Investigating Novel Therapeutic Approaches for Idiopathic Short Stature: Targeting siRNA and Growth Hormone Delivery to the Growth Plate Using Exosome Nanoparticles.

Idiopathic short stature (ISS) is a common childhood condition with largely unknown underlying causes. Recent research highlights the role of circulating exosomes in the pathogenesis of various disorders, but their connection to ISS remains unexplored. In the experiments, human chondrocytes are cocultured with plasma exosomes from ISS patients, leading to impaired chondrocyte growth and bone formation. Elevated levels of a specific long non-coding RNA (lncRNA), ISSRL, are identified as a distinguishing factor in ISS, boasting high specificity and sensitivity. Silencing ISSRL in ISS plasma exosomes reverses the inhibition of chondrocyte proliferation and bone formation. Conversely, overexpression of ISSRL in chondrocytes impedes their growth and bone formation, revealing its mechanism of action through the miR-877-3p/GZMB axis. Subsequently, exosomes (CT-Exo-siISSRL-oeGH) with precise cartilage-targeting abilities are engineered, loaded with customized siRNA for ISSRL and growth hormone. This innovative approach offers a therapeutic strategy to address ISS by rectifying abnormal non-coding RNA expression in growth plate cartilage and delivering growth hormone with precision to promote bone growth. This research provides valuable insights into ISS diagnosis and treatment, highlighting the potential of engineered exosomes.

Plasma exosome miRNA-26b-3p derived from idiopathic short stature impairs longitudinal bone growth via the AKAP2/ERK1/2 axis.

BACKGROUND: Currently, the etiology of idiopathic short stature (ISS) is still unclear. The poor understanding of the molecular mechanisms of ISS has largely restricted this strategy towards safe and effective clinical therapies. METHODS: The plasma exosomes of ISS children were co-cultured with normal human chondrocytes. The differential expression of exosome miRNA between ISS and normal children was identified via high-throughput microRNA sequencing and bioinformatics analysis. Immunohistochemistry, In situ hybridization, RT-qPCR, western blotting, luciferase expression, and gene overexpression and knockdown were performed to reveal the key signaling pathways that exosome miRNA of aberrant expression in ISS children impairs longitudinal bone growth. RESULTS: Chondrocytes proliferation and endochondral ossification were suppressed after coculture of ISS plasma exosomes with human normal chondrocytes. High-throughput microRNA sequencing and RT-qPCR confirmed that plasma exosome miR-26b-3p was upregulated in ISS children. Meanwhile, exosome miRNA-26b-3p showed a high specificity and sensitivity in discriminating ISS from normal children. The rescue experiment showed that downregulation of miR-26b-3p obviously improved the repression of chondrocyte proliferation and endochondral ossification caused by ISS exosomes. Subsequently, miR-26b-3p overexpression inhibited chondrocyte proliferation and endochondral ossification once again. In situ hybridization confirmed the colocalization of miR-26b-3p with AKAP2 in chondrocytes. In vitro and in vivo assay revealed exosome miRNA-26b-3p impairs longitudinal bone growth via the AKAP2 /ERK1/2 axis. CONCLUSIONS: This study is the first to confirm that miR-26b-3p overexpression in ISS plasma exosomes leads to disorders in proliferation and endochondral ossification of growth plate cartilage via inhibition of AKAP2/ERK1/2 axis, thereby inducing ISS. This study provides a new research direction for the etiology and pathology of ISS and a new idea for the biological treatment of ISS.

Human cytomegalovirus infection perturbs neural progenitor cell fate via the expression of viral microRNAs.

Human cytomegalovirus (HCMV) preferentially targets neural progenitor cells (NPCs) in congenitally infected fetal brains, inducing neurodevelopmental disorders. While HCMV expresses several microRNAs (miRNAs) during infection, their roles in NPC infection are unclear. Here, we characterized expression of cellular and viral miRNAs in HCMV-infected NPCs during early infection by microarray and identified seven differentially expressed cellular miRNAs and six significantly upregulated HCMV miRNAs. Deep learning approaches were used to identify potential targets of significantly upregulated HCMV miRNAs against differentially expressed cellular messenger RNA (mRNAs), and the associations with miRNA-mRNA expression changes were observed. Gene ontology enrichment analysis indicated cellular gene targets were significantly enriched in pathways involved in neurodevelopment and cell-cycle processes. Viral modulation of selected miRNAs and cellular gene targets involved in neurodevelopmental processes were further validated by real-time quantitative reverse transcription polymerase chain reaction. Finally, a predicted 3' untranslated region target site of hcmv-miR-US25-1 in Jag1, a factor important for neurogenesis, was confirmed by mutagenesis. Reduction of Jag1 RNA and protein levels in NPCs was observed in response to transient expression of hcmv-miR-US25-1. A hcmv-miR-US25-1 mutant virus (ΔmiR-US25) displayed limited ability to downregulate Jag1 mRNA levels and protein levels during the early infection stage compared with the wild type virus. Our collective experimental and computational investigation of miRNAs and cellular mRNAs expression in HCMV-infected NPCs yields new insights into the roles of viral miRNAs in regulating NPC fate and their contributions to HCMV neuropathogenesis.

Dissecting and targeting noncanonical functions of EZH2 in multiple myeloma via an EZH2 degrader.

Multiple myeloma (MM) is the second most common hematological malignancy with poor prognosis. Enhancer of zeste homolog 2 (EZH2) is the enzymatic subunit of polycomb repressive complex 2 (PRC2), which catalyzes trimethylation of histone H3 lysine 27 (H3K27me3) for transcriptional repression. EZH2 have been implicated in numerous hematological malignancies, including MM. However, noncanonical functions of EZH2 in MM tumorigenesis are not well understood. Here, we uncovered a noncanonical function of EZH2 in MM malignancy. In addition to the PRC2-mediated and H3K27me3-dependent canonical function, EZH2 interacts with cMyc and co-localizes with gene activation-related markers, promoting MM tumorigenesis in a PRC2- and H3K27me3-independent manner. Both canonical EZH2-PRC2 and noncanonical EZH2-cMyc complexes can be effectively depleted in MM cells by MS177, an EZH2 degrader we reported previously, leading to profound activation of EZH2-PRC2-associated genes and simultaneous suppression of EZH2-cMyc oncogenic nodes. The MS177-induced degradation of both canonical EZH2-PRC2 and noncanonical EZH2-cMyc complexes also reactivated immune response genes in MM cells. Phenotypically, targeting of EZH2's both canonical and noncanonical functions by MS177 effectively suppressed the proliferation of MM cells both in vitro and in vivo. Collectively, this study uncovers a new noncanonical function of EZH2 in MM tumorigenesis and provides a novel therapeutic strategy, pharmacological degradation of EZH2, for treating EZH2-dependent MM.

Tumor hypoxia: From basic knowledge to therapeutic implications.

Diminished oxygen availability, termed hypoxia, within solid tumors is one of the most common characteristics of cancer. Hypoxia shapes the landscape of the tumor microenvironment (TME) into a pro-tumorigenic and pro-metastatic niche through arrays of pathological alterations such as abnormal vasculature, altered metabolism, immune-suppressive phenotype, etc. In addition, emerging evidence suggests that limited efficacy or the development of resistance towards antitumor therapy may be largely due to the hypoxic TME. This review will focus on summarizing the knowledge about the molecular machinery that mediates the hypoxic cellular responses and adaptations, as well as highlighting the effects and consequences of hypoxia, especially for angiogenesis regulation, cellular metabolism alteration, and immunosuppressive response within the TME. We also outline the current advances in novel therapeutic implications through targeting hypoxia in TME. A deep understanding of the basics and the role of hypoxia in the tumor will help develop better therapeutic avenues in cancer treatment.

Exploring the immune microenvironment of osteosarcoma through T cell exhaustion-associated gene expression: a study on prognosis prediction.

Background: Osteosarcoma is a highly aggressive type of bone cancer with a poor prognosis. In the tumor immune microenvironment, T-cell exhaustion can occur due to various factors, leading to reduced tumor-killing ability. The purpose of this study was to construct a prognostic model based on T-cell exhaustion-associated genes in osteosarcoma. Methods: Patient data for osteosarcoma were retrieved from the TARGET and GEO databases. Consensus clustering was employed to identify two novel molecular subgroups. The dissimilarities in the tumor immune microenvironment between these subgroups were evaluated using the "xCell" algorithm. GO and KEGG analyses were conducted to elucidate the underlying mechanisms of gene expression. Predictive risk models were constructed using the least absolute shrinkage and selection operator algorithm and Cox regression analysis. To validate the prognostic significance of the risk gene expression model at the protein level, immunohistochemistry assays were performed on osteosarcoma patient samples. Subsequently, functional analysis of the key risk gene was carried out through in vitro experimentation. Results: Four gene expression signatures (PLEKHO2, GBP2, MPP1, and VSIG4) linked to osteosarcoma prognosis were identified within the TARGET-osteosarcoma cohort, categorizing patients into two subgroups. The resulting prognostic model showed strong predictive capability, with area under the receiver operating characteristic curve (AUC) values of 0.728/0.740, 0.781/0.658, and 0.788/0.642 for 1, 3, and 5-year survival in both training and validation datasets. Notably, patients in the low-risk group had significantly higher stromal, immune, and ESTIMATE scores compared to high-risk counterparts. Additionally, a nomogram was developed, exhibiting high accuracy in predicting the survival outcome of osteosarcoma patients. Immunohistochemistry, Kaplan-Meier, and time-dependent AUC analyses consistently supported the prognostic value of the risk model within our osteosarcoma patient cohort. In vitro experiments provided additional validation by demonstrating that the downregulation of GBP2 promoted the proliferation, migration, and invasion of osteosarcoma cells while inhibiting apoptosis. Conclusion: The current study established a prognostic signature associated with TEX-related genes and elucidated the impact of the pivotal gene GBP2 on osteosarcoma cells via in vitro experiments. Consequently, it introduces a fresh outlook for clinical prognosis prediction and sets the groundwork for targeted therapy investigations in osteosarcoma.

Differential Diagnosis of Mimicking Tumor Discs Using Coronal Magnetic Resonance Imaging of Three-Dimensional Fast-Field Echo with Water-Selective Excitation: A Single Center Retrospective Study.

OBJECTIVE: As disc fragment completely loses contact with the parent disc and can migrate in all directions of the epidural space, making it appear similar to schwannoma, it is fairly difficult to make a definitive diagnosis for mimicking tumor discs. The aim of this research is to differentially diagnose mimicking tumor discs and schwannomas using coronal magnetic resonance imaging (MRI) of three-dimensional fast-field echo with water-selective excitation (CMRI). METHODS: Among 76 patients (38 men and 38 women; mean age, 52.88 ± 15.80 [range, 18-78 years]) who were retrospectively examined in this study, 38 were primarily diagnosed with schwannomas and pathologically diagnosed with mimicking tumor discs after surgery, and 38 were primarily diagnosed with neurogenic tumors and pathologically diagnosed with schwannomas after surgery. Open surgery was performed in all the patients between March 2016 and April 2020. The preliminary diagnosis of all patients was considered an intraspinal tumor based on conventional two-dimensional MRI sequences. After open surgery, the final diagnosis was confirmed to mimic a tumor disc or schwannoma based on postoperative pathology reports. The sensitivity, specificity, and reliability of CMRI and conventional MRI for identifying mimicking tumor discs and schwannomas were compared. Chi-square and McNemar tests were used for statistical analyses. RESULTS: Symptoms were considerably relieved in all the patients after surgery. Seven patients had grade 1 extensor digitorum longus, triceps surae, or quadriceps femoris muscle strength prior to surgery. No nerve root injury was observed in any of the patients. CMRI showed significantly higher sensitivity (94.74%) and specificity (94.74%) than conventional MRI (71.05% and 92.11%, respectively; p = 0.012 < 0.05, and p = 1 > 0.05, respectively) for differential identification between mimicking tumor discs and schwannomas. Moreover, CMRI showed a higher reliability (kappa value = 0.787) than conventional MRI (kappa value = 0.374). CONCLUSIONS: CMRI is a better non-invasive technology for the identification of intraspinal lesions, especially for differentiating between mimicking tumor discs and schwannomas.

Histone H3 proline 16 hydroxylation regulates mammalian gene expression.

Histone post-translational modifications (PTMs) are important for regulating various DNA-templated processes. Here, we report the existence of a histone PTM in mammalian cells, namely histone H3 with hydroxylation of proline at residue 16 (H3P16oh), which is catalyzed by the proline hydroxylase EGLN2. We show that H3P16oh enhances direct binding of KDM5A to its substrate, histone H3 with trimethylation at the fourth lysine residue (H3K4me3), resulting in enhanced chromatin recruitment of KDM5A and a corresponding decrease of H3K4me3 at target genes. Genome- and transcriptome-wide analyses show that the EGLN2-KDM5A axis regulates target gene expression in mammalian cells. Specifically, our data demonstrate repression of the WNT pathway negative regulator DKK1 through the EGLN2-H3P16oh-KDM5A pathway to promote WNT/ß-catenin signaling in triple-negative breast cancer (TNBC). This study characterizes a regulatory mark in the histone code and reveals a role for H3P16oh in regulating mammalian gene expression.

Hsa_circ_0008870 suppresses bone formation of growth plate through inhibition of miR-185-3p/ MAPK1 axis in idiopathic short stature.

Idiopathic short stature (ISS) is the most common clinical cause of the short stature with an unclear aetiology and a lack of effective treatment. Circular RNAs have been shown to play a significant regulatory role through various signal transduction pathways in a variety of diseases in recent years. However, the role of circular RNAs on ISS is not yet well-understood and requires a special attention. The differentially expressed circular RNAs were screened by microarray chip analysis, and RT-qPCR was used to verify the expression of hsa_circ_0008870 in ISS patients. Subsequently, in vitro and in vivo experiments were conducted to determine the biological functions of hsa_circ_0008870 in ISS. The authors first confirmed that hsa_ circ_0008870 was downregulated in ISS children. Meanwhile, we also observed that the downregulated hsa_circ _0008870 significantly inhibited chondrocyte proliferation and endochondral ossification in vivo and in vitro. Mechanistically, hsa_circ_0008870 regulates MAPK1 expression by sponge miR-185-3p. This mechanism of action was further verified through rescue experiments. Finally, the authors revealed that the silencing of hsa_circ_0008870 induces low expression of MAPK1 by impairing the sponge action of miR-185-3p, thereby inhibiting chondrocyte proliferation, hypertrophy, and endochondral ossification, which results in a short stature phenotype. In addition to these, we also observed an interesting phenomenon that upregulated of miR-185-3p can in turn inhibit the expression of hsa_circ_0008870 in chondrocytes. This suggests that hsa_circ_0008870 could potentially serve as a therapeutic target for the treatment of ISS.

USP13 promotes deubiquitination of ZHX2 and tumorigenesis in kidney cancer.

Clear cell renal cell carcinoma (ccRCC) is characterized by the loss of tumor suppressor Von Hippel Lindau (VHL) function. VHL is the component of an E3 ligase complex that promotes the ubiquitination and degradation of hypoxia inducible factor α (HIF-α) (including HIF1α and HIF2α) and Zinc Fingers And Homeoboxes 2 (ZHX2). Our recent research showed that ZHX2 contributed to ccRCC tumorigenesis in a HIF-independent manner. However, it is still unknown whether ZHX2 could be modified through deubiquitination even in the absence of pVHL. Here, we performed a deubiquitinase (DUB) complementary DNA (cDNA) library binding screen and identified USP13 as a DUB that bound ZHX2 and promoted ZHX2 deubiquitination. As a result, USP13 promoted ZHX2 protein stability in an enzymatically dependent manner, and depletion of USP13 led to ZHX2 down-regulation in ccRCC. Functionally, USP13 depletion led to decreased cell proliferation measured by two-dimensional (2D) colony formation and three-dimensional (3D) anchorage-independent growth. Furthermore, USP13 was essential for ccRCC tumor growth in vivo, and the effect was partially mediated by its regulation on ZHX2. Our findings support that USP13 may be a key effector in ccRCC tumorigenesis.

Blockade of STAT3/IL-4 overcomes EGFR T790M-cis-L792F-induced resistance to osimertinib via suppressing M2 macrophages polarization.

BACKGROUND: The mechanism of missense alteration at EGFR L792F in patients with non-small cell lung cancer resistant to osimertinib has not been sufficiently clarified. We aimed to explore the critical molecular events and coping strategies in osimertinib resistance due to acquired L792F mutation. METHODS: Circulating tumor DNA-based sequencing data of 1153 patients with osimertinib resistance were collected to illustrate the prevalence of EGFR L792F mutation. Sensitivity to osimertinib was tested with constructed EGFR 19Del/T790M-cis-L792F cell lines in vitro and in vivo. The correlation and linked pathways between M2 macrophage polarization and EGFR L792Fcis-induced osimertinib resistance were investigated. Possible interventions to suppress osimertinib resistance by targeting IL-4 or STAT3 were explored. FINDINGS: The concomitant EGFR L792F was identified as an independent mutation following the acquisition of T790M after osimertinib resistance, in that 5 of the 946 patients with osimertinib resistance harbored EGFR T790M-cis-L792F mutation. Transfected EGFR 19Del/T790M-cis-L792F in cell lines had decreased sensitivity to osimertinib and enhanced infiltrating macrophage with M2 polarization. Silico analyses confirmed the role of M2 polarization in osimertinib resistance induced by EGFR T790M-cis-L792F mutation. EGFR T790M-cis-L792F mutation upregulated phosphorylation of STAT3 Tyr705 and promoted its specific binding to IL4 promoter, enhancing IL-4 expression and secretion and inducing macrophage M2 polarization. Furthermore, blockade of STAT3/IL-4 (SH-4-54 or dupilumab) suppressed macrophage M2 polarization and regressed tumor sensitivity to osimertinib. INTERPRETATION: Our results proved that targeting EGFR T790M-cis-L792F/STAT3 Tyr705/IL-4 pathway could be a potential strategy to suppress osimertinib resistance in NSCLC. FUNDING: This work was supported by the National Natural Science Foundation of China (81871889, 82072586, 81902910), Beijing Natural Science Foundation (7212084, 7214249), the China National Natural Science Foundation Key Program (81630071), the National Key Research and Development Project (2019YFC1315704), CAMS Innovation Fund for Medical Sciences (CIFMS 2021-1-I2M-012), Aiyou Foundation (KY201701) and CAMS Key Laboratory of translational research on lung cancer (2018PT31035).

Circulating Exosomal circRNA_0063476 Impairs Expression of Markers of Bone Growth Via the miR-518c-3p/DDX6 Axis in ISS.

OBJECTIVES: Idiopathic short stature (ISS), a disorder of unknown cause, accounts for approximately 80% of the clinical diagnoses of children with short stature. Exosomal circular RNA in plasma has been implicated in various disease processes. However, the role of exosome-derived circRNA in ISS has not been elucidated yet. METHODS: Plasma exosomes of ISS and normal children were cocultured with human chondrocytes. Microarray analysis and RT-PCR identified the differential expression of circRNA in exosomes between ISS and normal children. Hsa_circ_0063476 was upregulated or downregulated in human chondrocytes. Subsequently, overexpression rats of hsa_circ_0063476 was constructed via adenoviral vector to further validate the role of hsa_circ_0063476 on longitudinal bone growth via in vivo experiment. RESULTS: The plasma exosome of ISS children suppressed the expression of markers of chondrocyte hypertrophy and endochondral ossification. Subsequently, upregulation of hsa_circ_0063476 in ISS exosome was identified. In vitro experiments demonstrated that chondrocyte proliferation, cell cycle and endochondral ossification were suppressed, and apoptosis was increased following hsa_circ_0063476 overexpression in human chondrocytes. Conversely, silencing hsa_circ_0063476 in human chondrocytes can show opposite outcomes. Our study further revealed hsa_circ_0063476 overexpression in vitro can enhance chondrocyte apoptosis and inhibit the expression of markers of chondrocyte proliferation and endochondral ossification via miR-518c-3p/DDX6 axis. Additionally, the rats with hsa_circ_0063476 overexpression showed a short stature phenotype. CONCLUSIONS: The authors identified a novel pathogenesis in ISS that exosome-derived hsa_circ_0063476 retards the expression of markers of endochondral ossification and impairs longitudinal bone growth via miR-518c-3p/DDX6 axis, which may provide a unique therapeutic avenue for ISS.

An oncogenic JMJD6-DGAT1 axis tunes the epigenetic regulation of lipid droplet formation in clear cell renal cell carcinoma.

Characterized by intracellular lipid droplet accumulation, clear cell renal cell carcinoma (ccRCC) is resistant to cytotoxic chemotherapy and is a lethal disease. Through an unbiased siRNA screen of 2-oxoglutarate (2-OG)-dependent enzymes, which play a critical role in tumorigenesis, we identified Jumonji domain-containing 6 (JMJD6) as an essential gene for ccRCC tumor development. The downregulation of JMJD6 abolished ccRCC colony formation in vitro and inhibited orthotopic tumor growth in vivo. Integrated ChIP-seq and RNA-seq analyses uncovered diacylglycerol O-acyltransferase 1 (DGAT1) as a critical JMJD6 effector. Mechanistically, JMJD6 interacted with RBM39 and co-occupied DGAT1 gene promoter with H3K4me3 to induce DGAT1 expression. JMJD6 silencing reduced DGAT1, leading to decreased lipid droplet formation and tumorigenesis. The pharmacological inhibition (or depletion) of DGAT1 inhibited lipid droplet formation in vitro and ccRCC tumorigenesis in vivo. Thus, the JMJD6-DGAT1 axis represents a potential new therapeutic target for ccRCC.

LCN2 is a new diagnostic biomarker and potential therapeutic target in idiopathic short stature.

Idiopathic short stature (ISS) is the most common paediatric endocrine disease. However, the underlying pathology of ISS remains unclear. Currently, there are no effective diagnostic markers or therapeutic strategies available for ISS. In this study, we aimed to identify differential plasma protein expression and novel biomarkers in patients with ISS, and elucidate the biological functions of candidate proteins in ISS pathogenesis. Four specimen pairs from four ISS children and age-/sex-matched control individuals were subjected to proteomics analysis, and 340 samples of children with a mean age 9.73 ± 0.24 years were utilized to further verify the differentially expressed proteins by enzyme-linked immunosorbent assay (ELISA). The receiver-operating characteristic (ROC) curve and the area under the ROC curve (AUC) were plotted. A total of 2040 proteins were identified, of which 84 were differentially expressed. In vitro and in vivo experiments confirmed the biological functions of these candidate proteins. LCN2 overexpression in ISS was verified using ELISA. Meanwhile, LCN2 showed high sensitivity and specificity in discriminating children with ISS from those with growth hormone deficiency, precocious puberty and normal control individuals. The upregulated expression of LCN2 not only suppressed food intake but also impaired chondrocyte proliferation and bone growth in chondrocytes and rats. As a result, the rats presented a short-stature phenotype. Subsequently, we found that bone growth inhibition recovered after LCN2 overexpression was stopped in immature rats. To our knowledge, this is the first study to report that LCN2 may be a significant target for ISS diagnosis and treatment.

MiR-224 promotes lymphatic metastasis by targeting ANGPTL1 in non-small-cell lung carcinoma.

BACKGROUND: MicroRNAs can regulate tumor metastasis either as oncomiRs or suppressor miRNAs. Here, we investigated the role of microRNA 224 (miR-224) in lymphatic metastasis of non-small-cell lung cancer (NSCLC). METHODS: The expression of miR-224 was demonstrated by a validation cohort of 156 lung cancer patients (77 cases with lymphatic metastasis) by quantitative polymerase chain reaction (qPCR). In vitro and in vivo experiments were performed to study the malignant phenotype after upregulation and inhibition of miR-224 expression. Furthermore, the direct target genes of miR-224 were determined by a luciferase reporter assay. RESULTS: First, miR-224 was identified as a highly expressed miRNA in tumor tissues with lymphatic metastasis, with an area under the curve (AUC) of 0.57 as determined by qPCR analysis of a validation cohort of 156 lung cancer patients. Then, in vitro and in vivo experiments indicated that forced expression of miR-224 in H1299 cells promoted not only cell viability, plate colony formation, migration and invasion in vitro but also tumor growth and lung metastasis in vivo. Consistently, inhibition of miR-224 suppressed malignant characteristics both in vitro and in vivo. Moreover, molecular mechanistic research suggested that miR-224 enhanced NSCLC by directly targeting the tumor suppressor angiopoietin-like protein (ANGPTL). CONCLUSIONS: Overall, the collective findings demonstrate that miR-224 is a potential biomarker for the prediction of lymphatic metastasis of NSCLC.

EZH2 noncanonically binds cMyc and p300 through a cryptic transactivation domain to mediate gene activation and promote oncogenesis.

Canonically, EZH2 serves as the catalytic subunit of PRC2, which mediates H3K27me3 deposition and transcriptional repression. Here, we report that in acute leukaemias, EZH2 has additional noncanonical functions by binding cMyc at non-PRC2 targets and uses a hidden transactivation domain (TAD) for (co)activator recruitment and gene activation. Both canonical (EZH2-PRC2) and noncanonical (EZH2-TAD-cMyc-coactivators) activities of EZH2 promote oncogenesis, which explains the slow and ineffective antitumour effect of inhibitors of the catalytic function of EZH2. To suppress the multifaceted activities of EZH2, we used proteolysis-targeting chimera (PROTAC) to develop a degrader, MS177, which achieved effective, on-target depletion of EZH2 and interacting partners (that is, both canonical EZH2-PRC2 and noncanonical EZH2-cMyc complexes). Compared with inhibitors of the enzymatic function of EZH2, MS177 is fast-acting and more potent in suppressing cancer growth. This study reveals noncanonical oncogenic roles of EZH2, reports a PROTAC for targeting the multifaceted tumorigenic functions of EZH2 and presents an attractive strategy for treating EZH2-dependent cancers.

A Novel Diagnostic Predictive Model for Idiopathic Short Stature in Children.

Objective: Idiopathic short stature (ISS), an endocrine-related disease, is difficult to diagnose. Previous studies have shown that many children with some inflammation-related diseases often have short stature, but whether inflammation is the underlying mechanism of ISS has not been studied. Here, we attempt to explore the role of inflammation in the occurrence and development of ISS and to demonstrate an available clinical diagnostic model of ISS. Methods: Frozen serum samples were collected from ISS patients (n = 4) and control individuals (n = 4). Isobaric tags for relative and absolute quantitation (iTRAQ) combined with LC-MS/MS analysis were applied to quantitative proteomics analysis. To assess clusters of potentially interacting proteins, functional enrichment (GO and KEGG) and protein-protein interaction network analyses were performed, and the crucial proteins were detected by Molecular Complex Detection (MCODE). Furthermore, serum levels of two selected proteins were measured by ELISA between ISS patients (n = 80) and controls (n = 80). In addition, experiments in vitro were used to further explore the effects of crucial proteins on endochondral ossification. Results: A total of 437 proteins were quantified, and 84 DEPs (60 upregulated and 24 downregulated) were identified between patients with ISS and controls. Functional enrichment analysis showed that the DEPs were primarily enriched in blood microparticle, acute inflammatory response, protein activation cascade, collagen-containing extracellular matrix, platelet degranulation, etc. According to the results of top 10 fold change DEPs and MCODE analysis, C1QA and C1QB were selected to further experiment. The expression levels of C1QA and C1QB were validated in serum samples. Based on the logistic regression analysis and ROC curve analysis, we constructed a novel diagnostic model by serum levels of C1QA and C1QB with a specificity of 91.2% and a sensitivity of 75% (AUC = 0.900, p <0.001). Finally, the western blotting analysis confirmed the expression levels of OCN, OPN, RUNX2, and Collagen X were downregulated in chondrocytes, and the outcome of Collagen II was upregulated. Conclusion: Our study is the first to demonstrate the significant role of inflammation in the development of ISS. In addition, we identify C1QA and C1QB as novel serum biomarkers for the diagnosis of ISS.

Downregulation of miR-1-3p expression inhibits the hypertrophy and mineralization of chondrocytes in DDH.

BACKGROUND: Developmental dysplasia of the hip (DDH) is a highly prevalent hip disease among children. However, its pathogenesis remains unclear. MicroRNAs (miRNA) are important regulators of cartilage development. In a previous study, high-throughput miRNA sequencing of tissue samples from an animal model of DDH showed a low level of miR-1-3p in the cartilage of the acetabular roof (ARC), but its role in DDH pathogenesis was not addressed. Therefore, our aim here was to investigate the effects of miR-1-3p in the ARC. METHODS: The diagnosis of acetabular dysplasia was confirmed with X-ray examination, while imaging and HE staining were conducted to further evaluate the ARC thickness in each animal model. FISH was employed to verify miR-1-3p expression in the ARC and chondrocytes. The miR-1-3p target genes were predicted by a bioinformatics database. A dual-luciferase reporter assay was used to confirm the targeting relationship between miR-1-3p and SOX9. The gene expression of miR-1-3p, SOX9, RUNX2 and collagen type X was evaluated by qPCR analysis. The protein expression of SOX9, RUNX2 and collagen type X was detected by western blot analysis. The levels of SOX9, RUNX2, and collagen type X in the ARC were further assessed via immunohistochemistry analysis. Finally, Alizarin Red S staining was used to observe the mineralized nodules produced by the chondrocytes. RESULTS: We observed low expression of miR-1-3p in the ARC of animals with DDH. SOX9 is a miR-1-3p target gene. Using miR-1-3p silencing technology in vitro, we demonstrated significantly reduced chondrocyte-generated mineralized nodules compared to those of the control. We also confirmed that with miR-1-3p silencing, SOX9 expression was upregulated, whereas the expression of genes associated with endochondral osteogenesis such as RUNX2 and collagen type X was downregulated. To confirm the involvement of miR-1-3p silencing in abnormal ossification through SOX9, we also performed a rescue experiment in which SOX9 silencing restored the low expression of RUNX2 and collagen type X produced by downregulated miR-1-3p expression. Finally, the elevated SOX9 levels and reduced RUNX2 and collagen type X levels in the ARC of rabbits with DDH were also verified using immunohistochemistry, RT-PCR, and western blots. CONCLUSION: The relatively low expression of miR-1-3p in the ARC may be the cause of abnormal endochondral ossification in the acetabular roof of animals with DDH.

GINS1 Induced Sorafenib Resistance by Promoting Cancer Stem Properties in Human Hepatocellular Cancer Cells.

Hepatocellular carcinoma (HCC) is characterized by a high rate of incidence and recurrence, and resistance to chemotherapy may aggravate the poor prognosis of HCC patients. Sorafenib resistance is a conundrum to the treatment of advanced/recurrent HCC. Therefore, studies on the molecular pathogenesis of HCC and the resistance to sorafenib are of great interest. Here, we report that GINS1 was highly expressed in HCC tumors, associated with tumor grades, and predicted poor patient survival using Gene Expression Omnibus (GEO) databases exploration. Cell cycle, cell proliferation assay and in vivo xenograft mouse model indicated that knocking down GINS1 induced in G1/S phase cell cycle arrest and decreased tumor cells proliferation in vitro and in vivo. Spheroid formation assay results showed that GINS1 promoted the stem cell activity of HCC tumor cells. Furthermore, GEO database (GSE17112) analysis showed that HRAS oncogenic gene set was enriched in GINS1 high-expressed cancer cells, and quantitative real-time PCR, and Western blot results proved that GINS1 enhanced HCC progression through regulating HRAS signaling pathway. Moreover, knocking down endogenous GINS1 with shGINS1 increased the sensitivity of HCC cells to sorafenib, and restoring HRAS or stem associated pathway partly recovered the sorafenib resistance. Overall, the collective findings highlight GINS1 functions in hepatocarcinogenesis and sorafenib resistance, and indicate its potential use of GINS1 in drug-resistant HCC.

Circular RNA circANAPC2 mediates the impairment of endochondral ossification by miR-874-3p/SMAD3 signalling pathway in idiopathic short stature.

Idiopathic short stature (ISS) is a main reason for low height among children. Its exact aetiology remains unclear. Recent findings have suggested that the aberrant expression of circRNAs in peripheral blood samples is associated with many diseases. However, to date, the role of aberrant circRNA expression in mediating ISS pathogenesis remains largely unknown. The up-regulated circANAPC2 was identified by circRNA microarray analysis and RT-qPCR. Overexpression of circANAPC2 inhibited the proliferation of human chondrocytes, and cell cycle was arrested in G1 phase. The expressions of collagen type X, RUNX2, OCN and OPN were significantly down-regulated following circANAPC2 overexpression. Moreover, Von Kossa staining intensity and alkaline phosphatase activity were also decreased. Luciferase reporter assay results showed that circANAPC2 could be targeted by miR-874-3p. CircANAPC2 overexpression in human chondrocytes inhibits the expression of miR-874-3p. The co-localization of circANAPC2 and miR-874-3p was confirmed in both human chondrocytes and murine femoral growth plates via in situ hybridization. The rescue experiment demonstrated that the high expression of miR-874-3p overexpression antagonized the suppression of endochondral ossification, hypertrophy and chondrocyte growth caused by circANAPC2 overexpression. A high-throughput screening of mRNA expression and RT-qPCR verified SMAD3 demonstrated the highest different expressions following overcircANAPC2. Luciferase reporter assay results indicated that miR-874-3p could be targeted by Smad3, thus down-regulating the expression of Smad3. Subsequent rescue experiments of SMAD3 further confirmed that circANAPC2 suppresses endochondral ossification, hypertrophy and chondrocyte growth through miR-874-3p/Smad3 axis. The present study provides evidence that circANAPC2 can serve as a promising target for ISS treatment.