RNA Immunoprecipitation Assay Research Articles

Berberine inhibits the progression of breast cancer by regulating METTL3-mediated m6A modification of FGF7 mRNA.

Berberine (BBR), an isoquinoline alkaloid from Coptidis rhizoma, has been found to have powerful activities against various human malignancies, including breast cancer. However, the underlying antitumor mechanisms of BBR in breast cancer remain poorly understood. Breast cancer cells were cultured and treated with different doses (0, 20, 40, and 60 μM) of BBR for 48 h. Cell viability, proliferation, apoptosis, invasion, and migration were assessed using 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT), 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, transwell, and wound healing assays. Fibroblast growth factor 7 (FGF7), methyltransferase-like 3 (METTL3), and insulin-like growth factor-2 mRNA-binding protein 3 (IGF2BP3) mRNA levels and protein levels were measured using real-time quantitative polymerase chain reaction (RT-qPCR) and western blot. Interaction between METTL3 and FGF7 m6A was assessed using methylated RNA immunoprecipitation (MeRIP)-qPCR and RNA immunoprecipitation (RIP) assay. Binding ability between IGF2BP3 and FGF7 mRNA was analyzed using RIP assay. BBR treatment hindered breast cancer cell proliferation, invasion, migration, and induced apoptosis. FGF7 expression was upregulated in breast cancer tissues, while its level was reduced in BBR-treated tumor cells. FGF7 upregulation relieved the repression of BBR on breast cancer cell malignant behaviors. In mechanism, METTL3 stabilized FGF7 mRNA through the m6A-IGF2BP3-dependent mechanism and naturally improved FGF7 expression. BBR treatment inhibited breast cancer growth in vivo. BBR treatment blocked breast cancer cell growth and metastasis partly by regulating METTL3-mediated m6A modification of FGF7 mRNA, providing a promising therapeutic target for breast cancer treatment.

PITPNA-AS1 Inhibits Cell Proliferation and Migration in Ovarian Cancer by Regulating the MIR-223-3p/RHOB Axis.

Ovarian cancer is a fatal gynecologic malignancy. Long non-coding RNA (lncRNA) has been verified to serve as key regulator in ovarian cancer tumorigenesis. The aim of the study was to study the functions and mechanism of lncRNA PITPNA-AS1 in ovarian cancer cellular process. Clinical ovarian cancer samples were collected and stored at an academic medical center. Cellular fractionation assays and fluorescence in situ hybridization were conducted to locate PITPNA-AS1 in OC cells. TUNEL staining, colony-forming assays, and Transwell assays were performed for evaluating cell apoptosis as well as proliferative and migratory abilities. Western blot was conducted for quantifying protein levels of epithelialmesenchymal transition markers. The binding relation between genes was verified by RNA pulldown, RNA immunoprecipitation, and luciferase reporter assays. Gene expression levels in ovarian cancer tissues and cells were subjected to RT-qPCR. PITPNA-AS1 level was downregulated in ovarian cancer samples and cells. PITPNA-AS1 overexpression contributed to the accelerated ovarian cancer cell apoptosis and inhibited cell migration, proliferation, and epithelial-mesenchymal transition process. In addition, PITPNA-AS1 interacted with miR-223-3p to regulate RHOB. RHOB knockdown partially counteracted the repressive impact of PITPNA-AS1 on ovarian cancer cell activities. PITPNA-AS1 inhibited ovarian cancer cellular behaviors by targeting miR-223-3p and regulating RHOB.

Exploring the regulatory role of FBXL19-AS1 in triple-negative breast cancer through the miR-378a-3p/OTUB2 axis.

The regulatory potential of long noncoding RNA (lncRNA) FBXL19-AS1 has been highlighted in various cancers, but its effect on triple-negative breast cancer (TNBC) remains unclear. Here, we aimed to elucidate the role of FBXL19-AS1 in TNBC and its underlying mechanism. RT-qPCR was employed to detect the expressions of FBXL19-AS1 and miR-378a-3p in tissues and cells. Immunohistochemical staining and western blot were utilized to detect the expression levels of proteins. Cell activities were detected using flow cytometry, CCK-8, and transwell assay. Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were deployed to investigate interactions of different molecules. Protein-protein interaction (PPI) network, gene ontology (GO), and Kyoto encyclopedia of genes and genomes (KEGG) pathways were used to analyze the downstream pathway. In vivo xenograft model was conducted to detect the effect of FBXL19-AS1 on tumor growth. FBXL19-AS1 was overexpressed in TNBC tissues and cell lines compared with counterparts. FBXL19-AS1 knockdown suppressed TNBC cell activities, whereas its overexpression exhibited the opposite effect. Mechanistically, FBXL19-AS1 was found to interact with miR-378a-3p. Further analysis revealed that miR-378a-3p exerted tumor-suppressive effects in TNBC cells. Additionally, miR-378a-3p targeted and downregulated the expression of ubiquitin aldehyde binding 2 (OTUB2), a deubiquitinase associated with TNBC progression. In vivo experiments substantiated the inhibitory effects of FBXL19-AS1 knockdown on TNBC tumorigenesis, and a miR-378a-3p inhibitor partially rescued these effects. The downstream pathway of the miR-378a-3p/OTUB2 axis was explored, revealing connections with proteins involved in modifying other proteins, removing ubiquitin molecules, and influencing signaling pathways, including the Hippo signaling pathway. Western blot analysis confirmed changes in YAP and TAZ expression levels, indicating a potential regulatory network. In summary, FBXL19-AS1 promotes exacerbation in TNBC by suppressing miR-378a-3p, leading to increased OTUB2 expression. The downstream mechanism may be related to the Hippo signaling pathway. These findings propose potential therapeutic targets for TNBC treatment.

Circular RNA KIAA0564 Serves as a Competitive Endogenous RNA for MicroRNA-424-5p, Mediating the Expression of Lysine Demethylase 4a, Thereby Facilitating Intervertebral Disc Degeneration.

A growing body of research has confirmed the involvement of circular RNAs (circRNAs) in the regulation of intervertebral disc degeneration (IDD) progression. However, the underlying molecular networks remain largely elusive. This study aimed to explore whether a novel circRNA, named circKIAA0564, affects nucleus pulposus (NP) cell injury and to elucidate its molecular mechanism. Both in vivo and in vitro IDD models were established, and the expression patterns of circKIAA0564/miR-424-5p/lysine demethylase 4a (KDM4A) were evaluated through quantitative reverse transcription PCR and Western blot analysis. Actinomycin D, RNase R, and Northern blotting were utilized to assess the circular structure of circKIAA0564. The Cell Counting Kit-8, flow cytometry, enzyme-linked immunosorbent assay, commercial assay kits, Western blotting, and reactive oxygen species (ROS) probes were employed to assess the inflammatory and oxidative stress status in NP cells and tissues. Hematoxylin and eosin and TUNEL staining were used to evaluate pathological damage in mouse NP tissues. RNA immunoprecipitation and dual-luciferase reporter assays were conducted to assess the direct targeting relationships among circKIAA0564, miR-424-5p, and KDM4A. CircKIAA0564 was found to be abnormally overexpressed in IDD, functioning as a novel circRNA. Knockdown of circKIAA0564 ameliorated interleukin-1 beta (IL-1β)-induced inflammation and oxidative stress in NP cells. The therapeutic effect of circKIAA0564 knockdown on NP cells was reversed by the silencing of miR-424-5p. Overexpression of circKIAA0564 exacerbated IL-1β-induced NP cell injury, a process that was reversed by knockdown of KDM4A. CircKIAA0564 activated the toll-like receptor 4 (TLR4)/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)/NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) signaling pathway by regulating the miR-424-5p/KDM4A axis. CircKIAA0564 exacerbates IL-1β-induced inflammation and oxidative stress in NP cells by competitively binding miR-424-5p, thereby mediating KDM4A and activating the TLR4/NF-κB/NLRP3 signaling pathway. These findings provide robust data support for targeted therapy of IDD and the development of future pharmaceuticals.

MiR-92b-3p Protects against Myocardial Ischemia-Reperfusion Injury by Inhibiting MAP3K2 in a Mouse Model.

MicroRNAs are well-known RNA regulators modulating biological functions in complex signaling networks. This work aims to explore the impact of microRNA-92b-3p (miR-92b-3p) on myocardial ischemia-reperfusion (I/R) injury. The I/R model was established by left anterior descending coronary artery ligation in mice. The hemodynamic parameters were detected through a multichannel physiological recorder. Myocardial injury markers: serum cardiac troponin I, myocardial kinase isoenzyme (creatine kinase-MB), and serum inflammatory factors (tumor necrosis factor-α, interleukin [IL]-1β, and IL-6) were evaluated by enzyme-linked immunosorbent assay. Cardiac tissue oxidative stress-related factors (malondialdehyde, glutathione peroxidase, total antioxidation capability, and superoxide dismutase) were assessed by colorimetry, myocardial pathology was observed by hematoxylin-eosin staining, and cardiomyocyte apoptosis was measured by triphosphate nick end-labeling staining, as well as the expression of miR-92b-3p and mitogen-activated protein kinase kinase kinase 2 (MAP3K2) in cardiac tissues were determined by reverse transcription quantitative polymerase chain reaction or western blot assay. The targeting relationship between miR-92b-3p and MAP3K2 was verified by bioinformatics, RNA immunoprecipitation, and luciferase reporter assays. miR-92b-3p was lowly expressed and MAP3K2 was highly expressed in myocardial I/R injury mice. Upregulation of miR-92b-3p improved hemodynamic indices, decreased serum levels of myocardial injury biomarkers, inhibited serum inflammatory response, alleviated cardiac tissue oxidative stress, relieved myocardial pathology, and reduced cardiomyocyte apoptosis during the myocardial I/R injury in mice. MAP3K2 was a direct target gene of miR-92b-3p. This research suggests that miR-92b-3p protects against myocardial I/R injury by inhibiting MAP3K2, which may provide novel candidates for treatment of myocardial I/R injury.

CircST6GAL1 knockdown alleviates pulmonary arterial hypertension by regulating miR-509-5p/multiple C2 and transmembrane domain containing 2 axis.

Hypertension is a main contributing factor of cardiovascular diseases; deregulated circular RNAs are involved in the pathogenesis of pulmonary arterial hypertension (PAH). Herein, we evaluated the function and mechanism of circST6GAL1 in PAH process. Human pulmonary artery smooth muscle cells (HPASMCs) were cultured in hypoxic environment for functional analysis. The cell counting kit-8, 5-ethynyl-2'-deoxyuridine, wound healing, and flow cytometry assays were used to investigate cell proliferation, migration, and apoptosis. qRT-PCR and Western blotting analyses were used for level measurement of genes and proteins. The binding between miR-509-5p and circST6GAL1 or multiple C2 and transmembrane domain containing 2 (MCTP2) was analyzed by dual-luciferase reporter, RNA immunoprecipitation, and pull-down assays. The monocrotaline (MCT)-induced PAH mouse models were established for in vivo assay. CircST6GAL1 was highly expressed in PAH patients and hypoxia-induced HPASMCs. Functionally, circST6GAL1 deficiency reversed hypoxia-induced proliferation and migration, as well as apoptosis arrest in HPASMCs. Mechanistically, circST6GAL1 directly targeted miR-509-5p, and MCTP2 was a target of miR-509-5p. Rescue assays showed that the regulatory effects of circST6GAL1 deficiency on hypoxia-induced HPASMCs were abolished. Moreover, forced expression of miR-509-5p suppressed HPASMC proliferation and migration and induced cell apoptosis under hypoxia stimulation, while these effects were abolished by MCTP2 overexpression. Moreover, circST6GAL1 silencing improved MCT-induced pulmonary vascular remodeling and PAH. CircST6GAL1 deficiency reversed hypoxia-induced proliferation and migration, as well as apoptosis arrest in HPASMCs, and alleviated pulmonary vascular remodeling in MCT-induced PAH mouse models through the miR-509-5p/MCTP2 axis, indicating a potential therapeutic target for PAH.

METTL3-mediated m6A methylation of SLC38A1 stimulates cervical cancer growth

The objective of this study was to better characterize the role of the glutamine transporter SLC38A1 in cervical cancer and explore the underlying mechanisms. Data from public databases and clinical cervical cancer tissue samples were used to assess the expression of SLC38A1 and its prognostic significance. Immunohistochemical staining, qRT-PCR, and Western blotting were used to evaluate the expression of relevant genes and proteins. Cell viability, cell cycle, apoptosis, and intracellular glutamine content were measured using CCK-8, flow cytometry, and biochemical assays. Additionally, the RNA immunoprecipitation (RIP) assay was used to examine the impact of METTL3/IGF2BP3 on the m6A modification of the SLC38A1 3′UTR. Both cervical cancer specimens and cells showed significantly increased expression of SLC38A1 and its expression correlated with an unfavorable prognosis. Knockdown of SLC38A1 inhibited cell viability and cell cycle progression, induced apoptosis, and suppressed tumor growth in vivo. Glutaminase-1 inhibitor CB-839 reversed the effects of SLC38A1 overexpression. METTL3 promoted m6A modification of SLC38A1 and enhanced its mRNA stability through IGF2BP3 recruitment. Moreover, METTL3 silencing inhibited cell viability, cell cycle progression, intracellular glutamine content, and induced apoptosis, but these effects were reversed by SLC38A1 overexpression. In conclusion, METTL3-mediated m6A methylation of SLC38A1 stimulates cervical cancer progression. SLC38A1 inhibition is a potential therapeutic strategy for cervical cancer.

CRISPR Screen Identifies the RNA-Binding Protein Eef1a1 as a Key Regulator of Myogenesis.

Skeletal muscle myogenesis hinges on gene regulation, meticulously orchestrated by molecular mechanisms. While the roles of transcription factors and non-coding RNAs in myogenesis are widely known, the contribution of RNA-binding proteins (RBPs) has remained unclear until now. Therefore, to investigate the functions of post-transcriptional regulators in myogenesis and uncover new functional RBPs regulating myogenesis, we employed CRISPR high-throughput RBP-KO (RBP-wide knockout) library screening. Through this approach, we successfully identified Eef1a1 as a novel regulatory factor in myogenesis. Using CRISPR knockout (CRISPRko) and CRISPR interference (CRISPRi) technologies, we successfully established cellular models for both CRISPRko and CRISPRi. Our findings demonstrated that Eef1a1 plays a crucial role in promoting proliferation in C2C12 myoblasts. Through siRNA inhibition and overexpression methods, we further elucidated the involvement of Eef1a1 in promoting proliferation and suppressing differentiation processes. RIP (RNA immunoprecipitation), miRNA pull-down, and Dual-luciferase reporter assays confirmed that miR-133a-3p targets Eef1a1. Co-transfection experiments indicated that miR-133a-3p can rescue the effect of Eef1a1 on C2C12 myoblasts. In summary, our study utilized CRISPR library high-throughput screening to unveil a novel RBP, Eef1a1, involved in regulating myogenesis. Eef1a1 promotes the proliferation of myoblasts while inhibiting the differentiation process. Additionally, it acts as an antagonist to miR-133a-3p, thus modulating the process of myogenesis.

Ferroptosis-related lncRNA NRAV affects the prognosis of hepatocellular carcinoma via the miR-375-3P/SLC7A11 axis

Ferroptosis has important value in cancer treatment. It is significant to explore the new ferroptosis-related lncRNAs prediction model in Hepatocellular carcinoma (HCC) and the potential molecular mechanism of ferroptosis-related lncRNAs. We constructed a prognostic multi-lncRNA signature based on ferroptosis-related differentially expressed lncRNAs in HCC. qRT-PCR was applied to determine the expression of lncRNA in HCC cells. The biological roles of NRAV in vitro and in vivo were determined by performing a series of functional experiments. Furthermore, dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were used to confirm the interaction of NRAV with miR-375-3P. We identified 6 differently expressed lncRNAs associated with the prognosis of HCC. Kaplan–Meier analyses revealed the high-risk lncRNAs signature associated with poor prognosis of HCC. Moreover, the AUC of the lncRNAs signature showed utility in predicting HCC prognosis. Further functional experiments show that the high expression of NRAV can strengthen the viciousness of HCC. Interestingly, we found that NRAV can enhance iron export and ferroptosis resistance. Further study showed that NRAV competitively binds to miR-375-3P and attenuates the inhibitory effect of miR-375-3P on SLC7A11, affecting the prognosis of patients with HCC. In conclusion, We developed a novel ferroptosis-related lncRNAs prognostic model with important predictive value for the prognosis of HCC. NRAV is important in ferroptosis induction through the miR-375-3P/SLC7A11 axis.

RNA splicing modulates the postharvest physiological deterioration of cassava storage root.

Rapid postharvest physiological deterioration (PPD) of cassava (Manihot esculenta Crantz) storage roots is a major constraint that limits the potential of this plant as a food and industrial crop. Extensive studies have been performed to explore the regulatory mechanisms underlying the PPD processes in cassava to understand their molecular and physiological responses. However, the exceptional functional versatility of alternative splicing (AS) remains to be explored during the PPD process in cassava. Here, we identified several aberrantly spliced genes during the early PPD stage. An in-depth analysis of AS revealed that the abscisic acid (ABA) biosynthesis pathway might serve as an additional molecular layer in attenuating the onset of PPD. Exogenous ABA application alleviated PPD symptoms through maintaining ROS generation and scavenging. Interestingly, the intron retention transcript of MeABA1 (ABA DEFICIENT 1) was highly correlated with PPD symptoms in cassava storage roots. RNA yeast 3-hybrid and RNA immunoprecipitation (RIP) assays showed that the serine/arginine-rich protein MeSCL33 (SC35-like splicing factor 33) binds to the precursor mRNA of MeABA1. Importantly, overexpressing MeSCL33 in cassava conferred improved PPD resistance by manipulating the AS and expression levels of MeABA1 and then modulating the endogenous ABA levels in cassava storage roots. Our results uncovered the pivotal role of the ABA biosynthesis pathway and RNA splicing in regulating cassava PPD resistance and proposed the essential roles of MeSCL33 for conferring PPD resistance, broadening our understanding of SR proteins in cassava development and stress responses.

Cancer-associated fibroblasts-derived exosomal METTL3 promotes the proliferation, invasion, stemness and glutaminolysis in non-small cell lung cancer cells by eliciting SLC7A5 m6A modification.

Cancer-associated fibroblasts (CAFs) can promote the crosstalk between cancer cells and tumor microenvironment by exosomes. METTL3-mediated N6-methyladenine (m6A) modification has been proved to promote the progression of non-small cell lung cancer (NSCLC). Here, we focused on the impacts of CAFs-derived exosomes and METTL3-mediated m6A modification on NSCLC progression. Functional analyses were conducted using Cell Counting Kit-8, EdU, colony formation, sphere formation and transwell assays, respectively. Glutamine metabolism was evaluated by detecting glutamate consumption, and the production of intercellular glutamate and α-ketoglutarate (α-KG). qRT-PCR and western blotting analyses were utilized to measure the levels of genes and proteins. Exosomes were isolated by kits. The methylated RNA immunoprecipitation assay detected the m6A modification profile of Amino acid transporter LAT1 (SLC7A5) mRNA. The NSCLC mouse model was established to conduct in vivo experiments. We found that CAFs promoted the proliferation, invasion, stemness and glutaminolysis in NSCLC cells. METTL3 was enriched in CAFs and was packaged into exosomes. After knockdown of METTL3 in CAF exosomes, it was found the oncogenic effects of CAFs on NSCLC cells were suppressed. CAFs elevated m6A levels in NSCLC cells. Mechanistically, exosomal METTL3-induced m6A modification in SLC7A5 mRNA and stabilized its expression in NSCLC cells. Moreover, SLC7A5 overexpression abolished the inhibitory effects of exosomal METTL3-decreased CAFs on NSCLC cells. In addition, METTL3 inhibition in CAF exosomes impeded NSCLC growth in vivo. In all, CAFs-derived exosomal METTL3 promoted the proliferation, invasion, stemness and glutaminolysis in NSCLC cells by inducing SLC7A5 m6A modification.

WTAP-mediated N6-methyladenine Modification of circEEF2 Promotes Lung Adenocarcinoma Tumorigenesis by Stabilizing CANT1 in an IGF2BP2-dependent Manner.

N6-methyladenosine (m6A) is a common posttranscriptional RNA modification and plays an important role in cancer biology. Circular RNAs (circRNAs) are also reported to participate in lung adenocarcinoma (LUAD) progression. Here, we aimed to investigate the functions of Wilms tumor 1-associating protein (WTAP) methyltransferase and circEEF2 in LUAD cell tumorigenesis, and probe whether circEEF2 functioned through WTAP-induced m6A modification and its potential mechanisms. Functional analyses were conducted by tube formation, sphere formation, 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, and transwell assays in vitro as well as tumor formation experiments in mice, respectively. The N6-methyladenine (m6A) modification in circEEF2 mRNA was determined by RNA immunoprecipitation (Me-RIP) assay. The interaction between IGF2BP2 (Insulin Like Growth Factor 2 MRNA-Binding Protein 2) and circEEF2 or Calcium-activated nucleotidase 1 (CANT1) mRNA was confirmed using RIP assay. LUAD tissues and cells showed high circEEF2 expression, and the deficiency of circEEF2 suppressed LUAD cell angiogenesis, stemness, proliferation, migration, and invasion. WTAP induced circEEF2 m6A modification. WTAP silencing repressed the oncogenic phenotypes of LUAD cells via stabilizing circEEF2 in an m6A-dependent manner. IGF2BP2 interacted with circEEF2 and CANT1, and WTAP and circEEF2 could regulate CANT1 expression through IGF2BP2. The inhibition of LUAD cell oncogenic phenotypes caused by circEEF2 deficiency was abolished by CANT1 overexpression. In addition, WTAP silencing impeded LUAD growth via modulating circEEF2 and CANT1 in vivo. WTAP-mediated m6A modification of circEEF2 promotes lung adenocarcinoma growth and tumorigenesis by stabilizing CANT1 through IGF2BP2.

N6-Methyladenosine–Modified KREMEN2 Promotes Tumorigenesis and Malignant Progression of High-Grade Serous Ovarian Cancer

High-grade serous ovarian cancer (HGSOC) remains the most lethal female cancer by far. Herein, clinical HGSOC samples had higher N6-methyladenosine (m6A) modification than normal ovarian tissue, and its dysregulation had been reported to drive aberrant transcription and translation programs. However, Kringle-containing transmembrane protein 2 (KREMEN2) and its m6A modification have not been fully elucidated in HGSOC. In this study, the data from the high-throughput messenger RNA (mRNA) sequencing of clinical samples were processed using the weighted correlation network analysis and functional enrichment analysis. Results revealed that KREMEN2 was a driver gene in the tumorigenesis of HGSOC and a potential target of m6A demethylase fat-mass and obesity-associated protein (FTO). KREMEN2 and FTO levels were upregulated and downregulated, respectively, and correlation analysis showed a significant negative correlation in HGSOC samples. Importantly, upregulated KREMEN2 was remarkably associated with lymph node metastasis, distant metastasis, peritoneal metastasis, and high International Federation of Gynecology and Obstetrics stage (Ⅲ/Ⅳ), independent of the age of patients. KREMEN2 promoted the growth of HGSOC in vitro and in vivo, which was dependent on FTO. The methylated RNA immunoprecipitation qPCR and RNA immunoprecipitation assays were performed to verify the m6A level and sites of KREMEN2. FTO overexpression significantly decreased m6A modification in the 3′ and 5′ untranslated regions of KREMEN2 mRNA and downregulated its expression. In addition, we found that FTO-mediated m6A modification of KREMEN2 mRNA was recognized and stabilized by the m6A reader IGF2BP1 rather than by IGF2BP2 or IGF2BP3. This study highlights the m6A modification of KREMEN2 and extends the importance of RNA epigenetics in HGSOC.

LncRNA ZFAS1 promotes invasion of medullary thyroid carcinoma by enhancing EPAS1 expression via miR-214-3p/UCHL1 axis.

lncRNA ZFAS1 was identified to facilitate thyroid cancer, but its role in medullary thyroid carcinoma (MTC) remains unknown. This study aimed to unravel the potential function of this lncRNA in MTC by investigating the involvement of the lncRNA ZFAS1 in a ceRNA network that regulates MTC invasion. Proliferation, invasion, and migration of cells were evaluated using EdU staining and Transwell assays. Immunoprecipitation (IP) assays, dual-fluorescence reporter, and RNA IP assays were employed to examine the binding interaction among genes. Nude mice were used to explore the role of lncRNA ZFAS1 in MTC in vivo. ZFAS1 and EPAS1 were upregulated in MTC. Silencing ZFAS1 inhibited MTC cell proliferation and invasion under hypoxic conditions, which reduced EPAS1 protein levels. UCHL1 knockdown increased EPAS1 ubiquitination. ZFAS1 positively regulated UCHL1 expression by binding to miR-214-3p. Finally, silencing ZFAS1 significantly repressed tumor formation and metastasis in MTC. LncRNA ZFAS1 promotes invasion of MTC by upregulating EPAS1 expression via the miR-214-3p/UCHL1 axis.

WTAP-MEDIATED M6A MODIFICATION OF KLF6 AGGRAVATES HYPOXIA/REOXYGENATION-INDUCED HUMAN CARDIOMYOCYTE INJURY.

Background: Myocardial infarction (MI) is a severe condition that typically results from the ischemia and necrosis of heart muscle. Kruppel-like factor 6 (KLF6) can aggravate myocardial ischemia/reperfusion injury. This work aims to reveal its role and mechanism in hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury. Methods: Human cardiomyocyte (AC16) was exposed to hypoxic treatment to mimic MI-like cell injury. mRNA expression levels of KLF6 and WT1-associated protein (WTAP) were detected by quantitative real-time polymerase chain reaction. Protein expression was detected by western blotting assay. Cell viability was assessed by CCK-8 assay. Cell apoptosis and cell cycle were investigated by flow cytometry. Enzyme-linked immunosorbent assays were conducted to detect IL-1β, TNF-α and IL-6 levels. Fe 2+ colorimetric assay kit was used to detect Fe 2+ level. MDA Content Assay Kit was used to detect MDA level. Cellular ROS Assay kit was applied to assess ROS level. The association of KLF6 and WTAP was identified by RNA immunoprecipitation assay and dual-luciferase reporter assay. Results: KLF6 and WTAP expression at mRNA and protein levels were significantly upregulated in serum samples of MI patients and H/R-induced AC16 cells when compared with control groups. KLF6 silencing attenuated H/R-induced AC16 cell apoptosis, inflammatory response, oxidative stress, and ferroptosis. Additionally, WTAP stabilized KLF6 mRNA by regulating its m6A modification. Furthermore, WTAP knockdown rescued H/R-induced AC16 cell apoptosis, inflammatory response, oxidative stress, and ferroptosis by decreasing KLF6 expression. Conclusion: WTAP-mediated m6A modification of KLF6 aggravated hypoxia/reoxygenation-induced apoptosis, inflammatory response, oxidative stress, and ferroptosis of human cardiomyocytes, providing a therapeutic strategy for MI.

YBX1 Promotes Esophageal Squamous Cell Carcinoma Progression via m5C‐Dependent SMOX mRNA Stabilization

The modification and recognition of 5-methylcytosine (m5C) are involved in the initiation and progression of various tumor types. However, the precise role and potential mechanism of Y-box-binding protein 1 (YBX1) in esophageal squamous cell carcinoma (ESCC) remains unclear. Here, it is found that YBX1 is frequently upregulated in ESCC compared with matched nontumor tissues. Gain- and loss-of-function assays show that YBX1 promoted the proliferation and metastasis of ESCC cells both in vitro and in vivo. Functional studies revealed that NOP2/Sun RNA methyltransferase family member 2 (NSUN2) is a critical RNA methyltransferase that facilitates YBX1-mediated ESCC progression. Mechanistically, integrated analysis based on RNA immunoprecipitation sequencing (RIP-seq) and m5C methylated RNA immunoprecipitation and sequencing (MeRIP-seq) assays identified spermine oxidase (SMOX) as a target gene containing an m5C site in its coding sequence (CDS) region, which coincided well with the binding site of YBX1. Overexpression of SMOX-WT but not SMOX-Mut partially restored the proliferation and invasion ability of ESCC cells curbed by YBX1 knockdown. Moreover, YBX1 activated the mTORC1 signaling pathway by stabilizing SMOX mRNA. The study reveals that YBX1 promotes ESCC development by stabilizing SMOX mRNA in an m5C-dependent manner, thus providing a valuable therapeutic target for ESCC.

M6A-dependent upregulation of DDX21 by super-enhancer-driven IGF2BP2 and IGF2BP3 facilitates progression of acute myeloid leukaemia.

Acute myeloid leukaemia (AML) is a haematological malignancy with unfavourable prognosis. Despite the effectiveness of chemotherapy and targeted therapy, relapse or drug resistance remains a major threat to AML patients. N6-methyladenosine (m6A) RNA methylation and super-enhancers (SEs) are extensively involved in the leukaemogenesis of AML. However, the potential relationship between m6A and SEs in AML has not been elaborated. Chromatin immunoprecipitation (ChIP) sequencing data from Gene Expression Omnibus (GEO) cohort were analysed to search SE-related genes. The mechanisms of m6 A-binding proteins IGF2BP2 and IGF2BP3 on DDX21 were explored via methylated RNA immunoprecipitation (MeRIP) assays, RNA immunoprecipitation (RIP) assays and luciferase reporter assays. Then we elucidated the roles of DDX21 in AML through functional assays in vitro and in vivo. Finally, co-immunoprecipitation (Co-IP) assays, RNA sequencing and ChIP assays were performed to investigate the downstream mechanisms of DDX21. We identified two SE-associated transcripts IGF2BP2 and IGF2BP3 in AML. High enrichment of H3K27ac, H3K4me1 and BRD4 was observed in IGF2BP2 and IGF2BP3, whose expression were driven by SE machinery. Then IGF2BP2 and IGF2BP3 enhanced the stability of DDX21 mRNA in an m6A-dependent manner. DDX21 was highly expressed in AML patients, which indicated a poor survival. Functionally, knockdown of DDX21 inhibited cell proliferation, promoted cell apoptosis and led to cell cycle arrest. Mechanistically, DDX21 recruited transcription factor YBX1 to cooperatively trigger ULK1 expression. Moreover, silencing of ULK1 could reverse the promoting effects of DDX21 overexpression in AML cells. Dysregulation of SE-IGF2BP2/IGF2BP3-DDX21 axis facilitated the progression of AML. Our findings provide new insights into the link between SEs and m6A modification, elucidate the regulatory mechanisms of IGF2BP2 and IGF2BP3 on DDX21, and reveal the underlying roles of DDX21 in AML.

CircRNA circRREB1 promotes tumorigenesis and progression of breast cancer by activating Erk1/2 signaling through interacting with GNB4

Current investigations have illuminated the essential roles played by circular RNAs (circRNAs) in driving breast cancer (BC) tumorigenesis. However, the functional implications and molecular underpinnings of most circRNAs in BC are not well characterized. Here, Circular RNA (circRNA) expression profiles were analyzed in four surgically resected BC cases along with adjacent non-cancerous tissues applying RNA microarray analysis. The levels and prognostic implications of circRREB1 in BC were subjected to quantitative real-time PCR (qRT-PCR) and in situ hybridization (ISH). Experimental manipulation of circRREB1 levels in both in vivo and in vitro settings further delineated its role in BC cell growth, invasion, and metastasis. The mechanical verification of circRREB1's interaction with GNB4 was established through RNA pull-down, mass spectrometry, Western blot analysis, RNA immunoprecipitation assays (RIP), fluorescence ISH (FISH), and rescue experiments. We found that circRREB1 exhibited significant upregulation in BC tissues and cells, implicating its association with an unfavorable prognosis in BC patients. CircRREB1 knockdown elicited anti-proliferative, anti-migratory, anti-invasive, and pro-apoptotic effects in BC cells, whereas its upregulation exerted opposing influences. Follow-up mechanistic examinations suggested that circRREB1 might interact with GNB4 directly, inducing the activation of Erk1/2 signaling and driving BC progression. Our findings collectively indicate that the interplay of circRREB1 with GNB4 promotes Erk1/2 signaling, thereby fostering BC progression, and positioning circRREB1 as a candidate therapeutic target for intervention in BC.

CircNRD1 elevates THAP domain containing 11 through sequestering microRNA-421 to inhibit gastric cancer growth and tumorigenesis.

We explored the role and mechanism of circular RNAcircNRD1 in gastric cancer (GC) progression, aiming to identify new bio-markers for the treatment and prognosis of GC patients. The RNA expression was examined by reverse transcription-quantitative polymerase chain reaction. Cell proliferation, migration and invasion were analyzed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay, scratch assay and transwell assay. Western blot assay was conducted for protein expression measurement. Dual-luciferase reporter, RNA immunoprecipitation, and RNA pull-down assays were conducted to verify the interaction between microRNA-421 (miR-421) and circNRD1 or THAP domain containing 11 (THAP11). Xenograft tumor model was established to perform in vivo experiments. CircNRD1 was notably downregulated in GC tissues and cell lines. Additionally, decreased circNRD1 level was closely associated with advanced tumor stage and dismal prognosis in GC patients. CircNRD1 overexpression suppressed the proliferation and metastasis of GC cells. CircNRD1 acted as a molecular sponge for miR-421 in GC cells, and the antitumor impacts of circNRD1 overexpression in GC cells could be alleviated by miR-421 overexpression. miR-421 directly targeted THAP11, and circNRD1 could up-regulate THAP11 expression in GC cells through sponging miR-421. THAP11 knockdown reversed circNRD1 overexpression-induced tumor suppressing effects in GC cells. CircNRD1 overexpression significantly blocked tumor growth in vivo. CircNRD1 suppressed the proliferation and metastasis of GC cells in vitro and blocked tumor growth in vivo via modulating miR-421/THAP11 axis.

METTL3 mediates SOX5 m6A methylation in bronchial epithelial cells to attenuate Th2 cell differentiation in T2 asthma

ObjectiveAsthma, a chronic inflammatory disease in which type 2 T helper cells (Th2) play a causative role in the development of T2 asthma. N6-methyladenosine (m6A) modification, an mRNA modification, and methyltransferase-like 3 (METTL3) is involved in the development of T2 asthma by inhibiting Th2 cell differentiation. Sex determining region Y-box protein 5 (SOX5) is involved in regulating T cell differentiation, but its role in T2 asthma was unclear. The objective of this study was to explore the role of METTL3 and SOX5 in T2 asthma and whether there is an interaction between the two. Materials and methodsAdults diagnosed with T2 asthma (n = 14) underwent clinical information collection and pulmonary function tests. In vivo and in vitro T2 asthma models were established using female C57BL/6 mice and human bronchial epithelial cells (HBE). The expressions of METTL3 and SOX5 were detected by Western blot and qRT-PCR and Western blot. Th2 cell differentiation was determined by flow cytometry and IL-4 level was detected by ELISA. m6A methylation level was determined by m6A quantitative assay. The relationship between METTL3 expression and clinical parameters was determined by Spearman rank correlation analysis. The function of METTL3 and SOX5 genes in asthma was investigated in vitro and in vivo. The RNA immunoprecipitation assay detected the specific interaction between METTL3 and SOX5. ResultsPatients with T2 asthma displayed lower METTL3 levels compared to healthy controls. Within this group, a negative correlation was observed between METTL3 and Th2 cells, while a positive correlation was noted between METTL3 and clinical parameters as well as Th1 cells. In both in vitro and in vivo models representing T2 asthma, METTL3 levels decreased significantly, while SOX5 levels showed the opposite trend. Overexpression of METTL3 gene in HBE cells significantly inhibited Th2 cell differentiation and increased m6A methylation activity. From a mechanism perspective, low METTL3 negatively regulates SOX5 expression through m6A modification dependence, while high SOX5 expression is positively associated with T2 asthma severity. Cell transfection experiments confirmed that METTL3 regulates Th2 cell differentiation and IL-4 release through SOX5. ConclusionsOverall, our results indicate that METTL3 alleviates Th2 cell differentiation in T2 asthma by modulating the m6A methylation activity of SOX5 in bronchial epithelial cells. This mechanism could potentially serve as a target for the prevention and management of T2 asthma.