OBJECTIVE: To identify the role of gene silencing or overexpression of Nemo-like kinase (NLK) during the process of neural differentiation of human mesenchymal stem cells (hBMSCs), and to explore the effect of NLK downregulation by transfection of small interfering RNA (siRNA) on promoting neuralized tissue engineered bone regeneration. METHODS: NLK-knockdown hBMSCs were established by transfection of siRNA (the experimental group was transfected with siRNA silencing the NLK gene, the control group was transfected with control siRNA and labeled as negative control group), and NLK-overexpression hBMSCs were established using lentivirus vector transfection technique (the experimental group was infected with lentivirus overexpressing the NLK gene, the control group was infected with an empty vector lentivirus and labeled as the empty vector group). After neurogenic induction, quantitative real-time polymerase chain reaction (qPCR) was used to detect the expression of neural-related gene, and Western blot as well as immunofluorescence staining about several specific neural markers were used to evaluate the neural differentiation ability of hBMSCs.6-week-old male nude mice were divided into 4 groups: ① β-tricalcium phosphate (β-TCP) group, ② β-TCP+ osteogenic induced hBMSCs group, ③ β-TCP+ siRNA-negative control (siRNA-NC) transfection hBMSCs group, ④ β-TCP+ siRNA-NLK transfection hBMSCs group. Four weeks after the subcutaneous ectopic osteogenesis models were established, the osteogenesis and neurogenesis were detected by hematoxylin-eosin (HE) staining, Masson staining and tissue immunofluorescence assay. Statistical analysis was conducted by independent sample t test. RESULTS: After gene silencing of NLK by siRNA in hBMSCs, neural-related genes, including the class Ⅲ β-tubulin (TUBB3), microtubule association protein-2 (MAP2), soluble protein-100 (S100), nestin (NES), NG2 proteoglycan (NG2) and calcitonin gene-related peptide (CGRP), were increased significantly in NLK-knockdown hBMSCs compared with the negative control group(P < 0.05), and the expression levels of TUBB3 and MAP2 of the NLK silencing group were also increased. Oppositely, after NLK was overexpressed using lentivirus vector transfection technique, TUBB3, MAP2, S100 and NG2 were significantly decreased in NLK-overexpression hBMSCs compared with the empty vector group (P < 0.05), and the expression level of TUBB3 was also decreased. 4 weeks after the subcutaneous ectopic osteogenesis model was established, more mineralized tissues were formed in the β-TCP+ siRNA-NLK transfection hBMSCs group compared with the other three groups, and the expression of BMP2 and S100 was higher in the β-TCP+ siRNA-NLK transfection hBMSCs group than in the other groups. CONCLUSION Gene silencing of NLK by siRNA promoted the ability of neural differentiation of hBMSCs in vitro and promoted neuralized tissue engineered bone formation in subcutaneous ectopic osteogenic models in vivo in nude mice.
Bone Regeneration/genetics* ; Animals ; Mesenchymal Stem Cells/cytology* ; Humans ; RNA, Small Interfering/genetics* ; Tissue Engineering/methods* ; Cell Differentiation ; Mice, Nude ; Gene Silencing ; Mice ; Male ; Protein Serine-Threonine Kinases/genetics* ; Intracellular Signaling Peptides and Proteins/genetics* ; Transfection ; Cells, Cultured ; Lentivirus/genetics*

Objective:To investigate the expression of EIF3B and its role in the development of laryngeal carcinoma. Methods:Immunohistochemistry, cell culture, cell transfection, qRT-PCR, Western Blot and other techniques were used to determine the expression difference of EIF3B in laryngeal cancer and adjacent tissues, and analyze the relationship between EIF3B and the size and TNM stage of laryngeal cancer. By constructing a laryngeal carcinoma cell model with EIF3B knocked down, the cell function was studied, and the regulatory effect of EIF3B on laryngeal carcinoma cells was proved in vitro. Finally, the effect of EIF3B on laryngeal carcinoma growth in vivo was studied by subcutaneous xenograft tumor model in nude mice. Results:The signal intensity of EIF3B in laryngeal carcinoma tissues was significantly stronger than that in adjacent tissues, and the expression level of EIF3B was positively correlated with patient age, TNM stage, lymph node metastasis, tumor size and clinical stage. Knocking down EIF3B can significantly inhibit the proliferation, migration and aggregation of cancer cells, and promote apoptosis. In vivo experiments with nude mice also showed that down-regulating EIF3B expression could inhibit tumor formation in vivo. Conclusion:The expression of EIF3B in laryngeal cancer is significantly increased, and it is closely related to the pathological characteristics of laryngeal cancer, which can be used as a diagnostic index of laryngeal cancer. In terms of function, EIF3B knockdown can inhibit the proliferation, migration and tumor formation of laryngeal cancer cells in vitro and in vivo, and may become a candidate target for targeted therapy of laryngeal cancer in the future.
Laryngeal Neoplasms/pathology* ; Humans ; Eukaryotic Initiation Factor-3/metabolism* ; Animals ; Mice, Nude ; Mice ; Cell Line, Tumor ; Cell Proliferation ; Apoptosis ; Cell Movement ; Neoplasm Staging ; Male ; Transfection ; Female ; Middle Aged ; Gene Expression Regulation, Neoplastic

OBJECTIVES: To investigate the effect of high glucose on macrophage polarization and the role of immune-responsive gene 1 (IRG1) in mediating its effect. METHODS: RAW264.7 cells were transfected with IRG1-overexpressing plasmid or IRG1 siRNA via electroporation and cultured in either normal or high glucose for 72 h to observe the changes in cell viability and morphology using CCK-8 assay and phase contrast microscopy. The protein levels of IRG1, iNOS, Arg-1, IL-1β and IL-10 in the treated cells were detected with Western blotting, and the fluorescence intensities of iNOS and Arg-1 were detected using immunofluorescence assay. The protein levels of IL-1β and IL-10 in the culture medium were determined with ELISA. RESULTS: High glucose exposure significantly reduced IRG1 and Arg-1 expressions, increased iNOS and IL-1β expressions and IL-1β secretion, and decreased IL-10 level in RAW264.7 cells. Transfection with the IRG1-overexpressing plasmid provided the cells with obvious resistance to high glucose-induced changes in iNOS, Arg-1, IL-1β and IL-10, whereas IRG1 knockdown further enhanced the effects of high glucose exposure on Arg-1 expression and the expression and secretion of IL-10. CONCLUSIONS High glucose promotes M1 polarization of the macrophages possibly through a mechanism to inhibit the expression of IRG1 protein, thus leading to chronic inflammatory response.
Animals ; Mice ; Macrophages/drug effects* ; Glucose/pharmacology* ; Interleukin-10/metabolism* ; Nitric Oxide Synthase Type II/metabolism* ; RAW 264.7 Cells ; Interleukin-1beta/metabolism* ; Arginase/metabolism* ; RNA, Small Interfering/genetics* ; Transfection ; Inflammation

OBJECTIVES: To investigate the regulatory effect of LINC00837/miR-671-5p/SERPINE2 functional axis on pathological processes of fibroblast-like synovial cells (FLS) in rheumatoid arthritis (RA). METHODS: RA-FLS were transfected with a LINC00837 overexpression plasmid (pcDNA3.1-LINC00837), a LINC00837 interference plasmid (siRNA-LINC00837), or their respective negative control plasmids (pcDNA3.1-NC and siRNA-NC). Dual luciferase was used to verify the targeting relationship between LINC00837 and miR-671-5p and between miR-671-5p and SERPINE2. RT-qPCR was used to detect the expression levels of LINC00837, miR-671-5p and SERPINE2 in normal FLS or the transfected cells, whose proliferation and migration abilities were assessed using Edu assay and scratch healing assay and by detecting the expression levels of Ki-67, PCNA, E-cadherin and N-cadherin with Western blotting. The changes in cellular secretion of the inflammatory cytokines (TNF‑α, IL-17, IL-4 and IL-10) were examined using ELISA. RESULTS: Dual luciferase reporter gene assay showed that LINC00837 was capable of binding to the 3'-UTR of miR-671-5p, and the latter bound to the 3-UTR of SERPINE2 at specific binding sites between them. Compared with normal FLS, RA-FLS showed significantly increased expressions of LINC00837 and SERPINE2, lowered miR-671-5p expression and enhanced proliferation and migration abilities with increased expressions of pro-inflammatory cytokines and reduced expressions of anti-inflammatory cytokines. Transfection of RA-FLS with pcDNA-LINC00837 further enhanced cell proliferation and migration and the changes in the inflammatory cytokines, while transfection with si-LINC00837 produced the opposite changes. CONCLUSIONS RA-FLS have a LINC00837/miR-671-5p/SERPINE2 functional axis, which regulates cell proliferation, migration and secretion of inflammatory factors, and interventions targeting LINC00837 may provide a potential strategy to regulate the pathological processes in RA-FLS.
Arthritis, Rheumatoid/metabolism* ; MicroRNAs/metabolism* ; Humans ; Cell Proliferation ; Cell Movement ; Synovial Membrane/pathology* ; RNA, Long Noncoding/genetics* ; Fibroblasts/metabolism* ; Synoviocytes/metabolism* ; Cells, Cultured ; Transfection

OBJECTIVES: To investigate the effect of BRD4 inhibition on migration of esophageal squamous cell carcinoma (ESCC) cells with low acetyl-CoA carboxylase 1 (ACC1) expression. METHODS: ESCC cell lines with lentivirus-mediated ACC1 knockdown or transfected with a negative control sequence (shNC) were treated with DMSO, JQ1 (a BRD4 inhibitor), co-transfection with shNC-siBRD4 or siNC with additional DMSO or C646 (an ahistone acetyltransferase inhibitor) treatment, or JQ1combined with 3-MA (an autophagy inhibitor). BRD4 mRNA expression in the cells was detected using RT-qPCR. The changes in cell proliferation, migration, autophagy, and epithelial-mesenchymal transition (EMT) were examined with CCK8 assay, Transwell migration assay, and Western blotting. RESULTS: ACC1 knockdown did not significantly affect BRD4 expression in the cells but obviously increased their sensitivity to JQ1. JQ1 treatment at 1 and 2 μmol/L significantly inhibited ESCC cell proliferation, while JQ1 at 0.2 and 2 μmol/L promoted cell migration. The cells with ACC1 knockdown and JQ1 treatment showed increased expresisons of vimentin and Slug and decreased expression of E-cadherin. BRD4 knockdown promoted migration of ESCC cells, and co-transfection with shACC1 and siBRD4 resulted in increased vimentin and Slug expressions and decreased E-cadherin expression in the cells. C646 treatment of the co-transfected cells reduced acetylation levels, decreased vimentin and Slug expressions, and increased E-cadherin expression. Treatment with JQ1 alone obviously increased LC3A/B-II levels in the cells either with or without ACC1 knockdown. In the cells with ACC1 knockdown and JQ1 treatment, additional 3-MA treatment significantly decreased the expressions of vimentin, Slug and LC3A/B-II and increased the expression of E-cadherin. CONCLUSIONS BRD4 inhibition promotes autophagy of ESCC cells via a histone acetylation-dependent mechanism, thereby enhancing EMT and ultimately increasing cell migration driven by ACC1 deficiency.
Humans ; Cell Movement ; Transcription Factors/metabolism* ; Esophageal Neoplasms/metabolism* ; Cell Line, Tumor ; Cell Cycle Proteins ; Azepines/pharmacology* ; Epithelial-Mesenchymal Transition ; Carcinoma, Squamous Cell/metabolism* ; Esophageal Squamous Cell Carcinoma ; Triazoles/pharmacology* ; Nuclear Proteins/genetics* ; Cell Proliferation ; Acetyl-CoA Carboxylase/genetics* ; Transfection ; Autophagy ; Bromodomain Containing Proteins

Successful cloning through somatic cell nuclear transfer (SCNT) faces significant challenges due to epigenetic obstacles. Recent studies have highlighted the roles of H3K4me3 and H3K27me3 as potential contributors to these obstacles. However, the underlying mechanisms remain largely unclear. In this study, we generated genome-wide maps of H3K4me3 and H3K27me3 in mouse pre-implantation NT embryos. Our analysis revealed that aberrantly over-represented broad H3K4me3 domain and H3K27me3 signal lead to increased bivalent marks at gene promoters in NT embryos compared with naturally fertilized (NF) embryos at the 2-cell stage, which may link to relatively low levels of H3K36me3 in NT 2-cell embryos. Notably, the overexpression of Setd2, a H3K36me3 methyltransferase, successfully restored multiple epigenetic marks, including H3K36me3, H3K4me3, and H3K27me3. In addition, it reinstated the expression levels of ZGA-related genes by reestablishing H3K36me3 at gene body regions, which excluded H3K27me3 from bivalent promoters, ultimately improving cloning efficiency. These findings highlight the excessive bivalent state at gene promoters as a potent barrier and emphasize the removal of these barriers as a promising approach for achieving higher cloning efficiency.
Animals ; Mice ; Histone-Lysine N-Methyltransferase/biosynthesis* ; Histones/genetics* ; Nuclear Transfer Techniques ; Female ; Gene Expression Regulation, Developmental ; Promoter Regions, Genetic ; Epigenesis, Genetic ; Embryo, Mammalian/metabolism*

OBJECTIVE: To investigate the role of telomerase reverse transcriptase (TERT) in alleviating doxorubicin (DOX)-induced cardiotoxicity. METHODS: (1) Cell experiments: rat H9c2 cardiomyocytes were divided into control group (CON group), null adenovirus transfection group (NC group), TERT overexpression adenovirus transfection group (TERT group), DOX group (treated with 1 μmol/L DOX for 12 hours), DOX+NC group, and DOX+TERT group (null adenovirus or TERT overexpression adenovirus were transfected for 24 hours and then treated with 1 μmol/L DOX for 12 hours). The mRNA expression of TERT in cardiomyocytes was detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). The level of mitochondrial membrane potential was detected by immunofluorescence. The expression levels of intracellular Bax, Bcl-2, microtubule-associated protein 1 light chain 3 (LC3) and p62 were detected by Western blotting. (2) Animal experiments: male C57BL/6 mice were randomly divided into a sham operation group (Sham group), DOX group (acute cardiotoxicity model was constructed by intraperitoneal injection of DOX 15 mg/kg), DOX+NC group and DOX+TERT group (modeled after transfection with airborne adenovirus or TERT overexpression adenovirus for 7 days). After 7 days of modeling, the area of myocardial fibrosis was detected by Sirius scarlet staining, and cardiac function was detected by echocardiography. RESULTS: (1) Cellular experiments: the mRNA expression level of TERT was significantly higher in the TERT group compared with the CON and NC groups. Compared with the CON group, the TERT mRNA expression level of cardiomyocytes in the DOX group and the DOX+NC group were significantly lower, the level of mitochondrial membrane potential was significantly lower, the protein expressions of Bax and LC3 were significantly increased, and the protein expressions of Bcl-2 and p62 were significantly decreased. No significant differences were found between the DOX group and DOX+NC group. Compared with the DOX group and DOX+NC group, the TERT mRNA expression level was increased in the DOX+TERT group (relative expression: 1.02±0.10 vs. 0.61±0.05, 0.54±0.03, both P < 0.05), the level of mitochondrial membrane potential was significantly increased (1.14±0.05 vs. 0.96±0.01, 0.96±0.01, both P < 0.05), the protein expressions of Bax and LC3 were significantly decreased, and the protein expressions of Bcl-2 and p62 were significantly increased (Bax/β-actin: 0.88±0.01 vs. 1.31±0.02, 1.26±0.01; LC3-II/I: 2.16±0.05 vs. 2.64±0.06, 2.58±0.02; Bcl-2/β-actin: 0.65±0.01 vs. 0.40±0.01, 0.41±0.01; p62/β-actin: 0.45±0.01 vs. 0.23±0.02, 0.29±0.01; all P < 0.05). (2) Animal experiments: compared with the Sham group, the percentage of myocardial fibrosis area was significantly increased and left ventricular ejection fraction (LVEF) and fractional shortening (FS) were significantly decreased in the DOX group and DOX+NC group. Compared with the DOX group and DOX+NC group, the percentage of myocardial fibrotic area was significantly decreased in the DOX+TERT group (%: 2.33±0.06 vs. 3.76±0.07, 3.87±0.06, both P < 0.05), and the LVEF and FS were significantly increased [LVEF (%): 67.00±1.14 vs. 54.60±1.57, 53.40±2.18; FS (%): 38.60±0.51 vs. 30.60±1.10, 30.00±0.71; all P < 0.05]. CONCLUSION Up-regulation of TERT expression can inhibit DOX-induced cardiomyocyte autophagy and apoptosis, attenuate DOX-induced myocardial fibrosis in mice, improve cardiac function, and thus alleviate DOX-induced cardiotoxicity.
Animals ; Doxorubicin/toxicity* ; Telomerase/metabolism* ; Myocytes, Cardiac/metabolism* ; Rats ; Male ; Cardiotoxicity ; Mice, Inbred C57BL ; Mice ; Membrane Potential, Mitochondrial ; Adenoviridae ; bcl-2-Associated X Protein/metabolism* ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Transfection ; Apoptosis

Sepsis is a life-threatening organ dysfunction caused by the host's dysregulated response to infection, with a complex pathogenesis and high mortality rate. Currently, there are no clear and effective treatment drugs available. Epigenetic modification serves as a major mechanism regulating gene expression under pathological and physiological conditions, and it has been shown to play a critical role in regulating the occurrence and development of sepsis. Histone acetylation modification, as a sophisticated epigenetic modification mechanism, plays a crucial regulatory role in many aspects of life. It can jointly regulate the acetylation status of histones through histone acetyltransferase (HAT) and histone deacetylase (HDAC), thereby changing DNA expression and dynamically regulating sepsis related gene expression at the epigenetic level. Previous studies have shown that histone acetylation can participate in the progression of sepsis by regulating inflammatory mediators, nuclear factor-ΚB (NF-ΚB) signaling pathway, autophagy, efferocytosis, ferroptosis, pyroptosis. These mechanisms are promising targets for novel sepsis treatments. In addition, with the deepening of research, it has been found that various selective/non selective histone deacetylase inhibitors (HDACI) can regulate histone acetylation status by acting on different HDAC targets, which has been shown to alleviate organ damage caused by sepsis and improve prognosis in septic animal models. This article further summarizes the role and potential applications of histone acetylation in sepsis, providing new ideas for the treatment of sepsis.
Sepsis/metabolism* ; Acetylation ; Humans ; Histones/metabolism* ; Histone Acetyltransferases/metabolism* ; Histone Deacetylase Inhibitors ; Epigenesis, Genetic ; Histone Deacetylases/metabolism* ; Signal Transduction ; NF-kappa B/metabolism* ; Animals

With the continuous development of messenger RNA (mRNA) technology, mRNA-based drugs have shown broad application prospects in recent years. Since mRNA is easy to be degraded and difficult to enter cells directly, the mRNA delivery vectors have always been one of the focuses in the development of mRNA-based drugs. Although lipid nanoparticles (LNPs) have been widely used for the delivery of mRNA, they tend to accumulate in the liver, and repeated administration can easily induce inflammatory response which leads to tissue damage. Compared with LNPs, virus-like particles (VLPs) have the advantages of high biocompatibility and safety, being expected to offer new solutions for mRNA delivery. Based on the practical application requirements, this review summarized the research progress in VLPs according to the mRNA delivery steps: particle assembly, delivery into cells, and intracellular release. We hope to provide a basis and design ideas for the development of new VLPs as delivery vectors, promote the application of VLPs in mRNA delivery, and provide new possibilities for the research and application of mRNA-based therapeutics.
RNA, Messenger/administration & dosage* ; Humans ; Nanoparticles/chemistry* ; Genetic Vectors ; Lipids/chemistry* ; Drug Delivery Systems/methods* ; Virion ; Animals ; Gene Transfer Techniques ; Liposomes

The CRISPR/dCas9 system is a gene editing tool that has proven to be highly efficient and precise. By utilizing transcriptional activators, such as VP64, p65, and Rta, the system can effectively and stably activate target genes. Sox17, a transcription factor belonging to the SOX family, plays a crucial role in the differentiation of the germ layers and the determination of cell fates during the early stages of embryonic development. Sheep embryonic stem cells (sESCs) are characterized by their capacity for self-renewal and multidirectional differentiation, serving as a significant in vitro model for studying the mechanisms of cell differentiation during early embryonic development. However, the importing of exogenous genes into sESCs is challenging due to their unique growth characteristics. The objective of this study was to investigate the conditions necessary for successfully activating Sox17 in sESCs. To this end, we employed the CRISPR/dCas9 system along with liposome transfection, lentivirus invasion, and electroporation to activate Sox17 in sESCs. The expression of Sox17 was then determined by fluorescence quantitative PCR, on the basis of which the performance of different transfection methods was compared. The results indicated that the electroporation group had the best transfection effect and the highest Sox17 expression among the three transfection methods. The efficient and stable gene activation protocol will provide a reference for embryonic stem cell research in other species, especially livestock animals, and lay the foundation for the subsequent study of gene function and realization of precise cell fate regulation by regulating gene expression in sheep embryonic stem cells.
Animals ; CRISPR-Cas Systems/genetics* ; Sheep ; SOXF Transcription Factors/genetics* ; Embryonic Stem Cells/cytology* ; Genetic Vectors/genetics* ; Cell Differentiation/genetics* ; Transfection ; Gene Editing/methods*