Anti-nuclear valosin-containing protein-like autoantibody is associated with calcinosis and higher risk of cancer in systemic sclerosis.

OBJECTIVES: Systemic sclerosis (SSc)-specific autoantibodies allow the diagnosis and predict the prognosis of SSc patients with different clinical characteristics. The aim of this study was to describe new SSc-related autoantibodies by a novel protein immunoprecipitation (IP) assay. METHODS: Serum samples and clinical data were collected from 307 SSc patients. Antinuclear autoantibodies were tested in all patients by indirect immunofluorescence (IIF) on HEp-2 cells. SSc-specific autoantibodies were evaluated with a commercial immunoblot and chemiluminescence immunoassay, and traditional RNA-IP. Patients negative for all these autoantibodies (n = 51) were further tested with a non-radioactive protein IP assay. Protein bands detected on SDS-PAGE were then analysed by mass spectrometry (MS) and confirmed by western blot (WB). Additional 56 patients with nucleolar pattern by IIF were tested by protein IP-WB. RESULTS: Five patients who underwent protein IP testing showed a 110-115kDa molecular weight band on SDS-PAGE and a homogeneous nucleolar pattern by IIF. MS identified the bands as nuclear valosin-containing protein-like (NVL). An additional positive patient was detected by IP-WB. As compared with the remaining 101 negative patients, anti-NVL positive patients showed a greater prevalence of calcinosis (100% vs 18.9%, p< 0.001), and cancer (66.7% vs 8.9%, p= 0.002), with a particular association with synchronous cancer (OR = 16.3; p= 0.024). CONCLUSION: We identified NVL as a new autoantibody target by a novel protein IP assay in SSc patients with a homogeneous nucleolar IIF pattern, testing negative for all known SSc-specific autoantibodies by commercial assays and RNA IP. Anti-NVL identifies a new clinical phenotype, characterized by calcinosis and cancer.

Antigen Reactivity and Clinical Significance of Autoantibodies Directed Against the Pyruvate Dehydrogenase Antigen Complex in Patients With Connective Tissue Disease.

Introduction: Antimitochondrial antibodies (AMAs) are the hallmark of primary biliary cholangitis (PBC) but can be identified also in patients with connective tissue disease, namely, systemic sclerosis (SSc). Protein immunoprecipitation (IP) and IP-Western blot (WB) can be used to confirm AMA positivity directed at the pyruvate dehydrogenase complex (PDC) subunits E1α, E1ß, E2/E3, and E3BP in patients showing a cytoplasmic reticular pattern at indirect immunofluorescence when performed in a screening setting before the onset of overt cholestasis in rheumatic patients. Patients and Methods: We studied sera from 285 patients affected by connective tissue disease [SSc, n = 144; dermato/polymyositis (DM/PM), n = 56; and undifferentiated connective tissue disease (UCTD), n = 85] by indirect immunofluorescence (IIF), protein-IP, and IP-WB to identify specific PDC subunits recognized by AMA. Results: Twenty percent (57/285) of sera from patients with connective tissue disease had a cytoplasmic reticular pattern at IIF, and in 77% (44/57, including 20 SSc, 12 PM/DM, and 12 UCTD) of these, we detected different titers of autoantibodies against the PDC subunits, specifically against PDC-E2. Among these sera, 4 (9%) tested positive for anti-E1α, 15 (34%) for anti-E1ß, and 16 (36%) for anti-E3BP. Four of the 20 AMA-positive SSc cases (20%) had been already diagnosed with PBC, and all were positive for autoantibodies against the subunits PDC-E2, E3, and E3BP. Conclusions: Using IIF and IP, we confirm that autoantibodies against the PDC components are detected in rheumatic patients with PBC or without liver dysfunction. In view of the strong predictive value of AMA for PBC, a strict follow-up of these latter patients is warranted for an early diagnosis of the disease.

lncRNA RP11-624L4.1 Is Associated with Unfavorable Prognosis and Promotes Proliferation via the CDK4/6-Cyclin D1-Rb-E2F1 Pathway in NPC.

Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors in southern China and southeast Asia. Emerging evidence revealed that long noncoding RNAs (lncRNAs) might play important roles in the development and progression of many cancers, including NPC. The functions and mechanisms of the vast majority of lncRNAs involved in NPC remain unknown. In this study, a novel lncRNA RP11-624L4.1 was identified in NPC tissues using next-generation sequencing. In situ hybridization (ISH) was used to analyze the correlation between RP11-624L4.1 expression and the clinicopathological features or prognosis in NPC patients. RNA-Protein Interaction Prediction (RPISeq) predictions and RNA-binding protein immunoprecipitation (RIP) assays were used to identify RP11-624L4.1's interactions with cyclin-dependent kinase 4 (CDK4). As a result, we found that RP11-624L4.1 is hyper-expressed in NPC tissues, which was associated with unfavorable prognosis and clinicopathological features in NPC. By knocking down and overexpressing RP11-624L4.1, we also found that it promotes the proliferation ability of NPC in vitro and in vivo through the CDK4/6-Cyclin D1-Rb-E2F1 pathway. Overexpression of CDK4 in knocking down RP11-624L4.1 cells can partially rescue NPC promotion, indicating its role in the RP11-624L4.1-CDK4/6-Cyclin D1-Rb-E2F1 pathway. Taken together, RP11-624L4.1 is required for NPC unfavorable prognosis and proliferation through the CDK4/6-Cyclin D1-Rb-E2F1 pathway, which may be a novel therapeutic target and prognostic in patients with NPC.

Inhibition of long noncoding RNA HIF1A-AS2 confers protection against atherosclerosis via ATF2 downregulation.

INTRODUCTION: In atherosclerotic lesions, extensive inflammation of the vessel wall contributes to plaque instability. Long noncoding RNAs (lncRNAs) play important roles in diverse biological processes in atherosclerosis. OBJECTIVES: Here, we aim to identify the functional role and regulatory mechanisms of lncRNA hypoxia-inducible factor 1 alpha-antisense RNA 2 (HIF1A-AS2) in atherosclerotic inflammation. METHODS: An atherosclerotic mouse model was induced in ApoE-/- mice by high fat diet (HFD). Endothelial cells (ECs), human aortic smooth muscle cells (SMCs) or human coronary artery endothelial cells (HCAECs) were exposed to ox-LDL to develop the in vitro model. The effects of lncRNA HIF1A-AS2 on inflammation were evaluated by determining levels of inflammatory factors tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) and levels of adhesion molecules vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), and macrophage cationic peptide 1 (MCP-1). RESULTS: It was established that lncRNA HIF1A-AS2 and ATF2 were highly expressed in atherosclerotic ApoE-/- mice. Downregulating lncRNA HIF1A-AS2 in ox-LDL-exposed ECs, SMCs and HCAECs inhibited inflammation by reducing levels of pro-inflammatory factors and adhesion molecules. LncRNA HIF1A-AS2 bound to the transcription factor USF1 to elevate ATF2 expression. USF1 overexpression counteracted the suppressive effect of lncRNA HIF1A-AS2 silencing on ox-LDL-induced inflammation. Knockdown of lncRNA HIF1A-AS2 or ATF2 could also attenuate inflammation in atherosclerotic mice. Collectively, the present study demonstrates that downregulation of lncRNA HIF1A-AS2 represses the binding of USF1 to the ATF2 promoter region and then inhibits ATF2 expression, thereby suppressing atherosclerotic inflammation. CONCLUSION: This study suggests lncRNA HIF1A-AS2 as an promising therapeutic target for atherosclerosis.

Lin28 promotes dental pulp cell proliferation via upregulation of cyclin-dependent proteins and interaction with let-7a/IGF2BP2 pathways.

Caries, pulpitis, and trauma are the main causes of dental pulp damage. The regeneration capacity of dental pulp declines with age. Lin28 is a conserved RNA-binding protein in higher eukaryotes that regulates several important cellular functions associated with development, glucose metabolism, differentiation, and pluripotency. Conditional reactivation of Lin28 gene in adult mice markedly accelerates the wound-healing process in injured digits. However, little is known about its functions and molecular mechanism in human dental pulp. The aim of this study was to investigate the effects and mechanism of overexpression of Lin28 gene on the proliferation of human dental pulp cells (HDPCs). For this purpose, a number of molecular and biochemical analytical techniques, including the ethynyl-2'-deoxyuridine (EdU) incorporation assay, RNA-protein immunoprecipitation (RIP) analysis, and luciferase assays, were used for detailed characterization. In addition, factors regulating HDPCs activation were explored through gain-of-function and loss-of-function analyses. The results demonstrate that Lin28 promotes cell proliferation and the S-G2/M transition of HDPCs and directly binds to a group of cell cycle regulatory mRNAs in HDPCs. Through bioinformatics analysis and luciferase assays, we confirmed that let-7a targets IGF2BP2. Silencing of IGF2BP2 showed similar cellular and molecular effects as let-7a. Similarly, restoration of IGF2BP2 counteracted the effects of let-7a expression. In conclusion, Lin28 promotes cell proliferation by regulation of both mRNA translation (let-7-independent) and miRNA biogenesis (let-7-dependent). Lin28 can promote the expression of pro-proliferative genes by directly enhancing their translation to maintain a tight control over HDPC proliferation.

Calcinosis and malignancy are rare in Chinese adult patients with myositis and nuclear matrix protein 2 antibodies identified by an unlabeled immunoprecipitation assay.

Autoantibodies in patients with myositis may associate with specific clinical manifestations. This study aimed to identify a subset of patients with myositis carrying antinuclear matrix protein 2 (anti-NXP-2) antibodies using an unlabeled immunoprecipitation (IP) assay, and clarify the features of these patients in a Chinese cohort. We developed novel methods for unlabeled protein IP and immunoblotting of Myc-tagged truncated NXP-2 fragments for anti-NXP-2 detection. The sera of 120 Chinese adult patients with myositis were screened for anti-NXP-2 by IP and immunoblot. Anti-NXP-2 antibodies were detected in 10 of the 120 patients (8.3%) using the established unlabeled protein IP and immunoblotting, with 70% (7/10) exhibiting either heliotrope rash or Gottron's papules. All 10 anti-NXP-2-positive patients exhibited myopathy and 60% complained of dysphagia. Severe diffuse calcinosis (10%) and nasopharyngeal carcinoma (10%) were each only present in single anti-NXP-2-positive patients with myositis. Antibodies against Ro-52 were found in four living but not in three deceased anti-NXP-2-positive patients. A comprehensive review of 13 anti-NXP-2 studies demonstrated markedly lower anti-NXP-2 prevalence among adult patients with myositis and lower association of anti-NXP-2 with calcinosis in Japan, China, and Hungary than in the USA and Italy. Anti-NXP-2 antibody association with internal malignancy in adult patients varied from 0 to 50% across different studies. A novel IP assay was developed to detect patients with myositis expressing anti-NXP-2. Calcinosis and malignancy are rare in Chinese adult patients with myositis positive for anti-NXP-2. Literature review indicated highest anti-NXP-2 prevalence and association of anti-NXP-2 with calcinosis in US and Italian myositis cohorts.