RNA m6A甲基化参与肾脏纤维化进展的实验研究

    Role of RNA m6A expression profile in progression of renal fibrosis

    • 摘要: 目的 本研究探讨肾脏纤维化过程中RNA m6A水平的变化,为深入探讨和研究RNA m6A在肾脏纤维化中的变化及作用机制提供实验基础和理论依据。方法 采用C57BL/6小鼠单侧输尿管结扎方法建立肾脏纤维化小鼠模型(unilateral ureteral obstruction,UUO)。随机分为假手术(Sham)组和UUO组。首先运用质谱法检测两组肾脏RNA m6A含量变化,进一步运用RNA甲基化免疫共沉淀(methylated RNA Immunoprecipitation,MeRIP)测序方法检测两组肾脏RNA m6A具体修饰位点的变化情况。在明确纤维化肾脏存在RNA m6A异常修饰后,我们通过RT-PCR方法,进一步筛选肾脏RNA m6A相关的调控酶。结果 与Sham组相比,UUO组肾脏RNA m6A含量显著升高。MeRIP测序分析发现甲基化水平差异>1.5倍的位点共1 352个,其中上调位点906个,下调位点446个,包括细胞连接、炎症反应、转分化、钙离子结合、细胞黏附等相关的基因。RT-PCR筛选结果提示甲基转移酶样蛋白(methyltransferase-like protein,METTL)14可能为关键调控酶。结论 RNA m6A表观遗传调控参与肾脏纤维化发生,METTL14可能是关键调控酶。

       

      Abstract: Objective To investigate the expression profile of RNA m6A in renal fibrosis model of unilateral ureteral obstruction (UUO) mice and conduct its related bioinformatics analysis,so as to provide theoretical basis and experimental basis for further study on the change and mechanism of RNA m6A in renal fibrosis.Methods C57BL/6 UUO mouse model of renal fibrosis was established by unilateral ureteral ligation.They were randomly divided into sham and UUO group.At first,mass-spectrography was used to detect the changes of RNA m6A contents in the kidney in the two groups.Then,methylated RNA immunoprecipitation (MeRIP) sequencing method was used to detect the changes of the modified sites of RNA m6A in the two groups.After identifying presence of abnormal modification of RNA m6A in renal fibrosis,we further screened out regulation enzymes associated with RNA m6A in the kidney through real quantitative RT-PCT.Results Compared with the sham group,the RNA m6A content in the kidney in the UUP group was elevated significantly.MeRIP sequencing analysis showed a total of 1352 sites with methylation level differences >1.5 times among them,including 906 up-regulating ones and 446 down-regulating ones,involved the genes associated with cell junction,inflammatory response,transdifferentiation,calcium binding,cell adhesion,vascular calcification,etc.RT-PCR results suggested that RNE methylase METTl14 might be a key regulatory enzyme.Conclusions The epigenetic regulation of RNA m6A is involved in the development of renal fibrosis,and METTl14 may be the key regulatory enzyme.

       

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