RNA-binding protein SYNCRIP contributes to neuropathic pain through stabilising CCR2 expression in primary sensory neurones.

BACKGROUND: Nerve injury-induced changes in gene expression in the dorsal root ganglion (DRG) contribute to the genesis of neuropathic pain. SYNCRIP, an RNA-binding protein, is critical for the stabilisation of gene expression. Whether SYNCRIP participates in nerve injury-induced alterations in DRG gene expression and nociceptive hypersensitivity is unknown. METHODS: The expression and distribution of SYNCRIP in mouse DRG after chronic constriction injury (CCI) of the unilateral sciatic nerve were assessed. Effect of microinjection of Syncrip small interfering RNA into the ipsilateral L3 and L4 DRGs on the CCI-induced upregulation of chemokine (C-C motif) receptor 2 (CCR2) and nociceptive hypersensitivity were examined. Additionally, effects of microinjection of adeno-associated virus 5 expressing full length Syncrip mRNA (AAV5-Syncrip) on basal DRG CCR2 expression and nociceptive thresholds were observed. RESULTS: SYNCRIP is expressed predominantly in DRG neurones, where it co-exists with CCR2. Levels of Syncrip mRNA and SYNCRIP protein in injured DRG increased time-dependently on days 3-14 after CCI. Blocking this increase through microinjection of Syncrip small interfering RNA into injured DRG attenuated CCI-induced upregulation of DRG CCR2 and development and maintenance of nociceptive hypersensitivities. Mimicking this increase through DRG microinjection of AAV5-Syncrip elevated CCR2 expression in microinjected DRGs, enhanced the responses to mechanical, heat, and cold stimuli, and induced ongoing pain in naive mice. Mechanistically, SYNCRIP bound to 3-UTR of Ccr2 mRNA and stabilised its expression in DRG neurones. CONCLUSIONS: SYNCRIP contributes to the induction and maintenance of neuropathic pain likely through stabilising expression of CCR2 in injured DRG. SYNCRIP may be a potential target for treating this disorder.

LINC00987 knockdown inhibits the progression of acute myeloid leukemia by suppressing IGF2BP2-mediated PA2G4 expression.

This study aimed to investigate the role and potential mechanisms of LINC00987 in acute myeloid leukemia (AML) progression. The expression of LINC00987 in bone marrow specimens of AML patients and cell lines was measured by quantitative reverse transcription PCR (RT-qPCR). Small interfering RNA targeting LINC00987 (si-LINC00987) was transfected into AML cell lines HL-60 and KG-1, and the proliferation, invasion and apoptosis were detected with Cell Counting Kit-8 (CCK-8), Transwell and flow cytometry, respectively. Moreover, the binding between LINC00987 and insulin like growth factor 2 mRNA binding protein 2 (IGF2BP2) was validated with an RNA pull-down assay. Co-immunoprecipitation assay was used to verify the binding between IGF2BP2 and proliferation-associated 2G4 (PA2G4). Then rescue experiments were performed to explore the effects of LINC00987/IGF2BP2/PA2G4 axis on HL-60 and KG-1 cell functions. Additionally, HL-60 cells transfected with si-LINC00987 were injected into mice, followed by the evaluation of xenograft tumor growth. LINC00987 was upregulated in AML patient specimens and cell lines. LINC00987 knockdown inhibited proliferation and invasion and promoted apoptosis in AML cells. LINC00987 could bind with IGF2BP2 and promote its expression, and IGF2BP2 overexpression reversed the effects of LINC00987 knockdown on the proliferation, invasion and apoptosis in AML cells. Besides, IGF2BP2 could bind with PA2G4. IGF2BP2 knockdown inhibited proliferation and invasion, and promoted apoptosis in AML cells, whereas PA2G4 overexpression reversed these effects. Additionally, the LINC00987 knockdown inhibited the xenograft tumor growth of AML in vivo. Knockdown of LINC00987 inhibits AML cell proliferation and invasion, and promotes apoptosis in vitro and reduces tumor growth in vivo by suppressing IGF2BP2-mediated PA2G4 expression.

Mcrs1 interacts with Six1 to influence early craniofacial and otic development.

The Six1 transcription factor plays a major role in craniofacial development. Mutations in SIX1 and its co-factor, EYA1, are causative for about 50% of Branchio-otic/Branchio-oto-renal syndrome (BOR) patients, who are characterized by variable craniofacial, otic and renal malformations. We previously screened for other proteins that might interact with Six1 to identify additional genes that may play a role in BOR, and herein characterize the developmental role of one of them, Microspherule protein 1 (Mcrs1). We found that in cultured cells, Mcrs1 bound to Six1 and in both cultured cells and embryonic ectoderm reduced Six1-Eya1 transcriptional activation. Knock-down of Mcrs1 in embryos caused an expansion of the domains of neural plate genes and two genes expressed in both the neural plate and neural crest (zic1, zic2). In contrast, two other genes expressed in pre-migratory neural crest (foxd3, sox9) were primarily reduced. Cranial placode genes showed a mixture of expanded and diminished expression domains. At larval stages, loss of Mcrs1 resulted in a significant reduction of otic vesicle gene expression concomitant with a smaller otic vesicle volume. Experimentally increasing Mcrs1 above endogenous levels favored the expansion of neural border and neural crest gene domains over cranial placode genes; it also reduced otic vesicle gene expression but not otic vesicle volume. Co-expression of Mcrs1 and Six1 as well as double knock-down and rescue experiments establish a functional interaction between Mcrs1 and Six1 in the embryo, and demonstrate that this interaction has an important role in the development of craniofacial tissues including the otic vesicle.

Expression of transduced nucleolin promotes the clearance of accumulated α-synuclein in rodent cells and animal model.

Alpha-synuclein (αSyn) is a major component of Lewy bodies, which are a known pathogenic marker of Parkinson's disease (PD). The dysfunction of protein degradation machinery causes αSyn accumulation. The reinforcement of αSyn degradation is a potential therapeutic target for PD because accumulated αSyn is responsible for the pathogenesis of PD. Nucleolin (NCL) is essential in the formation of the nucleolar structure. The function of NCL is correlated with oxidative stress-mediated cell death. A previous study demonstrated that NCL overexpression alleviated rotenone-induced neurotoxic effects, whereas knockdown of NCL had the opposite effect. These results suggest that NCL malfunction would exacerbate PD pathology. Thus, it was hypothesized that the introduction of ectopic NCL could rescue α-synucleinopathy in PD. This study investigated whether the ectopic expression of NCL facilitates αSyn clearance. Ectopic expression of NCL was accomplished via the transfection of green fluorescent protein (GFP) or GFP-NCL in mouse embryonic fibroblasts (MEF) or transduction of GFP or GFP-NCL using lentivirus in rat primary cortical neurons and mouse substantia nigra. NCL overexpression enhanced the clearance of accumulated or aggregated αSyn in MEFs and rat primary cortical neurons. The activity of the autophagy-lysosome pathway was enhanced by NCL expression. NCL transduction in the substantia nigra, which was co-injected with αSyn fibrils, rescued PD manifestation. The elevation of NCL levels may reflect a therapeutic strategy for α-synucleinopathy in PD.

CircRNA circBACH1 (hsa_circ_0061395) serves as a miR-656-3p sponge to facilitate hepatocellular carcinoma progression through increasing SERBP1 expression.

Hsa_circ_0061395(circBACH1) and SERBP1(SERPINE1 mRNA binding protein 1) have been reported to play a carcinogenic role in HCC.In this study, circBACH1, microRNA(miR)-656-3p, and SERBP1 expression levels with quantitative real-time polymerase chain reaction (qRT-PCR) in HCC tissue specimens and cells.The protein levels of SERBP1, E-Cadherin, vimentin, and N-Cadherin were detected with western blotting.Cell proliferation, migration, invasion, and apoptosis were determined with CCK-8, colony formation, transwell, and flow cytometry assays.The targeting relatio-nship between circBACH1 or SERBP1 and miR-656-3p was verified by dual-lucifer- ase reporter assay.The role of circBACH1 was validated by xenograft assay.CircBAC- H1 and SERBP1 were upregulated in HCC tissues and cells.Both circBACH1 and SERBP1 knockdown constrained proliferation, migration, invasion, and EMT(epithel-ial-mesenchymal transition), and facilitated apoptosis of HCC cells in vitro.Knockdo-wn of circBACH1 reduced HCC growth in vivo. SERBP1 overexpression partially neutralized the repressive effect of circBACH1 silencing on malignant behaviors of HCC cells.CircBACH1 sponged miR-656-3p to elevate SERBP1 expression, thereby accelerating the progression of HCC.The research provided a new evidence to support the role of circBACH1 in HCC.

Expression regulation of cold-inducible protein RBM3 by FAK/Src signaling for neuroprotection against rotenone under mild hypothermia.

Mild hypothermia is a well-established technique for alleviating neurological injuries in clinical surgery. RNA-binding protein motif 3 (RBM3) has been identified as a crucial factor in mediating hypothermic neuroprotection, providing its induction as a promising strategy for mimicking therapeutic hypothermia. However, little is known about molecular control of RBM3 and signaling pathways affected by hypothermia. In the present study, human SH-SY5Y neuroblastoma cells were used as a neural cell model. Screening of signaling pathways showed that cold exposure led to inactivation of ERK and AMPK pathways, and activation of FAK and PLCγ pathways, with activities of p38, JNK and AKT pathways moderately changed. Next, various small molecule inhibitors specific to these signaling pathways were applied. Interestingly, only FAK-specific inhibitor exhibited a significant inhibitory effect on hypothermia-induced RBM3 gene transcription and protein expression. Likewise, FAK silencing using siRNA technique significantly abrogated the induction of RBM3 by hypothermia. Moreover, FAK inhibition accounted for an inactivation of Src, a known kinase downstream of FAK. Next, either the silencing of Src by siRNA or its inactivation by a chemical inhibitor, strongly blocked the induction of RBM3 by cooling. Notably, in HEK293 and PC12 cells, FAK/Src activation was also shown to be indispensable for hypothermia-stimulated RBM3 expression. Lastly, the CCK8 and Western blot assays showed that both FAK/Src inacitivation and their knockdown substantially abrogate the neuroprotective effects of mild hypothermia against rotenone in SH-SY5Y cells. These data suggest that FAK/Src signaling axis regulates the transcription of Rbm3 gene and mediates neuroprotective effects of mild hypothermia.

Shep regulates Drosophila neuronal remodeling by controlling transcription of its chromatin targets.

Neuronal remodeling is crucial for formation of the mature nervous system and disruption of this process can lead to neuropsychiatric diseases. Global gene expression changes in neurons during remodeling as well as the factors that regulate these changes remain poorly defined. To elucidate this process, we performed RNA-seq on isolated Drosophila larval and pupal neurons and found upregulated synaptic signaling and downregulated gene expression regulators as a result of normal neuronal metamorphosis. We further tested the role of alan shepard (shep), which encodes an evolutionarily conserved RNA-binding protein required for proper neuronal remodeling. Depletion of shep in neurons prevents the execution of metamorphic gene expression patterns, and shep-regulated genes correspond to Shep chromatin and/or RNA-binding targets. Reduced expression of a Shep-inhibited target gene that we identified, brat, is sufficient to rescue neuronal remodeling defects of shep knockdown flies. Our results reveal direct regulation of transcriptional programs by Shep to regulate neuronal remodeling during metamorphosis.

IGF2BP1 is the first positive marker for anaplastic thyroid carcinoma diagnosis.

Anaplastic thyroid carcinomas (ATC) are rare, but represent the most lethal malignancy of the thyroid. Selective molecular markers and drivers distinguishing ATC from other thyroid carcinomas of follicular origin remain largely unknown, limiting advances in diagnosis and treatment. In a retrospective study, we analyzed gene expression in 36 ATC, 18 poorly differentiated, 132 papillary, and 55 follicular thyroid carcinoma, as well as 124 paired and unpaired normal thyroid tissues in three independent cohorts by RNA-sequencing and immunohistochemistry. RNA-sequencing data in the test cohort suggested selective ATC protein biomarkers. Evaluation of these revealed that ATCs are characterized by the de novo expression of various testis antigens, including melanoma-associated antigen A3 (MAGEA3), but most importantly the oncofetal IGF2 mRNA binding protein 1 (IGF2BP1). Shallow whole genome sequencing essentially excluded that IGF2BP1 upregulation results from gene copy number alterations. Immunohistochemical analyses in all three tumor cohorts confirmed the selective de novo expression of IGF2BP1 protein in ATC. In sum, 75% (27/36) of all tested ATC and 0.5% (1/204) of poorly and well-differentiated thyroid carcinoma tissue samples were positive for IGF2BP1 protein. This indicates that IGF2BP1 protein expression identifies ATC with a diagnostic odds ratio of 612 (95% CI: 74.6-5021). In addition, we found that MAGEA3 is exclusively, although less consistently upregulated in ATC, presenting with an odds ratio of 411 (95% CI: 23.8-7098.7). Importantly, we provide confirmatory evidence that IGF2BP1 and MAGEA3 expression distinguishes ATC from poorly differentiated thyroid carcinoma. IGF2BP1 furthermore identified ATC foci within low-grade follicular thyroid carcinoma. In conclusion, IGF2BP1 represents the most promising single-gene marker available for ATC, followed by MAGEA3, improving on current techniques. Robust markers are essential to help distinguish this high-grade malignancy from other thyroid carcinomas, to guide surgical decision making, therapy and post-resection/therapy monitoring strategies.

Upregulated microRNA-132 in T helper 17 cells activates hepatic stellate cells to promote hepatocellular carcinoma cell migration in vitro.

MicroRNAs play an important role in the modulation of the immune system. T helper 17 (Th17) cells are involved in the modulation of the tumour microenvironment. However, the function of miRNA in Th17 cells in the tumour microenvironment is unclear. In this study, we analysed miR-132 expression in Th17 cells and assessed the function of miR-132 on Th17 cell differentiation. In addition, the effect of miR-132 on Th17 cells in the tumour microenvironment, especially hepatic stellate cells (HSCs), was confirmed. CD4+ IL-17 ∓ cells were isolated from hepatocellular carcinoma (HCC) tumour tissues. The expression of miR-132 was higher in CD4+ IL-17 + cells than in CD4+ IL-17- cells. Human primary CD4+ T cells were used for Th17 cell differentiation. Compared with primary CD4+ T cells, Th17 cells expressed high levels of miR-132. During Th17 cell differentiation, a miR-132 mimic and inhibition were applied. After treatment with the miR-132 mimic, the differentiation of Th17 cells accelerated, showing a a higher percentage of Th17 cells and the expression and secretion of IL-17 and IL-22. Smad nuclear interacting protein 1 (SNIP1), as one of the targets of miR-132, decreased during Th17 cell differentiation-related Th17 differentiation and IL-17 expression. The conditioned medium of miR-132-overexpressing Th17 cells could increase the activation of the HSCs, which strongly promoted HCC cell migration and epithelial-mesenchymal transition (EMT). In summary, miR-132 positively regulates Th17 cell differentiation and improves the function of Th17 on HSCs for their tumour-promoting effects.

Single cell RNA sequencing identifies IGFBP5 and QKI as ciliated epithelial cell genes associated with severe COPD.

BACKGROUND: Whole lung tissue transcriptomic profiling studies in chronic obstructive pulmonary disease (COPD) have led to the identification of several genes associated with the severity of airflow limitation and/or the presence of emphysema, however, the cell types driving these gene expression signatures remain unidentified. METHODS: To determine cell specific transcriptomic changes in severe COPD, we conducted single-cell RNA sequencing (scRNA seq) on n = 29,961 cells from the peripheral lung parenchymal tissue of nonsmoking subjects without underlying lung disease (n = 3) and patients with severe COPD (n = 3). The cell type composition and cell specific gene expression signature was assessed. Gene set enrichment analysis (GSEA) was used to identify the specific cell types contributing to the previously reported transcriptomic signatures. RESULTS: T-distributed stochastic neighbor embedding and clustering of scRNA seq data revealed a total of 17 distinct populations. Among them, the populations with more differentially expressed genes in cases vs. controls (log fold change >|0.4| and FDR = 0.05) were: monocytes (n = 1499); macrophages (n = 868) and ciliated epithelial cells (n = 590), respectively. Using GSEA, we found that only ciliated and cytotoxic T cells manifested a trend towards enrichment of the previously reported 127 regional emphysema gene signatures (normalized enrichment score [NES] = 1.28 and = 1.33, FDR = 0.085 and = 0.092 respectively). Among the significantly altered genes present in ciliated epithelial cells of the COPD lungs, QKI and IGFBP5 protein levels were also found to be altered in the COPD lungs. CONCLUSIONS: scRNA seq is useful for identifying transcriptional changes and possibly individual protein levels that may contribute to the development of emphysema in a cell-type specific manner.

IFIT Proteins Are Involved in CXCL10 Expression in Human Glomerular Endothelial Cells Treated with a Toll-Like Receptor 3 Agonist.

INTRODUCTION: Various viruses including a novel coronavirus (SARS-CoV-2) can infect the kidney. When viruses invade the glomeruli from the bloodstream, glomerular endothelial cells (GECs) initiate the innate immune reactions. We investigated the expression of interferon (IFN)-induced protein with tetratricopeptide repeats (IFIT) 1/2/3, antiviral molecules, in human GECs treated with a toll-like receptor (TLR) 3 agonist. Role of IFIT1/2/3 in the expression of C-X-C motif chemokine ligand 10 (CXCL10) was also examined. METHODS: Human GECs were cultured and stimulated with polyinosinic-polycytidylic acid (poly IC), a synthetic TLR3 agonist. Real-time qPCR, Western blotting, and ELISA were used to examine the expression of IFIT1/2/3, IFN-ß, and CXCL10. RNA interference against IFN-ß or IFIT1/2/3 was also performed. RESULTS: Expression of IFIT1/2/3 and CXCL10 was induced by poly IC in GECs. The inductions were inhibited by RNA interfering of IFN-ß. Knockdown of IFIT1/2/3 decreased the CXCL10 expression. Knockdown of IFIT3 decreased the expression of IFIT1 and IFIT2 proteins. CONCLUSION: IFIT1/2/3 and CXCL10 were induced by poly IC via IFN-ß in GECs. IFIT1/2/3 may increase the expression of CXCL10 which induces lymphocyte chemotaxis and may inhibit the replication of infected viruses. These molecules may play a role in GEC innate immune reactions in response to viruses.

Drosophila Sister-of-Sex-lethal reinforces a male-specific gene expression pattern by controlling Sex-lethal alternative splicing.

In Drosophila, female development is governed by a single RNA-binding protein, Sex-lethal (Sxl), that controls the expression of key factors involved in dosage compensation, germline homeostasis and the establishment of female morphology and behaviour. Sxl expression in female flies is maintained by an auto-regulatory, positive feedback loop with Sxl controlling splicing of its own mRNA. Until now, it remained unclear how males prevent accidental triggering of the Sxl expression cascade and protect themselves against runaway protein production. Here, we identify the protein Sister-of-Sex-lethal (Ssx) as an inhibitor of Sxl auto-regulatory splicing. Sxl and Ssx have a comparable RNA-binding specificity and compete for binding to RNA regulatory elements present in the Sxl transcript. In cultured Drosophila cells, Sxl-induced changes to alternative splicing can be reverted by the expression of Ssx. Moreover, in adult male flies ablation of the ssx gene results in a low level of productive Sxl mRNA splicing and Sxl protein production in isolated, clonal cell populations. In sum, this demonstrates that Ssx safeguards male animals against Sxl protein production to reinforce a stable, male-specific gene expression pattern.

Upregulation of Rpn10 promotes tumor progression via activation of the NF-κB pathway in clear cell renal cell carcinoma.

The ubiquitin-proteasome system (UPS) plays a central role in regulating protein homeostasis in tumor progression. The proteasome subunit Rpn10 is associated with the progression of several tumor types. However, little is known regarding the role of Rpn10 in clear cell renal cell carcinoma (ccRCC). In this study, we found that overexpression of Rpn10 increased ccRCC cell proliferation, migration, and invasion. Silencing Rpn10 expression resulted in decreased cell proli-feration, migration, and invasion in ccRCC cells. Knockdown of Rpn10 inhibits tumor growth and cell proliferation in vivo. Furthermore, we demonstrated that Rpn10 increased cell proliferation, migration, and invasion via regulation of the nuclear factor kappa B (NF-κB) pathway. Rpn10 directly promoted inhibitor of nuclear factor-kappa B alpha (IκBα) degradation through the UPS. Moreover, we observed that upregulation of Rpn10 or downregulation of IκBα in ccRCC was associated with poor prognosis. We found that the combination of these two parameters was a more powerful predictor of poor prognosis than either parameter alone. Collectively, these findings provide evidence that Rpn10 promotes the progression of ccRCC by regulation of the NF-κB pathways and is a prognostic indicator for patients with ccRCC.

Circ_0055625 knockdown inhibits tumorigenesis and improves radiosensitivity by regulating miR-338-3p/MSI1 axis in colon cancer.

BACKGROUND: Radiotherapy is a main therapeutic method for cancers, including colon cancer. In the current study, we aim to explore the effects of circular RNA (circRNA) circ_0055625 in the progression and radiosensitivity of colon cancer and the underlying mechanism. METHODS: The expression of circ_0055625 and musashi homolog 1 (MSI1) mRNA was detected by quantitative real-time polymerase chain reaction (qRT-PCR). MSI1 protein expression was determined by Western blot. Cell proliferation was assessed by cell counting kit-8 (CCK-8) and colony formation assays. Cell survival fraction, apoptosis, and invasion were investigated by colony formation assay, flow cytometry analysis, and transwell invasion assay, respectively. Cell migration was detected by wound-healing and transwell migration assays. The binding relationship between microRNA-338-3p (miR-338-3p) and circ_0055625 or MSI1 was predicted by online databases and identified by Dual-Luciferase Reporter Assay. The effects of circ_0055625 silencing on the tumor formation and radiosensitivity of colon cancer in vivo were explored by in vivo tumor formation assay. RESULTS: Circ_0055625 and MSI1 were upregulated in colon cancer tissues and cells relative to control groups. Radiation treatment apparently increased the expression of circ_0055625 and MSI1 in colon cancer cells. Circ_0055625 knockdown or MSI1 silencing repressed cell proliferation, migration, and invasion and promoted cell apoptosis and radiosensitivity in colon cancer. Also, circ_0055625 silencing-mediated effects were attenuated by MSI1 overexpression. Additionally, circ_0055625 silencing reduced MSI1 expression, which could be attenuated by miR-338-3p inhibitor. Mechanically, circ_0055625 acted as a sponge for miR-338-3p to regulate MSI1. Furthermore, circ_0055625 knockdown hindered tumor growth and improved radiosensitivity in vivo. CONCLUSION: Circ_0055625 repression inhibited the progression and radioresistance of colon cancer by downregulating MSI1 through sponging miR-338-3p. This result might provide a theoretical basis for improving the therapy of colon cancer with radiation.

LINC00941 promotes oral squamous cell carcinoma progression via activating CAPRIN2 and canonical WNT/ß-catenin signaling pathway.

Dysregulation of long non-coding RNAs (lncRNAs) has been implicated in many cancer developments. Previous studies showed that lncRNA LINC00941 was aberrantly expressed in oral squamous cell carcinoma (OSCC). However, its role in OSCC development remains elusive. In this study, we demonstrated that in OSCC cells, EP300 activates LINC00941 transcription through up-regulating its promoter H3K27ac modification. Up-regulated LINC00941 in turn activates CAPRIN2 expression by looping to CAPRIN2 promoter. Functional assays suggest that both LINC00941 and CAPRIN2 play pivotal roles in promoting OSCC cell proliferation and colony formation. In vivo assay further confirmed the role of LINC00941 in promoting OSCC cell tumour formation. Lastly, we showed that the role of LINC00941 and CAPRIN2 in OSCC progression was mediated through activating the canonical WNT/ß-catenin signaling pathway. Thus, LINC00941/CAPRIN2/ WNT/ß-catenin signaling pathway provides new therapeutic targets for OSCC treatment.

The extracellular domain of epithelial cell adhesion molecule (EpCAM) enhances multipotency of mesenchymal stem cells through EGFR-LIN28-LET7 signaling.

Mesenchymal stem cells (MSCs) are widely considered to be an attractive cell source for regenerative therapies, but maintaining multipotency and self-renewal in cultured MSCs is especially challenging. Hence, the development and mechanistic description of strategies that help promote multipotency in MSCs will be vital to future clinical use. Here, using an array of techniques and approaches, including cell biology, RT-quantitative PCR, immunoblotting, immunofluorescence, flow cytometry, and ChIP assays, we show that the extracellular domain of epithelial cell adhesion molecule (EpCAM) (EpEX) significantly increases the levels of pluripotency factors through a signaling cascade that includes epidermal growth factor receptor (EGFR), signal transducer and activator of transcription 3 (STAT3), and Lin-28 homolog A (LIN28) and enhances the proliferation of human bone marrow MSCs. Moreover, we found that EpEX-induced LIN28 expression reduces the expression of the microRNA LET7 and up-regulates that of the transcription factor high-mobility group AT-hook 2 (HMGA2), which activates the transcription of pluripotency factors. Surprisingly, we found that EpEX treatment also enhances osteogenesis of MSCs under differentiation conditions, as evidenced by increases in osteogenic markers, including Runt-related transcription factor 2 (RUNX2). Taken together, our results indicate that EpEX stimulates EGFR signaling and thereby context-dependently controls MSC states and activities, promoting cell proliferation and multipotency under maintenance conditions and osteogenesis under differentiation conditions.

Genetic variations on 31 and 450 residues of influenza A nucleoprotein affect viral replication and translation.

BACKGROUND: Influenza A viruses cause epidemics/severe pandemics that pose a great global health threat. Among eight viral RNA segments, the multiple functions of nucleoprotein (NP) play important roles in viral replication and transcription. METHODS: To understand how NP contributes to the virus evolution, we analyzed the NP gene of H3N2 viruses in Taiwan and 14,220 NP sequences collected from Influenza Research Database. The identified genetic variations were further analyzed by mini-genome assay, virus growth assay, viral RNA and protein expression as well as ferret model to analyze their impacts on viral replication properties. RESULTS: The NP genetic analysis by Taiwan and global sequences showed similar evolution pattern that the NP backbones changed through time accompanied with specific residue substitutions from 1999 to 2018. Other than the conserved residues, fifteen sporadic substitutions were observed in which the 31R, 377G and 450S showed higher frequency. We found 31R and 450S decreased polymerase activity while the dominant residues (31 K and 450G) had higher activity. The 31 K and 450G showed better viral translation and replication in vitro and in vivo. CONCLUSIONS: These findings indicated variations identified in evolution have roles in modulating viral replication in vitro and in vivo. This study demonstrates that the interaction between variations of NP during virus evolution deserves future attention.

Quaking Inhibits Doxorubicin-Mediated Cardiotoxicity Through Regulation of Cardiac Circular RNA Expression.

RATIONALE: RBPs (RNA-binding proteins) have been described to be expressed and regulated in various organs including the heart. Little is known about the role of RBPs in heart failure induced by the chemotherapy drug doxorubicin and their interaction with circular RNAs. OBJECTIVE: We aimed to identify key RBPs involved in doxorubicin-mediated heart failure and to elucidate their function. METHODS AND RESULTS: Global transcriptome profiling from murine myocardium exposed to doxorubicin identified 5 differentially expressed RBPs. Expression of the RBP QKI (Quaking) in response to doxorubicin was strongly downregulated in rodent cardiomyocytes and human induced pluripotent stem cell-derived cardiomyocytes in vitro and in vivo in mice. Knockdown of Qki in primary cardiomyocytes increased apoptosis and atrophy after treatment with doxorubicin, whereas lentiviral mediated overexpression of Qki5 inhibited the doxorubicin-induced apoptosis in cardiomyocytes. In vivo, AAV9 (adeno-associated virus serotype 9)-mediated cardiac overexpression of Qki5 prevented cardiac apoptosis and cardiac atrophy induced by doxorubicin and improved cardiac function. Mechanistically, by lentiviral-based overexpression and CRISPR/Cas9-mediated silencing of Qki5, we identified regulated expression of specific circular RNAs derived from Ttn (Titin), Fhod3 (Formin homology 2 domain containing 3), and Strn3 (Striatin, calmodulin-binding protein 3). Moreover, inhibition of Ttn-derived circular RNA increased the susceptibility of cardiomyocytes to doxorubicin. CONCLUSIONS: We here show that overexpression of Qki5 strongly attenuates the toxic effect of doxorubicin via regulating a set of circular RNAs. Qki5 is, thus, an interesting target molecule to combat doxorubicin-induced cardiotoxicity.

Airn Regulates Igf2bp2 Translation in Cardiomyocytes.

RATIONALE: Increasing evidence indicates the presence of lncRNAs in various cell types. Airn is an imprinting gene transcribed from the paternal chromosome. It is in antisense orientation to the imprinted, but maternally derived, Igf2r gene, on which Airn exerts its regulation in cis. Although Airn is highly expressed in the heart, functions aside from imprinting remain unknown. OBJECTIVE: Here, we studied the functions of Airn in the heart, especially cardiomyocytes. METHODS AND RESULTS: Silencing of Airn via siRNAs augmented cell death, vulnerability to cellular stress, and reduced cell migration. To find the cause of such phenotypes, the potential binding partners of Airn were identified via RNA pull-down followed by mass spectrometry, which indicated Igf2bp2 (insulin-like growth factor 2 mRNA-binding protein 2) and Rpa1 (replication protein A1) as potential binding partners. Further experiments showed that Airn binds to Igf2bp2 to control the translation of several genes. Moreover, silencing of Airn caused less binding of Igf2bp2 to other mRNAs and reduced translation of Igf2bp2 protein. CONCLUSIONS: Our study uncovers a new function of Airn and demonstrates that Airn is important for the physiology of cardiomyocytes.

The SnRK2 kinases modulate miRNA accumulation in Arabidopsis.

MicroRNAs (miRNAs) regulate gene expression and play critical roles in growth and development as well as stress responses in eukaryotes. miRNA biogenesis in plants requires a processing complex that consists of the core components DICER-LIKE 1 (DCL1), SERRATE (SE) and HYPONASTIC LEAVES (HYL1). Here we show that inactivation of functionally redundant members of the SnRK2 kinases, which are the core components of abscisic acid (ABA) and osmotic stress signaling pathways, leads to reduction in miRNA accumulation under stress conditions. Further analysis revealed that the steady state level of HYL1 protein in plants under osmotic stress is dependent on the SnRK2 kinases. Additionally, our results suggest that the SnRK2 kinases physically associate with the miRNA processing components SE and HYL1 and can phosphorylate these proteins in vitro. These findings reveal an important role for the SnRK2 kinases in the regulation of miRNA accumulation and establish a mechanism by which ABA and osmotic stress signaling is linked to miRNA biogenesis.