The role of a lncRNA (TCONS_00044595) in regulating pineal CLOCK expression after neonatal hypoxia–ischemia brain injury

Highlights

• Profiling of pineal lncRNA changes post neonatal HIBD.

• lncRNA TCONS_00044595 directly interacts with miR-182.

• lncRNA TCONS_00044595 knockdown alleviates pineal CLOCK activation post HIBD.

Abstract

A common, yet often neglectable, feature of neonatal hypoxic-ischemic brain damage (HIBD) is circadian rhythm disorders resulted from pineal gland dysfunction. Our previous work demonstrated that miRNAs play an important role in regulating key circadian genes in the pineal gland post HIBD [5,21]. In current study, we sought out to extend our investigation by profiling expression changes of pineal long non-coding RNAs (lncRNAs) upon neonatal HIBD using RNA-Seq. After validating lncRNA changes, we showed that one lncRNA: TCONS_00044595 is highly enriched in the pineal gland and exhibits a circadian expression pattern. Next, we performed bioinformatic analysis to predict the lncRNA-miRNA regulatory network and identified 168 miRNAs that potentially targetlncRNA TCONS_00044595. We further validated the bona fide interaction between one candidate miRNA: miR-182, a known factor to regulate pineal Clock expression, and lncRNA TCONS_00044595. Finally, we showed that suppression of lncRNA TCONS_00044595 alleviated the CLOCK activation both in the cultured pinealocytes under OGD conditions and in the pineal gland post HIBD in vivo. Our study thus shed light into novel mechanisms of pathophysiology of pineal dysfunction post neonatal HIBD.

Introduction

Neonatal hypoxic-ischemic brain damage (HIBD) leads to a broad spectrum of post-injury deficits [3,15]. While extensive studies have focused on the cognitive and motor impairments, little attention has been paid to some common, yet “minor” symptoms. For example, a substantial population of HIBD neonates exhibit abnormal, discontinuous sleep character, along with delayed onset of sleep-wake cycle development [14,19,20]. However, the underlying mechanisms of HIBD evoked sleep circadian disorders remain largely unknown. To tackle this problem, we started with clinical observation and discovered that patients with mild, but not severe, HIBD is closely associated with pineal cysts and sleep circadian dysfunction [4]. Thus, the dysfunction of the pineal gland, a critical organ for vertebrate circadian rhythm control, might play a key role in circadian dysfunction post neonatal HIBD.

Accumulating evidence revealed that post-transcriptional mechanisms play indispensable roles in regulating the expression of pineal circadian genes [11]. For example, several heterogeneous nuclear ribonucleoproteins (hnRNPs) were reported to interact with the mRNA of Aanat, a rate limiting enzyme for melatonin synthesis in the pineal gland, to regulate its stability and translation [9,10]. In addition, non-coding RNAs are reported to modulate two master circadian regulators: Clock and Aanat [2,5,21].

In addition to RNA binding proteins and miRNA, long non-coding RNAs (lncRNAs) is an important component of the post-transcriptional mechanisms [22]. Different from mRNAs, lncRNAs, although typically transcribed by RNA polymerase II, often lacks the open reading frame [[6], [7], [8]]. lncRNAs are capable to regulate gene expression at epigenetic, transcriptional and post-transcriptional levels. For example, they can either serve as platform to recruit proteins or molecular complexes to specific DNA loci, or serve as molecular sponge of miRNAs to trigger post-transcriptional regulation [12]. Previous studies revealed extensive alteration of cerebral lncRNA expression profiles in rat with neonatal HIBD [23]. However, it remains unknown whether and how expression levels of lncRNAs are changed in the pineal gland to affect circadian rhythm.

In this study, we sought out to perform a high-throughput screening of expression changes of pineal lncRNAs at different time points post neonatal HIBD. Among verified lncRNAs, a lncRNA (TCONS_00044595) is enriched in the pineal gland and striatum and exhibits a circadian expression pattern within the pineal gland. Bioinformatic prediction and follow-up biochemical measures demonstrated that lncRNATCONS_00044595 interacts with miR-182, a microRNA known to regulate pineal Clock [5]. In cultured pinealocytes, suppression of lncRNATCONS_00044595, working synergistically with miR-182, significantly alleviated the over-activation of CLOCK post oxygen-glucose deprivation (OGD) condition. Furthermore, the overactivation of pineal CLOCK post HIBD was significantly reduced when lncRNATCONS_00044595 up-regulation was blocked. Our results therefore shed lights into the pathophysiological mechanisms of pineal dysfunction post neonatal HIBD and the design of effective therapeutic targets to interevent circadian disorders in patients with HIBD.