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ABSTRACTAlthough Argonaute (AGO) proteins have been the focus of microRNA (miRNA) studies, we observed AGO-free mature miRNAs directly interacting with RNA-binding proteins, implying the sophisticated nature of fine-tuning gene regulation by miRNAs. To investigate microRNA-binding proteins (miRBPs) globally, we analyzed PAR-CLIP data sets to identify RBP quaking (QKI) as a novel miRBP for let-7b. Potential existence of AGO-free miRNAs were further verified by measuring miRNA levels in genetically engineered AGO-depleted human and mouse cells. We have shown that QKI regulates miRNA-mediated gene silencing at multiple steps, and collectively serves as an auxiliary factor empowering AGO2/let-7b-mediated gene silencing. Depletion of QKI decreases interaction of AGO2 with let-7b and target mRNA, consequently controlling target mRNA decay. This finding indicates that QKI is a complementary factor in miRNA-mediated mRNA decay. QKI, however, also suppresses the dissociation of let-7b from AGO2, and slows the assembly of AGO2/miRNA/target mRNA complexes at the single-molecule level. We also revealed that QKI overexpression suppresses cMYC expression at post-transcriptional level, and decreases proliferation and migration of HeLa cells, demonstrating that QKI is a tumour suppressor gene by in part augmenting let-7b activity. Our data show that QKI is a new type of RBP implicated in the versatile regulation of miRNA-mediated gene silencing.

Abstract Background The lncRNA growth arrest-specific 5 (GAS5) is involved in regulating breast cancer progression. In this study, we aimed to elucidate the function and mechanism of GAS5 in breast cancer. Methods The expressions of GAS5, fat mass and obesity-associated protein (FTO), insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2), and Quaking (QKI) were assessed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blot. The m6A modification level of GAS5 was detected using m6A immunoprecipitation assay (MeRIP). The interaction between IGF2BP2 and GAS5 or QKI was detected using RNA immunoprecipitation assay (RIP) and dual luciferase reporter assay. Cell proliferation was measured using the Cell Counting Kit-8 (CCK-8) assay. The biological functions of the FTO/GAS5/IGF2BP2/QKI axis was assessed using the tumor xenograft assay. Results LncRNA GAS5 expression decreased in breast cancer and was regulated by FTO-mediated m6A modification in an IGF2BP2-dependent manner, resulting in decreased GAS5 stability and expression. GAS5 recruited IGF2BP2 to target QKI and upregulated QKI expression in breast cancer cells. GAS5 suppressed breast cancer growth via IGF2BP2/QKI, and this inhibitory effect was modulated by FTO both in vitro and in vivo. Conclusions GAS5 regulated by FTO-mediated m6A modification represses the growth of breast cancer via the IGF2BP2/QKI pathway, suggesting that the FTO/GAS5/IGF2BP2/QKI pathway can be a potential target for breast cancer treatment.

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]

Background: The lncRNA growth arrest-specific 5 (GAS5) is involved in regulating breast cancer progression. In this study, we aimed to elucidate the function and mechanism of GAS5 in breast cancer. Methods: The expressions of GAS5, fat mass and obesity-associated protein (FTO), insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2), and Quaking (QKI) were assessed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blot. The m6A modification level of GAS5 was detected using m6A immunoprecipitation assay (MeRIP). The interaction between IGF2BP2 and GAS5 or QKI was detected using RNA immunoprecipitation assay (RIP) and dual luciferase reporter assay. Cell proliferation was measured using the Cell Counting Kit-8 (CCK-8) assay. The biological functions of the FTO/GAS5/IGF2BP2/QKI axis was assessed using the tumor xenograft assay. Results: LncRNA GAS5 expression decreased in breast cancer and was regulated by FTO-mediated m6A modification in an IGF2BP2-dependent manner, resulting in decreased GAS5 stability and expression. GAS5 recruited IGF2BP2 to target QKI and upregulated QKI expression in breast cancer cells. GAS5 suppressed breast cancer growth via IGF2BP2/QKI, and this inhibitory effect was modulated by FTO both in vitro and in vivo. Conclusions: GAS5 regulated by FTO-mediated m6A modification represses the growth of breast cancer via the IGF2BP2/QKI pathway, suggesting that the FTO/GAS5/IGF2BP2/QKI pathway can be a potential target for breast cancer treatment. [ABSTRACT FROM AUTHOR]

Abstract Background Circular RNAs (circRNAs) play important roles in the occurrence and development of cancer and chemoresistance. DNA damage repair contributes to the proliferation of cancer cells and resistance to chemotherapy-induced apoptosis. However, the role of circRNAs in the regulation of DNA damage repair needs clarification. Methods RNA sequencing analysis was applied to identify the differentially expressed circRNAs. qRT-PCR was conducted to confirm the expression of hsa_circ_0007919, and CCK-8, FCM, single-cell gel electrophoresis and IF assays were used to analyze the proliferation, apoptosis and gemcitabine (GEM) resistance of pancreatic ductal adenocarcinoma (PDAC) cells. Xenograft model and IHC experiments were conducted to confirm the effects of hsa_circ_0007919 on tumor growth and DNA damage in vivo. RNA sequencing and GSEA were applied to confirm the downstream genes and pathways of hsa_circ_0007919. FISH and nuclear-cytoplasmic RNA fractionation experiments were conducted to identify the cellular localization of hsa_circ_0007919. ChIRP, RIP, Co-IP, ChIP, MS-PCR and luciferase reporter assays were conducted to confirm the interaction among hsa_circ_0007919, FOXA1, TET1 and the LIG1 promoter. Results We identified a highly expressed circRNA, hsa_circ_0007919, in GEM-resistant PDAC tissues and cells. High expression of hsa_circ_0007919 correlates with poor overall survival (OS) and disease-free survival (DFS) of PDAC patients. Hsa_circ_0007919 inhibits the DNA damage, accumulation of DNA breaks and apoptosis induced by GEM in a LIG1-dependent manner to maintain cell survival. Mechanistically, hsa_circ_0007919 recruits FOXA1 and TET1 to decrease the methylation of the LIG1 promoter and increase its transcription, further promoting base excision repair, mismatch repair and nucleotide excision repair. At last, we found that GEM enhanced the binding of QKI to the introns of hsa_circ_0007919 pre-mRNA and the splicing and circularization of this pre-mRNA to generate hsa_circ_0007919. Conclusions Hsa_circ_0007919 promotes GEM resistance by enhancing DNA damage repair in a LIG1-dependent manner to maintain cell survival. Targeting hsa_circ_0007919 and DNA damage repair pathways could be a therapeutic strategy for PDAC.

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]

Background: Circular RNAs (circRNAs) play important roles in the occurrence and development of cancer and chemoresistance. DNA damage repair contributes to the proliferation of cancer cells and resistance to chemotherapy-induced apoptosis. However, the role of circRNAs in the regulation of DNA damage repair needs clarification. Methods: RNA sequencing analysis was applied to identify the differentially expressed circRNAs. qRT-PCR was conducted to confirm the expression of hsa_circ_0007919, and CCK-8, FCM, single-cell gel electrophoresis and IF assays were used to analyze the proliferation, apoptosis and gemcitabine (GEM) resistance of pancreatic ductal adenocarcinoma (PDAC) cells. Xenograft model and IHC experiments were conducted to confirm the effects of hsa_circ_0007919 on tumor growth and DNA damage in vivo. RNA sequencing and GSEA were applied to confirm the downstream genes and pathways of hsa_circ_0007919. FISH and nuclear-cytoplasmic RNA fractionation experiments were conducted to identify the cellular localization of hsa_circ_0007919. ChIRP, RIP, Co-IP, ChIP, MS-PCR and luciferase reporter assays were conducted to confirm the interaction among hsa_circ_0007919, FOXA1, TET1 and the LIG1 promoter. Results: We identified a highly expressed circRNA, hsa_circ_0007919, in GEM-resistant PDAC tissues and cells. High expression of hsa_circ_0007919 correlates with poor overall survival (OS) and disease-free survival (DFS) of PDAC patients. Hsa_circ_0007919 inhibits the DNA damage, accumulation of DNA breaks and apoptosis induced by GEM in a LIG1-dependent manner to maintain cell survival. Mechanistically, hsa_circ_0007919 recruits FOXA1 and TET1 to decrease the methylation of the LIG1 promoter and increase its transcription, further promoting base excision repair, mismatch repair and nucleotide excision repair. At last, we found that GEM enhanced the binding of QKI to the introns of hsa_circ_0007919 pre-mRNA and the splicing and circularization of this pre-mRNA to generate hsa_circ_0007919. Conclusions: Hsa_circ_0007919 promotes GEM resistance by enhancing DNA damage repair in a LIG1-dependent manner to maintain cell survival. Targeting hsa_circ_0007919 and DNA damage repair pathways could be a therapeutic strategy for PDAC. [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]

Abstract Background Terminal 6q deletions are rare, and the number of well-defined published cases is limited. Since parents of children with these aberrations often search the internet and unite via international social media platforms, these dedicated platforms may hold valuable knowledge about additional cases. The Chromosome 6 Project is a collaboration between researchers and clinicians at the University Medical Center Groningen and members of a Chromosome 6 support group on Facebook. The aim of the project is to improve the surveillance of patients with chromosome 6 aberrations and the support for their families by increasing the available information about these rare aberrations. This parent-driven research project makes use of information collected directly from parents via a multilingual online questionnaire. Here, we report our findings on 93 individuals with terminal 6q deletions and 11 individuals with interstitial 6q26q27 deletions, a cohort that includes 38 newly identified individuals. Results Using this cohort, we can identify a common terminal 6q deletion phenotype that includes microcephaly, dysplastic outer ears, hypertelorism, vision problems, abnormal eye movements, dental abnormalities, feeding problems, recurrent infections, respiratory problems, spinal cord abnormalities, abnormal vertebrae, scoliosis, joint hypermobility, brain abnormalities (ventriculomegaly/hydrocephaly, corpus callosum abnormality and cortical dysplasia), seizures, hypotonia, ataxia, torticollis, balance problems, developmental delay, sleeping problems and hyperactivity. Other frequently reported clinical characteristics are congenital heart defects, kidney problems, abnormalities of the female genitalia, spina bifida, anal abnormalities, positional foot deformities, hypertonia and self-harming behaviour. The phenotypes were comparable up to a deletion size of 7.1 Mb, and most features could be attributed to the terminally located gene DLL1. Larger deletions that include QKI (> 7.1 Mb) lead to a more severe phenotype that includes additional clinical characteristics. Conclusions Terminal 6q deletions cause a common but highly variable phenotype. Most clinical characteristics can be linked to the smallest terminal 6q deletions that include the gene DLL1 (> 500 kb). Based on our findings, we provide recommendations for clinical follow-up and surveillance of individuals with terminal 6q deletions.

Background: Terminal 6q deletions are rare, and the number of well-defined published cases is limited. Since parents of children with these aberrations often search the internet and unite via international social media platforms, these dedicated platforms may hold valuable knowledge about additional cases. The Chromosome 6 Project is a collaboration between researchers and clinicians at the University Medical Center Groningen and members of a Chromosome 6 support group on Facebook. The aim of the project is to improve the surveillance of patients with chromosome 6 aberrations and the support for their families by increasing the available information about these rare aberrations. This parent-driven research project makes use of information collected directly from parents via a multilingual online questionnaire. Here, we report our findings on 93 individuals with terminal 6q deletions and 11 individuals with interstitial 6q26q27 deletions, a cohort that includes 38 newly identified individuals. Results: Using this cohort, we can identify a common terminal 6q deletion phenotype that includes microcephaly, dysplastic outer ears, hypertelorism, vision problems, abnormal eye movements, dental abnormalities, feeding problems, recurrent infections, respiratory problems, spinal cord abnormalities, abnormal vertebrae, scoliosis, joint hypermobility, brain abnormalities (ventriculomegaly/hydrocephaly, corpus callosum abnormality and cortical dysplasia), seizures, hypotonia, ataxia, torticollis, balance problems, developmental delay, sleeping problems and hyperactivity. Other frequently reported clinical characteristics are congenital heart defects, kidney problems, abnormalities of the female genitalia, spina bifida, anal abnormalities, positional foot deformities, hypertonia and self-harming behaviour. The phenotypes were comparable up to a deletion size of 7.1 Mb, and most features could be attributed to the terminally located gene DLL1. Larger deletions that include QKI (> 7.1 Mb) lead to a more severe phenotype that includes additional clinical characteristics. Conclusions: Terminal 6q deletions cause a common but highly variable phenotype. Most clinical characteristics can be linked to the smallest terminal 6q deletions that include the gene DLL1 (> 500 kb). Based on our findings, we provide recommendations for clinical follow-up and surveillance of individuals with terminal 6q deletions. [ABSTRACT FROM AUTHOR]

Simple Summary: Meat goats are highly valued as excellent sources of lean dietary muscle-protein. However, the molecular mechanisms by which muscle cells and tissues are generated and the genes and signaling pathways regulating this process in meat goats are poorly understood. The present study applied currently conventional molecular-genetics methods and determined that the major isoform of the QKI gene drive the conversion of embryonic myoblast cells into mature muscle fiber and tissue. These genes activate other genetic factors and signaling pathways that are all implicated in the differentiation of muscle progenitor cells and the formation of mature, differentiated muscle tissue. Although future investigations will be necessary to elucidate the entire molecular mechanism involved, we believe that the discoveries reported herein will facilitate the genetic engineering of novel goat lines with enhanced musculature and, by extension, superior meat yield and quality, breeder profitability, and consumer satisfaction. The QKI genes encode RNA-binding proteins regulating cell proliferation, differentiation, and apoptosis. The Goat QKI has six isoforms, but their roles in myogenesis are unclear. In this study, the six isoforms of the QKI gene were overexpressed in goat myoblast. Immunofluorescence, qPCR and Western blot were used to evaluate the effect of QKI on the differentiation of goat myoblast. An RNA-Seq was performed on the cells with the gain of the function from the major isoforms to screen differentially expressed genes (DEGs). The results show that six isoforms had different degrees of deletion in exons 6 and 7, and caused the appearance of different types of encoded amino acids. The expression levels of the QKI-1 and QKI-5 groups were upregulated in the biceps femoris and latissimus dorsi muscle tissues compared with those of the QKI-4, QKI-7, QKI-3 and QKI-6 groups. After 6 d of myoblast differentiation, QKI-5 and the myogenic differentiators MyoG, MyoD, and MyHC were upregulated. Compared to the negative control group, QKI promoted myotube differentiation and the myoblasts overexpressing QKI-5 formed large, abundant myotubes. In summary, we identified that the overexpression of the QKI gene promotes goat-myoblast differentiation and that QKI-5 is the major isoform, with a key role. The RNA-Seq screened 76 upregulated and 123 downregulated DEGs between the negative control and the QKI-5-overexpressing goat myoblasts after d 6 of differentiation. The GO and KEGG analyses associated the downregulated DEGs with muscle-related biological functions. Only the pathways related to muscle growth and development were enriched. This study provides a theoretical basis for further exploring the regulatory mechanism of QKI in skeletal-muscle development in goats. [ABSTRACT FROM AUTHOR]

Very-low-density lipoprotein receptor (VLDLR) involves in ocular neovascularization, a major cause of severe vision loss. However, the underlying molecular mechanisms were not completely clarified. Here, we aimed to investigate roles of circular RNAs (circRNAs) in VLDLR-associated ocular neovascularization. Vldlr knockout (Vldlr -/-, ko), Robo4 knockout (Robo4 -/-, ko) and wild-type (WT) mice were used. Mouse model of oxygen induced retinopathy (OIR) and high-throughput sequence were performed to profile the differential expression of circRNA and transcripts. RNase R treatment, Sanger PCR sequencing and quantitative polymerase chain reaction (qPCR) were used to validate candidate circRNAs and their expression patterns. Choroidal sprouting assay ex vivo and laser induction choroid neovascularization were used to determine the expression and functions of QKI/CircSlc17a5 on choroidal neovascularization. In macrophage and ocular tissues derived from Vldlr (Vldlr-/-,Vldlr ko) or Robo4 (Robo4-/-,Robo4 ko) deficiency as well as wild-type (WT) mice, Quaking (Qki) expression was significantly down-regulated in Vldlr deficiency compared to WT and Robo4 deficiency groups. Ectopic VLDLR expression or Reelin stimulation increased expression of QKI in bEnd.3 cells. Circular RNA sequencing uncovered that VLDLR regulated the biogenesis of certain circular RNAs, including the circSlc17a5. The number of Circular RNAs increased in mice treated with OIR. QKI mediated the biogenesis of circSlc17a5, which was an important regulator of choroidal angiogenesis. CircSlc17a5 regulated by VLDLR/QKI plays important roles in the choroidal angiogenesis. • VLDLR regulates QKI expression. • VLDLR regulates the biogenesis of circular RNAs. • VLDLR regulated circSlc17a5 expression depending on QKI. • CircSlc17a5 inhibits angiogenesis. [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]

Early and specific detection of cancer provides an opportunity for appropriate treatment. Although studies have suggested that QKI is a tumor suppressor gene, no studies have evaluated the diagnostic utility of QKI methylation in colorectal cancer (CRC). Here, we evaluated the methylation status of QKI by integrating the methylation data of tissues and cell lines of multiple cancer types. The diagnostic performance of QKI was analyzed in the discovery dataset from the TCGA CRC 450K array (n = 440) and tested in the test sets (n = 845) from the GEO. The methylation level of QKI was further validated in our independent dataset (n = 388) using targeted bisulfite sequencing. All detected CpG sites in the QKI promoter showed CRC-specific hypermethylation in 31 types of tumor tissues. In the discovery dataset, six consecutive CpG sites achieved high diagnostic performances, with AUCs ranging from 0.821 to 0.930. In the test set, a region (chr6: 163,834,452-163,834,924) including four consecutive CpG sites had robust diagnostic ability in distinguishing CRC and adenoma from normal samples. In the validation dataset, similar robust results were observed in both early- and advanced-stage CRC patients. In addition, QKI exhibited hypermethylation in the cfDNA of patients with CRC (n = 14). Collectively, the QKI promoter is a CRC-specific methylation biomarker and holds great promise for improving the diagnosis using minimally invasive biopsy. [ABSTRACT FROM AUTHOR]

Argonaute (AGO), a component of RNA-induced silencing complexes (RISCs), is a representative RNA-binding protein (RBP) known to bind with mature microRNA (miRNA) and is directly involved in post-transcriptional gene silencing. However, despite the biological significance of miRNA, the roles of other micro RNA-binding proteins (miRBPs) remain unclear in regulation of miRNA loading, dissociation from RISC, and extracellular release. In this study, we perform protein arrays to profile miRBPs and identify 118 RNA-binding proteins directly binding with miRNAs. Among those proteins, RBP quaking (QKI) inhibits extracellular release of mature microRNA let-7b by controlling the loading of let-7b into extracellular vesicles via additional miRBPs such as hnRNPD/AUF1 and hnRNPK. The enhanced extracellular release of let-7b after QKI depletion activates the Toll-like Receptor 7 (TLR7) and promotes the production of proinflammatory cytokines in recipient cells, leading to brain inflammation in mouse cortex. Thus, this study reveals contribution of QKI to the inhibition of brain inflammation via regulation of extracellular let-7b release., (© 2024. Published by The Company of Biologists Ltd.)

Alternative splicing (AS) plays a crucial role in the hallmarks of cancer and can open new avenues for targeted therapies. However, the aberrant AS events and the metastatic cascade in papillary thyroid carcinoma (PTC) remain largely unclear. Here, we identify the splicing factor, quaking protein (QKI), which was significantly downregulated in PTC and correlated with poor survival outcomes in patients with PTC. Functional studies indicated that low expression of QKI promoted the PTC cell growth and metastasis in vitro and in vivo. Mechanistically, low QKI induced exon 14 retention of extended synaptotagmin 2 (E-Syt2) and produced a long isoform transcript (termed E-Syt2L) that acted as an important oncogenic factor of PTC metastasis. Notably, overexpression of long non-coding RNA eosinophil granule ontogeny transcript (EGOT) physically binds to QKI and suppressed its activity by inhibiting ubiquitin specific peptidase 25 (USP25) mediated deubiquitination and subsequent degradation of QKI. Collectively, these data demonstrate the novel mechanistic links between the splicing factor QKI and splicing event in PTC metastasis and support the potential utility of targeting splicing events as a therapeutic strategy for PTC., Competing Interests: Declaration of competing interest The authors declared no competing interests. All authors have seen and approved the manuscript and consent publication., (Copyright © 2024. Published by Elsevier B.V.)

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.)

Glioblastoma (GBM) is a high-grade, aggressive brain tumor with dismal median survival time of 15 months. Chromosome 6q (Ch6q) is a hotspot of genomic alterations, which is commonly deleted or hyper-methylated in GBM. Two neighboring genes in this region, QKI and PRKN have been appointed as tumor suppressors in GBM. While a genetically modified mouse model (GEMM) of GBM has been successfully generated with Qk deletion in the central nervous system (CNS), in vivo genetic evidence supporting the tumor suppressor function of Prkn has not been established. In the present study, we generated a mouse model with Prkn-null allele and conditional Trp53 and Pten deletions in the neural stem cells (NSCs) and compared the tumorigenicity of this model to our previous GBM model with Qk deletion within the same system. We find that Qk but not Prkn is the potent tumor suppressor in the frequently altered Ch6q region in GBM.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal types of cancer. Various microRNAs have been identified to play an important role in PDAC. The study aimed to explore the role of miR-429 in PDAC. PATIENTS AND METHODS: The expression and prognostic value of miR-429 were first analyzed using The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Next, miR-429 expression was evaluated in the tissues and serum of 90 patients with PDAC. CCK8, SRB, wound healing and transwell assays were used to determine the effect of miR-429 on the proliferation, invasion, and migration of PDAC cells, respectively. Weighted gene co-expression network analysis (WGCNA), correlation analysis, TargetScan, and miRDB databases were used to screen and identify the target genes of miR-429. RESULTS: The results revealed that the expression of miR-429 was downregulated in PDAC tissues and the serum compared with those in normal tissues and the serum of healthy volunteers, respectively. The decreased expression of miR-429 was significantly associated with shorter overall survival. The overexpression of miR-429 significantly inhibited the proliferation, invasion, and migration of PDAC cells. Potential target genes of miR-429 were identified using WGCNA and bioinformatics analysis, and the results showed that cadherin 11 (CDH11), inositol polyphosphate-4-phosphatase type I (INPP4A), laminin gamma 1 (LAMC1), low density lipoprotein receptor-related protein 1 (LRP1), and quaking (QKI) were potential target genes of miR-429 in PDAC. Lower expression of CDH11 and QKI was associated with a more favorable prognosis in patients with PDAC. The overexpression of miR-429 could inhibit the expression of CDH11 and QKI. A nomogram model, involving miR-429, CDH11, and QKI, was subsequently constructed to determine their ability to accurately predict overall and disease-free survival in patients with PDAC. CONCLUSIONS: Taken together, miR-429 is involved in the development and progress of PDAC. MiR-429 could be recommended as a prognostic biomarker and therapeutic indicator in PDAC diagnosis. [ABSTRACT FROM AUTHOR]

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.

• QKI gene expression was increased in tumor tissues and associated with poor prognosis of HCC patients. • QKI protein interacted with EGOT RNA and promoted its expression. • Overexpression of QKI gene promoted the malignancy of HCC cells by promoting EGOT expression in vitro and in vivo. • Overexpression of QKI or EGOT gene activated the SAPK/JNK pathway in HCC cells. 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. 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. 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. Our findings indicated that QKI exerted promotive effects on the malignant phenotypes of HCC through its interaction with EGOT. [ABSTRACT FROM AUTHOR]

The QKI genes encode RNA-binding proteins regulating cell proliferation, differentiation, and apoptosis. The Goat QKI has six isoforms, but their roles in myogenesis are unclear. In this study, the six isoforms of the QKI gene were overexpressed in goat myoblast. Immunofluorescence, qPCR and Western blot were used to evaluate the effect of QKI on the differentiation of goat myoblast. An RNA-Seq was performed on the cells with the gain of the function from the major isoforms to screen differentially expressed genes (DEGs). The results show that six isoforms had different degrees of deletion in exons 6 and 7, and caused the appearance of different types of encoded amino acids. The expression levels of the QKI-1 and QKI-5 groups were upregulated in the biceps femoris and latissimus dorsi muscle tissues compared with those of the QKI-4, QKI-7, QKI-3 and QKI-6 groups. After 6 d of myoblast differentiation, QKI-5 and the myogenic differentiators MyoG, MyoD, and MyHC were upregulated. Compared to the negative control group, QKI promoted myotube differentiation and the myoblasts overexpressing QKI-5 formed large, abundant myotubes. In summary, we identified that the overexpression of the QKI gene promotes goat-myoblast differentiation and that QKI-5 is the major isoform, with a key role. The RNA-Seq screened 76 upregulated and 123 downregulated DEGs between the negative control and the QKI-5-overexpressing goat myoblasts after d 6 of differentiation. The GO and KEGG analyses associated the downregulated DEGs with muscle-related biological functions. Only the pathways related to muscle growth and development were enriched. This study provides a theoretical basis for further exploring the regulatory mechanism of QKI in skeletal-muscle development in goats.

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.)

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. [ABSTRACT FROM AUTHOR]

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.

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.

RNA binding proteins (RBPs) have a broad biological and physiological function and are critical in regulating pre-mRNA posttranscriptional processing, intracellular migration, and mRNA stability. QKI, also known as Quaking, is a member of the signal transduction and activation of RNA (STAR) family, which also belongs to the heterogeneous nuclear ribonucleoprotein K- (hnRNP K-) homology domain protein family. There are three major alternatively spliced isoforms, QKI-5, QKI-6, and QKI-7, differing in carboxy-terminal domains. They share a common RNA binding property, but each isoform can regulate pre-mRNA splicing, transportation or stability differently in a unique cell type-specific manner. Previously, QKI has been known for its important role in contributing to neurological disorders. A series of recent work has further demonstrated that QKI has important roles in much broader biological systems, such as cardiovascular development, monocyte to macrophage differentiation, bone metabolism, and cancer progression. In this mini-review, we will focus on discussing the emerging roles of QKI in regulating cardiac and vascular development and function and its potential link to cardiovascular pathophysiology.

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.

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. [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.

The poor outcome of patients with lung adenocarcinoma (LUAD) highlights the importance to identify novel effective prognostic markers and therapeutic targets. Long noncoding RNAs (lncRNAs) have generally been considered to serve important roles in tumorigenesis and the development of various types of cancer, including LUAD. Here, we aimed to investigate the role of ENTPD3-AS1 (ENTPD3 Antisense RNA 1) in LUAD and to explore its potential mechanisms by performing comprehensive bioinformatic analyses. The regulatory effect of ENTPD3-AS1 on the expression of NR3C1 was validated by siRNA-based silencing. The effect of miR-421 on the modulation of NR3C1 was determined by miRNA mimics and inhibitors transfection. ENTPD3-AS1 was expressed at lower levels in tumor parts and negatively correlated with unfavorable prognosis in LUAD patients. It exerted functions as a tumor suppressor gene by competitively binding to oncomir, miR-421, thereby attenuating NR3C1 expression. Transfection of lung cancer A549 cells with miR-421 mimics decreased the expression of NR3C1 . Transfection of lung cancer A549 cells with miR-421 inhibitors increased the expression of NR3C1 with lower cellular functions as proliferation and migration via epithelial-mesenchymal transition. In addition, inhibition of ENTPD3-AS1 by siRNA transfection decreased the levels of NR3C1 , supporting the ENTPD3-AS1 /miR-421/NR3C1 cascade. Moreover, the bioinformatic analysis also showed that ENTPD3-AS1 could interact with the RNA-binding proteins (RBPs), CELF2 and QKI, consequently regulating RNA expression and processing. Taken together, we identified that ENTPD3-AS1 and its indirect target NR3C1 can act as novel biomarkers for determining the prognosis of patients with LUAD, and further study is required., Competing Interests: None., (AJCR Copyright © 2024.)

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]

In this study, we found miR-362-5p was upregulated in bladder cancer tissues and we predicted that QKI is potential a target of miR-362-5p and MBNL1-AS1 might be able to directly target to miR-362-5p. We attempted to evaluate whether miR-362-5p could play its roles in bladder cancer through regulating QKI (quaking) and whether the expression and function of miR-362-5p could be mediated by lncRNA MBNL1-AS1. We performed the gain- and loss-function experiments to explore the association between miR-362-5p expression and bladder cancer proliferation. In vivo, the nude mice were injected with miR-362-5p knockdown SW780 cells to assess the effects of miR-362-5p on tumor growth. The results showed upregulation of miR-362-5p promoted cell proliferation of bladder cancer cells. MBNL1-AS1 and QKI could directly bind with miR-362-5p, and knockdown of MBNL1-AS1 or QKI could abrogate the regulatory effects of miR-362-5p on bladder cancer cell proliferation. Furthermore, downregulation of miR-362-5p inhibited bladder tumor growth and increased QKI expression. Our data unveiled that miR-362-5p may play an oncogenic role in bladder cancer through QKI and MBNL1-AS1 might function as a sponge to mediate the miR-362-5p expression and function.

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. [ABSTRACT FROM AUTHOR]

In this study, we found miR-362-5p was upregulated in bladder cancer tissues and we predicted that QKI is potential a target of miR-362-5p and MBNL1-AS1 might be able to directly target to miR-362-5p. We attempted to evaluate whether miR-362-5p could play its roles in bladder cancer through regulating QKI (quaking) and whether the expression and function of miR-362-5p could be mediated by lncRNA MBNL1-AS1. We performed the gain- and loss-function experiments to explore the association between miR-362-5p expression and bladder cancer proliferation. In vivo , the nude mice were injected with miR-362-5p knockdown SW780 cells to assess the effects of miR-362-5p on tumor growth. The results showed upregulation of miR-362-5p promoted cell proliferation of bladder cancer cells. MBNL1-AS1 and QKI could directly bind with miR-362-5p, and knockdown of MBNL1-AS1 or QKI could abrogate the regulatory effects of miR-362-5p on bladder cancer cell proliferation. Furthermore, downregulation of miR-362-5p inhibited bladder tumor growth and increased QKI expression. Our data unveiled that miR-362-5p may play an oncogenic role in bladder cancer through QKI and MBNL1-AS1 might function as a sponge to mediate the miR-362-5p expression and function. [ABSTRACT FROM AUTHOR]

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. [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]

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. [ABSTRACT FROM AUTHOR]

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.

Although Argonaute (AGO) proteins have been the focus of microRNA (miRNA) studies, we observed AGO-free mature miRNAs directly interacting with RNA-binding proteins, implying the sophisticated nature of fine-tuning gene regulation by miRNAs. To investigate microRNA-binding proteins (miRBPs) globally, we analyzed PAR-CLIP data sets to identify RBP quaking (QKI) as a novel miRBP for let-7b. Potential existence of AGO-free miRNAs were further verified by measuring miRNA levels in genetically engineered AGO-depleted human and mouse cells. We have shown that QKI regulates miRNA-mediated gene silencing at multiple steps, and collectively serves as an auxiliary factor empowering AGO2/let-7b-mediated gene silencing. Depletion of QKI decreases interaction of AGO2 with let-7b and target mRNA, consequently controlling target mRNA decay. This finding indicates that QKI is a complementary factor in miRNA-mediated mRNA decay. QKI, however, also suppresses the dissociation of let-7b from AGO2, and slows the assembly of AGO2/miRNA/target mRNA complexes at the single-molecule level. We also revealed that QKI overexpression suppresses cMYC expression at post-transcriptional level, and decreases proliferation and migration of HeLa cells, demonstrating that QKI is a tumour suppressor gene by in part augmenting let-7b activity. Our data show that QKI is a new type of RBP implicated in the versatile regulation of miRNA-mediated gene silencing.

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. [ABSTRACT FROM AUTHOR]

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. What's new? The epithelial‐mesenchymal transition is controlled by various transcription factors and non‐coding RNAs. The miR‐200 family of microRNAs inhibit the EMT by targeting certain transcription factors. In this study, the authors identified the gene QKI as a novel target of miR‐200 in oral and lung cancer cells. These microRNAs suppressed QKI expression, and QKI knockdown reduced miR‐200 expression, suggesting the two interact in a negative feedback loop. QKI knockdown also promoted cell growth, EMT, and invasion. On the other hand, microarray data from surgical samples showed that high QKI expression correlated with good outcomes for patients with HSNCC and lung cancer. [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.

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.

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]

N 7-methylguanosine (m7G) modification, routinely occurring at mRNA 5′ cap or within tRNAs/rRNAs, also exists internally in messenger RNAs (mRNAs). Although m7G-cap is essential for pre-mRNA processing and protein synthesis, the exact role of mRNA internal m7G modification remains elusive. Here, we report that mRNA internal m7G is selectively recognized by Quaking proteins (QKIs). By transcriptome-wide profiling/mapping of internal m7G methylome and QKI-binding sites, we identified more than 1,000 high-confidence m7G-modified and QKI-bound mRNA targets with a conserved "GANGAN (N = A/C/U/G)" motif. Strikingly, QKI7 interacts (via C terminus) with the stress granule (SG) core protein G3BP1 and shuttles internal m7G-modified transcripts into SGs to regulate mRNA stability and translation under stress conditions. Specifically, QKI7 attenuates the translation efficiency of essential genes in Hippo signaling pathways to sensitize cancer cells to chemotherapy. Collectively, we characterized QKIs as mRNA internal m7G-binding proteins that modulate target mRNA metabolism and cellular drug resistance. [Display omitted] • Quaking proteins (QKIs) bind to internal m7G-modified mRNAs with GA-rich motifs • QKI7 interacts with G3BP1, a stress granule protein • QKI7 shuttles internal m7G-modified mRNAs into stress granules under stress conditions • QKI7 regulates mRNA metabolism and sensitizes cancer cells to chemotherapy drugs The Quaking protein QKI7 binds to internal m7G-modified mRNAs and directs them to stress granules to suppress their translation under stress conditions. [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 RNA binding protein quaking ( QKI), a key member of the STAR family, as an upstream gene could involve in much process including cell proliferation, apoptosis, differentiation and so on. However, the roles of QKI in germ cell, especially in swine testis (ST) cells, was not clear currently. And apoptosis plays important roles in the growth and development. The purpose of the present study was to clarify the relationship between QKI and apoptosis in ST cells. Firstly, our results showed that pEF1α-QKI and shQKI3 have clear effects on expression levels of QKI. Secondly, we established that QKI directly binds to WT1 3′UTR by binding with QRE-1 (2046-2052 bp, ACTAAC) only. Furthermore, QKI overexpression significantly increased the expression levels of WT1 and Bcl-2. QKI also has the effect on delaying the degradation of WT1 mRNA. In addition, we verified that QKI had a significantly suppressed apoptosis in ST cells. Finally, pBI-WT1 could make up for shQKI3-induced decrease in WT1, Bcl-2 mRNA levels and suppress apoptosis in ST cells. The results demonstrated that QKI was an important regulatory factor that affects apoptosis by targeting WT1 gene. [ABSTRACT FROM AUTHOR]