Title : microRNA Network Changes in the Brainstem Underlie the Development of Hypertension

Hypertension is a major chronic disease whose molecular mechanisms remain poorly 32 understood. We compared neuroanatomical patterns of microRNAs in the brainstem of the 33 spontaneous hypertensive rat (SHR) to the Wistar Kyoto rat (WKY; control). We quantified 419 34 well-annotated microRNAs in the nucleus of the solitary tract (NTS) and rostral ventrolateral 35 medulla (RVLM), from SHR and WKY rats, during three main stages of hypertension 36 development. Changes in microRNA expression were stageand region-dependent, with a 37 majority of SHR vs. WKY differential expression occurring at the hypertension onset stage in 38 NTS versus at the prehypertension stage in RVLM. Our analysis identified 24 microRNAs 39 showing time-dependent differential expression in SHR compared to WKY in at least one brain 40 region. We predicted potential gene regulatory targets corresponding to catecholaminergic 41 processes, neuroinflammation and neuromodulation using the miRWALK and RNA22 42 databases, and we tested those bioinformatics predictions using high-throughput qPCR to 43 evaluate correlations of differential expression between the microRNAs and their predicted gene 44 targets. We found a novel regulatory network motif consisting of microRNAs likely down45 regulating a negative regulator of pro-hypertensive processes such as angiotensin II (Ang II) 46 signaling and leukotriene-based inflammation. Our results provide new evidence on the 47 dynamics of microRNA expression in the development of hypertension and predictions of 48 microRNA-mediated regulatory networks playing a region-dependent role in potentially altering 49 brainstem cardiovascular control circuit function leading to the development of hypertension. 50 51