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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.

Backgrounds: A number of genetic and biological phenomena imply that tumorigenesis of clear cell renal cell carcinoma (ccRCC) is highly correlated with hypoxia-induced factor-1a (HIF-1α). Recently, research focusing on the post-transcriptional regulation of HIF-1α has provided a new perspective for ccRCC therapy. In this study, we observed the expression pattern of the RNA-binding protein QKI, which could regulate HIF expression in ccRCC both in vitro and in vivo. Methods: Tissue microarraywas subjected to immunohistochemistry and tumour cell lines and nude mice were used for in vitro and in vivo assays. QKI overexpression or knockdown was assessed in renal cancer cells. Results: The overexpression of QKI inhibited the proliferation of the 786-0 and caki-1 cells, blocked the cells' entry into the S phase, and promoted apoptosis. In ectopic-implantation nude mice model, QKI depletion significantly increased tumor sizes and initiation rates. Tissue microarrays showed that the expression of QKI genes, and especially QKI-6, was significantly decreased in tumor tissues compared with these in normal kidney tissues. Moreover, decreased QKI expression was closely correlated with high tumor grade, poor differentiation, and poor survival. Conclusions: QKI may be useful as a novel, independent diagnostic and biological marker for ccRCC.

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.

Hepatocellular carcinoma and cholangiocarcinoma are the most common liver primary tumors and showed an increased incidence and mortality over the last decades. Due to a frequent late diagnosis, liver cancer is associated with limited curative therapeutic options. Characterizing new molecular determinants of liver carcinogenesis is required to develop innovative treatments and to identify clinically relevant biomarkers. Recently, circular RNAs (circRNAs) emerged as promising regulatory molecules involved in cancer onset and progression, thus opening new opportunities. Mechanistically, circRNAs are currently mainly described for their capability to sponge and to regulate the activity of microRNAs and RNA binding proteins, although other functions are emerging (e.g. transcriptional and post-transcriptional regulation, protein scaffolding). In liver cancer, circRNAs are described to regulate tumor cell proliferation, migration, invasion and cell death resistance. Their role in regulating angiogenesis, genome instability, immune surveillance and metabolic switching is emerging. Importantly, circRNAs are detected in body fluids. Due to their circular structure, circRNAs are often more stable than mRNAs or miRNAs and could therefore serve as promising biomarkers quantifiable with high specificity and sensitivity though minimally invasive methods. This review focuses on the role and the clinical relevance of circRNAs in liver cancer, including the development of innovative biomarkers and therapeutic strategies.

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.

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.

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.

Cardiovascular disease is the leading cause of death amongst diabetic individuals. Atherosclerosis is the prominent driver of diabetic vascular complications, which is triggered by the detrimental effects of hyperglycemia and oxidative stress on the vasculature. Research has extensively shown diabetes to result in the malfunction of the endothelium, the main component of blood vessels, causing severe vascular complications. The pathogenic mechanism in which diabetes induces vascular dysfunction, however, remains largely unclear. Alternative splicing of protein coding pre-mRNAs is an essential regulatory mechanism of gene expression and is accepted to be intertwined with cellular physiology. Recently, a role for alternative splicing has arisen within vascular health, with aberrant mis-splicing having a critical role in disease development, including in atherosclerosis. This review focuses on the current knowledge of alternative splicing and the roles of alternatively spliced isoforms within the vasculature, with a particular focus on disease states. Furthermore, we explore the recent elucidation of the alternatively spliced QKI gene within vascular cell physiology and the onset of diabetic vasculopathy. Potential therapeutic strategies to restore aberrant splicing are also discussed.

// Bo Li 1, * , Xinzhe Li 1, * , Haojun Xiong 1 , Peng Zhou 1 , Zhenhong Ni 1 , Teng Yang 1 , Yan Zhang 1 , Yijun Zeng 1 , Jintao He 2 , Fan Yang 1 , Nan Zhang 1 , Yuting Wang 1 , Yingru Zheng 3 and Fengtian He 1 1 Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Third Military Medical University, Chongqing 400038, China 2 Battalion 17 of Students, College of Preventive Medicine, Third Military Medical University, Chongqing 400038, China 3 Department of Obstetrics and Gynecology, Daping Hospital and Research Institute of Surgery, Third Military Medical University, Chongqing 400042, China * These authors have contributed equally to this work Correspondence to: Yingru Zheng, email: zyrdaping@aliyun.com Fengtian He, email: hefengtian06@aliyun.com Keywords: dichloroacetate, COX2, celecoxib, QKI, cervical cancer Received: February 07, 2017 Accepted: May 06, 2017 Published: June 16, 2017 ABSTRACT Dichloroacetate (DCA), a traditional mitochondria-targeting agent, has shown promising prospect as a sensitizer in fighting against malignancies including cervical cancer. But it is unclear about the effect of DCA alone on cervical tumor. Moreover, previous reports have demonstrated that the increased cyclooxygenase-2 (COX2) expression is associated with chemoresistance and poor prognosis of cervical cancer. However, it is still unknown whether COX2 can affect the sensitivity of DCA in cervical cancer cells. In this study, we found that cervical cancer cells were insensitive to DCA. Furthermore, we for the first time revealed that DCA could upregulate COX2 which impeded the chemosensitivity of DCA in cervical cancer cells. Mechanistic study showed that DCA reduced the level of RNA binding protein quaking (QKI), leading to the decay suppression of COX2 mRNA and the subsequent elevation of COX2 protein. Inhibition of COX2 using celecoxib could sensitize DCA in repressing the growth of cervical cancer cells both in vitro and in vivo . These results indicate that COX2 is a novel resistance factor of DCA, and combination of celecoxib with DCA may be beneficial to the treatment of cervical cancer.

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.

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.

CMT1A patients commonly share PMP22 genetic overloading but they show phenotypic heterogeneity and variability in PMP22 mRNA and protein expression. Moreover, PMP22 mRNA levels do not correlate with clinical outcome measures in these patients, suggesting their uselessness as a disease biomarker. Thus, in-depth analysis of PMP22 transcription and translation might help to define its pathogenic role in CMT1A. We focused on the alternative splicing of PMP22 gene to verify whether mRNA processing is altered in CMT1A. We identified three new PMP22 transcripts enriched in human sural nerve biopsies. One of them was an untranslated variant, whereas the other two originated from a PMP22 undescribed exon and encoded for a new putative protein localized in the endoplasmic reticulum. As splicing events in the PMP22 gene are differently regulated in tissues and during development, we analyzed the levels of PMP22 transcripts and their splicing pattern in human and experimental CMT1A. We found an altered PMP22 splicing ratio in the CMT1A rat. In addition, we showed a remarkable derangement in rat QKI expression, which is a critical regulator of splicing during myelination. Overall, our data suggest that an alteration of mRNA processing could be a pathogenic mechanism in CMT1A.

Background Inflammatory cytokines and transforming growth factor-β (TGF-β) are mutually inhibitory. However, hyperactivation of nuclear factor-κB (NF-κB) and TGF-β signaling both emerge in glioblastoma. Here, we report microRNA-148a (miR-148a) overexpression in glioblastoma and that miR-148a directly suppressed Quaking (QKI), a negative regulator of TGF-β signaling. Methods We determined NF-κB and TGF-β/Smad signaling activity using pNF-κB-luc, pSMAD-luc, and control plasmids. The association between an RNA-induced silencing complex and QKI, mitogen-inducible gene 6 (MIG6), S-phase kinase–associated protein 1 (SKP1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was tested with microribonucleoprotein immunoprecipitation and real-time PCR. Xenograft tumors were established in the brains of nude mice. Results QKI suppression induced an aggressive phenotype of glioblastoma cells both in vitro and in vivo. Interestingly, we found that NF-κB induced miR-148a expression, leading to enhanced-strength and prolonged-duration TGF-β/Smad signaling. Notably, these findings were consistent with the significant correlation between miR-148a levels with NF-κB hyperactivation and activated TGF-β/Smad signaling in a cohort of human glioblastoma specimens. Conclusions These findings uncover a plausible mechanism for NF-κB–sustained TGF-β/Smad activation via miR-148a in glioblastoma, and may suggest a new target for clinical intervention in human cancer. Electronic supplementary material The online version of this article (doi:10.1186/1476-4598-14-2) contains supplementary material, which is available to authorized users.