Abstract

Bipolar disorder (BD) is a mental disorder that leads to abnormal swings in mood, energy, activity level, attention, and the capability to accomplish daily tasks. Several long non-coding RNAs (lncRNAs) are dysregulated in BD patients. We have compared expression levels of five NF-κB-associated lncRNAs, namely ANRIL, CEBPA-DT, H19, NKILA and HNF1A-AS1in blood samples of BD patients compared with controls. While ANRIL, CEBPA-DT and HNF1-AS1 were significantly under-expressed in BD patients compared with controls, NKILA levels were higher in patients versus controls. Among differentially expressed genes, HFN1A-AS1exhibited the best diagnostic parameters in the separation of patients from controls (AUC ± SD = 0.86 ± 0.03, sensitivity = 0.82, specificity = 0.82, P value < 0.0001). AUC values for NKILA, ANRIL and CEBPA-DT were 0.71, 0.68 and 0.65, respectively. In accordance with the previously reported participation of NF-ƙB in the pathophysiology of BD, the current study provides evidence for dysregulation of NF-κB-associated lncRNAs in BD.

Introduction

Bipolar disorder (BD) is a mental disorder that leads to abnormal swings in mood, energy, activity level, attention, and the capability to accomplish daily tasks¹. BD can be classified into two main categories, namely BD type I and type II with severe and persistent mood elevation in the former type but less severe mood elevation in the latter type¹. BD, particular type I has been shown to have a strong genetic background². Based on the results of family studies, it seems that a small number of genes with modest effects establish genetic background of BD². In addition, a number of linked chromosomal regions as well as candidate genes have been identified². Moreover, expression assays have shown dysregulation of several genes in the circulation of BD patients or in the postmortem brain samples^3,4,5. Ubiquitin cycle, synaptic function³, apoptosis regulators⁵ and vitamin D related genes⁶ are among dysregulated genes in BD.

Nuclear factor kappa B (NF-κB) is an important transcription factor which regulates inflammatory signals. Expression of this transcription factor has been dysregulated in BD. Elevation in the spontaneous levels of NF-κB have been reported in several types of immune cells in adolescents with BD. Moreover, BD patients have exhibited greater upsurges in the NF-κB levels in monocytes after induction with TNF-α. Most notably, the latter observation has been associated with the contemporary severity of depressive symptoms⁷.

Several non-coding RNAs have exhibited functional association with NF-κB⁸. Association of number of NF-κB-interacting long non-coding RNAs (lncRNAs) with human disorders has been more investigated. LncRNAs represent a group of transcripts with sizes more than 200 nucleotides that contribute in the regulation of gene regulation. These transcripts participate in the pathogenesis of several human disorders through changing expression of genes and influencing activity of signaling pathways^9,10. Among these lncRNAs are ANRIL, CEBPA-DT, H19, NKILA and HNF1A-AS1 whose participation in the pathogenesis of Parkinson's disease¹¹ and autism spectrum disorder¹² has been assessed. In the current study, we measured expression levels of these lncRNAs in the blood samples of BD patients compared with controls to appraise their possible contribution in this disorder. We hypothesized that expression of these lncRNAs has been changed in the peripheral blood of BD patients due to the abnormalities in gene regulation pathways. Since they are associated with NF-κB signaling, it is possible that they contribute to the pathoetiology of BD. For instance, ANRIL as a constituent of NF-κB pathway can regulate inflammatory response¹³. H19 has been shown to promote atherosclerosis through regulation of MAPK and NF-κB pathways¹⁴. NKILA has also been identified as an inhibitor of NF-κB, since it inhibits phosphorylation of IκBα and suppresses nuclear translocation of p65¹⁵. CEBPA-DT is another lncRNA that is transcribed from up-stream of the CEBPA gene regulating its expression in cis¹⁶. Notably, expression of CEBPA has also been shown to be regulated by NF-κB p50 ¹⁷. Besides, CEBPA contributes to the relocation of histone deacetylases from NF-κB p50 homodimers and induction of expression of NF-κB target genes¹⁸. Finally, HNF1A-AS1 is an NF-κB-regulated lncRNA that enhances the phosphatase activity of SHP-1¹⁹. Notably, this phosphatase has a role in the pathogenesis of immune-related disorders²⁰.

Material and methods

Subjects

The Ethical Committee of Shahid Beheshti University of Medical Sciences has confirmed the study protocol (IR.SBMU.MSP.REC.1400.620). All enrolled cases and controls signed the written informed consent. Blood samples of 50 type I BD patients and 50 normal subjects were collected from Imam Hussein hospital during 2016–2019. Cases were recruited consecutively from Imam Hossein hospital during the study period and were examined in this hospital. BD cases were diagnosed according to the Diagnostic and Statistical Manual of Mental Disorders-5²¹. Moreover, depressive and mania symptoms and the presence of euthymia were assessed using the Hamilton Depression Rating Scale (HAM-D)²²and Young Mania Rating Scale (YMRS)²³. Patients were also assessed using a semi-structured interview which was based on asking questions within a prearranged thematic structure by skilled specialists who were trained through passing relevant courses. Clinical data including disease duration/ onset and drug history were collected. All recruited patients were not responsive to first-line mood stabilizers and took standard dose of Carbamazepine (200 mg, 2 times a day). In fact, most of patients who were not responsive to the first-line mood stabilizers in the mentioned clinic were under treatment with carbamazepine. So, in order to have a homogenized cohort of patients, we just included these patients. With the purpose of decreasing heterogeneity of the patients’ cohort, cases who took other drugs were excluded. History of head trauma, encephalitis or other mental illnesses and systemic disorders were regarded as exclusion criteria. Control subjects were assessed by a specialist to rule out the presence of signs or symptoms related with psychiatric disorders.

Sample collection and RNA extraction

Five ml of peripheral blood was collected from all cases and controls.

https://www.nature.com/articles/s41598-022-25670-9