The Regulation Of Expanded Mutant ATXN3Gene Expression By MiR-25

Objective:In the previous work, we have already screened four dysregulated miRNAs in SCA3/MJD patients, including miR-25, miR-29a, miR-125b, and miR-34b. In addition, we discovered that miR-25could significantly reduce the wild-type ataxin-3protein expression levels without affecting its mRNA expression, while the other three miRNAs have no significant effect on wild-type ATXN3gene expression levels. And the targets of miR-25was the first259-266bases of the3’UTR of ATXN3gene mRNA. Based on the previous work, we are going to further explore the influence on polyQ-expanded mutant ATXN3mRNA and ataxin-3protein expression by miR-25, and to further explore the influence on cell viability of the SCA3/MJD transgenic cells by miR-25. Thus, we are going to provide new ideas for exploring the molecular pathogenesis of polyQ diseases such as SCA3/MJD, and to provide new clues for exploring the treatment of polyQ diseases such as SCA3/MJD.Methods:(1)The western blot technique was to detect the effect on polyQ-expanded mutant ATXN3mRNA expression levels by miR-25;(2) The qRT-PCR technique was usd to detect the effect on polyQ-expanded mutant ataxin-3protein expression levels by miR-25;(3) The MTT technique was used to detect the effect on the viability of SCA3/MJD transgenic cells by miR-25;(4) The flow cytometry technique was used to detect the effect on the early apoptotic rates of SCA3/MJD transgenic cells by miR-25; (5) The immunofluorescence technique was used to detect the effect on the nucleus inclusion of SCA3/MJD transgenic cells by miR-25.Results:(1) qRT-PCR results showed that miR-25didn’t affect the expanded mutant ATXN3mRNA expression;(2) Western blot results showed that miR-25could significantly reduce the level of polyQ-expanded mutant ataxin-3protein expression;(3) MTT results showed that miR-25could significantly increase the cell viability of SCA3/MJD transgenic cell models, and target of miR-25was the3’UTR region.;(4) Flow cytometry results showed that miR-25could significantly decrease early apoptosis rate of SCA3/MJD transgenic cell models, and target of miR-25was the3’UTR region;(5) The immunofluorescence results showed that miR-25could significantly reduced the positive cell rate of neuronal intranuclear inclusions, and targets for the3’UTR region.Conclusions:(1) miR-25regulated expanded mutant ATXN3expression by post-transcriptional mechanism;(2) miR-25could significantly improve the cell viability of SCA3/MJD transgenic cells;(3) miR-25could significantly dcrease the positive cell rate of neuronal intranuclear inclusions in SCA3/MJD transgenic cell models.