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The long noncoding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) is involved in a variety of human cancers. Two overlapping NEAT1 isoforms, NEAT1_1 and NEAT1_2, are produced through mutually exclusive alternative 3' end formation. Previous studies extensively investigated NEAT1 dysregulation in tumors, but often failed to achieve distinct quantification of the two NEAT1 isoforms. Moreover, molecular mechanisms governing the biogenesis of NEAT1 isoforms and the functional impacts of their dysregulation in tumorigenesis remain poorly understood. In this study, we employed an isoform-specific quantification assay and found differential dysregulation of NEAT1 isoforms in patient-derived glioblastoma multiforme cells. We further showed usage of the NEAT1 proximal polyadenylation site (PAS) is a critical mechanism that controls glioma NEAT1 isoform production. CRISPR-Cas9-mediated PAS deletion reduced NEAT1_1 and reciprocally increased NEAT1_2, which enhanced nuclear paraspeckle formation in human glioma cells. Moreover, the utilization of the NEAT1 PAS is facilitated by the RNA-binding protein quaking (QKI), which binds to the proximal QKI recognition elements. Functionally, we identified transcriptomic changes and altered biological pathways caused by NEAT1 isoform imbalance in glioma cells, including the pathway for the regulation of cell migration. Finally, we demonstrated the forced increase of NEAT1_2 upon NEAT1 PAS deletion is responsible for driving glioma cell migration and promoting the expression of genes implicated in the regulation of cell migration. Together, our studies uncovered a novel mechanism that regulates NEAT1 isoforms and their functional impacts on the glioma transcriptome, which affects pathological pathways of glioma, represented by migration., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: RNA-binding proteins are revealed to play important roles during the progression of hepatocellular carcinoma (HCC). However, the regulatory mechanisms of RNA-binding protein Quaking (QKI) in the expression and role of long non-coding RNAs (lncRNAs) in HCC cells remain not well understood., Methods: Cell Counting Kit-8, wound-healing, Transwell and colony-forming assays were performed to evaluate the effects of QKI and lncRNA EGOT on proliferation and migration of HCC cells. Tumor growth of HCC was analyzed using a mouse xenograft model. Immunoprecipitation (RIP) assay was used to investigate the interaction between QKI and EGOT., Results: The expression of QKI was significantly upregulated in HCC tissues and the higher QKI level was significantly associated with a poorer prognosis. Overexpression of QKI promoted the proliferation, migration, and colony-forming ability of HCC cells in vitro and tumor growth of HCC in vivo. Mechanistically, QKI protein could bind to EGOT RNA and increase its expression. Inhibition of EGOT attenuated the effects of QKI on the malignant phenotypes of HCC cells. In addition, both QKI and EGOT could activate the SAPK/JNK signaling pathway in HCC cells., Conclusions: Our findings indicated that QKI exerted promotive effects on the malignant phenotypes of HCC through its interaction with EGOT., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background/aim: The RNA binding protein quaking (QKI) is associated with the development and progression of tumor suppressors in various cancers. However, the clinical implications of QKI expression have not yet been fully elucidated. In this study, we aimed to investigate the clinicopathological and prognostic significance of QKI expression in hepatocellular carcinoma (HCC)., Materials and Methods: We performed QKI, Zinc finger E-box-binding homeobox 1 (ZEB1), E-cadherin, and glutathione peroxidase 4 (GPX4) immunohistochemical staining on 166 HCC patient tissue samples. X-tile bioinformatics software was used to set the cut-off value for high QKI expression. Correlations between QKI expression and various clinicopathological parameters were assessed., Results: The best cut-off value for high QKI expression was 12.5. High QKI expression was observed in 28 of 166 patients (16.9%) and was an independent prognostic factor for inferior recurrence-free survival (RFS). In addition, high ZEB1 and GPX4 expression correlated with high QKI expression, but not with the loss of E-cadherin expression., Conclusion: High QKI expression was identified in HCCs and associated with poor RFS. QKI might be a prognostic biomarker of HCCs associated with epithelial-to-mesenchymal transition and a potential candidate therapeutic target., (Copyright © 2024, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Objectives: To investigate the role of circRNA regulators MBNL1 and QKI in the progression of esophageal squamous cell carcinoma., Background: MBNL1 and QKI are pivotal regulators of pre-mRNA alternative splicing, crucial for controlling circRNA production - an emerging biomarker and functional regulator of tumor progression. Despite their recognized roles, their involvement in ESCC progression remains unexplored., Methods: The expression levels of MBNL1 and QKI were examined in 28 tissue pairs from ESCC and adjacent normal tissues using data from the GEO database. Additionally, a total of 151 ESCC tissue samples, from stage T1 to T4, consisting of 13, 43, 87, and 8 cases per stage, respectively, were utilized for immunohistochemical (IHC) analysis. RNA sequencing was utilized to examine the expression profiles of circRNAs, lncRNAs, and mRNAs across 3 normal tissues, 3 ESCC tissues, and 3 pairs of KYSE150 cells in both wildtype (WT) and those with MBNL1 or QKI knockouts. Transwell, colony formation, and subcutaneous tumorigenesis assays assessed the impact of MBNL1 or QKI knockout on ESCC cell migration, invasion, and proliferation., Results: ESCC onset significantly altered MBNL1 and QKI expression levels, influencing diverse RNA species. Elevated MBNL1 or QKI expression correlated with patient age or tumor invasion depth, respectively. MBNL1 or QKI knockout markedly enhanced cancer cell migration, invasion, proliferation, and tumor growth. Moreover, the absence of either MBNL1 or QKI modulated the expression profiles of multiple circRNAs, causing extensive downstream alterations in the expression of numerous lncRNAs and mRNAs. While the functions of circRNA and lncRNA among the top 20 differentially expressed genes remain unclear, mRNAs like SLCO4C1, TMPRSS15, and MAGEB2 have reported associations with tumor progression., Conclusions: This study underscores the tumor-suppressive roles of MBNL1 and QKI in ESCC, proposing them as potential biomarkers and therapeutic targets for ESCC diagnosis and treatment., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

QKI is a vital regulator in RNA splicing and maturation, but its role in cervical cancer (CC) is little known. In this study, we found that QKI is decreased in human CC, and overexpression of QKI inhibits HeLa cell proliferation and promotes the apoptosis of cancer cells. We identified hundreds of endogenous QKI-regulated alternative splicing events (ASEs) and differentially expressed genes (DEGs) in QKI-overexpressed HeLa cells by RNA-seq and selectively validated their expression by quantitative reverse-transcription polymerase chain reaction. The gene ontology and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis showed that QKI-regulated ASEs and DEGs were closely related to cancer, apoptosis, and transcriptional regulatory functions. In short, QKI may affect the occurrence and development of CC by regulating gene expression through AS.

Accumulating evidence indicates that the alternative splicing program undergoes extensive changes during cancer development and progression. The RNA-binding protein QKI-5 is frequently downregulated and exhibits anti-tumor activity in lung cancer. Howeve-r, little is known about the functional targets and regulatory mechanism of QKI-5. Here, we report that upregulation of exon 14 inclusion of cytoskeletal gene Adducin 3 (ADD3) significantly correlates with a poor prognosis in lung cancer. QKI-5 inhibits cell proliferation and migration in part through suppressing the splicing of ADD3 exon 14. Through genome-wide mapping of QKI-5 binding sites in vivo at nucleotide resolution by iCLIP-seq analysis, we found that QKI-5 regulates alternative splicing of its target mRNAs in a binding position-dependent manner. By binding to multiple sites in an upstream intron region, QKI-5 represses the splicing of ADD3 exon 14. We also identified several QKI mutations in tumors, which cause dysregulation of the splicing of QKI targets ADD3 and NUMB. Taken together, our results reveal that QKI-mediated alternative splicing of ADD3 is a key lung cancer-associated splicing event, which underlies in part the tumor suppressor function of QKI., (© The Author(s) (2021). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS.)

Myelination depends on timely, precise control of oligodendrocyte differentiation and myelinogenesis. Cholesterol is the most abundant component of myelin and essential for myelin membrane assembly in the central nervous system. However, the underlying mechanisms of precise control of cholesterol biosynthesis in oligodendrocytes remain elusive. In the present study, we found that Qki depletion in neural stem cells or oligodendrocyte precursor cells in neonatal mice resulted in impaired cholesterol biosynthesis and defective myelinogenesis without compromising their differentiation into Aspa + Gstpi + myelinating oligodendrocytes. Mechanistically, Qki-5 functions as a co-activator of Srebp2 to control transcription of the genes involved in cholesterol biosynthesis in oligodendrocytes. Consequently, Qki depletion led to substantially reduced concentration of cholesterol in mouse brain, impairing proper myelin assembly. Our study demonstrated that Qki-Srebp2-controlled cholesterol biosynthesis is indispensable for myelinogenesis and highlights a novel function of Qki as a transcriptional co-activator beyond its canonical function as an RNA-binding protein., Competing Interests: XZ, SS, CH, QZ, MR, JR, CD, RZ, TS, LY, YW, YC, FL, JH No competing interests declared, (© 2021, Zhou et al.)

The role of RNA binding proteins in regulating the phagocytic and cytokine-releasing functions of microglia is unknown. Here, we show that microglia deficient for the QUAKING (QKI) RNA binding protein have increased proinflammatory cytokine release and defects in processing phagocytosed cargo. Splicing analysis reveals a role for QKI in regulating microexon networks of the Rho GTPase pathway. We show an increase in RhoA activation and proinflammatory cytokines in QKI-deficient microglia that are repressed by treating with a Rock kinase inhibitor. During the cuprizone diet, mice with QKI-deficient microglia are inefficient at supporting central nervous system (CNS) remyelination and cause the recruited oligodendrocyte precursor cells to undergo apoptosis. Furthermore, the expression of QKI in microglia is downregulated in preactive, chronic active, and remyelinating white matter lesions of multiple sclerosis (MS) patients. Overall, our findings identify QKI as an alternative splicing regulator governing a network of Rho GTPase microexons with implications for CNS remyelination and MS patients., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Cervical cancer is the fourth most common female malignancy worldwide and microRNA (miR)-574-5p is a candidate oncogene in multiple cancers. The present study aimed to investigate the role and mechanism of miR-574-5p in cervical cancer. miR-574-5p inhibitors or mimics were transfected into cervical cancer cells to study the function of miR-574-5p. The effects of miR-574-5p on cell growth, invasion, and chemosensitivity were evaluated using CCK8, flow cytometry, transwell, immunofluorescence, and Western blotting analysis. Further depletion or forced expression of QKI was performed to explore the regulatory mechanism of miR-574-5p in cervical cancer. Up-regulation of miR-547-5p and down-regulation of QKI were observed in 30 cervical cancer tissues versus 30 adjacent normal tissues. Silencing of miR-574-5p increased apoptosis, inhibited proliferation, cell cycle progression, and cell invasiveness, as well as enhanced chemosensitivity towards cisplatin and doxorubicin in cervical cancer cells. Overexpression of miR-574-5p exerted promoting effect on cancer progression and metastasis. Knockdown of miR-574-5p induced an up-regulation of E-cadherin and down-regulation of cyclinD1, N-cadherin, matrix metallopeptidase 9 (MMP-9), and β-catenin in cervical cancer cells Moreover, QKI was verified as a target of miR-574-5p and involved in regulation of miR-574-5p-induced cervical cancer cell progression and metastasis. miR-574-5p functions to be oncogenic in cervical cancer, and its inhibition suppresses cervical cancer progression and metastasis as well as enhances chemosensitivity by targeting QKI. Therefore, miR-574-5p is suggested as a potential therapeutic target for cervical cancer treatment.

Quaking (QKI) is an RNA-binding protein (RBP) involved in multiple aspects of RNA metabolism and many biological processes. Despite a known immune function in regulating monocyte differentiation and inflammatory responses, the degree to which QKI regulates the host interferon (IFN) response remains poorly characterized. Here we show that QKI ablation enhances poly(I:C) and viral infection-induced IFNβ transcription. Characterization of IFN-related signalling cascades reveals that QKI knockout results in higher levels of IRF3 phosphorylation. Interestingly, complementation with QKI-5 isoform alone is sufficient to rescue this phenotype and reduce IRF3 phosphorylation. Further analysis shows that MAVS, but not RIG-I or MDA5, is robustly upregulated in the absence of QKI, suggesting that QKI downregulates MAVS and thus represses the host IFN response. As expected, MAVS depletion reduces IFNβ activation and knockout of MAVS in the QKI knockout cells completely abolishes IFNβ induction. Consistently, ectopic expression of RIG-I activates stronger IFNβ induction via MAVS-IRF3 pathway in the absence of QKI. Collectively, these findings demonstrate a novel role for QKI in negatively regulating host IFN response by reducing MAVS levels.

Purpose: Glioblastoma (GBM) stem cells (GSCs) have been found to be the main cause of malignant GBM progression. It has also been found that Quaking homolog (QKI) plays a predominant role in driving GBM development. Here, we aimed to asses the role of QKI in maintaining GSC stemness and inducing the invasiveness of GBM cells., Methods: Public databases were used to assess the expression of QKI and its correlation with stemness markers in primary GBMs. The CRISPR-Cas9 technology was used to generate QKI knockout GBM cells, and RNA immunoprecipitation was used to assess QKI-GLI1 protein-mRNA interactions. In addition, in vitro and in vivo GBM cell proliferation, migration, xenografting and neurosphere formation assays were performed., Results: Using public GBM databases, QKI was identified as a potential GSC regulator. We found that QKI could inhibit stem-like cell (SLC) stemness and prolong the survival of xenografted mice. Mechanistically, we found that QKI knockout increased the GLI Family Zinc Finger 1 (GLI1) mRNA level, which is essential for maintaining the self-renewal ability of GSCs. In addition, we found that QKI knockout activated the Hedgehog signaling pathway via Tra-2 and GLI response element (TGE)-specific GLI1 mRNA disruption., Conclusion: Our data indicate that upregulation of GLI1 induced by QKI deficiency maintains GSC stemness and enhances the invasiveness of GBM cells, thereby hinting at new options for the treatment of GBM.

Quaking homolog (QKI) is a member of the RNA-binding signal transduction and activator of proteins family. Previous studies showed that QKI possesses the tumour suppressor activity in human cancers by interacting with the 3'-untraslated region (3'-UTR) of various gene transcripts via the STAR domain. This study first assessed the association of QKI-6 expression with clinicopathological and survival data from bladder cancer patients and then investigated the underlying molecular mechanisms. Bladder cancer tissues (n = 223) were subjected to immunohistochemistry, and tumour cell lines and nude mice were used for different in vitro and in vivo assays following QKI-6 overexpression or knockdown. QKI-6 down-regulation was associated with advanced tumour TNM stages and poor patient overall survival. QKI-6 overexpression inhibited bladder cancer cell growth and invasion capacity, but induced tumour cell apoptosis and cell cycle arrest. Furthermore, ectopic expression of QKI-6 reduced tumour xenograft growth and expression of proliferation markers, Ki67 and PCNA. However, knockdown of QKI-6 expression had opposite effects in vitro and in vivo. QKI-6 inhibited expression of E2 transcription factor 3 (E2F3) by directly binding to the E2F3 3'-UTR, whereas E2F3 induced QKI-6 transcription by binding to the QKI-6 promoter in negative feedback mechanism. QKI-6 expression also suppressed activity and expression of nuclear factor-κB (NF-κB) signalling proteins in vitro, implying a novel multilevel regulatory network downstream of QKI-6. In conclusion, QKI-6 down-regulation contributes to bladder cancer development and progression., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

The microRNA-200 (miR-200) family plays a major role in specifying epithelial phenotype by preventing expression of the transcription repressors ZEB1 and ZEB2, which are well-known regulators of the epithelial-to-mesenchymal transition (EMT) in epithelial tumors including oral squamous cell carcinoma (OSCC). Here, we elucidated whether miR-200 family members control RNA-binding protein quaking (QKI), a newly identified tumor suppressor that is regulated during EMT. We predicted that miR-200a and miR-200b could recognize QKI 3'-UTR by analyzing TargetScan and The Cancer Genome Atlas head and neck squamous cell carcinoma (HNSCC) dataset. Forced expression of miR-200b/a/429 inhibited expression of ZEB1/2 and decreased cell migration in OSCC cell lines CAL27 and HSC3. QKI expression was also suppressed by miR-200 overexpression, and the 3'-UTR of QKI mRNA was directly targeted by miR-200 in luciferase reporter assays. Interestingly, shRNA-mediated knockdown of QKI led to pronounced EMT and protumor effects in both in vitro and in vivo studies of OSCC. Furthermore, high expression of QKI protein is associated with favorable prognosis in surgically resected HNSCC and lung adenocarcinoma. In conclusion, QKI increases during EMT and is targeted by miR-200; while, it suppresses EMT and tumorigenesis. We suggest that QKI and miR-200 form a negative feedback loop to maintain homeostatic responses to EMT-inducing signals., (© 2019 UICC.)

The four dengue viruses (DENV1-4) are rapidly reemerging infectious RNA viruses. These positive-strand viral genomes contain structured 3' untranslated regions (UTRs) that interact with various host RNA binding proteins (RBPs). These RBPs are functionally important in viral replication, pathogenesis, and defense against host immune mechanisms. Here, we combined RNA chromatography and quantitative mass spectrometry to identify proteins interacting with DENV1-4 3' UTRs. As expected, RBPs displayed distinct binding specificity. Among them, we focused on quaking (QKI) because of its preference for the DENV4 3' UTR (DENV-4/SG/06K2270DK1/2005). RNA immunoprecipitation experiments demonstrated that QKI interacted with DENV4 genomes in infected cells. Moreover, QKI depletion enhanced infectious particle production of DENV4. On the contrary, QKI did not interact with DENV2 3' UTR, and DENV2 replication was not affected consistently by QKI depletion. Next, we mapped the QKI interaction site and identified a QKI response element (QRE) in DENV4 3' UTR. Interestingly, removal of QRE from DENV4 3' UTR abolished this interaction and increased DENV4 viral particle production. Introduction of the QRE to DENV2 3' UTR led to QKI binding and reduced DENV2 infectious particle production. Finally, reporter assays suggest that QKI reduced translation efficiency of viral RNA. Our work describes a novel function of QKI in restricting viral replication., (© 2018 Liao et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Chronic exposure to arsenite can cause various human tumors. For the initiation and recurrence of human liver cancer, the acquisition of CSC-like properties is essential. In various cancers, microRNAs (miRNAs) act as regulators in induction of CSC-like properties. Liver cancers over-express miR-155, but the mechanism relating miR-155 and arsenite-induced liver cancer is unknown. Here, we show that long-term exposure of L-02 cells to arsenite increases miR-155 levels by activation of NF-κB and leads to the acquisition of CSC-like properties. In spheroids formed from arsenite-transformed L-02 cells, the levels of miR-155 positively relate to the levels of CD90, EpCAM, and OCT4. Inhibition of miR-155, by reduction of SOX2 and OCT4, results in suppression of spheroid formation. Luciferase reporter assays indicate that QKI is a target of miR-155. Inhibition of QKI expression by miR-155 promotes arsenite-induced acquisition of CSC-like properties, whereas QKI over-expression has the opposite effect. Collectively, the findings demonstrate that miR-155, driven by NF-κB, reduces QKI expression and is involved in acquisition of the CSC-like phenotype during neoplastic transformation of hepatic cells induced by arsenite., (© 2017 Wiley Periodicals, Inc.)

Quaking protein isoforms arise from a single Quaking gene and bind the same RNA motif to regulate splicing, translation, decay, and localization of a large set of RNAs. However, the mechanisms by which Quaking expression is controlled to ensure that appropriate amounts of each isoform are available for such disparate gene expression processes are unknown. Here we explore how levels of two isoforms, nuclear Quaking-5 (Qk5) and cytoplasmic Qk6, are regulated in mouse myoblasts. We found that Qk5 and Qk6 proteins have distinct functions in splicing and translation, respectively, enforced through differential subcellular localization. We show that Qk5 and Qk6 regulate distinct target mRNAs in the cell and act in distinct ways on their own and each other's transcripts to create a network of autoregulatory and cross-regulatory feedback controls. Morpholino-mediated inhibition of Qk translation confirms that Qk5 controls Qk RNA levels by promoting accumulation and alternative splicing of Qk RNA, whereas Qk6 promotes its own translation while repressing Qk5. This Qk isoform cross-regulatory network responds to additional cell type and developmental controls to generate a spectrum of Qk5/Qk6 ratios, where they likely contribute to the wide range of functions of Quaking in development and cancer., (© 2017 Fagg et al.; Published by Cold Spring Harbor Laboratory Press.)

In this study, we tested the hypothesis that extracellular vesicles (EVs)-mediated transfer of miR-208a/b can exacerbate apoptosis of cardiomyocytes (CMs) induced by hypoxia/reoxygenation (H/R) injury by reducing the expression of the RNA-binding protein Quaking (QKI). EVs were isolated from culture medium of hypoxic H9c2 cells (EVs-H). In in vitro H9c2 cell model, the EVs-H could be taken up by normoxic CMs and exacerbated cell apoptosis induced by H/R injury. In addition, miR-208a and miR-208b were enriched in EVs-H. Suppression of miR-208a and miR-208b loading significantly suppressed the detrimental effect of EVs-H on H/R injury in H9c2 cells. Inhibition of endogenous miR-208a and miR-208b restored QKI5 and QKI6 after H/R treatment. Dual-luciferase assay confirmed direct bindings between miR-208a/b and QKI 3'UTR. Functionally, QKI5 overexpression significantly suppressed H/R-induced CM apoptosis and suppressed the enhancing effect of EVs-H on CM apoptosis. Therefore, we infer that EVs-mediated transfer of miR-208a/b can exaggerate H/R injury in CMs by reducing QKI expression. This represents a previously unrecognized pathway of H/R injury in CMs.

Background: This study investigated abnormal methylation in colorectal cancer (CRC) and the potential role of the Quaking RNA-binding protein (QKI) gene in tumorigenesis., Materials and Methods: Oligonucleotide microarray expression profiling was carried out on a panel of primary CRC specimens (n=17) and CRC cell lines (n=5), followed by methylation analysis using methylation-specific polymerase chain reaction. QKI expression levels were assessed in 156 primary CRCs by qRT-PCR and immunohistochemistry., Results: Low QKI expression was observed in 47.7% in CRCs. QKI promoter methylation was detected in 32.1% of patients with CRC, and in these patients mRNA expression in tumor tissue was significantly down-regulated compared to matched normal tissues (p=0.049). There was a significant relationship between low QKI expression and recurrence after surgery (p=0.004). Low QKI expression was an independent risk factor for recurrence after surgery in 153 patients with CRC without distant metastases (p=0.036)., Conclusion: Patients with tumors expressing low levels of QKI experienced significantly higher rates of tumor recurrence after curative surgery and worse prognoses. Methylation of the QKI promoter and concomitant reduced expression of QKI mRNA may be important for CRC initiation and progression. Loew QKI expression may be a useful clinical biomarker for predicting recurrence and prognosis., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Increasing interest has been devoted in recent years to the understanding of alternative splicing in cancer. In this study, we performed a genome-wide analysis to identify cancer-associated splice variants in non-small cell lung cancer. We discovered and validated novel differences in the splicing of genes known to be relevant to lung cancer biology, such as NFIB, ENAH or SPAG9. Gene enrichment analyses revealed an important contribution of alternative splicing to cancer-related molecular functions, especially those involved in cytoskeletal dynamics. Interestingly, a substantial fraction of the altered genes found in our analysis were targets of the protein quaking (QKI), pointing to this factor as one of the most relevant regulators of alternative splicing in non-small cell lung cancer. We also found that ESYT2, one of the QKI targets, is involved in cytoskeletal organization. ESYT2-short variant inhibition in lung cancer cells resulted in a cortical distribution of actin whereas inhibition of the long variant caused an increase of endocytosis, suggesting that the cancer-associated splicing pattern of ESYT2 has a profound impact in the biology of cancer cells. Finally, we show that low nuclear QKI expression in non-small cell lung cancer is an independent prognostic factor for disease-free survival (HR = 2.47; 95% CI = 1.11-5.46, P = 0.026). In conclusion, we identified several splicing variants with functional relevance in lung cancer largely regulated by the splicing factor QKI, a tumor suppressor associated with prognosis in lung cancer., (Copyright © 2016 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Quaking (QKI) is a tumor-suppressor gene encoding a conserved RNA-binding protein, whose expression is downregulated in several solid tumors. Here we report that QKI plays an important role in the immune response and suppression of leukemogenesis. We show that the expression of Qki is reduced in lipopolysaccharide (LPS)-challenged macrophages, suggesting that Qki is a key regulator of LPS signaling pathway. Furthermore, LPS-induced downregulation of Qki expression is miR-155-dependent. Qki overexpression impairs LPS-induced phosphorylation of JNK and particularly p38 MAPKs, in addition to increasing the production of anti-inflammatory cytokine IL-10. In contrast, Qki ablation decreases Fas expression and the rate of Caspase3/7 activity, while increasing the levels of IL-1α, IL-1β and IL-6, and p38 phosphorylation. Similarly, the p38 pathway is also a target of QKI activity in chronic lymphocytic leukemia (CLL)-derived MEC2 cells. Finally, B-CLL patients show lower levels of QKI expression compared with B cells from healthy donor, and Qki is similarily downregulated with the progression of leukemia in Eµ-miR-155 transgenic mice. Altogether, these data implicate QKI in the pathophysiology of inflammation and oncogenesis where miR-155 is involved.

Monocyte differentiation into macrophages results in upregulation of miR-29a and scavenger receptor A (SRA) expression, while the expression of RNA binding protein, QKI is suppressed. Since SRA is a functionally important protein in atherosclerosis, it is imperative to understand the various mechanisms involved in its regulation specially the mechanism involving miR-29a. There are individual studies linking miR-29a to SRA or QKI to monocyte differentiation but there is no evidence of any linkage among them. Therefore, we intend to investigate the association among these three, if any, in terms of regulation of SRA expression. Hence, in this study, the differentiated macrophages were initially transfected with miR-29a or its inhibitor and it was shown that QKI is a direct target of mir-29a. In addition, it was also observed by bioinformatics analysis that 3'UTR in SRA mRNA has QKI binding site. So, we attempted to further understand the role of QKI in SRA regulation. The macrophages were manipulated either with overexpression of QKI or by its ablation and it was observed that QKI suppressed SRA at the transcriptional level. Moreover, with the help of luciferase reporter vector, it was shown that QKI inhibited SRA transcription by binding to QRE region in its 3'UTR mRNA. Furthermore, to link the QKI mediated regulation of SRA expression with its functional activity; we analyzed lipid uptake capacity of macrophages transfected with either ectopic OKI plasmid or ablated for QKI. It was observed that, indeed, QKI upregulation inhibits lipid uptake by repressing SRA expression. Overall, our study demonstrates that miR-29a inhibits QKI, which in turn results in upregulation of SRA and lipid uptake., (Copyright © 2015. Published by Elsevier Inc.)

Cancer stem cells (CSCs) may contribute to tumor initiation, distant metastasis and chemo-resistance. One of RNA-binding proteins, Quaking (QKI), was reported to be a tumor suppressor. Here we showed that reduced QKI levels were observed in many human oral cancer samples. Moreover further reduction of QKI expression in CSCs was detected compared with non-CSCs in oral cancer cell lines. Overexpressing QKI in oral cancer cells significantly reduced CSC sphere formation and stem cell-associated genes. In tumor implanting nude mice model, QKI significantly impeded tumor initiation rates, tumor sizes and lung metastasis rates. As a contrast, knocking down QKI enhanced the above effects. Among the putative CSC target genes, SOX2 expression was negatively affected by QKI, mechanism study revealed that QKI may directly regulate SOX2 expression via specific binding with its 3'UTR in a cis element-dependent way. Loss of SOX2 even completely reversed the sphere forming ability in QKI knockdown cell line. Taken together, these data demonstrated that SOX2 is an important CSC regulator in oral cancer. QKI is a novel CSC inhibitor and impaired multiple oral CSC properties via partial repression of SOX2. Therefore, reduced expression of QKI may provide a novel diagnostic marker for oral cancer.

Background/Aim: The RNA binding protein quaking (QKI) is associated with the development and progression of tumor suppressors in various cancers. However, the clinical implications of QKI expression have not yet been fully elucidated. In this study, we aimed to investigate the clinicopathological and prognostic significance of QKI expression in hepatocellular carcinoma (HCC). Materials and Methods: We performed QKI, Zinc finger E-box-binding homeobox 1 (ZEB1), E-cadherin, and glutathione peroxidase 4 (GPX4) immunohistochemical staining on 166 HCC patient tissue samples. X-tile bioinformatics software was used to set the cut-off value for high QKI expression. Correlations between QKI expression and various clinicopathological parameters were assessed. Results: The best cut-off value for high QKI expression was 12.5. High QKI expression was observed in 28 of 166 patients (16.9%) and was an independent prognostic factor for inferior recurrence-free survival (RFS). In addition, high ZEB1 and GPX4 expression correlated with high QKI expression, but not with the loss of E-cadherin expression. Conclusion: High QKI expression was identified in HCCs and associated with poor RFS. QKI might be a prognostic biomarker of HCCs associated with epithelial-to-mesenchymal transition and a potential candidate therapeutic target. [ABSTRACT FROM AUTHOR]

The role of lncRNAs in the pathogenesis of cancer, including colorectal cancer (CRC), has repeatedly been demonstrated. However, very few lncRNAs have been well annotated functionally. Our study identified a novel lncRNA upregulated in CRC, NONHSAT136151, which was correlated with clinical progression. In functional assays, NONHSAT136151 significantly enhanced CRC cell proliferation, migration and invasion. Mechanistically, NONHSAT136151 interacted with RNA‐binding protein (RBP) QKI (Quaking) to interfere with QKI binding to target mRNAs and regulate their expression. As well, FOXP3 may be causally related to the dysregulation of NONHSAT136151 in CRC cells through its transcriptional activity. In conclusion, our findings identified a novel lncRNA regulated by FOXP3 participates in CRC progression through interacting with QKI, indicating a novel lncRNA‐RBP interaction mechanism is involved in CRC pathogenesis. [ABSTRACT FROM AUTHOR]

Pulmonary hypertension (PH) is a devastating disease characterized by progressive increases in pulmonary vascular resistance and remodeling, which eventually leads to right ventricular failure and death. The aim of this study was to identify novel molecular mechanisms involved in the hyperproliferation of pulmonary artery smooth muscle cells (PASMCs) in PH. In this study, we first demonstrated that the mRNA and protein expression amounts of QKI (Quaking), an RNA-binding protein, were elevated in human and rodent PH lung and pulmonary artery tissues and hypoxic human PASMCs. QKI deficiency attenuated PASMC proliferation in vitro and vascular remodeling in vivo. Next, we elucidated that QKI increases STAT3 (signal transducer and activator of transcription 3) mRNA stability by binding to its 3′ untranslated region. QKI inhibition reduced STAT3 expression and alleviated PASMC proliferation in vitro. Moreover, we also observed that the upregulated expression of STAT3 promoted PASMC proliferation in vitro and in vivo. In addition, as a transcription factor, STAT3 bound to microRNA (miR)–146b promoter to enhance its expression. We further showed that miR-146b promoted the proliferation of smooth muscle cells by inhibiting STAT1 and TET2 (Tet methylcytosine dioxygenase 2) during pulmonary vascular remodeling. This study has demonstrated new mechanistic insights into hypoxic reprogramming that arouses vascular remodeling, thus providing proof of concept for targeting vascular remodeling by directly modulating the QKI–STAT3–miR-146b pathway in PH. [ABSTRACT FROM AUTHOR]

Alternative splicing of mRNA precursors represents a key gene expression regulatory step and permits the generation of distinct protein products with diverse functions. In a genome-scale expression screen for inducers of the epithelial-to-mesenchymal transition (EMT), we found a striking enrichment of RNA-binding proteins. We validated that QKI and RBFOX1 were necessary and sufficient to induce an intermediate mesenchymal cell state and increased tumorigenicity. Using RNA-seq and eCLIP analysis, we found that QKI and RBFOX1 coordinately regulated the splicing and function of the actin-binding protein FLNB, which plays a causal role in the regulation of EMT. Specifically, the skipping of FLNB exon 30 induced EMT by releasing the FOXC1 transcription factor. Moreover, skipping of FLNB exon 30 is strongly associated with EMT gene signatures in basal-like breast cancer patient samples. These observations identify a specific dysregulation of splicing, which regulates tumor cell plasticity and is frequently observed in human cancer.

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. [ABSTRACT FROM AUTHOR]

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.

Early studies indicated that the testicular nuclear receptor 4 (TR4) might play key roles in altering prostate cancer (PCa) progression; however, its ability to alter PCa radiosensitivity remains unclear. Here, we found that suppressing TR4 expression promoted radiosensitivity and better suppressed PCa by modulating the protein quaking (QKI)/circZEB1/miR-141-3p/ZEB1 signaling pathway. Mechanism dissection studies revealed that TR4 could transcriptionally increase the RNA-binding protein QKI to increase circZEB1 levels, which then sponges the miR-141-3p to increase the expression of its host gene ZEB1. Preclinical studies with an in vivo mouse model further proved that combining radiation therapy (RT) with metformin promoted radiosensitivity to suppress PCa progression. Together, these results suggest that TR4 may play key roles in altering PCa radiosensitivity and show that targeting this newly identified TR4-mediated QKI/circZEB1/miR-141-3p/ZEB1 signaling pathway may help in the development of a novel RT to better suppress the progression of PCa. [ABSTRACT FROM AUTHOR]

Circular RNAs (circRNAs) have important regulatory roles in the development of various cancers. However, the biological functions and potential molecular mechanisms of circRNAs in hepatocellular carcinoma (HCC) are still unclear. In this study, we investigated the role of a new circRNA-circGSK3B (hsa_circ_0003763) and its molecular mechanism in HCC. We found that circGSK3B was highly expressed in HCC tissues and HCC cell lines. Additionally, the expression level of circGSK3B significantly correlated with HCC tumor size and vascular invasion. Functionally, we confirmed that circGSK3B can promote the proliferation, migration, and invasion of HCC cells in vivo and in vitro. In terms of mechanism, we demonstrated that circGSK3B acts as a miR-1265 sponge, positively regulates the target gene CAB39 , and promotes the reprogramming of glutamine metabolism, thereby promoting the progression of HCC. Finally, the classic RNA binding protein QKI was observed to participate in the biogenesis of circGSK3B. In summary, we proved that the circGSK3B-miR-1265- CAB39 axis can promote the proliferation, migration, invasion of HCC cells, indicating that circGSKB may serve as a promising diagnostic and prognostic marker in HCC. [ABSTRACT FROM AUTHOR]

Lung cancer is the leading cause of cancer-related deaths worldwide and non-small cell lung cancer (NSCLC) accounts for over 80% of lung cancer cases. The RNA binding protein, QKI, belongs to the STAR family and plays tumor-suppressive functions in NSCLC. QKI-5 is a major isoform of QKIs and is predominantly expressed in NSCLC. However, the underlying mechanisms of QKI-5 in NSCLC progression remain unclear. We found that QKI-5 regulated microRNA (miRNA), miR-196b-5p, and its expression was significantly up-regulated in NSCLC tissues. Up-regulated miR-196b-5p promotes lung cancer cell migration, proliferation, and cell cycle through directly targeting the tumor suppressors, GATA6 and TSPAN12. Both GATA6 and TSPAN12 expressions were down-regulated in NSCLC patient tissue samples and were negatively correlated with miR-196b-5p expression. Mouse xenograft models demonstrated that miR-196b-5p functions as a potent onco-miRNA, whereas TSPAN12 functions as a tumor suppressor in NSCLC in vivo. QKI-5 bound to miR-196b-5p and influenced its stability, resulting in up-regulated miR-196b-5p expression in NSCLC. Further analysis showed that hypomethylation in the promoter region enhanced miR-196b-5p expression in NSCLC. Our findings indicate that QKI-5 may exhibit novel anticancer mechanisms by regulating miRNA in NSCLC, and targeting the QKI5~miR-196b-5p~GATA6/TSPAN12 pathway may enable effectively treating some NSCLCs. [ABSTRACT FROM AUTHOR]

Circular RNAs (circRNAs) are class of non-coding RNAs formed by back-splicing events as loops, and could be found in all types of organisms. They play important and diverse roles in cell development, growth, and tumorigenesis, but functions of the majority of circRNAs remain enigmatic. Particularly functional phenotypes of great majority of circRNAs are not obvious. Here we randomly selected a circRNA circ-UBR5, which has no obvious functional phenotype in non-small cell lung cancer (NSCLC) cells from our previous research findings, to explore its potential function in cells. Differential expression of circ-UBR5 was detected in paired samples of tumorous tissues and adjacent nontumorous tissues from 59 patients with NSCLC by real-time quantitative reverse transcription-polymerase chain reactions (qRT-PCRs). Results showed circ-UBR5 expression was significantly downregulated in NSCLC tissues ( p  < 0.001) and was correlated with tumor differentiation (p  = 0.00126), suggesting circ-UBR5 might serve as an index of NSCLC differentiation. Our findings indicated circ-UBR5 could bind splicing regulatory factor QKI, KH domain containing RNA binding (QKI) and NOVA alternative splicing regulator 1 (NOVA1) and U1 small nuclear RNA (snRNA) in the nucleus, revealing circ-UBR5 might be a novel snRNA involved in RNA splicing regulatory process. Moreover, we first presented a highly efficient strategy for finding specific circRNA binding proteins using Human Protein Microarray (Huprot™ Protoarray). [ABSTRACT FROM AUTHOR]

Dysregulation of MSCs differentiation is associated with many pathophysiological processes. Genetically modified MSCs transplantation helps restore bone loss efficiently. BMSCs-specific QKI overexpressing and knockdown mice were built to explore QKI's role in bone formation and fat accumulation. Primary BMSCs with QKI overexpression and knockout were subjected to osteogenic and adipogenic differentiation. ALP staining and oil red O staining were performed to evaluate the differences between the groups. RNA immunoprecipitation was performed to identify the QKI-related pathway. QKI deficient BMSCs were transplanted into mice with glucocorticoid-induced osteoporosis to evaluate its therapeutic potential. Mice harboring BMSC-specific transgenic QKI exhibited reduced bone mass, while BMSC-specific QKI-deficient mice showed an increase in bone mass. Osteogenic differentiation of QKI deficient BMSCs was promoted and adipogenic differentiation was inhibited, while QKI overexpression in BMSCs displayed the opposite effects. To define the underlying mechanisms, RIP sequencing was performed. Wnt pathway-related genes were the putative direct target mRNAs of QKI, Canonical Wnt pathway activation was involved in QKI's effects on osteogenic differentiation. RNA immunoprecipitation quantitative real-time Polymerase Chain Reaction (PCR) and RNA fluorescence in situ hybridization experiments further validated that QKI repressed the expressions of Wnt5b, Fzd7, Dvl3 and β-catenin via direct binding to their putative mRNA specific sites. Glucocorticoid-induced osteoporotic mice transplanted with QKI deficient BMSCs exhibited less bone loss compared with mice transplanted with control BMSCs. QKI suppressed BMSCs osteogenic differentiation by downregulating the expressions of Wnt5b, Fzd7, Dvl3 and β-catenin. Loss of QKI in BMSCs transplantation may provide a new strategy for the treatment of orthopedic diseases such as osteoporosis. QKI binds to mRNA through QKI response elements (QREs), inhibiting expression of Wnt5b, Dishevelled 3, Frizzled 7 and β-catenin and suppressing osteogenic differentiation of BMSCs. [Display omitted] [ABSTRACT FROM AUTHOR]

the article informs that astrocytes are key players in Parkinson's disease where they can contribute to α-Synuclein pathology but also to neuroprotection via α-Synuclein clearance. In healthy conditions Qki recognizes its mRNA targets through Quaking Responsive Element Consensus sequence leading to mRNA degradation and inhibition of gene expression.