OBJECTIVE: Angiogenesis is a key event in the successful healing of pulp injuries, and hypoxia is the main stimulator of pulpal angiogenesis. In this study, we investigated the effect of hypoxia on the proangiogenic potential of human dental pulp stem cells (hDPSCs) and the role of miR-143-5p in the process.
METHODS: Human dental pulp stem cells were isolated, cultured and characterized in vitro. Cobalt chloride (CoCl2) was used to induce hypoxia in hDPSCs. CCK-8 and Transwell assays were used to determine the effect of hypoxia on hDPSCs proliferation and migration. Quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting (WB) and ELISA were performed to assess the mRNA and protein levels of HIF-1α and angiogenic cytokines in hDPSCs. The effect of hypoxia on hDPSCs proangiogenic potential was measured in vitro using Matrigel tube formation and chick chorioallantoic membrane (CAM) assays. Recombinant lentiviral vectors were constructed to stably overexpress or inhibit miR-143-5p in hDPSCs, and the proangiogenic effects were assessed using qRT-PCR, WB, and tube formation assays. miR-143-5p target genes were identified and verified using bioinformatics prediction tools, dual-luciferase reporter assays and RNA pull-down experiments. Finally, a subcutaneous transplantation model in nude mice was used to determine the effects of hypoxia treatment and miR-143-5p overexpression/inhibition in hDPSCs in dental pulp regeneration.
RESULTS: Hypoxia promotes hDPSCs proliferation, migration and proangiogenic potential. The in vivo experiments showed that hypoxia treatment (50 and 100 μM CoCl2) promoted pulp angiogenesis and dentine formation. In contrast to the levels of proangiogenic factors, miR-143-5p levels decreased with increasing CoCl2 concentration. miR-143-5p inhibition significantly promoted proangiogenic potential of hDPSCs, whereas miR-143-5p overexpression inhibited angiogenesis in vitro. Dual-luciferase reporter assay identified retinoic acid receptor-related orphan receptor alpha (RORA) as an miR-143-5p target gene in hDPSCs. RNA pull-down experiments demonstrated that HIF-1α and RORA were pulled down by biotin-labelled miR-143-5p, and the levels of HIF-1α and RORA bound to miR-143-5p in the hypoxia group were lower than those in the normoxia group. Inhibition of miR-143-5p expression in hDPSCs promoted ectopic dental pulp tissue regeneration.
CONCLUSIONS: CoCl2-induced hypoxia promotes hDPSCs-driven paracrine angiogenesis and pulp regeneration. The inhibition of miR-143-5p upregulates the proangiogenic potential of hDPSCs under hypoxic conditions by directly targeting HIF-1α and RORA.

Gene regulatory elements, such as enhancers, greatly influence cell identity by tuning the transcriptional activity of specific cell types. Dynamics of enhancer landscape during early human Th17 cell differentiation remains incompletely understood. Leveraging ATAC-seq-based profiling of chromatin accessibility and comprehensive analysis of key histone marks, we identified a repertoire of enhancers that potentially exert control over the fate specification of Th17 cells. We found 23 SNPs associated with autoimmune diseases within Th17-enhancers that precisely overlapped with the binding sites of transcription factors actively engaged in T-cell functions. Among the Th17-specific enhancers, we identified an enhancer in the intron of RORA and demonstrated that this enhancer positively regulates RORA transcription. Moreover, CRISPR-Cas9-mediated deletion of a transcription factor binding site-rich region within the identified RORA enhancer confirmed its role in regulating RORA transcription. These findings provide insights into the potential mechanism by which the RORA enhancer orchestrates Th17 differentiation.

RORα is a transcription factor encoded by RORA gene. This protein is involved in several metabolic conditions, including obesity. We assessed association between two polymorphisms within this gene (namely rs11639084 and rs4774388) and severe obesity in Iranian population. Both SNPs were associated with obesity in all models (P < 0.0001) except for over-dominant model. T allele of rs11639084 was associated with this trait with OR (95% CI) of 16.85 (13.11-21.67) and was considered as the risk allele. Allelic model best fit the data, since the AIC value for this model was the highest (AIC = 28.82). In the co-dominant model, TT genotype was associated with obesity with OR (95% CI) of 301.6 (137.4-662.1). This genotype was shown to be the risk genotype in the recessive model when compared with TC+CC (OR (95% CI) = 60.54 (30.35-120.7)). The C allele of rs4774388 was shown to be the risk allele with OR (95% CI) of 4.61 (3.72-5.71). In the recessive model, the CC genotype was associated with the mentioned trait with OR (95% CI) of 9.92 (6.62-14.8). This model best fit the data (AIC = 37.08). Cumulatively, the current study suggests contribution of RORα to the pathogenesis of obesity.

Heart failure (HF) is a complex clinical syndrome associated with significant morbidity and mortality. Dysregulation of long non-coding RNA (lncRNA) has been implicated in the pathogenesis of HF. The present study aims to investigate the role of lncRNA HOX transcript antisense RNA (HOTAIR) in cardiomyocyte pyroptosis in a murine HF model. A murine HF model was established through transverse aortic contraction surgery, and an in vitro HF cell model was developed by treating HL-1 cells with H2O2. HOTAIR was overexpressed in TAC mice and HL-1 cells via pcDNA3.1-HOTAIR transfection. Cardiac function was assessed in TAC mice, and myocardial changes were evaluated using HE staining. The expression of NLRP3 was examined by immunohistochemistry. Myocardial injury markers and pyroptosis-related inflammatory cytokines were quantified using ELISA. Protein levels of NLRP3, cleaved-caspase-1, and GSDMD-N were analyzed by Western blot. Dual-luciferase assays and RNA immunoprecipitation were employed to confirm the binding interactions between HOTAIR and miR-17-5p, miR-17-5p and RORA. Functional rescue experiments were conducted by overexpressing miR-17-5p or silencing RORA in HL-1 cells. HOTAIR exhibited reduced expression in TAC mice and H2O2-induced cardiomyocytes. Overexpression of HOTAIR ameliorated cardiac dysfunction, reduced myocardial pathological injury, enhanced cardiomyocyte viability, and decreased myocardial injury and pyroptosis. HOTAIR interacted with miR-17-5p to repress RORA transcription. Overexpression of miR-17-5p or silencing of RORA abolished the inhibitory effect of HOTAIR overexpression on cardiomyocyte pyroptosis. In conclusion, HOTAIR competitively bound to miR-17-5p, relieving its inhibition of RORA transcription and leading to increased RORA expression and suppressed cardiomyocyte pyroptosis in HF models.

In this study, the relationship between RORA 23bp indel genotype and allele frequency with twin pregnancy, fertility, live weight and milk yield in 106 female Akkaraman ewes raised in Elazığ province was investigated. In the study conducted in Elâzığ province, 10ml milk was collected from 106 Akkaraman sheep and DNA was extracted from these milk. In RORA 23bp indel genotype frequency, DD genotype was found more than ID and II genotypes and RORA 23bp indel ın allele frequency, the D allele was found to be higher than the I allele. In both the first and second parity, the twinning rate was found to be lower. In both the first and second parity, the twinning rate was higher in the DD genotype, and it was observed that this genotype promınated mıddle lıvestock weıght and mılk yıeld. According to the results of our study, mutations in the RORA gene, which is a gene affecting reproductive efficiency in sheep, do not have a positive effect on fertility and twinning rate in Akkaraman sheep. To sum, this study provided theoretical references for the comprehensively research of the function of RORA gene and the breeding of Akkaraman Sheep. The 23-bp indel variants can be considered as molecular markers for litter size of sheep for marker-assisted selection breeding.

Although the interaction between tumor cells and tumor-associated macrophages (TAMs) has been widely studied; however, the mechanism of osteosarcoma cells in regulating the polarization of TAMs remains unclear. Exosomes from SAOS-2 cells were isolated and validated by electron microscopy and Western blot. Transfection of indicated plasmids was applied to modify the expressions of miR-181a-5p and RAR-related orphan receptor alpha (RORA). Flow cytometric analysis was carried out to analyze M1/M2 macrophage polarization. Quantitative real-time PCR was performed to determine the levels of miR-181a-5p and RORA. Protein levels of CD63, CD81, RORA, CD163, CD206, IL-10, CXCL10, and IL-1β were evaluated by Western blot. The direct interaction of miR-181a-5p and RORA was validated by dual-luciferase activity assay. The expression of miR-181a-5p was upregulated in osteosarcoma tissues and presented in SAOS-2-derived exosomes. SAOS-2-derived exosomes promoted the polarization of M2 macrophages by transferring miR-181a-5p. In addition, RORA was downregulated in osteosarcoma tissues and showed a negative correlation with miR-181a-5p. RORA was found to be the downstream target of miR-181a-5p in SAOS-2 cells. Inhibition of RORA reversed the effects of miR-181a-5p knockdown on the polarization of M2 macrophages. The results showed that exosomal miR-181a-5p derived from osteosarcoma cells induced polarization of M2 macrophages via targeting RORA.

The mortality rate of patients with coronary artery disease (CAD) increases year by year, and the age of onset is decreasing, primarily because of the lack of an efficient and convenient diagnostic method for CAD. In the present study, we aimed to detect CAD-correlated biomarkers and the regulatory pathways involved through weighted co-expression network analysis. The microarray data originated from 93 CAD patients and 48 controls within the Gene Expression Omnibus (GEO) database. The gene network was implemented by weighted gene co-expression network analysis, and the genes were observed to fall into a range of modules. We took the intersection of genes in the modules most correlated with CAD with the differentially expressed genes of CAD, which were identified by applying the limma package. Lasso regression and support vector machine recursive feature elimination algorithms were used to determine CAD candidate signature genes. The biomarkers for diagnosing CAD were detected by validating candidate signature gene diagnostic capabilities (receiver operating characteristic curves) based on data sets from GEO. Three modules were selected, and 26 vital genes were identified. Eight of these genes were reported as the optimal candidate features in terms of CAD diagnosis. Through receiver operating characteristic curve analysis, we identified three genes (ERCC5, HES6 and RORA; area under the curve > 0.8) capable of distinguishing CAD from the control, and observed that these genes are correlated with the immune response. In summary, ERCC5, HES6 and RORA may have potential for diagnosis of CAD.

BACKGROUND: The G allele in retinoid-related orphan receptor alpha (RORA, rs8042149) gene is associated with post-traumatic stress disorder (PTSD) diagnosis and more severe symptoms, reported in the first genome-wide association study of PTSD and subsequent replication studies. Although recent MRI studies identified brain structural deficits in RORA rs8042149 risk G allele carriers, the neural mechanism underlying RORA-related brain structural changes in PTSD remains poorly understood.
METHODS: This study included 227 Han Chinese adults who lost their only child. Cortical thickness and subcortical volume were extracted using FreeSurfer, and PTSD severity was assessed using the Clinician-Administered PTSD Scale. Hierarchical linear regression was used to assess the interaction effect between RORA genotypes (T/T, G/T, and G/G) and PTSD severity on cortical and subcortical structures.
RESULTS: Significant genotype × PTSD symptom severity interaction effects were found for bilateral transverse temporal gyrus thickness. For individuals with the homozygous T/T genotype, current PTSD symptom severity was positively associated with bilateral transverse temporal gyrus thickness. For individuals with heterozygous G/T genotype, current PTSD symptom severity was negatively associated with the left transverse temporal gyrus thickness. No significant main or interaction effects were found in any subcortical regions.
CONCLUSIONS: Cross-sectional design of this study.
CONCLUSIONS: These findings suggest that the non-risk T/T genotype - but not the risk G allele carriers - has a potentially protective or compensatory role on temporal gyrus thickness in adults who lost their only child. These results highlight the moderation effect of RORA polymorphism on the relationship between PTSD symptom severity and cortical structural changes.

UNASSIGNED: The purpose of the study was to examine the association of long and short sleep duration with risk of Parkinson\'s disease (PD) across RORA rs2028122 genotypes.
UNASSIGNED: In the present prospective study with a large sized UK Biobank cohort, we performed multivariate logistic regression analyses, generalized additive model, interaction terms, stratification analysis, and mediation analysis to evaluate the association of long and short sleep duration with risk of PD across RORA rs2028122 genotypes.
UNASSIGNED: The GG genotype [1.16 (1.01, 1.33)], a short sleep duration [1.23 (1.10, 1.37)], and a long sleep duration [1.19 (1.03, 1.37)] were identified as the independent risk factors for PD. Sleep duration exhibited a curvilinear U-shaped correlation with the risk of PD; first, the risk of PD gradually decreased as the length of sleep increase, but then, the risk began to increase as the length of sleep increase. Among habitual long sleepers, AG carriers had a higher risk of PD compared with AA carriers [1.67 (1.09, 2.55)]. Among AG carriers, both habitual short [1.28 (1.09, 1.50)] and long [1.38 (1.13, 1.69)] sleepers increased the risk of PD compared with habitual normal sleepers. Among GG carriers, habitual short sleepers have a higher risk of PD [1.26 (1.06, 1.50)] compared with habitual normal sleepers. A mediation model suggested that the rs2028122 genotype partially mediated the causal pathway of sleep duration leading to the development of PD on a positive effect.
UNASSIGNED: Our study demonstrated that the association between sleep duration and PD risk varied across different RORA rs2028122 genotypes. Our findings could help individuals to identify their potential risk profile and take timely actions to prevent the PD.

Circular RNAs (circRNAs) are involved in the regulation of various diseases, including periodontitis. The objective of this study was to analyze the biological role and regulatory mechanism of circ_0066881 in LPS-induced periodontal ligament cells (PDLCs). Circ_0066881, microRNA-144-5p (miR-144-5p) and retinoid acid-related orphan receptor A (RORA) levels were determined using reverse transcription-quantitative PCR (RT-qPCR) assay. Cell viability detection was performed by Cell Counting Kit-8 assay. Cell apoptosis was assessed through flow cytometry and caspase-3 activity assay. The protein analysis was completed via Western blot. Inflammatory cytokines were measured by ELISA. The target interaction was validated by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. The level of circ_0066881 was down-regulated in periodontitis tissues. Overexpression of circ_0066881 relieved LPS-induced cell viability inhibition and apoptosis or inflammation promotion in PDLCs. Circ_0066881 could bind to miR-144-5p. The protective function of circ_0066881 was achieved by sponging miR-144-5p in PDLCs. Circ_0066881 acts as a miR-144-5p sponge to mediate the RORA level. Inhibition of miR-144-5p attenuated LPS-induced cell injury via targeting RORA. All these results demonstrated that circ_0066881 partly prevented LPS-evoked cell dysfunction in PDLCs through miR-144-5p-mediated up-regulation of RORA.