Anti-inflammatory role of APRIL by modulating regulatory B cells in antigen-induced arthritis.

APRIL (A Proliferation-Inducing Ligand), a member of the TNF superfamily, was initially described for its ability to promote proliferation of tumor cells in vitro. Moreover, this cytokine has been related to the pathogenesis of different chronic inflammatory diseases, such as rheumatoid arthritis. This study aimed to evaluate the ability of APRIL in regulating B cell-mediated immune response in the antigen-induced arthritis (AIA) model in mice. AIA was induced in previously immunized APRIL-transgenic (Tg) mice and their littermates by administration of antigen (mBSA) into the knee joints. Different inflammatory cell populations in spleen and draining lymph nodes were analyzed using flow cytometry and the assay was performed in the acute and chronic phases of the disease, while cytokine levels were assessed by ELISA. In the acute AIA, APRIL-Tg mice developed a less severe condition and a smaller inflammatory infiltrate in articular tissues when compared with their littermates. We also observed that the total cellularity of draining lymph nodes was decreased in APRIL-Tg mice. Flow cytometry analysis revealed an increase of CD19+IgM+CD5+ cell population in draining lymph nodes and an increase of CD19+CD21hiCD23hi (B regulatory) cells in APRIL-Tg mice with arthritis as well as an increase of IL-10 and CXCL13 production in vitro.

Probiotics induce intestinal IgA secretion in weanling mice potentially through promoting intestinal APRIL expression and modulating the gut microbiota composition.

Intestinal infections are strongly associated with infant mortality, and intestinal immunoglobulin A (IgA) is important to protect infants from intestinal infections after weaning. This study aims to screen probiotics that can promote the production of intestinal IgA after weaning and further explore their potential mechanisms of action. In this study, probiotics promoting intestinal IgA production were screened in weanling mouse models. The results showed that oral administration of Bifidobacterium bifidum (B. bifidum) FL228.1 and Bifidobacterium bifidum (B. bifidum) FL276.1 significantly enhanced IgA levels in the small intestine and upregulated the expression of a proliferation-inducing ligand (APRIL) and its upstream regulatory factor toll-like receptor 4 (TLR4). Furthermore, B. bifidum FL228.1 upregulated the relative abundance of Lactobacillus, while B. bifidum FL276.1 increased the relative abundance of Marvinbryantia and decreased Mucispirillum, further elevating intestinal IgA levels. In summary, B. bifidum FL228.1 and B. bifidum FL276.1 can induce IgA production in the intestinal tract of weanling mice by promoting intestinal APRIL expression and mediating changes in the gut microbiota, thus playing a significant role in enhancing local intestinal immunity in infants.

A Phase 2 Trial of Sibeprenlimab in Patients with IgA Nephropathy.

BACKGROUND: A proliferation-inducing ligand (APRIL) is implicated in the pathogenesis of IgA nephropathy. Sibeprenlimab is a humanized IgG2 monoclonal antibody that binds to and neutralizes APRIL. METHODS: In this phase 2, multicenter, double-blind, randomized, placebo-controlled, parallel-group trial, we randomly assigned adults with biopsy-confirmed IgA nephropathy who were at high risk for disease progression, despite having received standard-care treatment, in a 1:1:1:1 ratio to receive intravenous sibeprenlimab at a dose of 2, 4, or 8 mg per kilogram of body weight or placebo once monthly for 12 months. The primary end point was the change from baseline in the log-transformed 24-hour urinary protein-to-creatinine ratio at month 12. Secondary end points included the change from baseline in the estimated glomerular filtration rate (eGFR) at month 12. Safety was also assessed. RESULTS: Among 155 patients who underwent randomization, 38 received sibeprenlimab at a dose of 2 mg per kilogram, 41 received sibeprenlimab at a dose of 4 mg per kilogram, 38 received sibeprenlimab at a dose of 8 mg per kilogram, and 38 received placebo. At 12 months, the geometric mean ratio reduction (±SE) from baseline in the 24-hour urinary protein-to-creatinine ratio was 47.2±8.2%, 58.8±6.1%, 62.0±5.7%, and 20.0±12.6% in the sibeprenlimab 2-mg, 4-mg, and 8-mg groups and the placebo group, respectively. At 12 months, the least-squares mean (±SE) change from baseline in eGFR was -2.7±1.8, 0.2±1.7, -1.5±1.8, and -7.4±1.8 ml per minute per 1.73 m2 in the sibeprenlimab 2-mg, 4-mg, and 8-mg groups and the placebo group, respectively. The incidence of adverse events that occurred after the start of administration of sibeprenlimab or placebo was 78.6% in the pooled sibeprenlimab groups and 71.1% in the placebo group. CONCLUSIONS: In patients with IgA nephropathy, 12 months of treatment with sibeprenlimab resulted in a significantly greater decrease in proteinuria than placebo. (Funded by Visterra; ENVISION ClinicalTrials.gov number, NCT04287985; EudraCT number, 2019-002531-29.).

TACI and endogenous APRIL in B cell maturation.

While many of the genes and molecular pathways in the germinal center B cell response which initiate protective antibody production are known, the contributions of individual molecular players in terminal B cell differentiation remain unclear. We have previously investigated how mutations in TACI gene, noted in about 10% of patients with common variable immunodeficiency, impair B cell differentiation and often, lead to lymphoid hyperplasia and autoimmunity. Unlike mouse B cells, human B cells express TACI-L (Long) and TACI-S (Short) isoforms, but only TACI-S promotes terminal B cell differentiation into plasma cells. Here we show that the expression of intracellular TACI-S increases with B cell activation, and colocalizes with BCMA and their ligand, APRIL. We show that the loss of APRIL impairs isotype class switch and leads to distinct metabolic and transcriptional changes. Our studies suggest that intracellular TACI-S and APRIL along with BCMA direct long-term PC differentiation and survival.

Features of Isoforms of Human Soluble TACI.

The BAFF/APRIL-system with the two cytokines BAFF and APRIL and their three receptors, transmembrane activator and CAML interactor (TACI), BAFF receptor, and B-cell maturation Ag, is important for B cell maintenance. The BAFF/APRIL system is a therapeutic target in B cell-derived malignancies and autoimmune diseases. However, unexpected outcomes of clinical trials with atacicept (TACI-Fc) underline our incomplete understanding of this system. Shedding of the three receptors is one important regulatory element. In humans, TACI exists in two isoforms generated through alternative splicing in their extracellular portion: TACI-long (l) has two cysteine-rich domains, whereas TACI-short (s) lacks the first low-affinity one. In this study, we discriminated soluble (s) forms of TACI-l and TACI-s with newly generated mAbs and found that both were spontaneously released from activated human B cells, with a predominance of sTACI-l. Furthermore, sTACI-l was also the dominant isoform in human serum. Vaccination with the mRNA vaccine from BioNTech does not significantly affect the serum levels of sTACI-l. Both TACI-s and TACI-l were shed by a disintegrin and metalloproteinase domain-containing protein 10. TACI-l and TACI-s formed homo- and hetero-oligomers in soluble and membrane-bound forms. Both sTACI-l and sTACI-s acted as decoy receptors for BAFF, but only sTACI-l also efficiently inhibited APRIL. Dimerization of sTACI-l enhanced its decoy functions only slightly. Together, we extend our knowledge of the complexity of the BAFF/APRIL system by identifying and characterizing the two soluble isoforms of TACI.

BAFF and APRIL counterregulate susceptibility to inflammation-induced preterm birth.

Clinical evidence points to a function for B cell-activating factor (BAFF) in pregnancy. However, direct roles for BAFF-axis members in pregnancy have not been examined. Here, via utility of genetically modified mice, we report that BAFF promotes inflammatory responsiveness and increases susceptibility to inflammation-induced preterm birth (PTB). In contrast, we show that the closely related A proliferation-inducing ligand (APRIL) decreases inflammatory responsiveness and susceptibility to PTB. Known BAFF-axis receptors serve a redundant function in signaling BAFF/APRIL presence in pregnancy. Treatment with anti-BAFF/APRIL monoclonal antibodies or BAFF/APRIL recombinant proteins is sufficient to manipulate susceptibility to PTB. Notably, macrophages at the maternal-fetal interface produce BAFF, while BAFF and APRIL presence divergently shape macrophage gene expression and inflammatory function. Overall, our findings demonstrate that BAFF and APRIL play divergent inflammatory roles in pregnancy and provide therapeutic targets for mitigating risk of inflammation-induced PTB.

Spatial heterogeneity for APRIL production by eosinophils in the small intestine.

Eosinophils may reside in the lower intestine to play several homeostatic functions. Regulation of IgA+ plasma-cell (PC) homeostasis is one of these functions. Here, we assessed regulation of expression for a proliferation-inducing ligand (APRIL), a key factor from the TNF superfamily for PC homeostasis, in eosinophils from the lower intestine. We observed a strong heterogeneity, since duodenum eosinophils did not produce APRIL at all, whereas a large majority of eosinophils from the ileum and right colon produced it. This was evidenced both in the human and mouse adult systems. At these places, the human data showed that eosinophils were the only cellular sources of APRIL. The number of IgA+ PCs did not vary along the lower intestine, but ileum and right colon IgA+ PC steady-state numbers significantly diminished in APRIL-deficient mice. Use of blood cells from healthy donors demonstrated that APRIL expression in eosinophils is inducible by bacterial products. Use of germ-free and antibiotics-treated mice confirmed the dependency on bacteria for APRIL production by eosinophils from the lower intestine. Taken together, our study shows that APRIL expression by eosinophils is spatially regulated in the lower intestine with a consequence on the APRIL dependency for IgA+ PC homeostasis.

Developing high-affinity decoy receptors to treat multiple myeloma and diffuse large B cell lymphoma.

Disease relapse and treatment-induced immunotoxicity pose significant clinical challenges for patients with hematological cancers. Here, we reveal distinctive requirements for neutralizing TNF receptor ligands APRIL and BAFF and their receptor activity in MM and DLBCL, impacting protein translation and production in MM cells and modulating the translation efficiency of the ATM interactor (ATMIN/ACSIZ). Therapeutically, we investigated the use of BCMA decoy receptor (sBCMA-Fc) as an inhibitor of APRIL and BAFF. While wild-type sBCMA-Fc effectively blocked APRIL signaling in MM, it lacked activity in DLBCL due to its weak BAFF binding. To expand the therapeutic utility of sBCMA-Fc, we engineered an affinity-enhanced mutant sBCMA-Fc fusion molecule (sBCMA-Fc V3) 4- and 500-fold stronger in binding to APRIL and BAFF, respectively. The mutant sBCMA-Fc V3 clone significantly enhanced antitumor activity against both MM and DLBCL. Importantly, we also demonstrated an adequate toxicity profile and on-target mechanism of action in nonhuman primate studies.

Protein and functional isoform levels and genetic variants of the BAFF and APRIL pathway components in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is the prototype of an autoimmune disease. Belimumab, a monoclonal antibody targets BAFF, is the only biologic approved for SLE and active lupus nephritis. BAFF is a cytokine with a key-regulatory role in the B cell homeostasis, which acts by binding to three receptors: BAFF-R, TACI and BCMA. TACI and BCMA also bind APRIL. Many studies reported elevated soluble BAFF and APRIL levels in the sera of SLE patients, but other questions about the role of this system in the disease remain open. The study aimed to investigate the utility of the cytokine levels in serum and urine as biomarkers, the role of non-functional isoforms, and the association of gene variants with the disease. This case-control study includes a cohort (women, 18-60 years old) of 100 patients (48% with nephritis) and 100 healthy controls. We used ELISA assays to measure the cytokine concentrations in serum (sBAFF and sAPRIL) and urine (uBAFF and uAPRIL); TaqMan Gene Expression Assays to quantify the relative mRNA expression of ΔBAFF, ßAPRIL, and εAPRIL, and next-generation sequencing to genotype the cytokine (TNFSF13 and TNFSF13B) and receptor (TNFRSF13B, TNFRSF17 and TNFRSF13C) genes. The statistical tests used were: Kruskal-Wallis (qualitative variables), the Spearman Rho coefficient (correlations), the Chi-square and SKAT (association of common and rare genetic variants, respectively). As expected, sBAFF and sAPRIL levels were higher in patients than in controls (p ≤ 0.001) but found differences between patient subgroups. sBAFF and sAPRIL significantly correlated only in patients with nephritis (rs = 0.67, p ≤ 0.001) and ßAPRIL levels were lower in patients with nephritis (p = 0.04), and ΔBAFF levels were lower in patients with dsDNA antibodies (p = 0.04). Rare variants of TNFSF13 and TNFRSF13B and TNFSF13 p.Gly67Arg and TNFRSF13B p.Val220Ala were associated with SLE. Our study supports differences among SLE patient subgroups with diverse clinical features in the BAFF/APRIL pathway. In addition, it suggests the involvement of genetic variants in the susceptibility to the disease.

TNFSF13 Is a Novel Onco-Inflammatory Marker and Correlates With Immune Infiltration in Gliomas.

Existing therapeutic strategies for gliomas are restricted; hence, exploration for novel diagnostic indicator and treatment is essential. Here, we performed bioinformatic analyses for TNFSF13 (also known as APRIL), a proliferation-inducing ligand of the tumor necrosis factor (TNF) superfamily, aiming to assess its potential for predicting glioma patient's prognosis and targeted therapy. TNFSF13 expression was upregulated in the increase of tumor grades based on Xiangya cohort. In high TNFSF13 gliomas, somatic mutation was proved to correlate with amplification of EGFR and deletion of CDKN2A; while mutation of IDH1 was more frequently observed in low TNFSF13 group. We also confirmed the positive correlation between TNFSF13 and infiltrating immune and stromal cells in glioma microenvironment. Further, TNFSF13 was found to be involved in immunosuppression via diverse immunoregulation pathways and was associated with other immune checkpoints and inflammation. Single-cell sequencing revealed an abundant expression of TNFSF13 in neoplastic cells and M2 macrophages, which TNFSF13 might potentially regulate the cell communication via IL-8, C3, and CD44. Lastly, TNFSF13 mediated the activities of transcription factors including FOXO3, MEIS2, and IRF8. Our analyses demonstrated the relevance between TNFSF13 and glioma progress and indicated the potential of TNFSF13 as a novel diagnostic onco-inflammatory biomarker and immunotherapy target of gliomas.

Circulating CD138 enhances disease progression by augmenting autoreactive antibody production in a mouse model of systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a progressive autoimmune disease characterized by high levels of antibodies directed against nuclear antigens. Elevated serum CD138, a heparan sulfate-bearing proteoglycan, correlates with increased disease activity in patients with SLE, but the contribution of CD138 to lupus disease is not known. Corroborating patient data, we detected an increase in serum CD138 in MRL/MpJ-Faslpr/J (MRL/Lpr) mice (a model for SLE disease) parallel to disease activity. Although T-cell receptor ß (TCRß)+CD138+ T cells typically expand in MRL/Lpr mice as the disease progresses, surprisingly, TCRß+CD138- cells were the primary source of circulating CD138, as the transfer of TCRß+CD138- cells, but not TCRß+CD138+ cells, to young MRL/Lpr mice resulted in higher serum CD138 in the recipients. We found that trypsin was able to cleave CD138 from TCRß+CD138+ cells, and that trypsin was highly expressed in TCRß+CD138- cells. Moreover, trypsin inhibitors, the "defined trypsin inhibitor" and leupeptin, increased CD138 expression on TCRß+CD138- cells, suggesting a contribution of cleaved CD138 to the increase in blood CD138 levels. Furthermore, soluble CD138 was able to bind "a proliferation-inducing ligand" (APRIL) and enhance APRIL-mediated plasma cell generation and autoreactive antibody production through the phosphorylation of extracellular signal-regulated kinase in B cells. The APRIL receptor "transmembrane activator, calcium modulator, and cyclophilin ligand interactor" was involved in the enhancement of APRIL activity by CD138, as the synergistic effect of APRIL and CD138 was ablated in transmembrane activator, calcium modulator, and cyclophilin ligand interactor-deficient B cells. These findings indicate a regulatory role for soluble CD138 in B-cell differentiation and autoreactive antibody production in SLE disease.

The number 13 of the family: a proliferation inducing ligand.

The TNF superfamily member a proliferation inducing ligand (APRIL, TNFSF13) plays a late role in humoral immunity at the level of antibody-producing plasmocytes. The recent characterization of the first immunodeficient patient with an inactivating mutation in the APRIL gene provided the last piece of functional data lacking in the human system. Based on this function, APRIL has been considered as a valuable target to dampen unwanted antibody production. After reviewing the late data acquired on the physiological function of APRIL in humoral immunity, we will here review the state of the art regarding APRIL targeting in autoimmune diseases.

BAFF, APRIL and BAFFR on the pathogenesis of Immunoglobulin-A vasculitis.

BAFF, APRIL and BAFF-R are key proteins involved in the development of B-lymphocytes and autoimmunity. Additionally, BAFF, APRIL and BAFFR polymorphisms were associated with immune-mediated conditions, being BAFF GCTGT>A a shared insertion-deletion genetic variant for several autoimmune diseases. Accordingly, we assessed whether BAFF, APRIL and BAFFR represent novel genetic risk factors for Immunoglobulin-A vasculitis (IgAV), a predominantly B-lymphocyte inflammatory condition. BAFF rs374039502, which colocalizes with BAFF GCTGT>A, and two tag variants within APRIL (rs11552708 and rs6608) and BAFFR (rs7290134 and rs77874543) were genotyped in 386 Caucasian IgAV patients and 806 matched healthy controls. No genotypes or alleles differences were observed between IgAV patients and controls when BAFF, APRIL and BAFFR variants were analysed independently. Likewise, no statistically significant differences were found in the genotype and allele frequencies of BAFF, APRIL or BAFFR when IgAV patients were stratified according to the age at disease onset or to the presence/absence of gastrointestinal (GI) or renal manifestations. Similar results were disclosed when APRIL and BAFFR haplotypes were compared between IgAV patients and controls and between IgAV patients stratified according to the clinical characteristics mentioned above. Our results suggest that BAFF, APRIL and BAFFR do not contribute to the genetic network underlying IgAV.

A BAFF/APRIL axis regulates obesogenic diet-driven weight gain.

The impact of immune mediators on weight homeostasis remains underdefined. Interrogation of resistance to diet-induced obesity in mice lacking a negative regulator of Toll-like receptor signaling serendipitously uncovered a role for B cell activating factor (BAFF). Here we show that overexpression of BAFF in multiple mouse models associates with protection from weight gain, approximating a log-linear dose response relation to BAFF concentrations. Gene expression analysis of BAFF-stimulated subcutaneous white adipocytes unveils upregulation of lipid metabolism pathways, with BAFF inducing white adipose tissue (WAT) lipolysis. Brown adipose tissue (BAT) from BAFF-overexpressing mice exhibits increased Ucp1 expression and BAFF promotes brown adipocyte respiration and in vivo energy expenditure. A proliferation-inducing ligand (APRIL), a BAFF homolog, similarly modulates WAT and BAT lipid handling. Genetic deletion of both BAFF and APRIL augments diet-induced obesity. Lastly, BAFF/APRIL effects are conserved in human adipocytes and higher BAFF/APRIL levels correlate with greater BMI decrease after bariatric surgery. Together, the BAFF/APRIL axis is a multifaceted immune regulator of weight gain and adipose tissue function.

The Ancient Cytokine BAFF- and APRIL-like Molecule Regulates the Functionality of Teleost IgM+ B Cells Similarly to BAFF and APRIL.

TNF superfamily (TNFSF) members, such as BAFF and a proliferation-inducing ligand (APRIL), emerged in vertebrates as key regulators of B cell homeostasis and activation. Many cartilaginous and teleost fish contain an additional gene, designated as BAFF- and APRIL-like molecule (BALM), of unknown function and lost in tetrapods. In this study, we have performed a wide characterization of the functions of BALM on naive B cells for the first time, to our knowledge, in teleosts using rainbow trout (Oncorhynchus mykiss) as a model. Similar to BAFF and APRIL, BALM increased the survival and promoted the proliferation of peripheral blood IgM+ B cells and cooperated with BCR cross-linking to increase the proliferation rate of IgM+ B cells. BALM also seemed to be a differentiating factor for trout IgM+ B cells, as it increased IgM secretion and increased cell size. Additionally, BALM appeared to increase the Ag-presenting properties of IgM+ B cells, augmenting MHC class II surface expression and upregulating the phagocytic capacity of these cells. Finally, the fact that there was no synergy between BALM and BAFF/APRIL in any of these functions strongly suggests that BALM signals through the same receptors as BAFF and APRIL to carry out its functions. This hypothesis was further supported in competitive BALM binding assays. The results presented provide relevant information for understanding how these TNFSF members cooperate in teleost fish to regulate B cell functionality, helping us to interpret the evolutionary relations between molecules of this family.

Involvement of APRIL in Helicobacter pylori-related gastric cancer.

BACKGROUND/AIMS: A proliferation-inducing ligand (APRIL, also known as TNFSF13, CD256) is a member of the tumor necrosis factor (TNF) superfamily and involved in a diverse set of diseases. In this work, we explored the potential associations and underlying mechanism in patients suffered from gastric cancer between the expression of APRIL and H. pylori infection. METHODS: We analyzed APRIL expression levels in 200 GC tissue samples by immunohistochemistry staining. H. pylori infection was detected by modified Giemsa staining. The biological effects of APRIL on human GC cells in vitro and in vivo were tested by CCK-8 assay, colony formation, flow cytometry detection, transwell migration assay, matrigel invasion assay, and tumor xenograft assay in animals. RESULTS: APRIL reactivity was positively correlated with H. pylori infection in vitro and vivo. It turned out that the decrease of miR-145 expression was dose-dependent and time-dependent on H. pylori infection and in consistent with APRIL expression. MiR-145 significantly attenuated the effect of H. pylori infection on APRIL gene expression in SGC7901 and BGC823 cell lines. Furthermore, APRIL overexpression promoted the proliferation, migration, invasion, and transfer of GC cells and decreased apoptosis, while APRIL knockdown suppressed these effects. We confirmed that APRIL activated the canonical NF-κB pathway through phosphorylation of AKT. CONCLUSION: The expression of APRIL, which promoted the proliferation, migration, invasion, viability, and metastasis of GC cells, was upregulated in human H. pylori-infected GC through miR-145. Besides, APRIL-induced gastric tumorigenicity via activating NF-κB pathway. These results may provide a framework for the deeper analysis of APRIL in GC risk and prognosis.

Aberrant Expression of a Proliferation-Inducing Ligand Underlies Autoimmune Mechanisms in Immune Thrombocytopenia.

PURPOSE: To study the relationship between surface membrane-bound APRIL and ITP. METHODS: The peripheral blood of all subjects, 50 patients diagnosed with ITP and 25 healthy controls, was collected. Flow cytometry was used to detect the expression of membrane-bound APRIL on immune cells and platelets. ELISA was used to detect the content of soluble APRIL in plasma. RESULTS: Membrane-bound APRIL was only expressed on the surface of platelets in both ITP patients and controls. APRIL expression on the platelet surface was significantly lower in newly diagnosed (P < 0.001) and chronic (P < 0.001) ITP patients than in controls. Platelet surface APRIL level was significantly enhanced in patients with complete remission after treatment (P = 0.02) but not in those with no response after treatment. Platelet surface APRIL level in ITP patients was negatively correlated with serum APRIL level (r = -0.09765, P = 0.0424). CONCLUSIONS: Platelet surface APRIL may play a key immunoregulative role. Platelet surface APRIL is likely to be one source of the excessive serum APRIL in ITP patients. The effectiveness of treatment may be measured by determining the platelet surface APRIL levels in ITP patients.

Plasma cell dynamics in the bone marrow niche.

Using intravital imaging, we report that bone marrow (BM) plasma cells (PCs) are motile. BM PCs exhibit a unique migration pattern, characterized by intermittent periods of high motility and longer stretches of confined migration or arrest. BM PCs accumulate into clusters, which have reduced cell motility. APRIL promotes cluster formation and overall PC motility in the BM. Although CXCL12 and its receptor, CXCR4, promote PC motility in the BM, VLA4 activity promotes arrest. However, blocking either pathway promotes PC egress from the BM. Under steady-state conditions, BM PCs recirculate to other bones and spleen. In older mice, overall PC motility and recirculation increase, and this is correlated with increased CXCR4 expression, which depends on PC age or maturation rather than mouse age. Altogether, these results suggest that changes in PC motility and CXCR4 expression are linked with survival of long-lived PCs in the BM.

Diagnostic Value of Plasma miR-145 and miR-185 as Targeting of the APRIL Oncogene in the B-cell Chronic Lymphocytic Leukemia.

BACKGROUND: Chronic lymphocytic leukemia (CLL) is one of the most common hematologic malignancy in adults worldwide. This cancer has a poor prognosis at different stages. So, the identification of new biomarkers is important for diagnosis of B-CLL. Considering the oncogenic role of APRIL molecule in this leukemia as well as the regulatory role of miRNAs in different signaling pathways, the present study evaluated the miRNAs targeting APRIL gene in B-CLL. METHODS: The miRNAs were predicted and selected using bioinformatics algorithms. A total of 80 plasma samples were subjected to RNA extraction and synthesis of cDNA. The expressions levels of predicted miRNAs and APRIL gene in plasma of B-CLL patients and healthy individuals were assessed by Real time PCR analysis. ROC analysis was performed to investigate the role predicted miRNAs as novel biomarkers in diagnosis of B-CLL. RESULTS: The results of the prediction showed that miR-145-5p and miR-185-5p target the APRIL gene. The expression level of APRIL gene was strikingly higher in plasma of B-CLL patients than in the healthy individuals (102, P= 0.001). On the other hand, expression levels of miR-145-5p and miR-185-5p were strikingly lower in B-CLL patients than in the healthy individuals (0.07, P= 0.001) (0.29, P= 0.001). Also, ROC curve analyses demonstrated that miR-145-5p and miR-185-5p are specific and sensitive and may serve as new biomarkers for the detection of B-CLL. (AUC; 0.95, sensitivity; %90) (AUC; 0.87, sensitivity; %63). CONCLUSION: These data suggest that miR-145-5p and miR-185-5p target the APRIL gene and might have a role in diagnosis of B-CLL. Therefore, these two miRNAs can be served as a novel and potential biomarker for detection of B-CLL.

Circular ANRIL isoforms switch from repressors to activators of p15/CDKN2B expression during RAF1 oncogene-induced senescence.

Long non-coding RNAs (ncRNAs) are major regulators of gene expression and cell fate. The INK4 locus encodes the tumour suppressor proteins p15INK4b, p16INK4a and p14ARF required for cell cycle arrest and whose expression increases during senescence. ANRIL is a ncRNA antisense to the p15 gene. In proliferative cells, ANRIL prevents senescence by repressing INK4 genes through the recruitment of Polycomb-group proteins. In models of replicative and RASval12 oncogene-induced senescence (OIS), the expression of ANRIL and Polycomb proteins decreases, thus allowing INK4 derepression. Here, we found in a model of RAF1 OIS that ANRIL expression rather increases, due in particular to an increased stability. This led us to search for circular ANRIL isoforms, as circular RNAs are rather stable species. We found that the expression of two circular ANRIL increases in several OIS models (RAF1, MEK1 and BRAF). In proliferative cells, they repress p15 expression, while in RAF1 OIS, they promote full induction of p15, p16 and p14ARF expression. Further analysis of one of these circular ANRIL shows that it interacts with Polycomb proteins and decreases EZH2 Polycomb protein localization and H3K27me3 at the p15 and p16 promoters, respectively. We propose that changes in the ratio between Polycomb proteins and circular ANRIL isoforms allow these isoforms to switch from repressors of p15 gene to activators of all INK4 genes in RAF1 OIS. Our data reveal that regulation of ANRIL expression depends on the senescence inducer and underline the importance of circular ANRIL in the regulation of INK4 gene expression and senescence.