Meta-analysis Followed by Replication Identifies Loci in or near CDKN1B, TET3, CD80, DRAM1, and ARID5B as Associated with Systemic Lupus Erythematosus in Asians

Systemic lupus erythematosus (SLE) is a prototype autoimmune disease with a strong genetic involvement and ethnic differences. Susceptibility genes identified so far only explain a small portion of the genetic heritability of SLE, suggesting that many more loci are yet to be uncovered for this disease. In this study, we performed a meta-analysis of genome-wide association studies on SLE in Chinese Han populations and followed up the findings by replication in four additional Asian cohorts with a total of 5,365 cases and 10,054 corresponding controls. We identified genetic variants in or near CDKN1B, TET3, CD80, DRAM1, and ARID5B as associated with the disease. These findings point to potential roles of cell-cycle regulation, autophagy, and DNA demethylation in SLE pathogenesis. For the region involving TET3 and that involving CDKN1B, multiple independent SNPs were identified, highlighting a phenomenon that might partially explain the missing heritability of complex diseases

An artificial miRNA against HPSE suppresses melanoma invasion properties, correlating with a down-regulation of chemokines and MAPK phosphorylation.

Ribonucleic acid interference (RNAi) based on microRNA (miRNA) context may provide an efficient and safe therapeutic knockdown effect and can be driven by ribonucleic acid polymerase II (RNAP II). In this study, we designed and synthesized miR155-based artificial miRNAs against heparanase (HPSE) constructed with BLOCK-iT™ Pol II miR RNAi Expression Vector Kit. The expression levels of HPSE declined significantly in both the mRNA and protein levels in HPSE-miRNA transfected melanoma cells that exhibited reduction of adhesion, migration, and invasion ability in vitro and in vivo. We also observed that HPSE miRNA could inhibit the expressions of chemokines of interleukin-8 (IL8) and chemokine (C-X-C motif) ligand 1 (CXCL1), at both the transcriptional and translational levels. Further study on its probable mechanism declared that down-regulation of IL8 and CXCL1 by HPSE-miRNA may be correlated with reduced growth-factor simulated mitogen-activated kinase (MAPK) phosphorylation including p38 MAPK, c-Jun N-terminal kinase (JNK) and extracellular-signal-regulated kinase (ERK) 1 and 2, which could be rescued by miRNA incompatible mutated HPSE cDNA. In conclusion, we demonstrated that artificial miRNAs against HPSE might serve as an alterative mean of therapy to low HPSE expression and to block the adhesion, invasion, and metastasis of melanoma cells. Furthermore, miRNA-based RNAi was also a powerful tool for gene function study

Development and validation of the AF score for diagnosis of adult-onset Still's disease in fever of unknown origin

Objective: To develop and validate a diagnostic score to identify adult-onset Still's disease (AOSD) in fever of unknown origin (FUO). Methods: A single center, retrospective case-control study of inpatients with FUO from January 2018 to December 2021. Using clinical and laboratory data from 178 cases with AOSD and 486 cases with FUO, we developed an AOSD/FUO (AF) score with a Bayesian Model Averaging approach. AF score and Yamaguchi's criteria were evaluated by sensitivity, specificity, accuracy, and positive/negative predictive value for diagnosis of AOSD in developmental and validation samples. Results: Persistent pruritic eruptions (PPEs) in patients with rashes was higher in AOSD group than FUO group (52.3% vs 7.4%; P < 0.01). PPEs yielded a specificity of 97.5% and a sensitivity of 44.9%. AF score = PPEs × 3.795+Evanescent rash × 2.774+Serum ferritin × 1.678+Myalgia × 0.958+Neutrophil count × 0.185+Platelet count × 0.004. A cut-off value ≥ 5.245 revealed the maximizing sensitivity of 88.7% and specificity of 95.8% in discriminating AOSD from FUO in the validation group. And AF score improved the accuracy from 82.6% to 93.3% compared with Yamaguchi's criteria. Conclusions: We developed and validated a new score which can identify AOSD in FUO with higher classification accuracy than Yamaguchi's criteria. Future multi-centric prospective studies need to be designed to confirm the diagnosis value of AF score

HPSE miRNAs inhibited expression of <i>IL8</i> and <i>CXCL1</i> and its probable mechanism.

<p>(A) Differentially expressed chemokine genes in the HPSE-miRNA1 and HPSE-miRNA2 groups compared to the Neg-miRNA group (log₂ ratio≥1 or log₂ ratio≤−1 and <i>P</i><0.05). (B) Pathways including chemokines activity modulated by HPSE-miRNA1 or HPSE-miRNA2 were confirmed to be significant by gene-set enrichment analysis (<i>P</i><0.05). (C) Both the mRNA and protein levels of <i>IL8</i> and <i>CXCL1</i> in HPSE miRNA transfected A375 cells were decreased compared to either control group. (^†<i>P</i><0.001, compared with the parental cells; *<i>P</i><0.001, compared with the Neg-miRNA transfected cells). (D) Attenuation of the HPSE-induced phosphorylation of MAPKs by HPSE miRNA. Phosphorylation of MAPK p38 (second and third panel), JNK/SAPK (fourth and fifth panel), and ERK1/2 (sixth and seventh panel) was monitored by western blotting.</p

Effects of HPSE miRNAs on A375 cells <i>in vitro</i>.

<p>A375 cells transfected stably with HPSE-miRNA1, HPSE-miRNA2 or Neg-miRNA were selected by blasticidin. (A-B) Flow cytometric results showed that the percentages of apoptotic cells in both HPSE-miRNA groups were not different from those in the parental cells or the Neg-miRNA group (<i>P</i>>0.05). (C) MTT assay showed that HPSE miRNA attenuated the cellular viability of A375 cells at both 24 hours and 48 hours, compared to the parental cells or the Neg-miRNA transfected cells. (D) Cellular proliferation assay using cell counts showed that HPSE miRNA could also inhibit cellular proliferation of A375 cells at 48, 72 and 96 hours, compared to the parental cells or the Neg-miRNA transfected cells. (E) Cell-Matrigel adhesion assay. The adhesive ability of A375 cells transfected with HPSE miRNAs was obviously inhibited compared to control groups. (F) Diagram of migrative cells or invasive cells, as determined by the transwell migration assay or the Matrigel-invasion assay. The migrative or invasive number of A375 cells transfected with HPSE miRNAs was much less than that of either control group. (G) Representative images of migrative cells in the HPSE miRNAs groups or control groups in the transwell migration assay (H&E staining, magnification of 10×10). (H) Representative images of invasive cells in the HPSE miRNAs groups or control groups in Matrigel invasion assay(H&E staining, magnification of 10×10). (^†<i>P</i><0.05, compared with the parental cells; *<i>P</i><0.05, compared with the Neg-miRNA transfected cells).</p

Construction of of miR-155-based HPSE miRNAs and their impact on HPSE expression levels in A375 cells.

<p>A375 cells were transfected with HPSE-miRNAs or Neg-miRNA for 48 hours. (A) The sequences and predicted secondary structures of three designed pre-miRNAs targeting <i>HPSE</i> (HPSE-miRNA1, HPSE-miRNA2, and HPSE-miRNA3) and a negative control miRNA (Neg-miRNA), and the precise regions of the <i>HPSE</i> mRNA that they targeted. (B) Pre-miRNA double-stranded oligo inserted into the miRNA expression vector-pcDNA6.2-GW/EmGFP-miR. (C) Schematic representation of the process of ligation and transformation. (D) Inhibitory effects of HPSE miRNAs on HPSE protein expression. Representative blots are shown from three independent experiments with identical results. The expressions of HPSE protein in A375 cells transfected with HPSE miRNAs were obviously down-regulated obviously compared to the parental cells and the Neg-miRNA group. (E) Inhibitory effects of HPSE miRNAs on <i>HPSE</i> mRNA expression. Calculation of the respective <i>HPSE</i> mRNA expression in each group was relative to the Neg-miRNA group (%). Quantitative real-time PCR results showed that the expression of <i>HPSE</i> mRNA in A375 cells transfected with HPSE miRNAs were down-regulated compared to the parental cells or the Neg-miRNA transfected cells. (^†<i>P</i><0.05, compared with the parental cells; *<i>P</i><0.05, compared with the Neg-miRNA transfected cells).</p