apoptosis,and found that miR-23b-27b were increased in cortical neurons of embryonic mice resulting alleviating neuronal apoptosis by inhibiting Apaf-1 in hypoxia-induced pathological conditions.In the second section,we investigated the role of c-Myc in suppressing miR-23b and miR-27b transcription during fetal distress and regulating the sensitivity of neurons to hypoxia-induced apoptosis.Our previous studies and further study showd that the expression of mature miR-23b/27b and primary transcripts were both downregulated in embryonic brain cortex during hypoxia-induced neuronal apoptosis.We found the c-Myc protein level was significantly elevated in embryonic brain cortices under hypoxia in a mouse model of fetal distress.Forced overexpression or knockdown of c-Myc could suppress or increase the expression of miR-23b-27b cluster polynucleotides.We identified 2 conserved c-Myc binding sites(E-boxes)in the enhancer and promoter regions of miR-23b-27b cluster in the mouse genome by Chip assay and luciferase assay.Finally,we founded that elevated c-Myc expression led to an increase in the Apaf-1 level by suppressing miR-23b-27b cluster expression and that this enhanced neuronal sensitivity to apoptosis.In the third section,we investigated the role of SIDT1 in transporting exogenous miRNAs into mammals.We found that cell lines derived from human expressed SIDT1 with membrane localization.We also found that almost all kinds of tissues expressed SIDT1 and the stomach tissue had the highest expression level.Trough cell uptake assay,we found that cells could uptake exogenous double-strand miRNAs and those single-stranded miRNAs which could form dimers with partially double-stranded regions.We futher found that knockdown of SIDT1 expression could reduce the the ability of cells to absorb exogenous miRNAs.We constructed SIDT1 knockout mice,and found that the absorption efficiency of exogenous miRNAs in SIDT1 KO mice was significantly lower than that in wild-type mice.Together,we found that SIDT1 played an important role in transporting exogenous miRNAs into mammals.In summary,we studied the miRNA functions in regulating neuron apoptosis in the nervous system and the absorption mechanism of exogenous miRNAs by mammal animals.On one hand,we found that miRNA(miR-23-27)could regulate neuron apoptosis through targeting Apaf-1 in physiological and pathological process and c-Myc could suppress miR-23b and miR-27b transcription during fetal distress and regulating the sensitivity of neurons to hypoxia-induced apoptosis.These conclusions will be important in understanding the role of miRNAs in the nervous system.On the other hand,we found that SIDT1 participated in transporting exogenous miRNAs into mammal animals through gastrointestinal tract.This study first explained the underlying mechanism of exogenous miRNAs transporting into mammals and targeting animal genes.