MicroRNAs Regulate The Expression Of The Parkinson Disease Causitive Gene LRRK2

ABSTRACT OF SECTION1The Isolation of miRNAs to regulate the expression of the Parkinson Disease Causitive Gene LRRK2Parkinson’s disease （PD） is the second most globally prevalent neurodegenerative disorder. The cause of PD is complex and multifactorial, involving both hereditary and environmental factors. Recent progress in molecular genetic studies of familial PD has led to the identification of16susceptible loci and11genes responsible for PD. Mutations in LRRK2are thus far the most prevalent genetic cause associated with autosomal dominant and idiopathic PD. Although the mechanism of how PD-associated LRRK2mutations cause disease is yet unknown, several studies indicated that certain kinds of mutations including G2019S are likely associated with toxic gain of function. Moreover, to overexpress wildtype LRRK2was found to be toxic in cultured cells and transgenic fly model. Becides, LRRK2was found to be associated with abnormal protein deposition in the brains of patients with lowy body disease including PD. To overexpress wildtype LRRK2, G2019S mutant or kinase domain alone accelerate the formation of aggregates caused by α-synuclein A53T mutant. These results suggest that precise regulation of LRRK2expression and function is necessary for maintaining homeostasis of the organism, and the disruption of the regulation mechanism may cause disease. microRNAs （miRNAs） are a class of endogenous22base-long single-stranded, noncoding RNAs that regulate gene expression in a sequence-specific manner in plants and animals. miRNAs are derived from long precursor transcripts by the action of nucleases Drosha and Dicer, and the resulting mature functional miRNAs bind to their target sequence in the3’-untranslated region （UTR） of mRNA with imperfect complementarity and lead to repression of translation.Therefore, to elucidate the mechanism of LRRK2post-transcriptional regulation is helpful to understand PD aetiology. In this study, the promoter activity of the LRRK23’-untransation region （UTR） was analized, which is essenssial to understand the post-transcriptional regulation of LRRK2.Methods:Several on-line softwares were involved in our study to predict the miRNAs to regulate the expression of LRRK2.23miRNAs was selected base on the predictation results, and further constructed the luciferase reporter plasmids. The plasmids were then transfected transiently into the N2a cell lines, and the luciferase relative activity were analysed to identified regulation of miRNAs to LRRK2. Finelly, miRNAs were transfected transiently into the Hek293cell lines, and the expression of endougenous LRRK2protein were analysed to identified the repression of miRNAs to LRRK2.Results:The study Found243miRNAs bind potentially with the sites of LRRK23’UTR of RefSeq NM₁98578using several software to predict the targets of miRNAs.23miRNAs was selected base on the predictation results, and further constructed the luciferase reporter plasmids. The luciferase relative activity decreased after miR205and miR454were transfected transiently into N2a cell line with wild type LRRK23’UTR Luc report system or mutation the binding site ones,the luciferase relative activity decreased lightly after miR-301b were transfected transiently into N2a cell line with wild type LRRK23’UTR Luc report system or mutation the binding site ones. The expression of endougenous LRRK2obviously decreased when miR-205and miR-454transfected transiently into the HEK293cell line. The expression of endoug-enous LRRK2decreased lightly when miR-301b transfected transiently into the HEK293cell line. Conclution:In summary,miR-205can regulate the expression through to bind the site of5’-AUGAAGG-3’in LRRK23’UTR; miR-454can regulate the expression through to bind the site of5’-UUGCACU-3’in LRRK23’UTR; miR-301b probably regulate the expression through to bind the site of5’-UUGCACU-3’in LRRK23’UTR. ABSTRACT OF SECTION2Identify expression of miRNAs and LRRK2mRNA for embryo stem-cell Neuron differentiated cell line and fetal brain tissueThe hallmark of a stem cell is its ability to self-renew and to produce numerousdifferentiated cells. This unique property is controlled by dynamic interplays between extrinsic signalling, epigenetic, transcriptional and post-transcriptional regulations. Recent research indicates that microRNAs （miRNAs） have an important role in regulating stem cell self-renewal and differentiation by repressing the translation of selected mRNAs in stem cells and differentiating daughter cells. Such a role has been shown in embryonic stem cells, germline stem cells and various somatic tissue stem cells. These findings reveal a newdimension of gene regulation in controlling stem cell fate and behaviour. This is shown deregulated synthesis of E2F1/DP caused by the miRNA pathway impairment as a key event in LRRK2pathogenesis and suggest novel miRNA-based therapeutic strategies.Mutations in the LRRK2gene cause autosomal dominant, late-onset parkinsonism, which presents with pleomorphic pathology including alpha-synucleopathy. The expression and localization of LRRK2to various neuronal populations in brain regions implicated inPD including the cerebral cortex, caudate-putamen and substantia nigra pars compacta. The localization of LRRK2to key neuronal populations throughout the nigrostriatal dopaminergic pathway is consistent with the involvement of LRRK2in the molecular pathogenesis of familial and sporadic parkinsonism.Methods:Using quantitative Real-time PCR, we analyzed the relative copies of miR-454and miR-205for embryo stem cell-Neuron differentiated cell line and fetal brain tissue which from the fetus developed about26weeks. We analyzed the expression of LRRK2mRNA and protein in different fetal brain tissue by quantitative real-time PCR and western blotting.Results:The expression profile of miR-454and LRRK2mRNA were rather low in the embryo stem cell-neuron differentiated cell line,however,miR-454and miR-205and LRRK2mRNA shows very high expression in fetal brain tissue,the most highest exression in cerebral tissue composed cerebral cortex,medulla,basal ganlia and striatum, high expression in cerebral cortex, and relatively lower expression in cerebellum. MiR-205expressed in the embryo stem cell and embryo body, but could not be detected in NPC.Conclusion:MiR454and miR-205expressed in central neuronal system.MiR-205expressed in embryo stem cell-neuron differentiated cell line ABSTRACT OF SECTION3Identify expression of serum miRNAs for PD and Normal SubjectsDysregulated expression of microRNAs （miRNAs） in various tissues has been associated with a variety of diseases, including neurodegenerative disorders. MiRNAs are present in the serum and plasma of humans and other animals such as mice, rats, bovine fetuses, calves, and horses. The levels of miRNAs in serum are in a remarkably stable form that is protected from endogenous RNase activity, reproducible, and consistent among individuals of the same species. the study identified specific expression patterns of serum miRNAs for prostate cancer,lung cancer, colorectal cancer, and diabetes, providing evidence that serum miRNAs contain fingerprints for various diseases. Through these analyses, we conclude that serum miRNAs can serve as potential biomarkers for the detection of various cancers and other diseases.There has been tremendous effort by the field to find biomarkers and physiological tests that accurately diagnose PD. There is still no conclusive diagnostic test. The goal of this proposal is to test the utility of using a miRNA signature to selectively diagnose and track Parkinson’s disease. By the completion of this study, we will know if blood contains a better diagnostic miRNA signature for PD.Methods:Using quantitative Real-time PCR, we analyzed the relative copies of miR-205and miR-454in serum among16sopradic PD patients and20genetically unrelated control individuals.Results:The expression profile of serum miR-205in PD was significantly different from that of normal subjects, P value of T test was0.0001. The expression profile of serum miR-454in PD was significantly different from that of normal subjects, P value of T test was0.040.Conclusion:We conclude that miR-205is in all a probability as a biomarker to diagnosing Parkinson’s disease, and miR454is probability as a biomarker to diagnosing Parkinson’s disease.