OBJECTIVE: To analyze the clinical characteristics, treatment, and prognosis of seven pediatric patients with Acute myeloid leukemia (AML) positive for the t(16;21)(p11;q22) FUS::ERG fusion gene. METHODS: A retrospective analysis was carried out on the clinical data, treatment, and prognosis of seven AML patients with t(16;21)(p11;q22) FUS::ERG fusion gene admitted to Henan Children's Hospital between June 2015 and November 2024. Relevant literature was also reviewed. This study was approved by the Medical Ethics Committee of the Hospital (Ethics No.: 2024-102-001). RESULTS: Among 297 pediatric patients with AML, 7 cases (2.36%) were positive for the t(16;21)(p11;q22) FUS::ERG fusion gene, including 3 males and 4 females, with a median age of 11 years (range: 3 ~ 12 years). According to the FAB classification, these included 1 case of M2, 3 cases of M5, and 3 cases of AML-not otherwise specified (non-M3). All 7 patients were found to harbor the t(16;21)(p11;q22) translocation, with 3 cases showing additional chromosomal abnormalities. Immunophenotyping revealed universal expression of CD13, CD33, CD34, and CD117, with partial expression of CD56, CD4, CD64, CD123, CD15, CD38, CD11b, HLA-DR, cMPO, and CD16. One patient achieved complete remission (CR) after the first course of DAE (cytarabine + daunorubicin + etoposide) induction chemotherapy but relapsed and discontinued the treatment. Six patients received DAH (cytarabine + daunorubicin + homoharringtonine) induction therapy, of whom 2 achieved CR after two courses and underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT), resulting in an overall CR rate of 42.86%. Five children did not receive allo-HSCT and had a median overall survival of 9 months (range: 6 ~ 18 months). Two children who underwent transplantation achieved bone marrow morphological and molecular biological relapse at 6 and 9 months post-transplantation, respectively. After receiving combined chemotherapy and donor lymphocyte infusion, one child failed to achieve remission and died at 22 months post-transplantation, while the other has been followed up to date with positive fusion gene status. Their overall survival was 25 months and 30 months, respectively. CONCLUSION The t(16;21)(p11;q22) FUS::ERG fusion gene is rare in pediatric AML and associated with poor prognosis. Allo-HSCT may mitigate the adverse prognostic impact of the FUS::ERG fusion gene and contribute to prolonged survival.
Humans ; Male ; Child ; Female ; Leukemia, Myeloid, Acute/drug therapy* ; Oncogene Proteins, Fusion/genetics* ; Translocation, Genetic ; Retrospective Studies ; RNA-Binding Protein FUS/genetics* ; Chromosomes, Human, Pair 16/genetics* ; Adolescent ; Child, Preschool ; Chromosomes, Human, Pair 21/genetics* ; Prognosis ; Treatment Outcome

BACKGROUND: The main cause of restenosis after percutaneous transluminal angioplasty (PTA) is the excessive proliferation and migration of vascular smooth muscle cells (VSMCs). Lin28a has been reported to play critical regulatory roles in this process. However, whether CCAAT/enhancer-binding proteins β (C/EBPβ) binds to the Lin28a promoter and drives the progression of restenosis has not been clarified. Therefore, in the present study, we aim to clarify the role of C/EBPβ-Lin28a axis in restenosis. METHODS: Restenosis and atherosclerosis rat models of type 2 diabetes ( n   =  20, for each group) were established by subjecting to PTA. Subsequently, the difference in DNA methylation status and expression of C/EBPβ between the two groups were assessed. EdU, Transwell, and rescue assays were performed to assess the effect of C/EBPβ on the proliferation and migration of VSMCs. DNA methylation status was further assessed using Methyltarget sequencing. The interaction between Lin28a and ten-eleven translocation 1 (TET1) was analysed using co-immunoprecipitation (Co-IP) assay. Student's t -test and one-way analysis of variance were used for statistical analysis. RESULTS: C/EBPβ expression was upregulated and accompanied by hypomethylation of its promoter in restenosis when compared with atherosclerosis. In vitroC/EBPβ overexpression facilitated the proliferation and migration of VSMCs and was associated with increased Lin28a expression. Conversely, C/EBPβ knockdown resulted in the opposite effects. Chromatin immunoprecipitation assays further demonstrated that C/EBPβ could directly bind to Lin28a promoter. Increased C/EBPβ expression and enhanced proliferation and migration of VSMCs were observed after decitabine treatment. Further, mechanical stretch promoted C/EBPβ and Lin28a expression accompanied by C/EBPβ hypomethylation. Additionally, Lin28a overexpression reduced C/EBPβ methylation via recruiting TET1 and enhanced C/EBPβ-mediated proliferation and migration of VSMCs. The opposite was noted in Lin28a knockdown cells. CONCLUSION Our findings suggest that the C/EBPβ-Lin28a axis is a driver of restenosis progression, and presents a promising therapeutic target for restenosis.
Animals ; Cell Proliferation/genetics* ; Cell Movement/genetics* ; Muscle, Smooth, Vascular/metabolism* ; Rats ; DNA Methylation/physiology* ; CCAAT-Enhancer-Binding Protein-beta/genetics* ; Male ; Myocytes, Smooth Muscle/cytology* ; Rats, Sprague-Dawley ; RNA-Binding Proteins/genetics* ; Cells, Cultured ; Coronary Restenosis/metabolism*

OBJECTIVES: The mechanism of the odontogenic differentiation of apical papillary cells (APCs) stimulated by bioactive glass 45S5 is still unclear. This study aims to investigate the effect of autophagy on the odontogenic differentiation of APCs stimulated by bioactive glass 45S5. METHODS: APCs were isolated and cultured in vitro, and the cell origin was identified by flow cytometry. The culture medium was prepared with 1 mg/mL 45S5, and its pH and ion concentration were determined. The experiments were divided into control, 45S5, and 3-methyladenine (3-MA) 45S5 groups. In the 45S5 group, APCs were induced to culture with 1 mg/mL 45S5. In the 3-MA 45S5 group, the autophagy inhibitor 3-MA was added to 1 mg/mL 45S5. Protein immunoblotting assay (Western blot) was used to detect the expression of autophagy-associated proteins of microtubule-associated protein 1 light-chain 3β (LC3B) and P62 after 24 h of induction culture in each group. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of bone sialoprotein (BSP), Runt-related transcription factor 2 (Runx2), dentin sialophosphoprotein (DSPP), and dentin matrix protein-1 (DMP-1) after 7 d of induction culture. Cellular alkaline phosphatase (ALP) staining analyzed cellular ALP activity at 7 d of induction, and alizarin red staining evaluated the formation of mineralized nodules at 21 d of induction. RESULTS: The pH of the 45S5 extract culture medium was 8.65±0.01, which was not significantly different from that of the control group (P>0.05). The silicon ion concentration of the 45S5 induction culture medium was (1.56±0.07) mmol/L, which was higher than that of the control group (0.08±0.01) mmol/L (P<0.05). The calcium ion concentration of the 45S5 induction culture was (1.57±0.15) mmol/L, which was not significantly different from that of the control group (P>0.05). Western blot results showed that LC3B-Ⅱ/Ⅰ ratio increased and P62 expression decreased in the 45S5 group compared with those in the control group (P<0.05). By contrast, the ratio decreased and the expression increased in the 3-MA 45S5 group compared with those in the 45S5 group (P<0.05). RT-qPCR results showed that the expression of BSP, Runx2, DMP-1, and DSPP enhanced in the 45S5 group compared with that in the control group (P<0.05), but the expression decreased in the 3-MA 45S5 group compared with that in the 45S5 group (P<0.05). Semi-quantitative analysis of ALP staining and alizarin red staining showed that the ALP activity was enhanced, and the formation mineralized nodule increased in the 45S5 group compared with those in the control group. The ALP activity weakened, and the formation mineralized nodules were reduced in the 3-MA 45S5 group compared with that those in the 45S5 group. CONCLUSIONS Cell autophagy participates in the odontogenic differentiation of APCs induced by 1 mg/mL 45S5 in vitro.
Autophagy/drug effects* ; Cell Differentiation/drug effects* ; Odontogenesis/drug effects* ; Dental Papilla/cytology* ; Humans ; Microtubule-Associated Proteins/metabolism* ; Glass/chemistry* ; Cells, Cultured ; Core Binding Factor Alpha 1 Subunit/metabolism* ; Extracellular Matrix Proteins/metabolism* ; Ceramics/pharmacology* ; Adenine/pharmacology* ; Sialoglycoproteins/metabolism* ; Phosphoproteins/metabolism* ; Integrin-Binding Sialoprotein/metabolism* ; Alkaline Phosphatase/metabolism* ; RNA-Binding Proteins

N⁶-methyladenosine (m⁶A) modification plays a critical role in cell cycle regulation, while the mechanism of m⁶A in regulating mitosis remains underexplored. Here, we found that the total m⁶A modification level in cells increased during mitosis by the liquid chromatography-mass spectrometry/mass spectrometry and m⁶A dot blot assays. Silencing methyltransferase-like 3 (METTL3) or METTL14 results in delayed mitosis, abnormal spindle assembly, and chromosome segregation defects by the immunofluorescence. By analyzing transcriptome-wide m⁶A targets in HeLa cells, we identified polo-like kinase 1 (PLK1) as a key gene modified by m⁶A in regulating mitosis. Specifically, through immunoblotting and RNA pulldown, m⁶A modification inhibits PLK1 translation via YTH N⁶-methyladenosine RNA binding protein 1, thus mediating cell cycle homeostasis. Demethylation of PLK1 mRNA leads to significant mitotic abnormalities. These findings highlight the critical role of m⁶A in regulating mitosis and the potential of m⁶A as a therapeutic target in proliferative diseases such as cancer.
Humans ; Polo-Like Kinase 1 ; Cell Cycle Proteins/metabolism* ; Proto-Oncogene Proteins/metabolism* ; Protein Serine-Threonine Kinases/metabolism* ; Mitosis/physiology* ; HeLa Cells ; Adenosine/genetics* ; Methyltransferases/metabolism* ; RNA, Messenger/metabolism* ; RNA-Binding Proteins/metabolism*

Objective To investigate the effect of liquid-liquid phase separation(LLPS)of YTH domain family protein 2(YTHDF2)on the sodium arsenite-induced malignant transformation of skin cells,providing a new intervention target for the prevention and control of sodium arsenite-induced carcinogenesis.Methods The HaCaT cell model of malignant transformation was constructed by continuous treatment with 1 μmol/L sodium arsenite for 22 weeks,including cells with normal YTHDF2 LLPS(YTHDF2-wt)and cells with inhibited YTHDF2 LLPS(YTHDF2-mut).Confocal microscopy was employed to observe and characterize the LLPS droplets formed by YTHDF2 during sodium arsenite-induced malignant transformation of skin cells.Cell proliferation,scratch healing,and colony formation assays were performed to detect malignant phenotypes.Western blotting,quantitative reverse transcription PCR,and immunofluorescence experiments were conducted to examine the effects of YTHDF2 LLPS on the mRNA and protein levels of phosphatase and tensin homolog deleted on chromosome ten(PTEN)during sodium arsenite-induced malignant transformation of skin cells.Results After 4 weeks of sodium arsenite treatment,LLPS droplets of YTHDF2 appeared in YTHDF2-wt cells,and the number of droplets gradually increased as the treatment time was prolonged(F=35.252,P<0.001),while no phase-separated droplets were observed in YTHDF2-mut cells.Compared with YTHDF2-mut cells,YTHDF2-wt cells showed enhanced proliferation at the time points of 48 h(t=3.654,P=0.006)and 72 h(t=5.458,P<0.001)after 22 weeks of sodium arsenite treatment.The scratch healing rate of YTHDF2-wt cells was increased at the 8th(t=12.137,P<0.001)and 22th(t=4.484,P=0.011)weeks of sodium arsenite treatment.The number of colonies formed by YTHDF2-wt cells was higher at the 4th(t=3.365,P=0.027),8th(t=5.580,P=0.005),and 22th(t=3.328,P=0.029)weeks of sodium arsenite treatment.Compared with YTHDF2-mut cells,YTHDF2-wt cells showed down-regulated protein(t=-3.119,P=0.036)and mRNA(t=4.051,P=0.015) levels of PTEN after 22 weeks of sodium arsenite treatment.Immunofluorescence results showed that after 4 weeks of sodium arsenite treatment,YTHDF2 LLPS droplets in YTHDF2-wt cells were localized to stress granules,translation-related membrane-less organelles.Conclusions During sodium arsenite-induced malignant transformation of skin cells,YTHDF2 undergoes LLPS and localizes to stress granules,translation-related membrane-less organelles.YTHDF2 LLPS participates in sodium arsenite-induced malignant transformation of skin cells by down-regulating the mRNA level of the key tumor suppressor PTEN.
Arsenites/toxicity* ; Sodium Compounds/toxicity* ; Humans ; Cell Transformation, Neoplastic/drug effects* ; PTEN Phosphohydrolase/metabolism* ; Cell Proliferation ; Skin/cytology* ; RNA-Binding Proteins ; Skin Neoplasms/chemically induced* ; Cell Line

Objective To investigate the expression and correlation of LY86-AS1 and KHDRBS2 in glioblastoma (GBM), and their impacts on the prognosis of patients and immune cell infiltration. Methods Based on the GSE50161 dataset from the Gene Expression Omnibus (GEO) database, LY86-AS1 and KHDRBS2, which are closely related to the development of GBM, were identified by WGCNA and differential expression analysis. The Cancer Genome Atlas (TCGA) and the Chinese Glioma Genome Atlas (CGGA) databases were used to analyze the relationship between the expression of LY86-AS1 and KHDRBS2 and the prognosis of GBM patients. Multiple datasets were employed to analyze the correlation between the expression levels of LY86-AS1 and KHDRBS2 and its relationship with immune cell infiltration. Real-time quantitative PCR was used to verify the expression of LY86-AS1 and KHDRBS2 in GBM and normal brain tissues. The Human Protein Atlas (HPA) database was accessed to obtain the protein expression of KHDRBS2, and immunohistochemical staining was conducted to verify the protein expression of KHDRBS2. Results LY86-AS1 and KHDRBS2 were lowly expressed in GBM tissues and were closely related to the development of GBM, showing a significant positive correlation. Patients with low expression levels of LY86-AS1 and KHDRBS2 had a lower overall survival rate than those with high expression levels. LY86-AS1 was positively correlated with naive B cells, plasma cells, activated NK cells, M1 macrophages, activated mast cells and monocytes. KHDRBS2 was positively correlated with naive B cells, plasma cells, helper T cells, activated NK cells and monocytes. Conclusion The low expression levels of LY86-AS1 and KHDRBS2 in GBM, which is associated with poor prognosis, affect the tumor immune microenvironment and may serve as potential new biomarkers for the diagnosis of GBM and the prognosis assessment of patients.
Humans ; Glioblastoma/metabolism* ; Prognosis ; Brain Neoplasms/pathology* ; Gene Expression Regulation, Neoplastic ; RNA-Binding Proteins/metabolism*

OBJECTIVE: To investigate the role of nucleolin (NCL) in acute myeloid leukemia (AML) Kasumi-1 cells and its underlying mechanism. METHODS: The Kasumi-1 cells were infected with lentivirus carrying shRNA to downregulate NCL expression. Cell proliferation was detected by CCK-8 assay, and cell apoptosis and cell cycle were determined by flow cytometry. Transcriptome next-generation sequencing (NGS) was performed to predict associated signaling pathways, the expression levels of related genes were measured by RT-PCR. RESULTS: Down-regulation of NCL expression significantly inhibited the proliferation of Kasumi-1 cells (P <0.01) and markedly increased the apoptosis rate (P <0.001). Cell cycle analysis showed significant changes in the distribution of cells in the G₁ and S phases after NCL knockdown (P <0.05), while no significant difference was observed in the G₂ phase (P >0.05). Transcriptome sequencing analysis demonstrated that differentially expressed genes in Kasumi-1 cells with low expression of NCL were primarily enriched in key signaling pathways, including ribosome, spliceosome, RNA transport, cell cycle, and amino acid biosynthesis. qPCR validation showed that the expression of BAX, CASP3, CYCS, PMAIP1, TP53 , and CDKN1A was significantly upregulated after NCL downregulation (P <0.05), with CDKN1A exhibiting the most pronounced difference. CONCLUSION NCL plays a critical role in regulating the proliferation, apoptosis, and cell cycle progression of Kasumi-1 cells. The mechanism likely involves suppressing cell cycle progression through activation of the TP53-CDKN1A pathway and promoting apoptosis by upregulating apoptosis-related genes.
Humans ; Leukemia, Myeloid, Acute/pathology* ; Down-Regulation ; Cell Proliferation ; Apoptosis ; RNA-Binding Proteins/genetics* ; Nucleolin ; Cell Line, Tumor ; Phosphoproteins/metabolism* ; Cell Cycle ; Signal Transduction ; RNA, Small Interfering

BACKGROUND: Double-stranded RNA-specific adenosine deaminase 1 (ADAR1) binds to double-stranded RNA and catalyzes the deamination of adenosine (A) to inosine (I). The functional mechanism of ADAR1 in non-small cell lung cancer (NSCLC) remains incompletely understood. This study aimed to investigate the prognostic significance of ADAR1 in NSCLC and to elucidate its potential role in regulating tumor cell proliferation and migration. METHODS: Data from The Cancer Genome Atlas (TCGA) and cBioPortal were analyzed to assess the correlation between high ADAR1 expression and clinicopathological features as well as prognosis in lung cancer. We performed Western blot (WB), cell proliferation assays, Transwell invasion/migration assays, and nude mouse xenograft modeling to examine the phenotypic changes and molecular mechanisms induced by ADAR1 knockdown. Furthermore, the ADAR1 p150 overexpression model was utilized to validate the proposed mechanism. RESULTS: ADAR1 expression was significantly elevated in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) tissues compared with adjacent non-tumor tissues (LUAD: P=3.70×10-15, LUSC: P=0.016). High ADAR1 expression was associated with poor prognosis (LUAD: P=2.03×10-2, LUSC: P=2.81×10-2) and distant metastasis (P=0.003). Gene Set Enrichment Analysis (GSEA) indicated that elevated ADAR1 was associated with mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway activation, matrix metalloproteinase-9 (MMP-9) expression, and cell adhesion. ADAR1 and MMP-9 levels showed a strongly positive correlation (P=6.45×10-34) in 10 lung cancer cell lines, highest in H1581. Knockdown of ADAR1 in H1581 cells induced a rounded cellular morphology with reduced pseudopodia. Concomitantly, it suppressed cell proliferation, invasion, migration, and in vivo tumorigenesis. It also suppressed ERK phosphorylation and downregulated cellular Finkel-Biskis-Jinkins murine osteosarcoma viral oncogene homolog (c-FOS), MMP-9, N-cadherin, and Vimentin. Conversely, ADAR1 p150 overexpression in PC9 cells enhanced ERK phosphorylation and increased c-FOS and MMP-9 expression. CONCLUSIONS High ADAR1 expression is closely associated with poor prognosis and distant metastasis in NSCLC patients. Mechanistically, ADAR1 may promote proliferation, invasion, migration, and tumorigenesis in lung cancer cells via the ERK/c-FOS/MMP-9 axis.
Humans ; Lung Neoplasms/physiopathology* ; Adenosine Deaminase/genetics* ; Matrix Metalloproteinase 9/genetics* ; Cell Proliferation ; Carcinoma, Non-Small-Cell Lung/physiopathology* ; Cell Movement ; Animals ; Mice ; RNA-Binding Proteins/genetics* ; Female ; Male ; Cell Line, Tumor ; Proto-Oncogene Proteins c-fos/genetics* ; Middle Aged ; MAP Kinase Signaling System ; Gene Expression Regulation, Neoplastic ; Mice, Nude ; Extracellular Signal-Regulated MAP Kinases/genetics*

OBJECTIVES: Bladder cancer is a common malignancy with high incidence and poor prognosis. N⁶-methyladenosine (m⁶A) modification is widely involved in diverse physiological processes, among which the m⁶A recognition protein YTH N⁶-methyladenosine RNA binding protein F2 (YTHDF2) plays a crucial role in bladder cancer progression. This study aims to elucidate the molecular mechanism by which O-linked N-acetylglucosamine (O-GlcNAc) modification of YTHDF2 regulates its downstream target, period circadian regulator 1 (PER1), thereby promoting bladder cancer cell proliferation. METHODS: Expression of YTHDF2 in bladder cancer was predicted using The Cancer Genome Atlas (TCGA). Twenty paired bladder cancer and adjacent normal tissues were collected at the clinical level. Normal bladder epithelial cells (SV-HUC-1) and bladder cancer cell lines (T24, 5637, EJ-1, SW780, BIU-87) were examined by quantitative real-time PCR (RT-qPCR), Western blotting, and immunohistochemistry for expression of YTHDF2, PER1, and proliferation-related proteins [proliferating cell nuclear antigen (PCNA), minichromosome maintenance complex component 2 (MCM2), Cyclin D1]. YTHDF2 was silenced in 5637 and SW780 cells, and cell proliferation was assessed by Cell Counting Kit-8 (CCK-8), colony formation, and EdU assays. Bioinformatics was used to predict glycosylation sites of YTHDF2, and immunoprecipitation (IP) was performed to detect O-GlcNAc modification levels of YTHDF2 in tissues and cells. Bladder cancer cells were treated with DMSO, OSMI-1 (O-GlcNAc inhibitor), or Thiamet G (O-GlcNAc activator), followed by cycloheximide (CHX), to assess YTHDF2 ubiquitination by IP. YTHDF2 knockdown and Thiamet G treatment were further used to evaluate PER1 mRNA stability, PER1 m⁶A modification, and cell proliferation. TCGA was used to predict PER1 expression in tissues; SRAMP predicted potential PER1 m⁶A sites. Methylated RNA immunoprecipitation (MeRIP) assays measured PER1 m⁶A modification. Finally, the effects of knocking down YTHDF2 and PER1 on 5637 and SW780 cell proliferation were assessed. RESULTS: YTHDF2 expression was significantly upregulated in bladder cancer tissues compared with adjacent tissues (mRNA: 2.5-fold; protein: 2-fold), which O-GlcNAc modification levels increased 3.5-fold (P<0.001). YTHDF2 was upregulated in bladder cancer cell lines, and its knockdown suppressed cell viability (P<0.001), downregulated PCNA, MCM2, and CyclinD1 (all P<0.05), reduced colony numbers 3-fold (P<0.01), and inhibited proliferation. YTHDF2 exhibited elevated O-GlcNAc modification in cancer cells. OSMI-1 reduced YTHDF2 protein stability (P<0.01) and enhanced ubiquitination, while Thiamet G exerted opposite effects (P<0.001). Thiamet G reversed the proliferation-suppressive effects of YTHDF2 knockdown, promoting cell proliferation (P<0.01) and upregulating PCNA, MCM2, and CyclinD1 (all P<0.05). Mechanistically, YTHDF2 targeted PER1 via m⁶A recognition, promoting PER1 mRNA degradation. Rescue experiments showed that PER1 knockdown reversed the inhibitory effect of YTHDF2 knockdown on cell proliferation, upregulated PCNA, MCM2, and Cyclin D1 (all P<0.05), and promoted bladder cancer cell proliferation (P<0.001). CONCLUSIONS O-GlcNAc modification YTHDF2 promotes bladder cancer development by downregulating the tumor suppressor gene PER1 through m⁶A-mediated post-transcriptional regulation.
Humans ; Urinary Bladder Neoplasms/metabolism* ; RNA-Binding Proteins/genetics* ; Cell Proliferation ; Cell Line, Tumor ; Disease Progression ; Acetylglucosamine/metabolism* ; Adenosine/metabolism* ; Gene Expression Regulation, Neoplastic ; Genes, Tumor Suppressor

OBJECTIVES: To explore the mechanism of Circ-MYOCD back-splicing and its regulatory role in myocardial hypertrophy. METHODS: Sanger sequencing and RNase R assays were performed to verify the circularity and stability of Circ-MYOCD, whose subcellular distribution was determined by nuclear-cytoplasmic fractionation. Bioinformatics analysis and mass spectrometry from pull-down assays were conducted to predict the RNA-binding proteins (RBPs) interacting with Circ-MYOCD. In rat cardiomyocytes H9C2 cells, the effects of HNRNPH1 and HNRNPL knockdown and overexpression on Circ-MYOCD back-splicing were evaluated. In a H9C2 cell model of angiotensin II (Ang II)-induced myocardial hypertrophy, the expression of HNRNPH1 was detected, the effects of HNRNPH1 knockdown and overexpression on progression of myocardial hypertrophy were assessed, and the regulatory effect of HNRNPH1 on Circ-MYOCD back-splicing was analyzed. RESULTS: Sanger sequencing confirmed that the junction primers could amplify the correct Circ-MYOCD sequence. RNase R and nuclear-cytoplasmic fractionation assays showed that Circ-MYOCD was stable and predominantly localized in the cytoplasm. Bioinformatics analysis and mass spectrometry from the Circ-MYOCD pull-down assay identified HNRNPH1 and HNRNPL as the RBPs interacting with Circ-MYOCD. In H9C2 cells, HNRNPH1 knockdown significantly enhanced while its overexpression inhibited Circ-MYOCD back-splicing; HNRNPH1 overexpression obviously increased the expressions of myocardial hypertrophy markers ANP and BNP, while its knockdown produced the opposite effect. In Ang II-induced H9C2 cells, which exhibited a significant increase of HNRNPH1 expression and increased expressions of ANP and BNP, HNRNPH1 knockdown obviously increased Circ-MYOCD expression, decreased MYOCD expression and lowered both ANP and BNP expressions. CONCLUSIONS HNRNPH1 regulates Circ-MYOCD back-splicing to influence the progression of myocardial hypertrophy.
Animals ; Rats ; RNA, Circular/genetics* ; Cardiomegaly/metabolism* ; Myocytes, Cardiac/metabolism* ; Heterogeneous-Nuclear Ribonucleoprotein Group F-H/metabolism* ; Cell Line ; RNA Splicing ; Angiotensin II ; RNA-Binding Proteins