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Abstract：
BACKGROUND: Previous studies have shown that peptides encoded by noncoding RNAs (ncRNAs) can be used as peptide drugs to alleviate diseases. We found that microRNA-31 (miR-31) is involved in the regulation of hypertension and that the peptide miPEP31, which is encoded by the primary transcript of miR-31 (pri-miR-31), can inhibit miR-31 expression. However, the role and mechanism of miPEP31 in hypertension have not been elucidated.
METHODS: miPEP31 expression was determined by western blot analysis. miPEP31-deficient mice (miPEP31-/-) were used, and synthetic miPEP31 was injected into Ang II-induced hypertensive mice. Blood pressure was monitored through the tail-cuff method. Histological staining was used to evaluate renal damage. Regulatory T (Treg) cells were assessed by flow cytometry. Differentially expressed genes were analysed through RNA sequencing. The transcription factors were predicted by JASPAR. Luciferase reporter and electrophoretic mobility shift assays (EMSAs) were used to determine the effect of pri-miR-31 on the promoter activity of miPEP31. Images were taken to track the entry of miPEP31 into the cell.
RESULTS: miPEP31 is endogenously expressed in target organs and cells related to hypertension. miPEP31 deficiency exacerbated but exogenous miPEP31 administration mitigated the Ang II-induced systolic blood pressure (SBP) elevation, renal impairment and Treg cell decreases in the kidney. Moreover, miPEP31 deletion increased the expression of genes related to Ang II-induced renal fibrosis. miPEP31 inhibited the transcription of miR-31 and promoted Treg differentiation by occupying the Cebpα binding site. The minimal functional domain of miPEP31 was identified and shown to regulate miR-31.
CONCLUSIONS: miPEP31 was identified as a potential therapeutic peptide for treating hypertension by promoting Treg cell differentiation in vivo. Mechanistically, we found that miPEP31 acted as a transcriptional repressor to specifically inhibit miR-31 transcription by competitively occupying the Cebpα binding site in the pri-miR-31 promoter. Our study highlights the significant therapeutic effect of miPEP31 on hypertension and provides novel insight into the role and mechanism of miPEPs.
METHODS: miPEP31 expression was determined by western blot analysis. miPEP31-deficient mice (miPEP31-/-) were used, and synthetic miPEP31 was injected into Ang II-induced hypertensive mice. Blood pressure was monitored through the tail-cuff method. Histological staining was used to evaluate renal damage. Regulatory T (Treg) cells were assessed by flow cytometry. Differentially expressed genes were analysed through RNA sequencing. The transcription factors were predicted by JASPAR. Luciferase reporter and electrophoretic mobility shift assays (EMSAs) were used to determine the effect of pri-miR-31 on the promoter activity of miPEP31. Images were taken to track the entry of miPEP31 into the cell.
RESULTS: miPEP31 is endogenously expressed in target organs and cells related to hypertension. miPEP31 deficiency exacerbated but exogenous miPEP31 administration mitigated the Ang II-induced systolic blood pressure (SBP) elevation, renal impairment and Treg cell decreases in the kidney. Moreover, miPEP31 deletion increased the expression of genes related to Ang II-induced renal fibrosis. miPEP31 inhibited the transcription of miR-31 and promoted Treg differentiation by occupying the Cebpα binding site. The minimal functional domain of miPEP31 was identified and shown to regulate miR-31.
CONCLUSIONS: miPEP31 was identified as a potential therapeutic peptide for treating hypertension by promoting Treg cell differentiation in vivo. Mechanistically, we found that miPEP31 acted as a transcriptional repressor to specifically inhibit miR-31 transcription by competitively occupying the Cebpα binding site in the pri-miR-31 promoter. Our study highlights the significant therapeutic effect of miPEP31 on hypertension and provides novel insight into the role and mechanism of miPEPs.
摘要：
背景：先前的研究表明，由非编码RNA（ncRNA）编码的肽可用作肽类药物来缓解疾病。我们发现microRNA-31（miR-31）参与高血压的调节，并且由miR-31（pri-miR-31）的初级转录物编码的肽miPEP31，可以抑制miR-31的表达。然而,miPEP31在高血压中的作用和机制尚未阐明。
方法:通过蛋白质印迹分析测定miPEP31表达。使用miPEP31缺陷小鼠（miPEP31-/-），将合成的miPEP31注射到AngII诱导的高血压小鼠体内。通过尾套法监测血压。组织学染色用于评估肾损伤。通过流式细胞术评估调节性T(Treg)细胞。通过RNA测序分析差异表达的基因。通过JASPAR预测转录因子。使用荧光素酶报告基因和电泳迁移率变化测定(EMSAs)来确定pri-miR-31对miPEP31的启动子活性的影响。拍摄图像以跟踪miPEP31进入细胞。
结果：miPEP31在与高血压相关的靶器官和细胞中内源性表达。miPEP31缺乏加剧，但外源性miPEP31给药减轻了AngII引起的收缩压（SBP）升高，肾损伤和Treg细胞减少。此外，miPEP31缺失增加了AngII诱导的肾纤维化相关基因的表达。miPEP31通过占据Cebpα结合位点抑制miR-31的转录并促进Treg分化。鉴定miPEP31的最小功能结构域并显示其调节miR-31。
结论：miPEP31被鉴定为通过促进体内Treg细胞分化治疗高血压的潜在治疗肽。机械上,我们发现miPEP31作为转录抑制因子,通过竞争性占据pri-miR-31启动子中的Cebpα结合位点,特异性抑制miR-31转录.我们的研究强调了miPEP31对高血压的显着治疗作用，并为miPEPs的作用和机制提供了新的见解。
方法:通过蛋白质印迹分析测定miPEP31表达。使用miPEP31缺陷小鼠（miPEP31-/-），将合成的miPEP31注射到AngII诱导的高血压小鼠体内。通过尾套法监测血压。组织学染色用于评估肾损伤。通过流式细胞术评估调节性T(Treg)细胞。通过RNA测序分析差异表达的基因。通过JASPAR预测转录因子。使用荧光素酶报告基因和电泳迁移率变化测定(EMSAs)来确定pri-miR-31对miPEP31的启动子活性的影响。拍摄图像以跟踪miPEP31进入细胞。
结果：miPEP31在与高血压相关的靶器官和细胞中内源性表达。miPEP31缺乏加剧，但外源性miPEP31给药减轻了AngII引起的收缩压（SBP）升高，肾损伤和Treg细胞减少。此外，miPEP31缺失增加了AngII诱导的肾纤维化相关基因的表达。miPEP31通过占据Cebpα结合位点抑制miR-31的转录并促进Treg分化。鉴定miPEP31的最小功能结构域并显示其调节miR-31。
结论：miPEP31被鉴定为通过促进体内Treg细胞分化治疗高血压的潜在治疗肽。机械上,我们发现miPEP31作为转录抑制因子,通过竞争性占据pri-miR-31启动子中的Cebpα结合位点,特异性抑制miR-31转录.我们的研究强调了miPEP31对高血压的显着治疗作用，并为miPEPs的作用和机制提供了新的见解。
