Реферат
SUMMARY: Overexpression of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in various tumor tissues and cell lines was found to promote tumor cell proliferation, migration, and invasion. However, the role of MALAT1 in gastric cancer (GC) is still unclear. We aimed to investigate the correlation between long-chain non-coding RNAs (lncRNAs), MALAT1, MicroRNAs (miRNA) and vascular endothelial growth factor A (VEGFA) in gastric cancer and to disclose underlying mechanism. The correlation between MALAT1 levels and clinical features was analyzed by bioinformatics data and human samples. The expression of MALAT1 was down regulated in AGS cells to detect the cell proliferation, migration, and invasion characteristics, as well as the effects on signal pathways. Furthermore, we validated the role of MALAT1/miR-330-3p axis in GC by dual luciferase reporter gene assays. Expression of MALAT1 was higher in cancer tissues than in para-cancerous tissues. The high MALAT1 level predicted malignancy and worse prognosis. Down-regulation of MALAT1 expression in AGS cells inhibited cell proliferation, migration, and invasion by targeting VEGFA. By dual luciferase reporter gene assay and miR-330-3p inhibitor treatment, we demonstrate that MALAT1 sponged miR-330-3p in GC, leading to VEGFA upregulation and activation of the mTOR signaling pathway. The MALAT1/miR-330-3p axis regulates VEGFA through the mTOR signaling pathway and promotes the growth and metastasis of gastric cancer.
Se descubrió que la sobreexpresión del transcrito 1 de adenocarcinoma de pulmón asociado a metástasis (MALAT1) en varios tejidos tumorales y líneas celulares promueve la proliferación, migración e invasión de células tumorales. Sin embargo, el papel de MALAT1 en el cáncer gástrico (CG) aún no está claro. Nuestro objetivo fue investigar la correlación entre los ARN no codificantes de cadena larga (lncRNA), MALAT1, los microARN (miARN) y el factor de crecimiento endotelial vascular A (VEGFA) en el cáncer gástrico y revelar el mecanismo subyacente. La correlación entre los niveles de MALAT1 y las características clínicas se analizó mediante datos bioinformáticos y muestras humanas. La expresión de MALAT1 se reguló negativamente en las células AGS para detectar las características de proliferación, migración e invasión celular, así como los efectos sobre las vías de señales. Además, validamos el papel del eje MALAT1/miR- 330-3p en GC mediante ensayos de genes indicadores de luciferasa dual. La expresión de MALAT1 fue mayor en tejidos cancerosos que en tejidos paracancerosos. El alto nivel de MALAT1 predijo malignidad y peor pronóstico. La regulación negativa de la expresión de MALAT1 en células AGS inhibió la proliferación, migración e invasión celular al apuntar a VEGFA. Mediante un ensayo de gen indicador de luciferasa dual y un tratamiento con inhibidor de miR-330-3p, demostramos que MALAT1 esponjaba miR-330-3p en GC, lo que lleva a la regulación positiva de VEGFA y la activación de la vía de señalización mTOR. El eje MALAT1/miR-330-3p regula VEGFA a través de la vía de señalización mTOR y promueve el crecimiento y la metástasis del cáncer gástrico.
Тема - темы
Humans , Stomach Neoplasms/genetics , Stomach Neoplasms/pathology , Vascular Endothelial Growth Factor A , TOR Serine-Threonine Kinases , RNA, Long Noncoding , RNA/genetics , Signal Transduction , Gene Expression Regulation, Neoplastic , Cell Movement , Blotting, Western , Apoptosis , Genes, Reporter , Cell Proliferation , Real-Time Polymerase Chain Reaction , Neoplasm InvasivenessРеферат
Photosynthesis in plants directly affects the synthesis and accumulation of organic matter, which directly influences crop yield. RNA-binding proteins (RBPs) are involved in the regulation of a variety of physiological functions in plants, while the functions of RBPs in photosynthesis have not been clearly elucidated. To investigate the effect of a glycine-rich RNA-binding protein (SlRBP1) in tomato on plant photosynthesis, a stably inherited SlRBP1 silenced plant in Alisa Craig was obtained by plant tissue culture using artificial small RNA interference. It turns out that the size of the tomato fruit was reduced and leaves significantly turned yellow. Chlorophyll(Chl) content measurement, Chl fluorescence imaging and chloroplast transmission electron microscopy revealed that the chloroplast morphology and structure of the leaves of tomato amiR-SlRBP1 silenced plants were disrupted, and the chlorophyll content was significantly reduced. Measurement of photosynthesis rate of wild-type and amiR-SlRBP1 silenced plants in the same period demonstrated that the photosynthetic rate of these plants was significantly reduced, and analysis of RNA-seq data indicated that silencing of SlRBP1 significantly reduced the expression of photosynthesis-related genes, such as PsaE, PsaL, and PsbY, and affected the yield of tomato fruits through photosynthesis.
Тема - темы
RNA , Solanum lycopersicum/genetics , Photosynthesis/genetics , Chlorophyll , RNA-Binding Proteins/geneticsРеферат
BACKGROUND@#The hepatitis B virus (HBV) vaccine has been efficiently used for decades. However, hepatocellular carcinoma caused by HBV is still prevalent globally. We previously reported that interferon (IFN)-induced tripartite motif-containing 25 (TRIM25) inhibited HBV replication by increasing the IFN expression, and this study aimed to further clarify the anti-HBV mechanism of TRIM25.@*METHODS@#The TRIM25-mediated degradation of hepatitis B virus X (HBx) protein was determined by detecting the expression of HBx in TRIM25-overexpressed or knocked-out HepG2 or HepG2-NTCP cells via Western blotting. Co-immunoprecipitation was performed to confirm the interaction between TRIM25 and HBx, and colocalization of TRIM25 and HBx was identified via immunofluorescence; HBV e-antigen and HBV surface antigen were qualified by using an enzyme-linked immunosorbent assay (ELISA) kit from Kehua Biotech. TRIM25 mRNA, pregenomic RNA (pgRNA), and HBV DNA were detected by quantitative real-time polymerase chain reaction. The retinoic acid-inducible gene I (RIG-I) and pgRNA interaction was verified by RNA-binding protein immunoprecipitation assay.@*RESULTS@#We found that TRIM25 promoted HBx degradation, and confirmed that TRIM25 could enhance the K90-site ubiquitination of HBx as well as promote HBx degradation by the proteasome pathway. Interestingly, apart from the Really Interesting New Gene (RING) domain, the SPRY domain of TRIM25 was also indispensable for HBx degradation. In addition, we found that the expression of TRIM25 increased the recognition of HBV pgRNA by interacting with RIG-I, which further increased the IFN production, and SPRY, but not the RING domain is critical in this process.@*CONCLUSIONS@#The study found that TRIM25 interacted with HBx and promoted HBx-K90-site ubiquitination, which led to HBx degradation. On the other hand, TRIM25 may function as an adaptor, which enhanced the recognition of pgRNA by RIG-I, thereby further promoting IFN production. Our study can contribute to a better understanding of host-virus interaction.
Тема - темы
Humans , Hepatitis B virus , DEAD Box Protein 58/metabolism , RNA , Liver Neoplasms , Virus Replication , Tripartite Motif Proteins/genetics , Transcription Factors , Ubiquitin-Protein Ligases/geneticsРеферат
BACKGROUND@#Angiogenesis is described as a complex process in which new microvessels sprout from endothelial cells of existing vasculature. This study aimed to determine whether long non-coding RNA (lncRNA) H19 induced the angiogenesis of gastric cancer (GC) and its possible mechanism.@*METHODS@#Gene expression level was determined by quantitative real-time polymerase chain reaction and western blotting. Cell counting kit-8, transwell, 5-Ethynyl-2'-deoxyuridine (EdU), colony formation assay, and human umbilical vein endothelial cells (HUVECs) angiogenesis assay as well as Matrigel plug assay were conducted to study the proliferation, migration, and angiogenesis of GC in vitro and in vivo . The binding protein of H19 was found by RNA pull-down and RNA Immunoprecipitation (RIP). High-throughput sequencing was performed and next Gene Ontology (GO) as well as Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was conducted to analyze the genes that are under H19 regulation. Methylated RIP (me-RIP) assay was used to investigate the sites and abundance among target mRNA. The transcription factor acted as upstream of H19 was determined through chromatin immunoprecipitation (ChIP) and luciferase assay.@*RESULTS@#In this study, we found that hypoxia-induced factor (HIF)-1α could bind to the promoter region of H19, leading to H19 overexpression. High expression of H19 was correlated with angiogenesis in GC, and H19 knocking down could inhibit cell proliferation, migration and angiogenesis. Mechanistically, the oncogenic role of H19 was achieved by binding with the N 6 -methyladenosine (m 6 A) reader YTH domain-containing family protein 1 (YTHDF1), which could recognize the m 6 A site on the 3'-untransated regions (3'-UTR) of scavenger receptor class B member 1 (SCARB1) mRNA, resulting in over-translation of SCARB1 and thus promoting the proliferation, migration, and angiogenesis of GC cells.@*CONCLUSION@#HIF-1α induced overexpression of H19 via binding with the promoter of H19, and H19 promoted GC cells proliferation, migration and angiogenesis through YTHDF1/SCARB1, which might be a beneficial target for antiangiogenic therapy for GC.
Тема - темы
Humans , Cell Line, Tumor , Cell Proliferation/genetics , Endothelial Cells/metabolism , Gene Expression Regulation , Gene Expression Regulation, Neoplastic/genetics , Hypoxia , MicroRNAs/genetics , RNA , RNA, Long Noncoding/metabolism , RNA-Binding Proteins/metabolism , Scavenger Receptors, Class B/metabolism , Stomach Neoplasms/geneticsРеферат
Lipid metabolism is a complex physiological process, which is closely related to nutrient regulation, hormone balance and endocrine function. It involves the interactions of multiple factors and signal transduction pathways. Lipid metabolism disorder is one of the main mechanisms to induce a variety of diseases, such as obesity, diabetes, non-alcoholic fatty liver disease, hepatitis, hepatocellular carcinoma and their complications. At present, more and more studies have found that the "dynamic modification" of N6-adenylate methylation (m6A) on RNA represents a new "post-transcriptional" regulation mode. m6A methylation modification can occur in mRNA, tRNA, ncRNA, etc. Its abnormal modification can regulate gene expression changes and alternative splicing events. Many latest references have reported that m6A RNA modification is involved in the epigenetic regulation of lipid metabolism disorder. Based on the major diseases induced by lipid metabolism disorders, we reviewed the regulatory roles of m6A modification in the occurrence and development of those diseases. These overall findings inform further in-depth investigations of the underlying molecular mechanisms regarding the pathogenesis of lipid metabolism disorders from the perspective of epigenetics, and provide reference for health prevention, molecular diagnosis and treatment of related diseases.
Тема - темы
Humans , Methylation , Epigenesis, Genetic , Lipid Metabolism/genetics , Lipid Metabolism Disorders/genetics , Liver Neoplasms , RNAРеферат
The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR associated proteins) system is an adaptive immune system of bacteria and archaea against phages, plasmids and other exogenous genetic materials. The system uses a special RNA (CRISPR RNA, crRNA) guided endonuclease to cut the exogenous genetic materials complementary to crRNA, thus blocking the infection of exogenous nucleic acid. According to the composition of the effector complex, CRISPR-Cas system can be divided into two categories: class 1 (including type Ⅰ, Ⅳ, and Ⅲ) and class 2 (including type Ⅱ, Ⅴ, and Ⅵ). Several CRISPR-Cas systems have been found to have very strong ability to specifically target RNA editing, such as type Ⅵ CRISPR-Cas13 system and type Ⅲ CRISPR-Cas7-11 system. Recently, several systems have been widely used in the field of RNA editing, making them a powerful tool for gene editing. Understanding the composition, structure, molecular mechanism and potential application of RNA-targeting CRISPR-Cas systems will facilitate the mechanistic research of this system and provide new ideas for developing gene editing tools.
Тема - темы
CRISPR-Cas Systems/genetics , RNA/genetics , Bacteria/genetics , Gene Editing , ArchaeaРеферат
There are a variety of post-transcriptional modifications in mRNA, which regulate the stability, splicing, translation, transport and other processes of mRNA, followed by affecting cell development, body immunity, learning and cognition and other important physiological functions. m6A modification is one of the most abundant post-transcriptional modifications widely existing in mRNA, regulating the metabolic activities of RNA and affecting gene expression. m6A modified homeostasis is critical for the development and maintenance of the nervous system. In recent years, m6A modification has been found in neurodegenerative diseases, mental diseases and brain tumors. This review summarizes the role of m6A methylation modification in the development, function and related diseases of the central nervous system in recent years, providing potential clinical therapeutic targets for neurological diseases.
Тема - темы
Methylation , Central Nervous System/metabolism , RNA, Messenger/metabolism , RNAРеферат
N6-methyladenosine (m6A) is a ubiquitous RNA modification in mammals. This modification is "written" by methyltransferases and then "read" by m6A-binding proteins, followed by a series of regulation, such as alternative splicing, translation, RNA stability, and RNA translocation. At last, the modification is "erased" by demethylases. m6A modification is essential for normal physiological processes in mammals and is also a very important epigenetic modification in the development of cancer. In recent years, cancer-related m6A regulation has been widely studied, and various mechanisms of m6A regulation in cancer have also been recognized. In this review, we summarize the changes of m6A modification in prostate cancer and discuss the effect of m6A regulation on prostate cancer progression, aiming to profile the potential relevance between m6A regulation and prostate cancer development. Intensive studies on m6A regulation in prostate cancer may uncover the potential role of m6A methylation in the cancer diagnosis and cancer therapy.
Тема - темы
Animals , Male , Humans , Methylation , Adenosine/metabolism , RNA/metabolism , Methyltransferases/metabolism , Prostatic Neoplasms , MammalsРеферат
OBJECTIVE@#To investigate whether circular RNA circRSF1 regulates radiation-induced inflammatory phenotype of hepatic stellate cells (HSCs) by binding to HuR protein and repressing its function.@*METHODS@#Human HSC cell line LX2 with HuR overexpression or knockdown was exposed to 8 Gy X-ray irradiation, and the changes in the expression of inflammatory factors (IL-1β, IL-6 and TNF-α) were detected by qRT-PCR. The expressions of IκBα and phosphorylation of NF-κB were detected with Western blotting. The binding of circRSF1 to HuR was verified by RNA pull-down assay and RNA-binding protein immunoprecipitation (RIP). The expressions of inflammatory factors, IκBα and the phosphorylation of NF-κB were detected after modifying the interaction between circRSF1 and HuR.@*RESULTS@#Knockdown of HuR significantly up- regulated the expressions of IL-1β, IL-6 and TNF-α, decreased IκBα expression and promoted NF-κB phosphorylation in irradiated LX2 cells, whereas overexpression of HuR produced the opposite changes (P < 0.05). Overexpression or knockdown of circRSF1 did not significantly affect the expression of HuR. RNA pull-down and RIP experiments confirmed the binding between circRSF1 and HuR. Overexpression of circRSF1 significantly reduced the binding of HuR to IκBα and down-regulated the expression of IκBα (P < 0.05). Overexpression of circRSF1 combined with HuR overexpression partially reversed the up-regulation of the inflammatory factors, down-regulated IκBα expression and increased phosphorylation of NFκB in LX2 cells, while the opposite effects were observed in cells with knockdown of both circRSF1 and HuR (P < 0.05).@*CONCLUSION@#circRSF1 reduces IκBα expression by binding to HuR to promote the activation of NF-κB pathway, thereby enhancing radiation- induced inflammatory phenotype of HSCs.
Тема - темы
Humans , Hepatic Stellate Cells/radiation effects , Interleukin-6 , NF-kappa B , NF-KappaB Inhibitor alpha , Phenotype , RNA , RNA, Circular/metabolism , Tumor Necrosis Factor-alpha , ELAV-Like Protein 1/metabolismРеферат
Saliva testing is a vital method for clinical applications, for its noninvasive features, richness in substances, and the huge amount. Due to its direct anatomical connection with oral, digestive, and endocrine systems, clinical usage of saliva testing for these diseases is promising. Furthermore, for other diseases that seeming to have no correlations with saliva, such as neurodegenerative diseases and psychological diseases, researchers also reckon saliva informative. Tremendous papers are being produced in this field. Updated summaries of recent literature give newcomers a shortcut to have a grasp of this topic. Here, we focused on recent research about saliva biomarkers that are derived from humans, not from other organisms. The review mostly addresses the proceedings from 2016 to 2022, to shed light on the promising usage of saliva testing in clinical diagnostics. We recap the recent advances following the category of different types of biomarkers, such as intracellular DNA, RNA, proteins and intercellular exosomes, cell-free DNA, to give a comprehensive impression of saliva biomarker testing.
Тема - темы
Humans , Saliva/metabolism , Biomarkers/metabolism , RNA , Exosomes/metabolismРеферат
The failure rate of dental implantation in patients with well-controlled type 2 diabetes mellitus (T2DM) is higher than that in non-diabetic patients. This due, in part, to the impaired function of bone marrow mesenchymal stem cells (BMSCs) from the jawbone marrow of T2DM patients (DM-BMSCs), limiting implant osseointegration. RNA N6-methyladenine (m6A) is important for BMSC function and diabetes regulation. However, it remains unclear how to best regulate m6A modifications in DM-BMSCs to enhance function. Based on the "m6A site methylation stoichiometry" of m6A single nucleotide arrays, we identified 834 differential m6A-methylated genes in DM-BMSCs compared with normal-BMSCs (N-BMSCs), including 43 and 790 m6A hypermethylated and hypomethylated genes, respectively, and 1 gene containing hyper- and hypomethylated m6A sites. Differential m6A hypermethylated sites were primarily distributed in the coding sequence, while hypomethylated sites were mainly in the 3'-untranslated region. The largest and smallest proportions of m6A-methylated genes were on chromosome 1 and 21, respectively. MazF-PCR and real-time RT-PCR results for the validation of erythrocyte membrane protein band 4.1 like 3, activity-dependent neuroprotector homeobox (ADNP), growth differentiation factor 11 (GDF11), and regulator of G protein signalling 2 agree with m6A single nucleotide array results; ADNP and GDF11 mRNA expression decreased in DM-BMSCs. Furthermore, gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses suggested that most of these genes were enriched in metabolic processes. This study reveals the differential m6A sites of DM-BMSCs compared with N-BMSCs and identifies candidate target genes to enhance BMSC function and improve implantation success in T2DM patients.
Тема - темы
Humans , Bone Marrow/metabolism , Bone Morphogenetic Proteins/metabolism , Dental Implants/adverse effects , Diabetes Mellitus, Type 2/metabolism , Growth Differentiation Factors/metabolism , Mesenchymal Stem Cells/metabolism , RNA/metabolism , RNA Processing, Post-TranscriptionalРеферат
Objective: To identify the expression profile of circular RNA (circRNA) in placenta of pre-eclampsia (PE) pregnant women by high-throughput sequencing, and to construct the circRNA-microRNA (miRNA)-messenger RNA (mRNA) interaction network, so as to reveal the related pathways and regulatory mechanisms of PE. Methods: The clinical data and placentas of 42 women with PE (PE group) and 30 normal pregnant women (control group) who delivered in West China Second University Hospital from November 2019 to June 2021 were collected. (1) High-throughput sequencing was used to establish the differentially expressed circRNA profiles in placental tissues of 5 pairs of PE group and the control group. (2) Real-time quantitative PCR (qRT-PCR) was used to verify the expression levels of 6 differentially expressed circRNAs in placental tissues of PE group and control group. (3) Bioinformatics analysis was used to predict the target miRNA and analyze the co-expressed mRNA to construct a competitive endogenous RNA (ceRNA) network. The differentially expressed circRNAs were analyzed by Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathways. (4) Logistic regression analysis, Pearson correlation and Kendall's tau-b correlation analysis were used to test the correlation between the three differentially expressed circRNAs and the risk of PE and clinical characteristics. (5) circRNA_05393 was selected for subsequent functional study. Small interfering RNA (siRNA) and overexpression plasmid were used to knock down or increase the expression level of circRNA_05393 in trophoblast cell line HTR-8/SVneo cells, respectively. Transwell assay was used to detect the migration and invasion ability of the trophoblasts in vitro. Cell counting kit-8 assay was used to detect the proliferation ability of the trophoblasts. Results: (1) Seventy-two differentially expressed circRNAs were identified by high-throughput sequencing, of which 35 were up-regulated and 37 were down-regulated. (2) qRT-PCR showed that compared with the control group, circRNA_00673 (1.306±0.168 vs 2.059±0.242; t=2.356, P=0.021) and circRNA_07796 (1.275±0.232 vs 1.954±0.230; t=2.018, P=0.047) were significantly increased, while circRNA_05393 (1.846±0.377 vs 0.790±0.094; t=3.138, P=0.002) was significantly decreased. (3) The circRNA-miRNA-mRNA interaction network contained 3 circRNAs, 8 miRNAs and 53 mRNAs. GO functional annotation analysis showed that the biological process was mainly enriched in iron ion homeostasis, membrane depolarization during action potential and neuronal action potential. In terms of cellular components, they were mainly enriched in cytoskeleton and membrane components. In terms of molecular function, they were mainly enriched in the activity of voltage-gated sodium channel and basic amino acid transmembrane transporter. KEGG pathway enrichment analysis showed that mRNAs in the interaction network were mainly enriched in complement and coagulation cascade, glycine, serine and threonine metabolism, p53 signaling pathway and peroxisome proliferators-activated receptors (PPAR) signaling pathway. (4) Logistic regression analysis showed that down-regulation of circRNA_05393 expression was a risk factor for PE (OR=0.044, 95%CI: 0.003-0.596; P=0.019). Correlation analysis showed that circRNA_05393 was significantly correlated with systolic blood pressure and diastolic blood pressure in PE pregnant women (both P<0.05). (5) Knock down or overexpression of circRNA_05393 significantly reduced or increased the migration and invasion abilities of HTR-8/SVneo cells (all P<0.05), but had no significant effect on the ability of tube formation and proliferation (all P>0.05). Conclusions: The construction of circRNA expression profile in placenta and the exploration of circRNA-miRNA-mRNA interaction network provide the possibility to reveal the regulatory mechanism of specific circRNA involved in PE. Inhibition of circRNA_05393 may induce the progression of PE by reducing the migration and invasion of trophoblasts.
Тема - темы
Female , Humans , Pregnancy , MicroRNAs/metabolism , RNA, Circular/metabolism , RNA, Messenger/metabolism , Pre-Eclampsia/metabolism , Placenta/metabolism , RNA/metabolism , RNA, Small Interfering , Gene Expression ProfilingРеферат
Objective: To investigate the effects of RNA m6A demethylase ALKBH5 gene deficiency on cerebellar morphology and function in the aged mice, and to explore the role of ALKBH5 in cerebellar degeneration. Methods: Western blot was performed to detect the protein level of ALKBH5 in the cerebellum of wild-type mice of various ages. The expression of NeuN, Calbindin-D28K, MAP2, GFAP and other proteins in the cerebella of middle-aged (12-month-old) and aged (18-month-old) wild-type mice and ALKBH5-/- mice was examined using immunohistochemistry. The balance beam test and gait analysis were performed to test the balance ability and motor coordination of the mice. Results: With aging of the mice, the expression of ALKBH5 in the cerebellum increased gradually in an age-dependent manner. In the aged mice, but not middle-aged mice, the body weight, whole brain weight and cerebellum weight of ALKBH5-/- mice decreased by 15%, 10% and 21%, respectively (P<0.05). The expression of ALKBH5 in the Purkinje cells was much higher than that in other types of neural cells. Correspondingly, ALKBH5-deficiency caused 40% reduction in the number of Purkinje cells, as well as the length and density of neuronal dendrites in the aged mice (P<0.01). In addition, the time for the aged ALKBH5-/- mice to pass the balance beam was 70% longer than that of the wild type mice of the same age, with unstable gaits (P<0.01). Conclusions: Gene deficiency of RNA m6A demethylase ALKBH5 causes cerebellar atrophy, Purkinje neuron loss and damage in the aged mice. These changes eventually affect mice's motor coordination and balance ability. These results suggest that imbalanced RNA m6A methylation may lead to neurodegenerative lesions in the cerebellum of mice.
Тема - темы
Animals , Mice , AlkB Homolog 5, RNA Demethylase/metabolism , Cerebellum/metabolism , Methylation , RNA/metabolismРеферат
OBJECTIVE@#To investigate the effect of titanium dioxide nanoparticles (TiO2 NPs) on the expression profile of circular ribonucleic acid (circRNA) in human hepatocytes through in vitro cell experiments, and to attempt to understand the potential mechanism of hepatotoxicity through bioinformatics analysis.@*METHODS@#TiO2 NPs were characterized from the aspects of particle size, shape and agglomeration state. The cell counting kit-8 (CCK8) was used to detect the cytotoxicity of TiO2 NPs against human hepatocellular carcinoma cells (HepG2) after exposure to 0, 1.56, 3.13, 6.25, 12.5, 25, 50, 100, and 200 mg/L TiO2 NPs for 24 h or 48 h. The cells were treated at doses of 0 mg/L TiO2 NPs (control group) and 100 mg/L TiO2 NPs (treatment group), and collected after exposure for 48 h, and then RNA from the extracted cell samples was collected and sequenced. The differential circRNAs between the control and the TiO2 NPs treatment groups were screened, and then the enrichment pathway of the differential circRNA target gene was analyzed by multivariate statistics. According to the sequencing results, significantly altered genes and important genes in the significant enrichment pathways were screened, and real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) was performed to verify the results.@*RESULTS@#TiO2 NPs were spherical anatase with a hydrated particle size of (323.50±85.44) nm and a Zeta potential of (-21.00±0.72) mV in a serum-free medium. The results of the CCK8 cytotoxicity assay showed that with the increase of TiO2 NPs concentration, cell viability gradually decreased. A total of 11 478 circRNAs were found by RNA sequencing. Compared with the control groups, TiO2 NPs treatment groups (100 mg/L) had a total of 89 differential circRNAs, of which 59 were up-regulated and 30 were down-regulated. Analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway showed that the targeted genes of differential circRNAs were mainly enriched in fatty acid degradation, Fanconi anemia pathway, and fatty acid metabolism. The expression levels of circRNA.6730, circRNA.3650 and circRNA.4321 were significantly different between the TiO2 NPs treatment group and the control group, which were consistent with the sequencing results.@*CONCLUSION@#TiO2 NPs can induce changes in circRNA expression profile, and epigenetics may play an important role in the mechanism of hepatotoxicity.
Тема - темы
Humans , RNA/genetics , RNA, Circular/genetics , Carcinoma, Hepatocellular/genetics , Liver Neoplasms/genetics , Titanium , Nanoparticles , Chemical and Drug Induced Liver Injury , Fatty AcidsРеферат
OBJECTIVE@#To establish a rapid detection and genotyping method for SARS-CoV-2 Omicron BA.4/5 variants using CRISPPR-Cas12a gene editing technology.@*METHODS@#We combined reverse transcription-polymerase chain reaction (RT-PCR) and CRISPR gene editing technology and designed a specific CRISPPR RNA (crRNA) with suboptimal protospacer adjacent motifs (PAM) for rapid detection and genotyping of SARS- CoV-2 Omicron BA.4/5 variants. The performance of this RT- PCR/ CRISPPR-Cas12a assay was evaluated using 43 clinical samples of patients infected by wild-type SARS-CoV-2 and the Alpha, Beta, Delta, Omicron BA. 1 and BA. 4/5 variants and 20 SARS- CoV- 2-negative clinical samples infected with 11 respiratory pathogens. With Sanger sequencing method as the gold standard, the specificity, sensitivity, concordance (Kappa) and area under the ROC curve (AUC) of RT-PCR/CRISPPR-Cas12a assay were calculated.@*RESULTS@#This assay was capable of rapid and specific detection of SARS- CoV-2 Omicron BA.4/5 variant within 30 min with the lowest detection limit of 10 copies/μL, and no cross-reaction was observed in SARS-CoV-2-negative clinical samples infected with 11 common respiratory pathogens. The two Omicron BA.4/5 specific crRNAs (crRNA-1 and crRNA-2) allowed the assay to accurately distinguish Omicron BA.4/5 from BA.1 sublineage and other major SARS-CoV-2 variants of concern. For detection of SARS-CoV-2 Omicron BA.4/5 variants, the sensitivity of the established assay using crRNA-1 and crRNA-2 was 97.83% and 100% with specificity of 100% and AUC of 0.998 and 1.000, respectively, and their concordance rate with Sanger sequencing method was 92.83% and 96.41%, respectively.@*CONCLUSION@#By combining RT-PCR and CRISPPR-Cas12a gene editing technology, we successfully developed a new method for rapid detection and identification of SARS-CoV-2 Omicron BA.4/5 variants with a high sensitivity, specificity and reproducibility, which allows rapid detection and genotyping of SARS- CoV-2 variants and monitoring of the emerging variants and their dissemination.
Тема - темы
Humans , COVID-19 , CRISPR-Cas Systems , Genotype , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2/genetics , RNA , COVID-19 TestingРеферат
Objective To establish an efficient method for isolating migrasomes from RAW264.7 macrophages and identifying these isolated migrasomes. Methods Scanning electron microscopy was used to observe the morphological characteristics of migrasomes produced by RAW264.7 cells. A 0.45 μm filter was employed for reverse filtration and elution to isolate the migrasomes. The morphological characteristics of the migrasomes were then observed using transmission electron microscopy. Western blot analysis was performed to determine the expression of characteristic markers of the migrasomes. The RNA carried by the migrasomes was analysed by using LabChip bioanalyzer. Results Scanning electron microscopy revealed that the migrasomes, with membranous structures, were attached to the tip or bifurcation of the retraction fiber formed in the tail of RAW264.7 cells. Transmission electron microscopy showed that the isolated migrasomes had a typical oval vesicle-like structure with wrinkled membrane surfaces. Western blot analysis confirmed the expression of the characteristic markers phosphatidylinositol glycan anchor biosynthesis class K (PIGK), epidermal growth factor domain-specific O-linked N-acetylglucosamine transferase (EOGT) and tetraspanin 4 (TSPAN4) in the migrasomes, while the EV (extracellular vesicle) markers tumor susceptibility gene 101 (TSG101) and Arabidopsis homolog of apoptosis-linked gene 2-interacting protein X (ALIX) were not detected. Furthermore, the isolated migrasomes were found to be rich in small RNA, which were approximately 25-200 nt in length. Conclusion A method for the extraction of well-structured and high quality migrasomes from macrophages is established.
Тема - темы
Extracellular Vesicles , Microscopy, Electron, Transmission , RNA , MacrophagesРеферат
RNA-based next-generation sequencing (NGS) has been recommended as a method for detecting fusion genes in non-small cell lung cancer (NSCLC) according to clinical practice guidelines and expert consensus. The primary targetable alterations in NSCLC consist of gene mutations and fusions, making the detection of gene mutations and fusions indispensable for assessing the feasibility of targeted therapies. Currently, the integration of DNA-based NGS and RNA-based NGS allows for simultaneous detection of gene mutations and fusions and has been partially implemented in clinical practice. However, standardized guidelines and criteria for the significance, application scenarios, and quality control of RNA-based NGS in fusion gene detection are still lacking in China. This consensus aims to provide further clarity on the practical significance, application scenarios, and quality control measures of RNA-based NGS in fusion gene detection. Additionally, it offers guiding recommendations to facilitate the clinical implementation of RNA-based NGS in the diagnosis and treatment of NSCLC, ultimately maximizing the benefits for patients from fusion gene detection. .
Тема - темы
Humans , Carcinoma, Non-Small-Cell Lung/drug therapy , Lung Neoplasms/drug therapy , RNA , Consensus , High-Throughput Nucleotide Sequencing/methodsРеферат
OBJECTIVE@#To explore the potential molecular mechanism of tetrahydropalmatine (THP) on acute myocardial ischemia (AMI).@*METHODS@#First, the target genes of THP and AMI were collected from SymMap Database, Traditional Chinese Medicine Database and Analysis Platform, and Swiss Target Prediction, respectively. Then, the overlapping target genes between THP and AMI were evaluated for Grene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and protein-protein interaction network analysis. The binding affinity between the protein and THP was assessed by molecular docking. Finally, the protective effects of THP on AMI model and oxygen and glucose deprivation (OGD) model of H9C2 cardiomyocyte were explored and the expression levels of target genes were detected by RT-qPCR in vivo and in vitro.@*RESULTS@#MMP9, PPARG, PTGS2, SLC6A4, ESR1, JAK2, GSK3B, NOS2 and AR were recognized as hub genes. The KEGG enrichment analysis results revealed that the potential target genes of THP were involved in the regulation of PPAR and hormone pathways. THP improved the cardiac function, as well as alleviated myocardial cell damage. Furthermore, THP significantly decreased the RNA expression levels of MMP9, PTGS2, SLC6A4, GSK3B and ESR1 (P<0.05, P<0.01) after AMI. In vitro, THP significantly increased H9C2 cardiomyocyte viability (P<0.05, P<0.01) and inhibited the RNA expression levels of PPARG, ESR1 and AR (P<0.05, P<0.01) in OGD model.@*CONCLUSIONS@#THP could improve cardiac function and alleviate myocardial injury in AMI. The underlying mechanism may be inhibition of inflammation, the improvement of energy metabolism and the regulation of hormones.
Тема - темы
Humans , Matrix Metalloproteinase 9 , Network Pharmacology , Cyclooxygenase 2 , Molecular Docking Simulation , PPAR gamma , Myocardial Ischemia/genetics , Glucose , RNA , Drugs, Chinese Herbal/therapeutic use , Serotonin Plasma Membrane Transport ProteinsРеферат
METTL3 and METTL14 are two components that form the core heterodimer of the main RNA m6A methyltransferase complex (MTC) that installs m6A. Surprisingly, depletion of METTL3 or METTL14 displayed distinct effects on stemness maintenance of mouse embryonic stem cell (mESC). While comparable global hypo-methylation in RNA m6A was observed in Mettl3 or Mettl14 knockout mESCs, respectively. Mettl14 knockout led to a globally decreased nascent RNA synthesis, whereas Mettl3 depletion resulted in transcription upregulation, suggesting that METTL14 might possess an m6A-independent role in gene regulation. We found that METTL14 colocalizes with the repressive H3K27me3 modification. Mechanistically, METTL14, but not METTL3, binds H3K27me3 and recruits KDM6B to induce H3K27me3 demethylation independent of METTL3. Depletion of METTL14 thus led to a global increase in H3K27me3 level along with a global gene suppression. The effects of METTL14 on regulation of H3K27me3 is essential for the transition from self-renewal to differentiation of mESCs. This work reveals a regulatory mechanism on heterochromatin by METTL14 in a manner distinct from METTL3 and independently of m6A, and critically impacts transcriptional regulation, stemness maintenance, and differentiation of mESCs.
Тема - темы
Animals , Mice , Methylation , Chromatin , Histones/metabolism , RNA, Messenger/genetics , Methyltransferases/metabolism , RNA/metabolismРеферат
Sepsis is a life-threatening organ dysfunction caused by infection that lead to dysregulation of the host response. Sepsis and septic shock with a high mortality threaten human health at present, which are important medical and health problems. Early diagnosis and treatment decision-making for sepsis and septic shock still need to be improved. Exosomes are extracellular vesicles with a diameter of 30-150 nm formed by the fusion of multi-vesicle bodies and cell membranes. Exosomes can effectively transport a variety of bioactive substances such as proteins, lipids, RNA, DNA, and participate in the regulation of inflammatory response, immune response, infection and other pathophysiological processes. In recent years, exosomes have become one of the important methods for the diagnosis and treatment of systemic inflammatory diseases. This article will focus on the basic and clinical research of sepsis, and focus on the research progress of exosomes in the diagnosis and targeted therapy of sepsis.