Clinical significance of long non-coding RNA NORAD in rheumatoid arthritis

Abstract Background Rheumatoid arthritis (RA) is a chronic autoimmune disease that may cause joint deformities and seriously affect the normal life of the patients. In order to enable patients to receive timely attention and treatment, this study developed new diagnostic markers by exploring the expression and molecular mechanism of the long non-coding RNA NORAD (NORAD) in RA. Methods Participants including 77 RA patients and 52 healthy persons were enrolled, and the corresponding clinical data and serum samples were obtained. The NORAD and miR-204-5p expression were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). The content of inflammatory cytokines (IL-6, TNF-α) were determined through enzyme-linked immunosorbent assay (ELISA). Luciferase activity reporter assay demonstrated the association between NORAD and miR-204-5p. In addition, receiver operating characteristic (ROC) curve was used to evaluate the diagnostic efficacy of NORAD, and Pearson's correlation analysis was applied for the correlation analysis. Results NORAD was enriched in RA serum with high diagnostic value. Simultaneously, IL-6 and TNF-α levels were also upregulated (P < 0.001). The C-reactive protein (CRP), rheumatoid factor (RF), erythrocyte sedimentation rate (ESR) and anti-cyclic citrullinated peptide antibody (Anti-CCP) levels in RA patients were generally elevated (P < 0.001). NORAD was positively correlated with the levels of clinical indicators and inflammatory factors (P < 0.0001). Mechanistically, NORAD may affect the progression of RA by targeting and negatively regulating miR-204-5p. Conclusions There is a correlation between NORAD and the processes of RA, and NORAD has the potential to predict and diagnose the occurrence of RA.

Effects of lncRNA-UCA1 targeting miR-204-5p on the proliferation, migration, apoptosis and immune escape of endometrial carcinoma cells / 中华肿瘤杂志

Objective: To investigate the effect of long non-coding RNA urothelial carcinoma-associated 1 (UCA1) gene on the proliferation, migration, apoptosis and immune escape of endometrial cancer cells and its molecular mechanism. Methods: Endometrial cancer tissues and adjacent normal tissues of patients with endometrioid adenocarcinoma who underwent total or partial hysterectomy in Henan Provincial People's Hospital from 2017 to 2019 were collected. The expressions of UCA1 and miR-204-5p were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), and the cell proliferation, migration and apoptosis were detected by cell counting kit 8 (CCK8) method, Transwell method, flow cytometry, and dual-luciferase reporter assay was used to explore the target relationship between UCA1 and miR-204-5p. HEC-1A-sh-NC or HEC-1A-sh-UCA1 cells were co-cultured with peripheral blood mononuclear cells (PBMC) or cytokine-induced killer cells in vitro to explore the role of UCA1 in immune escape. Results: The expression level of UCA1 in endometrial cancer tissue (17.08±0.84) was higher than that in adjacent normal endometrial tissue (3.00±0.37), and the expression level of miR-204-5p (0.98±0.16) was lower than that in adjacent normal endometrial tissue (2.00±0.20, P<0.05). Pearson correlation analysis showed that the expression of miR-204-5p was negatively correlated with the expression of UCA1 (r=-0.330, P=0.030). The expressions of UCA1 and miR-204-5p were associated with the International Federation of Gynecology and Obstetrics stage of endometrial cancer, lymph node metastasis and vascular invasion (P<0.05). The relative ratio of absorbance (0.58±0.11) and the number of cell migration [(199.68±18.44)] in the sh-UCA1 group were lower than those in the sh-NC group (1.24±0.17 and 374.76±24.83), respectively. The apoptosis rate of sh-UCA1 group [(28.64±7.80)%] was higher than that of sh-NC group [(14.27±4.38)%, P<0.05]. After different ratios of effector cells and target cells were cultured, the cell survival rate of HEC-1A-sh-UCA1 group was lower than that of HEC-1A-sh-NC group, and the difference was statistically significant (P<0.05). UCA1 had a binding site for miR-204-5p. The relative ratio of absorbance (1.74±0.08) and the number of cell migration (426.00±18.00) cells in the UCA1+ anti-miR-204-5p group were higher than those in the control group [1.00±0.03 and (284.00±8.00) cells, respectively]. The apoptosis rate of UCA1+ anti-miR-204-5p group [(5.42±0.93)%] was lower than that of control group [(14.82±1.48)%, P<0.05]. HEC-1A-sh-UCA1 cells could induce higher interferon gamma (IFN-γ) expression when co-cultured with PBMC, and the levels of IFN-γ expression in PHA group and PHA+ pre-miR-204-5p group cells were 2.42±0.49 and 1.88±0.26, which were higher than that in the PHA+ pre-NC group (0.85±0.10, P<0.05). When co-cultured with cytokine-induced killer cells (different ratios) in vitro, the HEC-1A-sh-UCA1 group and the HEC-1A-pre-miR-204-5p group had lower survival rates than that in the HEC-1A-pre-miR-204-5p group. In the HEC-1A-pre-NC group, the differences were statistically significant (P<0.05). Conclusion: UCA1/miR-204-5p may play an important role in human endometrial cancer.

LncRNA XIST knockdown ameliorates oxidative low-density lipoprotein-induced endothelial cells injury by targeting miR-204-5p/TLR4

Oxidative low-density lipoprotein (ox-LDL)-induced endothelial cell injury is a key contributor toatherosclerosis development. However, the role and mechanism of long noncoding RNA X-inactive specifictranscript (XIST) in atherosclerosis remain largely unknown. The ox-LDL-induced human umbilical veinendothelial cells (HUVECs) injury was analyzed by cell viability, apoptosis, inflammatory cytokines secretionand oxidative stress. The expression levels of XIST, microRNA-204-5p (miR-204-5p) and toll-like receptor 4(TLR4) were detected by quantitative real-time polymerase chain reaction and western blot, respectively. Thetarget interaction between miR-204-5p and XIST or TLR4 was explored by bioinformatics analysis, luciferaseassay and RNA immunoprecipitation. The expression of XIST was enhanced in ox-LDL-treatedHUVECs. Knockdown of XIST attenuated ox-LDL-induced viability inhibition, apoptosis production,inflammatory response and oxidative stress in HUVECs. XIST was validated as a sponge of miR-204-5pand TLR4 acted as a target of miR-204-5p. Knockdown of miR-204-5p reversed silence of XISTmediated suppressive role in ox-LDL-induced injury. TLR4 alleviated miR-204-5p-mediated inhibitiveeffect on ox-LDL-induced injury. Moreover, XIST could regulate TLR4 expression by spongingmiR-204-5p. In conclusion, silence of XIST displayed a protective role in ox-LDL-induced injury inHUVECs by regulating miR-204-5p/TLR4 axis, providing a novel mechanism for understanding thepathogenesis of atherosclerosis.