【摘要】 目的：通过甲基化RNA免疫沉淀测序（MeRIP-seq）和转录组测序（RNA-seq）绘制N6-甲基腺苷（m6A）修饰图谱，探讨环状RNA(circ RNAs)m6A甲基化修饰在有氧运动改善高脂饮食小鼠心脏病理性重塑中的作用。方法：C57BL/6J野生型小鼠随机分为正常饮食安静（normal diet-sedentary,ND-SED）组、正常饮食运动（normal diet-exercise,ND-EX）组、高脂饮食安静（high-fat diet-sedentary,HFD-SED）组和高脂饮食运动（high-fat diet-exercise,HFD-EX）组。NDSED组和ND-EX组小鼠给予普通饲料喂养，HFD-SED组和HFD-EX组小鼠给予60%脂肪供能高脂饲料喂养12周后，ND-EX组和HFD-EX组小鼠进行8周有氧运动。运动干预结束后，检测小鼠体重、体脂含量和心功能。采血、取心脏，检测血清TC和TG含量，酶联免疫吸附测定检测心脏组织TG含量，RT-qPCR检测m6A调节因子，Western blot检测METTL3和FTO蛋白表达，比色法检测心脏组织m6A RNA甲基化含量，MeRIP-seq检测HFD-SED组和HFDEX组小鼠心脏circRNAs m6A甲基化修饰，SRAMP预测发生m6A甲基化差异和表达差异的circRNA甲基化位点，MeRIP-qPCR验证其m6A水平。结果：8周有氧运动显著降低了高脂饮食小鼠体重和体脂含量，提高了左心室射血分数和短轴缩短率，抑制心脏纤维化和脂质沉积，改善心脏病理性重塑。有氧运动可以显著降低高脂饮食小鼠心脏m6A甲基化水平，甲基转移酶METTL3在其中发挥了重要作用。MeRIP-seq显示，HFD-SED组和HFD-EX组小鼠心脏受m6A修饰的circRNAs大部分都只具有1个m6A修饰位点，且m6A circRNAs主要来自重叠区和外显子。与HFDSED组比较，HFD-EX组小鼠心脏有13个circ RNAs发生了甲基化上调，19个circRNAs发生了甲基化下调，两组差异甲基化的circRNAs大部分来源于重叠区，长度主要富集在1～10 000 bps，大部分位于1号、7号和13号染色体上。RNA-seq显示，与HFD-SED组比较，HFD-EX组小鼠心脏有40个circRNAs发生差异表达，其中22个显著上调，18个显著下调。MeRIP-seq和RNA-seq联合分析发现，只有circ RNA＿4614|Chr7:127484542＿127486889＿+（以下简称“circ RNA＿4614”）同时发生m6A甲基化差异和表达差异，其m6A水平和表达水平均显著上调。SRAMP预测显示，circRNA＿4614存在8个潜在的m6A位点，其中1个位点具有非常高置信度，2个位点具有高置信度。Me RIP-q PCR验证显示，与HFD-SED组比较，HFD-EX组小鼠心脏circ RNA＿4614的m6A水平显著上调，其趋势与MeRIP-seq测序结果一致。结论：有氧运动可以显著降低高脂饮食小鼠心脏m6A甲基化水平，调节心脏circRNAs表达谱和m6A修饰谱。circRNA＿4614介导的m6A修饰在有氧运动改善肥胖性心脏重塑中可能发挥重要作用。更多还原

【Abstract】 Objective: To explore the role of N6-methyladenosine(m6A) methylation modified circular RNAs(circRNAs) in aerobic exercise-induced improvement of cardiac pathological remodeling in high-fat diet mice. Methods: C57BL/6J wild-type mice were randomly divided into four groups: Normal diet-sedentary(ND-SED) group, normal diet-exercise(ND-EX) group, high-fat dietsedentary(HFD-SED) group and high-fat diet-exercise(HFD-EX) group. The ND-SED and ND-EX groups were fed with normal diet, while the HFD-SED and HFD-EX groups were fed with 60% fat-enriched high-fat diet for 12 weeks, the ND-EX and HFD-EX groups were subjected to 8-week aerobic exercise. After the exercise intervention, the body weight, body fat content, cardiac function, serum TC and TG levelsand heart tissue TG content were measured. m6A regulatory factors were detected by using RTqPCR. Protein expressions of METTL3 and FTO were determined by using Western blot. m6A RNA methylation content in cardiac tissue was quantified using colorimetric methods. MeRIP-seq was used to detect circRNAs with m6A methylation modification in the heart. SRAMP was employed to predict circRNA methylation sites with m6A methylation difference and expression difference;and MeRIP-qPCR was performed to verify its m6A level. Results: 8-week aerobic exercise significantly reduced body weight/fat content of high-fat diet-fed mice, and increased left ventricular ejection fraction(EF) and fractional shortening(FS), inhibited cardiac fibrosis, lipid deposition, and pathological remodeling. Aerobic exercise also decreased m6A methylation level in the heart of high-fat diet-fed mice, while the methyltransferase METTL3 with played an important role in this process. MeRIP-seq analysis revealed that the majority of m6A-modified circRNAs in the mice heart of HFD-SED and HFD-EX groups had only one m6A modification site, with a significant enrichment in overlapping regions and exons. Compared with the HFD-SED group, there were 13 upregulated and 19 downregulated m6A-modified circRNAs in that of HFD-EX group. The differential methylation of these circRNAs was mainly observed in overlapping regions, with most of them ranging from 1 to 10 000 bps and being located on chromosomes 1, 7, and 13. Compared with the HFD-SED group, there were 40 circRNAs differentially expressed in the HFD-EX group, among which 22 circRNAs were significantly up-regulated and 18 circRNAs were significantly down-regulated. Combined analysis of MeRIP-seq and RNA-seq data revealed that only circRNA＿4614 exhibited both differential m6A methylation levels and expression levels, showing a significant increase in both aspects. SRAMP prediction indicated that circRNA＿4614 harbored eight potential m6A sites, among which one had high confidence while two had moderate confidence. Compared to the HFD-SED group,there was a significant increase in m6A methylation level for circRNA＿4614 in the HFD-EX group, consistent with MeRIP-seq sequencing results. Conclusions: Aerobic exercise could significantly reduce m6A methylation levels in high-fat diet-fed mice, and regulate the expression profiles and m6A modification patterns of cardiac circRNAs. The involvement of circRNA＿4614 mediated by m6A modification may play an important role in improving obesity-induced cardiac remodeling through aerobic exercise.更多还原