miR-25对扩展突变型ATXN3的表达调控
发布时间：2019-05-07 17:17
【摘要】：目的： 在前期工作中,本研究组成员已初步获得4个SCA3/MJD患者外周血清中异常表达的miRNAs,其中包括3个下调的miRNAs-miR-25, miR-29a及miR-125b,以及1个上调的miRNA-miR-34b。并且证实,4个miRNAs对SCA3/MJD的致病基因ATXN3mRNA均无明显影响,而miR-25可显著降低野生型ataxin-3蛋白表达水平,其作用靶点为ATXN3mRNA的3'UTR的第259-266碱基区域。在前期工作基础上,进一步了解miR-25对扩展突变型ATXN3mRNA及ataxin-3蛋白表达的影响；进一步研究miR-25对SCA3/MJD转基因细胞模型的活性的影响,为进一步阐明SCA3/MJD等polyQ疾病的分子发病机制提供新思路,为深入探讨SCA3/MJD等polyQ疾病的治疗提供新线索。 方法： (1)利用实时荧光定量PCR (qRT-PCR)技术检测miR-25对polyQ扩展突变型及ataxin-3蛋白表达水平的影响； (2)利用免疫印迹(Western blot)技术检测miR-25对扩展突变型ATXN3mRNA表达水平的影响； (3)利用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐比色法(MTT)检测miR-25对SCA3/MJD转基因细胞模型存活能力的影响； (4)利用流式细胞技术(FCM)检测miR-25对SCA3/MJD转基因细胞模型早期凋亡率的影响； (5)利用免疫荧光技术检测miR-25对SCA3/MJD转基因细胞模型核内包涵体的影响。 结果： (1) qRT-PCR结果显示：miR-25不影响扩展突变型ATXN3mRNA的表达； (2) Western blot结果显示：miR-25可显著降低polyQ扩展突变型ataxin-3蛋白的表达水平(P0.05)； (3)MTT结果显示：转染SCA3/MJD转基因细胞模型48小时后,miR-25可显著升高SCA3/MJD转基因细胞模型存活能力(P0.05),且作用靶点作用靶点为3'UTR区； (4)FCM技术结果显示：转染SCA3/MJD转基因细胞模型48小时后,miR-25可显著降低SCA3/MJD转基因细胞模型的早期凋亡率(P0.05),且作用靶点作用靶点为3'UTR区； (5)免疫荧光结果显示：转染SCA3/MJD转基因细胞模型48小时后,miR-25可显著降低SCA3/MJD转基因细胞模型的核内包涵体阳性细胞率(P0.05),且作用靶点作用靶点为3'UTR区；核内包涵体多数位于细胞核周围。 结论： (1)miR-25通过转录后水平影响扩展突变型ATXN3的表达； (2)miR-25可显著提高SCA3/MJD转基因细胞模型的细胞活性； (3)miR-25可显著降低SCA3/MJD转基因细胞模型核内包涵体的阳性细胞率。
[Abstract]:Objective: in the previous work, the members of the study group have obtained the abnormal expression of miRNAs, in the peripheral serum of 4 SCA3/MJD patients, including 3 down-regulated miRNAs-miR-25, miR-29a and miR-125b,. And an up-regulated miRNA-miR-34b.. Four miRNAs had no significant effect on ATXN3mRNA of SCA3/MJD, but miR-25 could significantly decrease the expression level of wild-type ataxin-3 protein. The target of miR-25 was 259-266bp region of 3'UTR of ATXN3mRNA. On the basis of previous work, the effect of miR-25 on the expression of extended mutant ATXN3mRNA and ataxin-3 protein was further studied. Further studies on the effect of miR-25 on the activity of SCA3/MJD transgenic cell model provide a new idea for further elucidating the molecular pathogenesis of polyQ diseases such as SCA3/MJD and providing new clues for further exploring the treatment of polyQ diseases such as SCA3/MJD. Methods: (1) Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the effect of miR-25 on polyQ extended mutation and ataxin-3 protein expression. (2) Western blot (Western blot) was used to detect the effect of miR-25 on the expression level of extended mutant ATXN3mRNA. (3) 3-(4,5-dimethylthiazole-2)-2,5-diphenyl tetrazolium bromide colorimetry (MTT) was used to detect the effect of miR-25 on the viability of SCA3/MJD transgenic cell model. (4) flow cytometry (FCM) was used to detect the effect of miR-25 on the early apoptosis rate of SCA3/MJD transgenic cell model. (5) the effect of miR-25 on the nuclear inclusion bodies of SCA3/MJD transgenic cell model was detected by immunofluorescence technique. Results: (1) the results of qRT-PCR showed that: (1) miR-25 did not affect the expression of extended mutant ATXN3mRNA (2) Western blot); (2) miR-25 significantly decreased the expression level of polyQ extended mutant ataxin-3 protein (P0.05); (3) the results of MTT showed that after 48 hours of transfection of SCA3/MJD transgenic cell model, miR-25 significantly increased the viability of SCA3/MJD transgenic cell model (P0.05), and the target was 3 'UTR region. (4) the results of FCM showed that after 48 hours of transfection of SCA3/MJD transgenic cell model, miR-25 could significantly reduce the early apoptosis rate of SCA3/MJD transgenic cell model (P0.05), and the target was 3 'UTR region. (5) the results of immunofluorescence showed that after 48 hours of transfection of SCA3/MJD transgenic cell model, miR-25 significantly decreased the rate of intracellular inclusion body positive cells in SCA3/MJD transgenic cell model (P0.05). The target of action was 3 'UTR region. Most of the inclusion bodies in the nucleus are located around the nucleus. Conclusion: (1) miR-25 affects the expression of extended mutant ATXN3 at post-transcriptional level, (2) miR-25 can significantly increase the cell activity of SCA3/MJD transgenic cell model. (3) miR-25 could significantly decrease the positive rate of inclusion bodies in the nucleus of SCA3/MJD transgenic cell model.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R744.7
本文编号：2471250
[Abstract]:Objective: in the previous work, the members of the study group have obtained the abnormal expression of miRNAs, in the peripheral serum of 4 SCA3/MJD patients, including 3 down-regulated miRNAs-miR-25, miR-29a and miR-125b,. And an up-regulated miRNA-miR-34b.. Four miRNAs had no significant effect on ATXN3mRNA of SCA3/MJD, but miR-25 could significantly decrease the expression level of wild-type ataxin-3 protein. The target of miR-25 was 259-266bp region of 3'UTR of ATXN3mRNA. On the basis of previous work, the effect of miR-25 on the expression of extended mutant ATXN3mRNA and ataxin-3 protein was further studied. Further studies on the effect of miR-25 on the activity of SCA3/MJD transgenic cell model provide a new idea for further elucidating the molecular pathogenesis of polyQ diseases such as SCA3/MJD and providing new clues for further exploring the treatment of polyQ diseases such as SCA3/MJD. Methods: (1) Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the effect of miR-25 on polyQ extended mutation and ataxin-3 protein expression. (2) Western blot (Western blot) was used to detect the effect of miR-25 on the expression level of extended mutant ATXN3mRNA. (3) 3-(4,5-dimethylthiazole-2)-2,5-diphenyl tetrazolium bromide colorimetry (MTT) was used to detect the effect of miR-25 on the viability of SCA3/MJD transgenic cell model. (4) flow cytometry (FCM) was used to detect the effect of miR-25 on the early apoptosis rate of SCA3/MJD transgenic cell model. (5) the effect of miR-25 on the nuclear inclusion bodies of SCA3/MJD transgenic cell model was detected by immunofluorescence technique. Results: (1) the results of qRT-PCR showed that: (1) miR-25 did not affect the expression of extended mutant ATXN3mRNA (2) Western blot); (2) miR-25 significantly decreased the expression level of polyQ extended mutant ataxin-3 protein (P0.05); (3) the results of MTT showed that after 48 hours of transfection of SCA3/MJD transgenic cell model, miR-25 significantly increased the viability of SCA3/MJD transgenic cell model (P0.05), and the target was 3 'UTR region. (4) the results of FCM showed that after 48 hours of transfection of SCA3/MJD transgenic cell model, miR-25 could significantly reduce the early apoptosis rate of SCA3/MJD transgenic cell model (P0.05), and the target was 3 'UTR region. (5) the results of immunofluorescence showed that after 48 hours of transfection of SCA3/MJD transgenic cell model, miR-25 significantly decreased the rate of intracellular inclusion body positive cells in SCA3/MJD transgenic cell model (P0.05). The target of action was 3 'UTR region. Most of the inclusion bodies in the nucleus are located around the nucleus. Conclusion: (1) miR-25 affects the expression of extended mutant ATXN3 at post-transcriptional level, (2) miR-25 can significantly increase the cell activity of SCA3/MJD transgenic cell model. (3) miR-25 could significantly decrease the positive rate of inclusion bodies in the nucleus of SCA3/MJD transgenic cell model.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R744.7
【参考文献】
相关期刊论文 前1条
1 王树奇;徐斌;刘建;王吉鹏;胡薇;;日本血吸虫醛糖还原酶基因RNA干扰效应的初步研究[J];中国寄生虫学与寄生虫病杂志;2014年01期
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