BACKGROUND: The RNA-binding protein Quaking (QKI) increases during epithelial-to-mesenchymal transition and its expression is controlled by microRNA-200 family members. Here, we aimed to describe the expression of QKI in the developing lungs of control and nitrofen-induced congenital diaphragmatic hernia lungs (CDH).
METHODS: To investigate the expression of QKI, we dissected lungs from control and nitrofen-induced CDH rats on embryonic day 15, 18, 21 (E15, E18, E21). We performed immunofluorescence (IF) and quantitative reverse transcription PCR (RT-qPCR) for QKI expression. Additionally, we assessed Interleukin-6 (IL-6) abundance using IF.
RESULTS: On E21, IF showed that the abundance of all three QKI isoforms and IL-6 protein was higher in CDH lungs compared to control lungs (QKI5: p = 0.023, QKI6: p = 0.006, QKI7: p = 0.014, IL-6: p = 0.045, respectively). Furthermore, RT-qPCR data showed increased expression of QKI5, QKI6, and QKI7 mRNA in E21 nitrofen lungs by 1.63 fold (p = 0.001), 1.63 fold (p = 0.010), and 1.48 fold (p = 0.018), respectively.
CONCLUSIONS: Our data show an increase in the abundance and expression of QKI at the end of gestation in nitrofen-induced CDH lungs. Therefore, a disruption in the regulation of QKI during the late stage of pregnancy could be associated with the pathogenesis of abnormal lung development in CDH.

Epithelial-mesenchymal transition (EMT) plays important roles in tumour progression and is orchestrated by dynamic changes in gene expression. While it is well established that post-transcriptional regulation plays a significant role in EMT, the extent of alternative polyadenylation (APA) during EMT has not yet been explored. Using 3\' end anchored RNA sequencing, we mapped the alternative polyadenylation (APA) landscape following Transforming Growth Factor (TGF)-β-mediated induction of EMT in human mammary epithelial cells and found APA generally causes 3\'UTR lengthening during this cell state transition. Investigation of potential mediators of APA indicated the RNA-binding protein Quaking (QKI), a splicing factor induced during EMT, regulates a subset of events including the length of its own transcript. Analysis of QKI crosslinked immunoprecipitation (CLIP)-sequencing data identified the binding of QKI within 3\' untranslated regions (UTRs) was enriched near cleavage and polyadenylation sites. Following QKI knockdown, APA of many transcripts is altered to produce predominantly shorter 3\'UTRs associated with reduced gene expression. These findings reveal the changes in APA that occur during EMT and identify a potential role for QKI in this process.

Exploration of the molecular mechanisms of mesenchymal stem cell (MSC) growth has significant clinical benefits. Long non-coding RNAs (lncRNAs) have been reported to play vital roles in the regulation of the osteogenic differentiation of MSCs. However, the mechanism by which lncRNA affects the proliferation and apoptosis of MSCs is unclear. In this study, sequencing analysis revealed that LINC00707 was significantly decreased in non-adherent human MSCs (non-AC-hMSCs) compared to adherent human MSCs. Moreover, LINC00707 overexpression promoted non-AChMSC proliferation, cell cycle progression from the G0/G1 phase to the S phase and inhibited apoptosis, whereas LINC00707 silencing had the opposite effect. Furthermore, LINC00707 interacted directly with the quaking (QKI) protein and enhanced the E3 ubiquitin-protein ligase ring finger protein 6 (RNF6)-mediated ubiquitination of the QKI protein. Additionally, the overexpression of QKI rescued the promotive effects on proliferation and inhibitory effects on apoptosis in non-AC-hMSCs induced by the ectopic expression of LINC00707. Thus, LINC00707 contributes to the proliferation and apoptosis in non-AChMSCs by regulating the ubiquitination and degradation of the QKI protein.

Circular RNAs (circRNAs) are a class of non-coding RNA molecules that are gaining increasing attention for their roles in various pathophysiological processes. The RNA-binding protein quaking (QKI) has been identified as a regulator of circRNA formation. In this study, we investigate the role of QKI in the formation of circRNAs in the heart by performing RNA-sequencing on Qki-knockout mice. Loss of QKI resulted in the differential expression of 17% of the circRNAs in adult mouse hearts. Interestingly, the majority of the QKI-regulated circRNAs (58%) were derived from genes undergoing QKI-dependent splicing, indicating a relationship between back-splicing and linear splicing. We compared these QKI-dependent circRNAs with those regulated by RBM20, another cardiac splicing factor essential for circRNA formation. We found that QKI and RBM20 regulate the formation of a distinct, but partially overlapping set of circRNAs in the heart. Strikingly, many shared circRNAs were derived from the Ttn gene, and they were regulated in an opposite manner. Our findings indicate that QKI not only regulates alternative splicing in the heart but also the formation of circRNAs.

In the heart, splicing factors orchestrate the functional properties of cardiomyocytes by regulating the alternative splicing of multiple genes. Work in embryonic stem cells has shown that the splicing factor Quaking (QKI) regulates alternative splicing during cardiomyocyte differentiation. However, the relevance and function of QKI in adult cardiomyocytes remains unknown. In this study, we aim to identify the in vivo function of QKI in the adult mouse heart.
We generated mice with conditional deletion of QKI in cardiomyocytes by the Cre-Lox system. Mice with cardiomyocyte-specific deletion of QKI died during the foetal period (E14.5), without obvious anatomical abnormalities of the heart. Adult mice with tamoxifen-inducible QKI deletion rapidly developed heart failure associated with severe disruption of sarcomeres, already 7 days after knocking out QKI. RNA sequencing revealed that QKI regulates the alternative splicing of more than 1000 genes, including sarcomere and cytoskeletal components, calcium-handling genes, and (post-)transcriptional regulators. Many of these splicing changes corresponded to the loss of muscle-specific isoforms in the heart. Forced overexpression of QKI in cultured neonatal rat ventricular myocytes directed these splicing events in the opposite direction and enhanced contractility of cardiomyocytes.
Altogether, our findings show that QKI is an important regulator of the muscle-specific alternative splicing program that builds the contractile apparatus of cardiomyocytes.

BACKGROUND: Lacrimal adenoid cystic carcinoma (LACC) is the most common orbital malignant epithelial neoplasm. LACC with high-grade transformation (LACC-HGT) has higher rates of recurrence, metastasis, and mortality than LACC without HGT. This study investigated the effects of microRNA-29a-3p (miR-29a-3p) in the pathogenesis of LACC-HGT.
METHODS: An Agilent human miRNA microarray was used to screen the differentially expressed miRNAs (DEMs) in LACC and LACC-HGT tumor tissues. Then, the primary cells obtained in previous studies were used to determine the effect of miR-29a-3p.
RESULTS: The expression of miR-29a-3p was abnormally lower in LACC-HGT than in LACC. miR-29a-3p can specifically target the 3\' UTR of Quaking mRNA and down-regulate Quaking expression, thereby inhibiting the proliferation, migration, and epithelial-mesenchymal transition of LACC cells.
CONCLUSIONS: This study illustrated that miR-29a-3p functions as a tumor suppressor by down-regulating the expression of Quaking to inhibit the tumorigenesis of LACC cells. This study may also reveal the pathogenesis of HGT in LACC cells and provide a reference for LACC-HGT targeted diagnosis.

BACKGROUND: Sepsis is a fatal condition commonly caused by Methicillin-resistant Staphylococcus aureus (MRSA) with a high death rate. Macrophages can protect the host from various microbial pathogens by recognizing and eliminating them. Earlier we found that Quaking (QKI), an RNA binding protein (RBP), was involved in differentiation and polarization of macrophages. However, the role of QKI in sepsis caused by pathogenic microbes, specifically MRSA, is unclear. This study aimed to investigate the role of QKI in regulation of host-pathogen interaction in MRSA-induced sepsis and explored the underlying mechanisms.
METHODS: Transmission electron microscope and immunofluorescence were used to observe the autophagy level in macrophages. Real-time PCR and western blot were used to analyzed the expression of mRNA and protein respectively. The potential protein interaction was analyzed by iTRAQ mass spectrometry and Immunoprecipitation. RNA fluorescence in situ hybridization, dual-luciferase reporter assay and RNA immunoprecipitation were used to explore the mechanism of QKI regulating mRNA of PI3K-p110β.
RESULTS: The mRNA level of QKI was aberrantly decreased in monocytes and PBMCs of septic patients with the increasing level of plasma procalcitonin (PCT). Then the mice with myeloid specific knockout of QKI was challenged with MRSA or Cecal Ligation and Puncture (CLP). Mice in these two models displayed higher survival rates and lower bacterial loads. Mechanistically, QKI deletion promoted phagocytosis and autophagic degradation of MRSA via activating p110β (a member of Class IA phosphoinositide 3-kinases) mediated autophagic response. QKI expression in macrophages led to the sequestration of p110β in mRNA processing (P) bodies and translational repression. Upon infection, the direct interaction of RNF6, a RING-type E3 ligase, mediated QKI ubiquitination degradation and facilitated PI3K-p110β related autophagic removal of pathogen. The administration of nanoparticles with QKI specific siRNA significantly protected mice from MRSA infection.
CONCLUSIONS: This study disclosed the novel function of QKI in the P body mRNA regulation during infection. QKI degradation in macrophage by RNF6 protects mice from MRSA infection via enhancing PI3K-p110β dependent autophagy. It suggested that QKI may serve as a potential theranostic marker in MRSA-induced sepsis.

Vascular diseases account for a significant number of deaths worldwide, with cardiovascular diseases remaining the leading cause of mortality. This ongoing, ever-increasing burden has made the need for an effective treatment strategy a global priority. Recent advances in regenerative medicine, largely the derivation and use of induced pluripotent stem cell (iPSC) technologies as disease models, have provided powerful tools to study the different cell types that comprise the vascular system, allowing for a greater understanding of the molecular mechanisms behind vascular health. iPSC disease models consequently offer an exciting strategy to deepen our understanding of disease as well as develop new therapeutic avenues with clinical translation. Both transcriptional and post-transcriptional mechanisms are widely accepted to have fundamental roles in orchestrating responses to vascular damage. Recently, iPSC technologies have increased our understanding of RNA-binding proteins (RBPs) in controlling gene expression and cellular functions, providing an insight into the onset and progression of vascular dysfunction. Revelations of such roles within vascular disease states have therefore allowed for a greater clarification of disease mechanisms, aiding the development of novel therapeutic interventions. Here, we discuss newly discovered roles of RBPs within the cardio-vasculature aided by iPSC technologies, as well as examine their therapeutic potential, with a particular focus on the Quaking family of isoforms.

OBJECTIVE: Percutaneous coronary intervention (PCI) is one of the important methods for the treatment of coronary artery disease (CAD). In-sent restenosis (ISR) after PCI for patients suffered from CAD is considered to be an essential factor affecting long-term outcomes and prognosis of this disease. This study aims to investigate the correlation between plasma Quaking (QKI) and cyclooxygenase-2 (COX-2) levels and ISR in patients with CAD.
METHODS: A total of 218 consecutive CAD patients who underwent coronary angiography and coronary arterial stenting from September 2019 to September 2020 in the Department of Cardiology of Xiangya Hospital of Central South University were enrolled in this study, and 35 matched individuals from the physical examination center were served as a control group. After admission, clinical data of these 2 groups were collected. Plasma QKI and COX-2 levels were measured by enzyme linked immunosorbent assay (ELISA). Follow-up angiography was performed 12 months after PCI. CAD patients were divided into a NISR group (n=160) and an ISR group (n=58) according to the occurrence of ISR based on the coronary angiography. The clinical data, coronary angiography, and stent features between the NISR group and the ISR group were compared, and multivariate logistic regression was used to explore the factors influencing ISR. The occurrence of major adverse cardiovascular events (MACE) 1 year after operation was recorded. Fifty-eight patients with ISR were divided into an MACE group (n=24) and a non-MACE group (n=34), classified according to the occurrence of MACE, and the plasma levels of QKI and COX-2 were compared between the 2 groups. Receiver operating characteristic (ROC) curves were utilized to analyze the diagnostic value of plamsa levels of QKI and COX-2 for ISR and MACE occurrences in patients after PCI.
RESULTS: Compared with control group, plasma levels of QKI and COX-2 in the CAD group decreased significantly (all P<0.001). Compared with the NISR group, the plasma levels of QKI and COX-2 also decreased obviously in the ISR group (all P<0.001), while the levels of high sensitivity C-reactive protein (hs-CRP) and glycosylated hemoglobin (HbAlc) significantly increased (all P<0.001). The level of COX-2 was negatively correlated with hs-CRP (r=-0.385, P=0.003). Multivariate logistic regression analysis showed that high level of plasma QKI and COX-2 were protective factors for in-stent restenosis after PCI, while hs-CRP was a risk factor. ROC curve analysis showed that the sensitivity and specificity of plasma QKI for evaluating the predictive value of ISR were 77.5% and 66.5%, respectively, and the sensitivity and specificity of plasma COX-2 for evaluating the predictive value of ISR were 80.0% and 70.7%, respectively. The sensitivity and specificity of plasma QKI combined with COX-2 for evaluating the predictive value of ISR were 81.3% and 74.1%, respectively. The sensitivity and specificity of plasma QKI for evaluating the prognosis of ISR were 75.0% and 64.7%, respectively. The sensitivity and specificity of plasma COX-2 for evaluating the prognosis of ISR were 75.0% and 70.6%, respectively. The sensitivity and specificity of plasma QKI combined with COX-2 for prognostic evaluation of ISR were 81.7% and 79.4%, respectively. The sensitivity and specificity of plasma COX-2 combined with QKI for evaluating ISR and MACE occurrences in patients after PCI were better than those of COX-2 or QKI alone (P<0.001).
CONCLUSIONS: High level of plasma QKI and COX-2 might be a protective factor for ISR, which can also predict ISR patient\'s prognosis.
目的: 经皮冠状动脉介入术(percutaneous coronary intervention，PCI)是治疗冠心病的重要手段之一，支架植入术后发生的支架内再狭窄(in-stent restenosis，ISR)是影响冠心病患者PCI术后远期疗效及预后的重要因素。本研究旨在探讨血浆Quaking(QKI)、环氧合酶-2(cyclooxygenase-2，COX-2)水平与冠心病患者PCI术后ISR的相关性。方法: 连续选取2019年9月至2020年9月在中南大学湘雅医院心血管内科行冠状动脉造影并行支架植入术的冠心病患者218例，选取同期在体检中心体检的与冠心病患者基线资料相匹配的35例个体作为对照组。入院后收集两组患者的临床资料，采用酶联免疫吸附试验(enzyme linked immunosorbent assay，ELISA)测定血浆QKI、COX-2水平。术后12个月复查冠心病患者的冠状动脉造影，根据是否发生ISR将218例冠心病患者分为无再狭窄组(NISR组，n=160)与再狭窄组(ISR组，n=58)。比较NISR组与ISR组的一般资料及冠状动脉临床指标，采用多因素logistic回归分析ISR的影响因素。记录1年内主要不良心血管事件(major adverse cardiovascular events，MACE)发生情况，并据此将58例PCI术后ISR患者分为未发生MACE组(34例)与发生MACE组(24例)，比较两组的血浆QKI、COX-2水平，采用受试者操作特征(receiver operating characteristic，ROC)曲线探究血浆QKI、COX-2水平对PCI术后ISR及MACE的诊断价值。结果: 与对照组相比，冠心病组COX-2及QKI水平明显下降(均P<0.001)。与NISR组相比，ISR组超敏C反应蛋白(high sensitivity C-reactive protein，hs-CRP)、糖化血红蛋白(glycosylated hemoglobin，HbAlc)显著升高(均P<0.001)，COX-2及QKI水平明显下降(均P<0.001)。血浆COX-2与hs-CRP水平呈负相关(r=-0.385，P=0.003)。多因素logistic回归分析发现血浆高水平QKI、COX-2是冠心病患者PCI术后ISR的保护因素，高水平hs-CRP是危险因素。ROC曲线分析显示：血浆QKI诊断冠心病患者PCI术后ISR的灵敏度为77.5%，特异度为65.5%；血浆COX-2的灵敏度为80.0%，特异度为70.7%；血浆QKI联合COX-2的灵敏度为81.3%，特异度为74.1%。血浆QKI预测PCI术后ISR患者发生MACE的灵敏度为75.0%，特异度为64.7%；血浆COX-2的灵敏度为75.0%，特异度为70.6%；血浆QKI联合COX-2的灵敏度为81.7%，特异度为79.4%。血浆QKI联合COX-2预测冠心病患者PCI术后发生ISR及MACE的灵敏度、特异度均优于单独应用QKI或COX-2。结论: 高水平的血浆QKI和COX-2可能是冠心病患者PCI术后ISR的保护因素；血浆QKI和COX-2水平可为冠心病患者PCI术后ISR的发生及患者预后提供一定的预测价值。.

Japanese encephalitis (JE) is a major reason to cause viral encephalitis, with 50% patients suffering from severe neuro-inflammation and permanent neural injury. Effective anti-viral treatment is urgently needed. Here, we found RNA binding protein quaking (QKI) was involved in the progression of JE by regulating migration and anti-viral response of macrophages. After JE virus (JEV) infection, QKI-deficient mice had lower viral loads in the brain and fewer neurological symptoms. In comparison with control mice, proinflammatory cytokines in the brain of QKI-deficient animals revealed distinct patterns, with lower levels of IL-6 (interleukin-6) and IFN-β (interferon-β) at the early stage but higher levels at the end of JE. Then we found infiltration of CCR2 positive ((C-C motif) receptor 2) peripheral macrophages and CCR2 expression on macrophages were inhibited in QKI-deficient mice, while the expression of CCR2 ligands was not changed. Bioinformatical analysis showed that a QRE (quaking response element) located on 3\'UTR (untranslated region) of Ccr2. We further verified that QKI was able to interact with Ccr2 mRNA and regulate its degradation in vitro. Additionally, since the IFN-β production was increased in QKI-ablation mice after JEV infection, the anti-viral response was analyzed. Results in QKI-silenced N9 cells showed that the expression of RIG-I (retinoic acid-inducible gene-I) and TBK1 (TANK binding kinase 1) was increased, thus further inducing IRF3 (interferon regulatory factor 3) phosphorylation and interferon activation. Overall, these results revealed QKI mediated the anti-viral process via interfering migration of macrophages to CNS (central nervous system) and enhancing RIG-I/IRF3/IFN-β pathway to restrict virus dissemination.