BACKGROUND: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: CircSlc17a5 regulated by VLDLR/QKI plays important roles in the choroidal angiogenesis.

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.

OBJECTIVE: 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).
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.
CONCLUSIONS: 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.

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.

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.

OBJECTIVE: 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.

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.

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.

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.
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.
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.
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.