LncRNA00492 is required for marginal zone B-cell development.

B-cell development undergoes a series of steps from the bone marrow to the secondary lymphoid organs. A defect in B-cell development can lead to immunodeficiency or malignant disorders, such as leukaemia or lymphoma. Long non-coding RNAs have been reported to act as important regulators of many pathological processes. However, very little is known regarding the role of lncRNAs during B-cell development and the regulation of their expression. In this study, we explored the expression and role of lncRNA Gme00492 in B-cell development. We observed that lnc00492 was highly expressed in B-cell development and primarily expressed in the nucleus. Lnc00492-deficient mice had fewer marginal zone B cells in the spleen, likely due to a developmental block. Importantly, lnc00492 interacts with CTBP1 and targets it for ubiquitination and degradation during B-cell development, whereas the transcriptional corepressor factor CTBP1 plays a critical role in Notch2 signalling. Thus, we identified a novel regulatory axis between lnc00492 and CTBP1 in B cells, suggesting that lnc00492 is essential for marginal zone B-cell development.

A novel BRET based genetic coded biosensor for apoptosis detection at deep tissue level in live animal.

ANNEXIN V belongs to a family of phospholipid binding proteins which is able to bind to negatively charged phospholipids such as phosphatidylserine (PS) in the presence of a high affinity Ca2+ ion. When apoptosis occurs, even at early stage, PS will be exposed to the outside of the cell surface from the cytoplasm side of membrane leaflets., Therefore ANNEXIN V has been suggested as a bio-marker for imaging early apoptotic events of various cell death including those in disease conditions. However, most ANNEXIN V-based apoptotic detecting techniques were in vitro approaches. Here, we presented a new BRET (Bioluminescence Resonance Energy Transfer) based genetic coded biosensor by fusing ANNEXIN V and a BRET version of NanoLuc (teLuc) for both in vitro and in vivo apoptosis detection. The BRET feature of this new sensor makes it convenient to be applied to both conventional fluorescent-based in vitro apoptosis detection and bioluminescence-based animal live imaging for in vivo study. Because of its robust bioluminescence signal, it is possible to perform the evaluation of the disease-induced apoptotic damage and recovery process directly at deep tissue level in live animal.

Down-regulation of LncRNA 2900052N01Rik inhibits LPS-induced B cell function in vitro.

B cells play a crucial role in immune responses. The main functions include B cell protective antibody production, inflammation reduction, activation and proliferation. Long non-coding RNAs (lncRNAs) have been reported to act as important regulators of many pathological processes. However, few lncRNAs have been reported to affect B cell function. In this study, we explored the expression and role of lncRNA 2900052N01Rik (lnc-290) in lipopolysaccharide (LPS)-induced B cells purified from mouse spleens in vitro. Here, we confirmed that lnc-290 was highly expressed in B cells stimulated by LPS. Knockdown of lnc-290 inhibited the expression of CD69/CD86 and the growth of B cells. Moreover, down-regulated lnc-290 reduced B cell differentiation and immunoglobulin production in vitro. In addition, we found that lnc-290 regulated LPS-induced B cell activation via the NF-κB/ERK pathways. Interestingly, abnormal lnc-290 expression did not alter the B cell activation or proliferation induced by IL-4 or CD40/CD40L. Accordingly, these results indicated, for the first time, that lnc-290 down-regulation inhibits LPS-induced B cell proliferation, activation and differentiation by blocking the LPS/TLR4 signaling pathway. Together, the in vitro data demonstrate that lnc-290 participated in the inflammation and tissue damage mediated by LPS-activated B cells.

MiR-315 is required for neural development and represses the expression of dFMR1 in Drosophila melanogaster.

AIM: The fragile X mental retardation protein (FMRP), the product of the FMR1 gene, is responsible for the fragile X syndrome (FXS). FMRP regulates miRNA expression and is involved in miRNA-mediated gene silencing. However, the question of whether FMRP is, in turn, regulated by miRNAs remains unanswered. MAIN METHODS: We detected the FMRP expression pattern by in situ hybridization. MiR-315 overexpression and knockout models were generated by germ-line transformation and ends-out homologous recombination, respectively. Western blotting and immunohistochemistry were used to detect Drosophila FMRP (dFMRP) and a Luciferase reporter assay was used to confirm the regulation of dfmr1 mRNA by mir-315. Synaptic structural quantification and electrophysiological methods were used to compare synaptic functions among groups. KEY FINDINGS: Here, we determined that the transcription product of dFMR1, the Drosophila homologue of FMR1, is a direct target of miR-315. MiR-315 is mainly expressed in the nervous system of Drosophila. Flies overexpressing miR-315 showed pupation defects and reduced hatching rates. A homozygous miR-315 knockout status is embryonic lethal in flies. These observations indicate that miR-315 is a key regulator of the Drosophila nervous system. Furthermore, computational prediction and cell-based luciferase and in vivo assays demonstrated that dfmr1 is directly targeted by miR-315. Lastly, using the neuromuscular junction as a model, we found that miR-315 regulates synaptic structure and transmission by targeting dfmr1. SIGNIFICANCE: These findings provide compelling evidence that miR-315 targets dfmr1 in the Drosophila nervous system, acting as a regulatory factor for the fine-tuned modulation of FMRP expression.

Circ_0005075 promotes hepatocellular carcinoma progression by suppression of microRNA-335.

Accumulating evidence suggests that noncoding RNAs play a vital role in cancer biology. Circular RNAs (circRNAs), a newly defined class of endogenously widespread noncoding RNAs, have been intensively reported to influence cell function and development, and even cancer prognosis by sponging microRNAs in various types of cancer. Nevertheless, the circRNAs research in hepatocellular carcinoma (HCC) still remains far insufficient. Herein, we investigated the role of a newly defined circRNAs, circ_0005075, in HCC development. We found circ_0005075 was upregulated in HCC tissues. HCC progression was suppressed by downregulation of circ_0005075 in vitro and in vivo, and the suppression was partially reversed by inhibition of microRNA-335 (miR-335) expression. Further, we found the expression of mitogen-activated protein kinase 1 (MAPK1) was substantially regulated by circ_0005075 and miR-335. Mechanically, it was demonstrated that circ_0005075 could directly bind to miR-335 and miR-335 could bind to MAPK1. Our data provide evidence that circ_0005705 promotes the HCC progression by sponging miR-335 and further regulating MAPK1 expression.

Critical role of CD4 T cells in PF4/heparin antibody production in mice.

Antibodies specific for platelet factor 4 (PF4)/heparin complexes are central to the pathogenesis of heparin-induced thrombocytopenia. Marginal zone B cells appear to be the source of such antibodies, but whether T-cell help is required is unclear. Here, we showed that induction of PF4/heparin-specific antibodies by PF4/heparin complexes was markedly impaired in mice depleted of CD4 T cells by anti-CD4 antibodies. Furthermore, Rag1-deficient recipient mice produced PF4/heparin-specific antibodies upon PF4/heparin challenge when reconstituted with a mixture of wild-type splenic B cells and splenocytes from B-cell-deficient (µMT) mice but not splenocytes from T- and B-cell-deficient (Rag1 knockout) mice. Lastly, mice with B cells lacking CD40, a B-cell costimulatory molecule that helps T-cell-dependent B-cell responses, displayed a marked reduction of PF4/heparin-specific antibody production following PF4/heparin challenge. Together, these findings show that helper T cells play a critical role in production of PF4/heparin-specific antibodies.

Rhythmic expressed clock regulates the transcription of proliferating cellular nuclear antigen in teleost retina.

Teleost fish continues to grow their eyes throughout life with the body size. In Astatotilapia burtoni, the fish retina increases by adding new retinal cells at the ciliary marginal zone (CMZ) and in the outer nuclear layer (ONL). Cell proliferation at both sites exhibits a daily rhythm in number of dividing cells. To understand how this diurnal rhythm of new cell production is controlled in retinal progenitor cells, we studied the transcription pattern of clock genes in retina, including clock1a, clock1b, bmal1a (brain and muscle ARNT-Like), and per1b (period1b). We found that these genes have a strong diurnal rhythmic transcription during light-dark cycles but not in constant darkness. An oscillation in pcna transcription was also observed during light-dark cycles, but again not in constant darkness. Our results also indicate an association between Clock proteins and the upstream region of pcna (proliferating cellular nuclear antigen) gene. A luciferase reporter assay conducted in an inducible clock knockdown cell line further demonstrated that the mutation on predicted E-Boxes in pcna promoter region significantly attenuated the transcriptional activation induced by Clock protein. These results suggested that the diurnal rhythmic expression of clock genes in A. burtoni retina could be light dependent and might contribute to the daily regulation of the proliferation of the retina progenitors through key components of cell cycle machinery, for instance, pcna.

Intronic miR-932 targets the coding region of its host gene, Drosophila neuroligin2.

Despite great progress for two decades in microRNAs (miRNAs), the direct regulation of host gene by intragenic (mostly intronic) miRNA is conceptually plausible but evidence-limited. Here, we report that intronic miR-932 could target its host gene via binding with coding sequence (CDS) region rather than regular 3'UTR. The conserved miR-932 is embedded in the fourth intron of Drosophila neuroligin2 (dnlg2), which encodes a synaptic cell adhesion molecule, DNlg2. In silico analysis predicted two putative miR-932 target sites locate in the CDS region of dnlg2 instead of regular 3'-UTR miRNA binding sites. Employing luciferase reporter assay, we further proved that the miR-932 regulates expression of its host gene dnlg2 via the binding CDS region of dnlg2. Consistently, we observed miR-932 downregulated expression of dnlg2 in S2 cell, and the repression of dnlg2 by miR-932 at both protein and RNA level. Furthermore, we found CDS-located site1 is dominant for regulating expression of host dnlg2 by miR-932. In addition to providing thorough examination of one intronic miRNA targeting the CDS region of its host gene, our genome-wide analysis indicated that nearly half of fruitfly and human intronic miRNAs may target their own host gene at coding region. This study would be valuable in elucidating the regulation of intronic miRNA on host gene, and provide new information about the biological context of their genomic arrangements and functions.

miR-92a-3p Exerts Various Effects in Glioma and Glioma Stem-Like Cells Specifically Targeting CDH1/ß-Catenin and Notch-1/Akt Signaling Pathways.

MicroRNAs (miRNAs) are implicated in the regulation of tumor progression and stemness of cancer stem-like cells. Recently, miR-92a-3p was reported to be up-regulated in human glioma samples. Nevertheless, the precise role of miR-92a-3p in glioma cells and glioma stem-like cells (GSCs) has not been fully elucidated. It is necessary to clarify the function of miR-92a-3p in glioma and GSCs to develop novel therapeutic approaches for glioma patients. In the present study, we applied methyl-thiazolyl-tetrazolium (MTT) assay and Transwell assay to measure the proliferation rate and metastatic potential of glioma cells. Meanwhile, the self-renewal ability of GSCs was detected by tumor sphere formation assay. The results revealed that down-regulation of miR-92a-3p suppressed the glioma cell malignancy in vitro. Moreover, knockdown of miR-92a-3p led to a reduction of tumorgenesis in vivo. Interestingly, we also observed that up-regulation of miR-92a-3p could inhibit the stemness of GSCs. Subsequent mechanistic investigation indicated that cadherin 1 (CDH1)/ß-catenin signaling and Notch-1/Akt signaling were the downstream pathways of miR-92a-3p in glioma cells and GSCs, respectively. These results suggest that miR-92a-3p plays different roles in glioma cells and GSCs through regulating different signaling pathways.

Critical role for mouse marginal zone B cells in PF4/heparin antibody production.

Heparin-induced thrombocytopenia (HIT) is an immune-mediated disorder that can cause fatal arterial or venous thrombosis/thromboembolism. Immune complexes consisting of platelet factor 4 (PF4), heparin, and PF4/heparin-reactive antibodies are central to the pathogenesis of HIT. However, the B-cell origin of HIT antibody production is not known. Here, we show that anti-PF4/heparin antibodies are readily generated in wild-type mice on challenge with PF4/heparin complexes, and that antibody production is severely impaired in B-cell-specific Notch2-deficient mice that lack marginal zone (MZ) B cells. As expected, Notch2-deficient mice responded normally to challenge with T-cell-dependent antigen nitrophenyl-chicken γ globulin but not to the T-cell-independent antigen trinitrophenyl-Ficoll. In addition, wild-type, but not Notch2-deficient, B cells plus B-cell-depleted wild-type splenocytes adoptively transferred into B-cell-deficient µMT mice responded to PF4/heparin complex challenge. PF4/heparin-specific antibodies produced by wild-type mice were IgG2b and IgG3 isotypes. An in vitro class-switching assay showed that MZ B cells were capable of producing antibodies of IgG2b and IgG3 isotypes. Lastly, MZ, but not follicular, B cells adoptively transferred into B-cell-deficient µMT mice responded to PF4/heparin complex challenge by producing PF4/heparin-specific antibodies of IgG2b and IgG3 isotypes. Taken together, these data demonstrate that MZ B cells are critical for PF4/heparin-specific antibody production.

MiR-124 inhibits cell proliferation in breast cancer through downregulation of CDK4.

Studies have shown that microRNAs (miRNAs) are involved in the malignant progression of human cancer. However, little is known about the potential role of miRNAs in breast carcinogenesis. miR-124 expression in breast cancer tissue was measured by quantitative real-time PCR (qRT-PCR). Target prediction algorithms and luciferase reporter gene assays were used to investigate the target of miR-124. Breast cancer cells growth was regulated by overexpression or knockdown miR-124. At the end of the study, tumor-bearing mice were tested to confirm the function of miR-124 in breast cancer. In this study, we demonstrated that the expression of miR-124 was significantly downregulated in breast cancer tissues compared with matched adjacent non-neoplastic tissues. We identified and confirmed that cyclin-dependent kinase 4 (CDK4) was a direct target of miR-124. Overexpression of miR-124 suppressed CDK4 protein expression and attenuated cell viability, proliferation, and cell cycle progression in MCF-7 and MDA-MB-435S breast cancer cells in vitro. Overexpression of CDK4 partially rescued the inhibitory effect of miR-124 in the breast cancer cells. Moreover, we found that miR-124 overexpression effectively repressed tumor growth in xenograft animal experiments. Our results demonstrate that miR-124 functions as a growth-suppressive miRNA and plays an important role in inhibiting tumorigenesis by targeting CDK4.

Bleomycin loaded exosomes enhanced antitumor therapeutic efficacy and reduced toxicity.

BACKGROUND: Bleomycin (BLM) is a chemotherapeutic agent with potent antitumor activity against the tumor. However, lung fibrosis is the main drawback that limits BLM use. Tumor targeted, safe, efficient and natural delivery of BLM is important to increase the effectiveness and reduce the toxic side effects. Although tumor derived Exosomes (Exo), provide a potential vehicle for in vivo drug delivery due to their cell tropism. This study primarily focuses on generating a natural delivery platform for Exo loaded with BLM and testing its therapeutic efficacy against cancer. METHODS: Exosomes were isolated from cancer cells and incubated with BLM. Exo were characterized by transmission electron microscopy, western blot analysis and nanoparticle tracking analysis. We performed in vitro and in vivo analyses to evaluate the effect of Exo-BLM. RESULTS: Exosomes loaded with BLM are highly cancer targeting and cause the cytotoxicity of tumor cells by ROS. The fluorescence images showed that Exo-BLM accumulated in cancer cells. The results revealed that Exo-BLM induces tumor cell apoptosis by the caspase pathway. In vivo, the treatment of Exo-BLM showed targeted ability and enhanced the antitumor activity. CONCLUSION: This study provides an avenue for specific BLM therapeutics with minimal side effects.

Neuroligin 2 is required for synapse development and function at the Drosophila neuromuscular junction.

Neuroligins belong to a highly conserved family of cell adhesion molecules that have been implicated in synapse formation and function. However, the precise in vivo roles of Neuroligins remain unclear. In the present study, we have analyzed the function of Drosophila neuroligin 2 (dnl2) in synaptic development and function. We show that dnl2 is strongly expressed in the embryonic and larval CNS and at the larval neuromuscular junction (NMJ). dnl2 null mutants are viable but display numerous structural defects at the NMJ, including reduced axonal branching and fewer synaptic boutons. dnl2 mutants also show an increase in the number of active zones per bouton but a decrease in the thickness of the subsynaptic reticulum and length of postsynaptic densities. dnl2 mutants also exhibit a decrease in the total glutamate receptor density and a shift in the subunit composition of glutamate receptors in favor of GluRIIA complexes. In addition to the observed defects in synaptic morphology, we also find that dnl2 mutants show increased transmitter release and altered kinetics of stimulus-evoked transmitter release. Importantly, the defects in presynaptic structure, receptor density, and synaptic transmission can be rescued by postsynaptic expression of dnl2. Finally, we show that dnl2 colocalizes and binds to Drosophila neurexin (dnrx) in vivo. However, whereas homozygous mutants for either dnl2 or dnrx are viable, double mutants are lethal and display more severe defects in synaptic morphology. Altogether, our data show that, although dnl2 is not absolutely required for synaptogenesis, it is required postsynaptically for synapse maturation and function.

The LncRNA RP11-301G19.1/miR-582-5p/HMGB2 axis modulates the proliferation and apoptosis of multiple myeloma cancer cells via the PI3K/AKT signalling pathway.

Long non-coding RNAs (lncRNAs) have recently been reported to act as crucial regulators and prognostic biomarkers of human tumorigenesis. Based on microarray data, RP11-301G19.1 was previously identified as an upregulated lncRNA during B cell development. However, the effect of RP11-301G19.1 on multiple myeloma (MM) cells remains unclear. In the present study, the effects of RP11-301G19.1 on tumour progression were ascertained both in vitro and in vivo. Our results demonstrated that RP11-301G19.1 was upregulated in MM cell lines and that its downregulation inhibited the proliferation and cell cycle progression and promoted the apoptosis of MM cells. Bioinformatic analysis and luciferase reporter assay results revealed that RP11-301G19.1 can upregulate the miR-582-5p-targeted gene HMGB2 as a competing endogenous RNA (ceRNA). Furthermore, Western blot results indicated that RP11-301G19.1 knockdown decreased the levels of PI3K and AKT phosphorylation without affecting their total protein levels. Additionally, in a xenograft model of human MM, RP11-301G19.1 knockdown significantly inhibited tumour growth by downregulating HMGB2. Overall, our data demonstrated that RP11-301G19.1 is involved in MM cell proliferation by sponging miR-582-5p and may serve as a therapeutic target for MM.

Therapeutical Significance of Serpina3n Subsequent Cerebral Ischemia via Cytotoxic Granzyme B Inactivation.

Ischemic stroke is a devastating CNS insult with few clinical cures. Poor understanding of underlying mechanistic network is the primary limitation to develop novel curative therapies. Extracellular accumulation of granzyme B subsequent ischemia promotes neurodegeneration. Inhibition of granzyme B can be one of the potent strategies to mitigate neuronal damage. In present study, we investigated the effect of murine Serpina3n and human (homolog) SERPINA3 against cerebral ischemia through granzyme B inactivation. Recombinant Serpina3n/SERPINA3 were expressed by transfected 293 T cells, and eluted proteins were examined for postischemic influence both in vitro and in vivo. During in vitro test, Serpina3n was found effective enough to inhibit granzyme B, while SERPINA3 was ineffectual to counter cytotoxic protease. Treatment of hypoxic culture with recombinant Serpina3n/SERPINA3 significantly increased cell viability in dosage-dependent manner, recorded maximum at the highest concentration (4 mM). Infarct volume analysis confirmed that 50 mg/kg dosage of exogenous Serpina3n was adequate to reduce disease severity, while SERPINA3 lacked behind in analeptic effect. Immunohistochemical test, western blot analysis, and protease activity assay's results illustrated successful diffusion of applied protein to the ischemic lesion and reactivity with the target protease. Taken together, our findings demonstrate therapeutic potential of Serpina3n by interfering granzyme B-mediated neuronal death subsequent cerebral ischemia.

Localized expression pattern of miR-184 in Drosophila.

MicroRNAs (miRNAs) are a kind of endogenous non-coding small RNAs whose specific functions in animals are generally important. Although functions of some miRNAs have been identified, the role of miR-184 remains unknown. Here, we determined the temporal and spatial expression pattern of miR-184 during the different development stages and tissues in Drosophila. Strikingly, miR-184 is expressed ubiquitously in Drosophila embryos, larvae and adults, its expression pattern shows a dynamic changes during the development of embryo, especially in the central nervous system. This expression profile suggests that miR-184 may act important function in Drosophila development.

Iguratimod-encapsulating PLGA-NPs induce human multiple myeloma cell death via reactive oxygen species and Caspase-dependent signalling.

Human multiple myeloma (MM) is a currently incurable haematopoietic malignancies. Our research investigate the anti-tumour effect of iguratimod (IGU) encapsulated in poly(lactic-co-glycolic acid) PLGA nanoparticles (IGU-PLGA-NPs) on MM cells in vitro and in vivo. A significant inhibitory effect of IGU-PLGA-NPs on MM cancer cells and MM CSCs was demonstrated by the Cell Counting Kit-8 (CCK-8) assay. Treatment with IGU-PLGA-NPs induced significant cell cycle arrest at G1 in MM cells and reduced tumour colony formation in MM CSCs. Mechanistically, IGU-PLGA-NPs increase apoptosis in MM cells by activating Caspase-dependent signalling pathway to increase the levels of bax, cytochrome c (cyt-c), caspase-9 and caspase-3 proteins. Moreover, IGU-PLGA-NPs effectively increase ROS production assayed using a DCFH-DA fluorescent probe in MM cells. The data indicate that IGU-PLGA-NPs induce a significant reduction in the tumour volume and a marked increase in the survival rate in a mouse model of multiple myeloma. Overall, our findings indicate that IGU-PLGA-NPs are a potential therapeutic strategy that may contribute to the therapy of MM and elimination of MM CSCs in future clinical trials.

The LncRNA RP11-279C4.1 Enhances the Malignant Behaviour of Glioma Cells and Glioma Stem-Like Cells by Regulating the miR-1273g-3p/CBX3 Axis.

Glioma is the most common type of solid tumour affecting the central nervous system, and the survival rate of patients with glioma is low. However, the mechanism associated with glioma progression remains unclear. Growing evidence suggests that lncRNAs play essential roles in the initiation and progression of tumours, including gliomas. In the present study, we identified and verified the expression of the novel lncRNA RP11-279C4.1 by analyzing the TANRIC database and performing qRT-PCR assays, the results of which revealed its upregulation in glioma tissues and cell lines. The results of multiple functional experiments demonstrated that RP11-279C4.1 knockdown inhibited glioma malignant phenotypes, including cell proliferation, migration, invasion and cell self-renew ability in vitro. In addition, RP11-279C4.1 downregulation suppressed tumour growth in vivo. Mechanistically, RP11-279C4.1 induced CBX3 activation via competitively sponging miR-1273g-3p, and rescue assay results confirmed the importance of the RP11-279C4.1/miR-1273g-3p/CBX3 axis. Overall, the results of our present study demonstrated that RP11-279C4.1 functions as an oncogene that promotes tumour progression by modulating the miR-1273g-3p/CBX3 axis in glioma, suggesting that RP11-279C4.1 may be a novel therapeutic target for glioma.

Serpina3n: Potential drug and challenges, mini review.

Serpina3n is a secretory serine protease inhibitor belonging to clade "a" exhibiting unique structural and physiological characteristics, playing significant roles ranging from complement cascade, apoptosis, wound healing to Alzheimer by inhibiting a wide range of proteases. Recently studies have reported its significant roles during various pathologies. Although its full range of potential applications are yet to reveal, its reported implications particularly in CNS insults are making it potential therapeutical approach. Here we aim to draw together the literature shedding light on the potential therapeutical applications of Serpina3n/SERPINA3 in various diseases and a brief comparison between murine Serpina3n and its human ortholog SERPINA3 (α1-antichymotrypsin) accounting their biological roles and significance.

A GAL4/UAS luciferase system to identify the miRNAs target mRNAs in Drosophila S2 cells.

MicroRNAs (miRNAs) have been shown to regulate gene expression through the sequence-specific base pairing with their target mRNAs. However, our understanding of the biological roles of miRNAs is still quite limited, and only a handful of miRNAs have been assigned by genetic analysis in part owing to the difficulty in the identification of their targets. Although computational methods have shown to be helpful in the prediction of miRNA targets, a major obstacle has been the lack of quick and efficient experimental procedures to verify these targets. In this report, we describe a UAS/GAL4-based reporter system for this purpose. Our data indicate it an assay of miRNA-target gene interaction, with greater sensitivity over the previously reported methods, and may be useful for more efficient identification/validation the miRNA targets in Drosophila cell lines.