METTL14-mediated epitranscriptome modification of MN1 mRNA promote tumorigenicity and all-trans-retinoic acid resistance in osteosarcoma.

BACKGROUND: Osteosarcoma (OS) is the most common primary malignant bone tumor in adolescents. The molecular mechanism behind OS progression and metastasis remains poorly understood, which limits the effectiveness of current therapies. RNA N6-methyladenosine (m6A) modification plays a critical role in influencing RNA fate. However, the biological significance of m6A modification and its potential regulatory mechanisms in the development of OS remain unclear. METHODS: Liquid chromatography-tandem mass spectrometry (LC-MS/MS), dot blotting, and colorimetric ELISA were used to detect m6A levels. Western blotting, quantitative real-time PCR (RT-qPCR) and immunohistochemistry (IHC) were used to investigate METTL14 expression levels. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and transcriptomic RNA sequencing (RNA-seq) were used to screen the target genes of METTL14. RNA pull-down and RNA immunoprecipitation (RIP) assays were conducted to explore the specific binding of target genes and relevant m6A "readers". RNA stability and polysome analysis assays were used to detect the half-lives and translation efficiencies of the downstream genes of METTL14. IHC and clinical data were applied to explore the clinical correlations of METTL14 and its downstream target genes with the prognosis of OS. FINDINGS: We observed the abundance of m6A modifications in OS and revealed that METTL14 plays an oncogenic role in facilitating OS progression. MeRIP-seq and RNA-seq revealed that MN1 is a downstream gene of METTL14. MN1 contributes to tumor progression and all-trans-retinoic acid (ATRA) chemotherapy resistance in OS. Mechanistically, MN1 is methylated by METTL14, specifically in the coding sequence (CDS) regions, and this modification is recognized by the specific m6A reader insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) to prevent MN1 mRNA degradation and promote it translation efficiency. IHC showed that MN1 expression was positively correlated with METTL14 and IGF2BP2 expression in OS tissues. The METTL14-IGF2BP2-MN1 panel demonstrated more promising prognostic value for OS patients than any of these molecules individually. INTERPRETATION: Our study revealed that METTL14 contributes to OS progression and ATRA resistance as an m6A RNA methylase by regulating the stability and translation efficiency of MN1 and thus provides both an underlying biomarker panel for prognosis prediction in OS patients. FUNDING: This work was supported by the National Natural Science Foundation of China (Grants 81972510 and 81772864).

Cordycepin augments the chemosensitivity of osteosarcoma to cisplatin by activating AMPK and suppressing the AKT signaling pathway.

BACKGROUND: Osteosarcoma is the most common primary bone tumor in children and adolescents. However, some patients with osteosarcoma develop resistance to chemotherapy, leading to a poor clinical prognosis. Hence, effective therapeutic agents that can improve the response to chemotherapy drugs to improve the prognosis of patients with osteosarcoma are urgently needed. Cordycepin has recently emerged as a promising antitumor drug candidate. This study aims to explore the effect of cordycepin in suppressing osteosarcoma in vivo and in vitro and the synergistic effect of cordycepin combined with cisplatin and to demonstrate the underlying molecular mechanism. METHODS: CCK-8 assay was performed to investigate the inhibition effect of cordycepin combined with cisplatin in osteosarcoma cell lines. The colony formation and invasion abilities were measured by colony formation assay and Transwell assay. Osteosarcoma cells apoptosis was detected by flow cytometry. Western blot analysis were used to detect the expression of cell apoptosis-related proteins and AMPK and AKT/mTOR signaling pathway-related proteins. Finally, we performed the in vivo animal model to further explore whether cordycepin and cisplatin exert synergistic antitumor effects. RESULTS: Notably, we found that treatment with cordycepin inhibited cell proliferation, invasion, and induced apoptosis in osteosarcoma cells in vitro and in vivo. Moreover, the combination of cordycepin and cisplatin led to marked inhibition of osteosarcoma cell proliferation and invasion and promoted osteosarcoma cell apoptosis in vitro and in vivo. Mechanistically, we demonstrated that cordycepin enhanced the sensitivity of osteosarcoma cells to cisplatin by activating AMPK and inhibiting the AKT/mTOR signaling pathway. CONCLUSIONS: In brief, this study provides comprehensive evidence that cordycepin inhibits osteosarcoma cell growth and invasion and induces osteosarcoma cell apoptosis by activating AMPK and inhibiting the AKT/mTOR signaling pathway and enhances the sensitivity of osteosarcoma cells to cisplatin, suggesting that cordycepin is a promising treatment for osteosarcoma.

Epigenetic inactivation of HOXD10 is associated with human colon cancer via inhibiting the RHOC/AKT/MAPK signaling pathway.

BACKGROUND: To examine the influence of HOXD10 on the metabolism and growth of colon carcinoma cells by suppressing the RHOC/AKT/MAPK pathway. METHODS: Thirty-seven paired colon cancer and its adjacent samples from The Cancer Genome Atlas (TCGA) were analyzed. Chip Analysis Methylation Pipeline (ChAMP) analysis was employed for differential methylated points (DMPs) and the differential methylation regions (DMRs) screening. The HOXD10 mRNA expression and DNA methylation levels were detected by RT-PCR. The Cell proliferation, migration, invasion and apoptosis were respectively measured by MTT assay, transwell assay, wound healing assay and flow cytometry assay in carcinoma cell lines after treated with 5-aza-2'-deoxycytidine (5-Aza-dC) or transfected with HOXD10-expressing plasmid. The expression of HOXD10 and RHOC was revealed by immunohistochemistry in disparate differentiation colon carcinoma tissues, and the dephosphorylation of AKT and MAPK pathways were detected by RT-PCR and western blot. RESULTS: The bioinformatics analysis demonstrated that HOXD10 was hypermethylated and low-expressed in colorectal cancer tissues. The detection of RT-PCR indicated the similar results in colorectal cancer cell lines and tissues. The induction of demethylation was recovered by treatment with 5-Aza-dC and the HOXD10 in colorectal cancer cell lines was re-expressed by transfection with a HOXD10 expression vector. The demethylation or overexpression of HOXD10 suppressed proliferation, migration, invasion and promoted apoptosis in colorectal cancer cells. HXOD10 suppressed the tumor growth and detected an opposite trend of protein RHOC. AKT and MAPK pathways were notably inactivated after the dephosphorylation due to the overexpression of HOXD10. CONCLUSIONS: HOXD10 was suppressed in colon adenocarcinoma cells, which down-regulated RHOC/AKT/MAPK pathway to enhance colon cancer cells apoptosis and constrain the proliferation, migration and invasion.

Involvement of NMDA Receptors, Nitric Oxide, and GABA in Rostral Ventrolateral Medulla in Acute Ethanol-Induced Cardiovascular Responses in Rats.

BACKGROUND: Consumption of ethanol (EtOH) (alcohol) has many effects on physiological functions, particularly those in the central nervous system (CNS) and cardiovascular system. Acute excessive intake of EtOH (alcohol intoxication) may cause hypotension and tachycardia. In this study, we examined the mechanistic involvement of glutamatergic N-methyl-d-aspartate (NMDA) receptors, nitric oxide (NO), and γ-aminobutyric acid (GABA) pathways in the CNS in acute EtOH-induced cardiovascular effects. METHODS: EtOH was administered by intraperitoneal (IP) injection in Sprague-Dawley rats. The blood pressure (BP) and heart rate (HR) were measured in conscious and in urethane-anesthetized rats. Inhibitors were applied by intracerebroventricular (ICV) injection or by microinjection into rostral ventrolateral medulla (RVLM). Microdialysis was used to determine the level of glutamate, NO, and GABA in the RVLM. RESULTS: IP injection of EtOH (3.2 g/kg) caused a significant decrease in BP in conscious and anesthetized rats and a late increase in HR in conscious rats. The cardiovascular effects of EtOH were significantly attenuated by ICV or by RVLM post treatment with ketamine (an NMDA receptor antagonist), N5-(nitroamidino)-L-2,5-diaminopentanoic acid (L-NNA; a NO synthase inhibitor), or bicuculline (a GABA receptor antagonist). EtOH caused an increase in the level of glutamate, NO, and GABA in the RVLM during the hypotensive responses. RVLM posttreatment with ketamine blocked the increase in NO and GABA levels; post treatment with L-NNA blocked the increase in GABA level. CONCLUSIONS: Our results indicate that EtOH augmentation of glutamatergic NMDA receptors/NO/GABA pathways in the RVLM may participate in the hypotensive effects induced by acute administration of EtOH.

Fluorescence Quenching Investigation of Methyl Red Adsorption on Aluminum-Based Metal-Organic Frameworks.

The adsorption of methyl red (MR) isomers (ortho, meta, and para) on metal-organic frameworks (MOFs) was investigated by using a fluorescence quenching technique. All three MR isomers were found to quench the fluorescence of MOFs effectively. Nonlinear fluorescence quenching trends were observed in Stern-Volmer plots. A modified nonlinear Stern-Volmer equation with the concepts of multiple adsorption sites, adsorption strength, and quencher accessibility was successfully adopted to fit the fluorescence quenching data. The fitted parameters were correlated with the structural properties of MRs and MOFs. The order of quenching efficiency was found to be m-MR > p-MR > o-MR for all MOFs. This indicates that MR molecules not only adsorb via carboxylate-metal bonding but also adsorb through π-π interactions between the aromatic rings of MR and linker molecules in MOFs. The position of the carboxylate group in MRs and the structure of the linkers in MOFs are the key factors affecting the fluorescence quenching efficiency.

[Fast automated finite element mesh generation of residual lower limb for clinical application].

Objective. To develop a model of fast automated mesh generation of residual lower limb for clinical application. Method. Actual geometry of the bone was represented as a standard pretreated model of bone, which was auto-located by employing an affine scaling transformation. Hexahedral or wedge elements were constructed by joining quadrilateral or triangular meshes generated by graph partitioning method and transport-mapping method from successive planar cross sections. Result. A model of fast automated mesh generation of residual lower limb was developed, which can be applied to different amputee and ensure the geometric integrality of bone. Auto-location of the bone was carried out. The mesh refinement can be controlled to adapt to different computational efficiency. The generated elements are hexahedral or wedge elements, both of which are more precise than tetrahedral element. Conclusion. The present method could be applied in finite element analysis method for prosthetic socket designing. Radioactive damage from those techniques such as CT or MRI to the amputee and the difficulty in image processing could be avoided.