Clinicopathologic Significance of Quaking Expression in Hepatocellular Carcinoma.

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.

RNA-binding protein QKI promotes the progression of HCC by interacting with long non-coding RNA EGOT.

BACKGROUND: RNA-binding proteins are revealed to play important roles during the progression of hepatocellular carcinoma (HCC). However, the regulatory mechanisms of RNA-binding protein Quaking (QKI) in the expression and role of long non-coding RNAs (lncRNAs) in HCC cells remain not well understood. METHODS: Cell Counting Kit-8, wound-healing, Transwell and colony-forming assays were performed to evaluate the effects of QKI and lncRNA EGOT on proliferation and migration of HCC cells. Tumor growth of HCC was analyzed using a mouse xenograft model. Immunoprecipitation (RIP) assay was used to investigate the interaction between QKI and EGOT. RESULTS: The expression of QKI was significantly upregulated in HCC tissues and the higher QKI level was significantly associated with a poorer prognosis. Overexpression of QKI promoted the proliferation, migration, and colony-forming ability of HCC cells in vitro and tumor growth of HCC in vivo. Mechanistically, QKI protein could bind to EGOT RNA and increase its expression. Inhibition of EGOT attenuated the effects of QKI on the malignant phenotypes of HCC cells. In addition, both QKI and EGOT could activate the SAPK/JNK signaling pathway in HCC cells. CONCLUSIONS: Our findings indicated that QKI exerted promotive effects on the malignant phenotypes of HCC through its interaction with EGOT.

GAS5 regulated by FTO-mediated m6A modification suppresses cell proliferation via the IGF2BP2/QKI axis in breast cancer.

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.

Involvement of circRNA Regulators MBNL1 and QKI in the Progression of Esophageal Squamous Cell Carcinoma.

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.

Downregulated antisense lncRNA ENTPD3-AS1 contributes to the development of lung adenocarcinoma.

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.

Mature microRNA-binding protein QKI promotes microRNA-mediated gene silencing.

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.

FOXP3-regulated lncRNA NONHSAT136151 promotes colorectal cancer progression by disrupting QKI interaction with target mRNAs.

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.

hsa_circ_0007919 induces LIG1 transcription by binding to FOXA1/TET1 to enhance the DNA damage response and promote gemcitabine resistance in pancreatic ductal adenocarcinoma.

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.

SIRT1 alleviates insulin resistance and respiratory distress in late preterm rats by activating QKI5-mediated PPARγ/PI3K/AKT pathway.

Neonatal respiratory distress syndrome (NRDS) is a common complication of gestational diabetes mellitus (GDM) and late preterm births. Research suggests that SIRT1 was involved in LPS-induced acute respiratory distress syndrome, but its mechanism remains to be further explored. Here, pregnant rats were intraperitoneally injected with 45 mg/Kg streptozotocin at day 0 of gestation to induce GDM and injected with LPS at day 17 of gestation to induce late preterm birth. Pioglitazone (a PPARγ agonist) was administered from day 17 to parturition in GDM group, and it was administered for 3 days before LPS injection in late preterm birth group. SRT1720 (a SIRT1 activator) was administered by oral gavage from day 0 to day 17 in both groups. Our data showed that activation of SIRT1 or PPARγ alleviated the abnormal blood glucose metabolism and lung tissue injury, downregulated expression of surfactant proteins (SP-B and SP-C), and decreased activation of the PI3K/AKT pathway induced by GDM and late preterm birth in neonatal rats. Moreover, an insulin resistance model was established by treating primary AT-II cells with insulin. Activation of SIRT1 reversed insulin-induced reduction in cell proliferation, glucose consumption, SP-B and SP-C expression, and the activity of the PI3K/AKT pathway and increase in cellular inflammation and apoptosis. Mechanistically, SIRT1 upregulated PPARγ expression via deacetylation of QKI5, an RNA binding protein that can stabilize its target mRNA molecules, and then activated the PI3K/AKT pathway. In conclusion, SIRT1 promotes the expression of PPARγ via upregulation of QKI5 and activates the PI3K/AKT pathway, thus mitigating NRDS caused by GDM and late preterm birth.

RNA-binding Protein QKI Inhibits Osteogenic Differentiation Via Suppressing Wnt Pathway.

BACKGROUND: Dysregulation of MSCs differentiation is associated with many pathophysiological processes. Genetically modified MSCs transplantation helps restore bone loss efficiently. METHODS: 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. RESULTS: 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 ß-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. CONCLUSIONS: QKI suppressed BMSCs osteogenic differentiation by downregulating the expressions of Wnt5b, Fzd7, Dvl3 and ß-catenin. Loss of QKI in BMSCs transplantation may provide a new strategy for the treatment of orthopedic diseases such as osteoporosis.

QKI shuttles internal m7G-modified transcripts into stress granules and modulates mRNA metabolism.

N7-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.

Case Report: Brainstem angiocentric glioma presenting in a toddler child-diagnostic and therapeutic challenges.

Introduction: Angiocentric gliomas (AG) in brainstem location are exceedingly rare and might cause differential diagnostic problems and uncertainty regarding the best therapeutic approach. Hereby, we describe the clinicopathological findings in a brainstem AG presenting in a toddler child and review the literature. Case report: A 2-year-old boy presented with 5 weeks history of gait disturbances, frequent falls, left-sided torticollis and swallowing problems. MRI head showed a T2-hyperintense, partly exophytic mass lesion centred in the pontomedullary region, raising the possibility of diffuse midline glioma. The exophytic component was partially resected by suboccipital craniotomy, leaving intact the infiltrative component. Ventriculoperitoneal shunt was implanted due to postoperative hydrocephalus. Histological examination revealed a moderately cellular tumour consisted of bland glial cells infiltrating the brain parenchyma and radially arranged around the blood vessels. By immunohistochemistry, the tumour strongly expressed S100 and GFAP in addition to intense nestin positivity, while OLIG2 was negative in the perivascular tumour cells. DNA methylation array profiled the tumour as "methylation class diffuse astrocytoma, MYB or MYBL1-altered subtype B (infratentorial)" and an in-frame MYB::QKI fusion was identified by RNA sequencing, confirming the diagnosis of angiocentric glioma. The patient has been initially treated with angiogenesis inhibitor and mTOR inhibitor, and now he is receiving palliative vinblastine. He is clinically stable on 9 months follow-up. Conclusion: Brainstem AG may cause a diagnostic problem, and the surgical and oncological management is challenging due to unresectability and lack of response to conventional chemo-radiation. In the future, genetically-tailored therapies might improve the prognosis.

DIPG-like MYB-altered diffuse astrocytoma with durable response to intensive chemotherapy.

Pontine gliomas represent difficult to treat entity due to the location and heterogeneous biology varying from indolent low-grade gliomas to aggressive diffuse intrinsic pontine glioma (DIPG). Making the correct tumor diagnosis in the pontine location is thus critical. Here, we report a case study of a 14-month-old patient initially diagnosed as histone H3 wild-type DIPG. Due to the low age of the patient, the MRI appearance of DIPG, and anaplastic astrocytoma histology, intensive chemotherapy based on the HIT-SKK protocol with vinblastine maintenance chemotherapy was administered. Rapid clinical improvement and radiological regression of the tumor were observed with nearly complete remission with durable effect and excellent clinical condition more than 6.5 years after diagnosis. Based on this unexpected therapeutic outcome, genome-wide DNA methylation array was employed and the sample was classified into the methylation class "Low-grade glioma, MYB(L1) altered." Additionally, RT-PCR revealed the presence of MYB::QKI fusion. Taken together, the histopathological classification, molecular-genetic and epigenetic features, clinical behavior, and pontine location have led us to reclassify the tumor as a pontine MYB-altered glioma. Our case demonstrates that more intensive chemotherapy can achieve long-term clinical effect in the treatment of MYB-altered pontine gliomas compared to previously used LGG-based regimens or radiotherapy. It also emphasizes the importance of a biopsy and a thorough molecular investigation of pontine lesions.

microRNA-497 slows esophageal cancer development and reverses chemotherapy resistance through its target QKI.

Esophageal cancer (EC) is considered one of the most lethal cancers in human beings, and multiple miRNAs have been investigated to be involved in EC development by targeting their target genes. However, the function and related mechanism of miRNA-497 on EC tumorigenesis remain uncertain. This study first demonstrated that the expression levels of miR-497 in esophageal cancer specimens and cells were down-regulated. Forced expression of miR-497 inhibited cell proliferation, tube formation and migration in EC cells. To further investigate the potential molecular mechanism of miR-497 suppression in regulating EC, we found that miR-497 directly binds to the 3'-untranslational region of QKI, miR-497 overexpression suppressed QKI expression. We further found that overexpression of miR-497 enhanced the effect of chemotherapy in EC cell lines, and prevented the tumor growth of EC in vivo. Our findings indicated that miR-497 suppression increased QKI expression and therapeutic resistance of esophageal cancer, which is likely to be a biomarker of EC progression and potential therapeutic target.

RNA Binding Protein Quaking Promotes Hypoxia-induced Smooth Muscle Reprogramming in Pulmonary Hypertension.

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.

The phenotypic spectrum of terminal 6q deletions based on a large cohort derived from social media and literature: a prominent role for DLL1.

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.

Overexpression of the QKI Gene Promotes Differentiation of Goat Myoblasts into Myotubes.

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.

miR-31/QKI-5 axis facilitates cell cycle progression of non-small-cell lung cancer cells by interacting and regulating p21 and CDK4/6 expressions.

BACKGROUND: RNA-binding protein Quaking-5 (QKI-5), a major isoform of QKIs, inhibits tumor progression in non-small cell lung cancer (NSCLC). However, the underlying molecular mechanisms of QKI-5 in the cell cycle of NSCLC are still largely unknown. METHODS: MTT, flow cytometry, and colony formation assays were used to investigate cellular phenotypic changes. Mice xenograft model was used to evaluate the antitumor activities of QKI-5. Co-immunoprecipitation, RNA immunoprecipitation (RIP), and RIP sequencing were used to investigate protein-protein interaction and protein-mRNA interaction. RESULTS: The QKI-5 expression was downregulated in NSCLC tissues compared with that in paired normal adjacent lung tissues. Overexpression of QKI-5 inhibited NSCLC cell proliferative and colony forming ability. In addition, QKI-5 induced cell cycle arrest at G0/G1 phase through upregulating p21Waf1/Cip1 (p21) expression and downregulating cyclin D1, cyclin-dependent kinase 4 (CDK4), and CDK6 expressions. Further analyses showed that QKI-5 interacts with p21 protein and CDK4, CDK6 mRNAs, suggesting a critical function of QKI-5 in cell cycle regulation. In agreement with in vitro study, the mouse xenograft models validated tumor suppressive functions of QKI-5 in vivo through altering cell cycle G1-phase-associated proteins. Moreover, we demonstrated that QKI-5 is a direct target of miR-31. The QKI-5 expression was anticorrelated with the miR-31 expression in NSCLC patient samples. CONCLUSION: Our results suggest that the miR-31/QKI-5/p21-CDK4-CDK6 axis might have critical functions in the progression of NSCLC, and targeting this axis could serve as a potential therapeutic strategy for NSCLC.

Quaking but not parkin is the major tumor suppressor in 6q deleted region in glioblastoma.

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.

Multiple circRNAs regulated by QKI5 conjointly spongemiR-214-3p to antagonize bisphenol A-inducedspermatocyte toxicity.

Although circular RNAs (circRNAs) are found to play important roles in many pathophysiological processes, the canonical theory that they act as microRNA sponges is now more and more challenged, given that most circRNAs only have few binding sites in a particular microRNA. Our previous study revealed that some up-regulated circRNAs play protective roles in bisphenol A (BPA)-induced toxicity in GC-2 germ cells. Here by CCK-8 assay, apoptosis assay, qRT-PCR and western blot analysis, we further discover that circRNAs (represented by circDcbld2, circMapk1 and circTbcld20) can cooperatively sponge miR-214-3p and then up-regulate AKT1 in ameliorating BPA-induced reproductive toxicity. They share binding sites with miR-214-3p and collectively reinforce the sponging effects. In addition, the upstream regulation mechanism, proven by bioinformatics analysis and in vitro gain- and loss-of-function study, shows that down-regulation of RNA binding protein QKI5 after BPA exposure can increase the expressions of these protective circRNAs, and thus activate the cell protective process. The QKI5-circDcbld2/circMapk1/circTblcd20-miR-214-3p-AKT1 axis ameliorates the toxic effect of BPA on GC-2 cells. Many other circRNAs up-regulated upon BPA treatment and QKI5 down-regulation also show binding sites with miR-214-3p. Thus the above axis may also be extrapolated to other circRNAs. Our results enrich the context of circRNA sponge mode and may provide new ideas in future multiple nucleic acid therapy.