MiR-26b-5p/TET3 regulates the osteogenic differentiation of human bone mesenchymal stem cells and bone reconstruction in female rats with calvarial defects.

BACKGROUND: MicroRNAs (miRNAs) play critical roles in the osteogenic differentiation of human bone mesenchymal stem cells (hBMSCs), but the mechanism by which miRNAs indirectly modulate osteogenesis remains unclear. Here, we explored the mechanism by which miRNAs indirectly modulate gene expression through histone demethylases to promote bone regeneration. METHODS AND RESULTS: Bioinformatics analysis was performed on hBMSCs after 7 days of osteogenic induction. The differentially expressed miRNAs were screened, and potential target mRNAs were identified. To determine the bioactivity and stemness of hBMSCs and their potential for bone repair, we performed wound healing, Cell Counting Kit-8 (CCK-8), real-time reverse transcription quantitative polymerase chain reaction (RT‒qPCR), alkaline phosphatase activity, alizarin red S (ARS) staining and radiological and histological analyses on SD rats with calvarial bone defects. Additionally, a dual-luciferase reporter assay was utilized to investigate the interaction between miR-26b-5p and ten-eleven translocation 3 (TET3) in human embryonic kidney 293T cells. The in vitro and in vivo results suggested that miR-26b-5p effectively promoted the migration, proliferation and osteogenic differentiation of hBMSCs, as well as the bone reconstruction of calvarial defects in SD rats. Mechanistically, miR-26b-5p bound to the 3' untranslated region of TET3 mRNA to mediate gene silencing. CONCLUSIONS: MiR-26b-5p downregulated the expression of TET3 to increase the osteogenic differentiation of hBMSCs and bone repair in rat calvarial defects. MiR-26b-5p/TET3 crosstalk might be useful in large-scale critical bone defects.

Graphene therapy-related lncRNAs as prognostic and immune microenvironmental biomarkers in hepatocellular carcinoma.

BACKGROUND: Graphene materials have the capacity to influence the tumor microenvironment and intracellular signaling responsiveness. However, the process of graphene-assisted liver cancer treatment still lacks specific biomarkers for assessing its efficacy. METHODS: We identified graphene therapy-related lncRNAs (GTLncRNAs) through gene analysis and correlation tests. Multivariate COX and LASSO regression analyses yielded significant lncRNAs for a risk score model. We evaluated clinicopathological factors and tumor microenvironment using ssGSEA. We scrutinized the pathways of immune function, the evasion of tumor immunity, and the potential for immunotherapy. GTLncRNAs with differential expression were subjected to GO/KEGG analysis, and prospective chemotherapy drugs were discerned utilizing the pRRophetic algorithm. The prognostic model was authenticated through the examination of the Imvigor210 cohort, and an analysis of mRNA stemness was executed. RESULTS: The researchers constructed a prognostic model based on 22 graphene therapy-related lncRNAs. Protective lncRNAs (AC010280.2, AL365361.1, and LINC01549) and negative lncRNAs (AC026412.3, AL031985.3, ELFN1-AS1, SNHG4, and EB2-AS1) were identified. Higher risk scores correlated with shorter survival. Low-risk immune pathways included Type_II_IFN_Reponse and cytolytic_activity. Subgroups differed significantly in TMB, TIDE, MDSC, exclusion, and dysfunction. Low TMB values correlated with longer survival. The high-risk subgroup showed increased sensitivity to screened compounds, and mRNAsi was higher in cancer tissue. CONCLUSIONS: Our GTLncRNAs-based model accurately predicted survival of HCC patients and underscored the influence of graphene therapy-related genes on the tumor microenvironment. Potential treatment compounds were identified, and the mRNAsi index demonstrated prognostic value.

MLL1 inhibits the neurogenic potential of SCAPs by interacting with WDR5 and repressing HES1.

Mesenchymal stem cell (MSC)-based therapy has emerged as a promising treatment for spinal cord injury (SCI), but improving the neurogenic potential of MSCs remains a challenge. Mixed lineage leukemia 1 (MLL1), an H3K4me3 methyltransferases, plays a critical role in regulating lineage-specific gene expression and influences neurogenesis. In this study, we investigated the role and mechanism of MLL1 in the neurogenesis of stem cells from apical papilla (SCAPs). We examined the expression of neural markers, and the nerve repair and regeneration ability of SCAPs using dynamic changes in neuron-like cells, immunofluorescence staining, and a SCI model. We employed a coimmunoprecipitation (Co-IP) assay, real-time RT-PCR, microarray analysis, and chromatin immunoprecipitation (ChIP) assay to investigate the molecular mechanism. The results showed that MLL1 knock-down increased the expression of neural markers, including neurogenic differentiation factor (NeuroD), neural cell adhesion molecule (NCAM), tyrosine hydroxylase (TH), ßIII-tubulin and Nestin, and promoted neuron-like cell formation in SCAPs. In vivo, a transplantation experiment showed that depletion of MLL 1 in SCAPs can restore motor function in a rat SCI model. MLL1 can combine with WD repeat domain 5 (WDR5) and WDR5 inhibit the expression of neural markers in SCAPs. MLL1 regulates Hairy and enhancer of split 1 (HES1) expression by directly binds to HES1 promoters via regulating H3K4me3 methylation by interacting with WDR5. Additionally, HES1 enhances the expression of neural markers in SCAPs. Our findings demonstrate that MLL1 inhibits the neurogenic potential of SCAPs by interacting with WDR5 and repressing HES1. These results provide a potential therapeutic target for promoting the recovery of motor function in SCI patients.

tRNA-derived small RNA changes in bone marrow stem cells under hypoxia and osteogenic conduction.

BACKGROUND: Tissue engineering using bone mesenchymal stem cells (BMSCs) transplantation is a promising therapeutic for bone regeneration. However, the effect of bone regeneration remains unsatisfactory due to the BMSCs' functional abnormality influenced by hypoxia. In this study, we attempt to explore the mechanism of osteogenic differentiation of BMSCs under hypoxic conditions from the perspective of non-coding RNA regulation. METHODS: The study employed BMSCs obtained from healthy donors and simulated hypoxia using CoCl2 stimulation. High-throughput sequencing technique was used to identify differential expression profiles of tRNA-derived small RNA (tsRNA) in three experimental groups: BMSCs-0d, BMSCs-7d and BMSCs-0d-CoCl2 . TargetScan and miRanda algorithms were used to determine tsRNA target genes, while Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis were employed for the prediction of biological functions. Real-time reverse transcriptase-polymerase chain reaction (Real-time RT-PCR) was carried out on four selected differentially expressed tsRNAs. RESULTS: After the osteogenic induction and CoCl2 stimulated separately, there were 19 tsRNAs differentially expressed in BMSCs, including 14 upregulated and five downregulated. According to the analysis of biological information, these tsRNAs may regulate 311 potential target genes and mainly enrich the pathways such as metabolic pathways, Wnt signalling pathway, osteoclast differentiation, cellular senescence and mTOR signalling pathway. The results of Real-time RT-PCR for 3'tiRNA-41-GlnTTG-6, 3'tiRNA-42-LysTTT-8, 5'tiRNA-35-CysACA-1 and tRF3a-AsnGTT-9 were consistent with small RNA sequencing data. CONCLUSION: We discovered the tsRNA that changes the process of osteogenesis and hypoxia, which provides new targets for promoting survival and regeneration functions after BMSCs transplantation.

KDM6B Negatively Regulates the Neurogenesis Potential of Apical Papilla Stem Cells via HES1.

Stem cells from the apical papilla (SCAPs) are used to regulate the microenvironment of nerve defects. KDM6B, which functions as an H3K27me3 demethylase, is known to play a crucial role in neurogenesis. However, the mechanism by which KDM6B influences the neurogenesis potential of SCAPs remains unclear. We evaluated the expression of neural markers in SCAPs by using real-time RT-PCR and immunofluorescence staining. To assess the effectiveness of SCAP transplantation in the SCI model, we used the BBB scale to evaluate motor function. Additionally, toluidine blue staining and Immunofluorescence staining of NCAM, NEFM, ß-III-tubulin, and Nestin were used to assess nerve tissue remodeling. Further analysis was conducted through Microarray analysis and ChIP assay to study the molecular mechanisms. Our results show that KDM6B inhibits the expression of NeuroD, TH, ß-III tubulin, and Nestin. In vivo studies indicate that the SCAP-KDM6Bsh group is highly effective in restoring spinal cord structure and motor function in rats suffering from SCI. Our findings suggest that KDM6B directly binds to the HES1 promoter via regulating H3K27me3 and HES1 expression. In conclusion, our study can help understand the regulatory role of KDM6B in neurogenesis and provide more effective treatments for nerve injury.

Research progress of functional motifs based on growth factors in cartilage tissue engineering: A review.

Osteoarthritis is a chronic degenerative joint disease that exerts significant impacts on personal life quality, and cartilage tissue engineering is a practical treatment in clinical. Various growth factors are involved in cartilage regeneration and play important roles therein, which is the focus of current cartilage repair strategy. To compensate for the purification difficulty, high cost, poor metabolic stability, and circulating dilution of natural growth factors, the concept of functional motifs (also known as mimetic peptides) from original growth factor was introduced in recent studies. Here, we reviewed the selection mechanisms, biological functions, carrier scaffolds, and modification methods of growth factor-related functional motifs, and evaluated the repair performance in cartilage tissue engineering. Finally, the prospects of functional motifs in researches and clinical application were discussed.

Statistical analysis of parameters for the ramus, teeth, and biomechanical stress: A retrospective study.

OBJECTIVE: To explore the correlation between the anterior border absorption of the mandibular ramus and the occurrence of supernumerary teeth, along with the biomechanical stress. DESIGN: The bone mass of mandibular ramus was evaluated on CBCT images by bone mass analysis. In addition, the number of supernumerary teeth and corresponding anatomical positions were recorded. RESULTS: The CBCT images of 5100 patients were analyzed. Along with eruption of mandibular permanent molars in sequence, the width of the ramus increased from 29.02 mm to 36.02 mm (P < 0.001). The proportion of the width from the nerve canal to the anterior border of the ramus in the whole ramus decreased from 52.51% to 43.79% (P < 0.001). Meanwhile, among the 5100 participants, 278 supernumerary teeth were identified in 209 patients (4.1%). The predilection loci were close to the regions of central incisors (72.66%) in the maxilla and of the premolars (11.51%) in the mandible. CONCLUSIONS: Biomechanical stress, mainly pressure during tooth development, was the internal force of anterior border absorption of the mandibular ramus. The release of pressure after tooth eruption is a possible reason for the occurrence of supernumerary teeth.

Identified Gefitinib Metabolism-Related lncRNAs can be Applied to Predict Prognosis, Tumor Microenvironment, and Drug Sensitivity in Non-Small Cell Lung Cancer.

Gefitinib has shown promising efficacy in the treatment of patients with locally advanced or metastatic EGFR-mutated non-small cell lung cancer (NSCLC). Molecular biomarkers for gefitinib metabolism-related lncRNAs have not yet been elucidated. Here, we downloaded relevant genes and matched them to relevant lncRNAs. We then used univariate, LASSO, and multivariate regression to screen for significant genes to construct prognostic models. We investigated TME and drug sensitivity by risk score data. All lncRNAs with differential expression were selected for GO/KEGG analysis. Imvigor210 cohort was used to validate the value of the prognostic model. Finally, we performed a stemness indices difference analysis. lncRNA-constructed prognostic models were significant in the high-risk and low-risk subgroups. Immune pathways were identified in both groups at low risk. The higher the risk score the greater the value of exclusion, MDSC, and CAF. PRRophetic algorithm screened a total of 58 compounds. In conclusion, the prognostic model we constructed can accurately predict OS in NSCLC patients. Two groups of low-risk immune pathways are beneficial to patients. Gefitinib metabolism was again validated to be related to cytochrome P450 and lipid metabolism. Finally, drugs that might be used to treat NSCLC patients were screened.

The Mechanisms of Ferroptosis and the Applications in Tumor Treatment: Enemies or Friends?

Ferroptosis, as a newly discovered non-apoptotic cell death mode, is beginning to be explored in different cancer. The particularity of ferroptosis lies in the accumulation of iron dependence and lipid peroxides, and it is different from the classical cell death modes such as apoptosis and necrosis in terms of action mode, biochemical characteristics, and genetics. The mechanism of ferroptosis can be divided into many different pathways, so it is particularly important to identify the key sites of ferroptosis in the disease. Herein, based on ferroptosis, we analyze the main pathways in detail. More importantly, ferroptosis is linked to the development of different systems of the tumor, providing personalized plans for the examination, treatment, and prognosis of cancer patients. Although some mechanisms and side effects of ferroptosis still need to be studied, it is still a promising method for cancer treatment.

3D Printing of PLLA/Biomineral Composite Bone Tissue Engineering Scaffolds.

Tissue engineering is one of the most effective ways to treat bone defects in recent years. However, current highly active bone tissue engineering (BTE) scaffolds are mainly based on the addition of active biological components (such as growth factors) to promote bone repair. High cost, easy inactivation and complex regulatory requirements greatly limit their practical applications. In addition, conventional fabrication methods make it difficult to meet the needs of personalized customization for the macroscopic and internal structure of tissue engineering scaffolds. Herein, this paper proposes to select five natural biominerals (eggshell, pearl, turtle shell, degelatinated deer antler and cuttlebone) with widely available sources, low price and potential osteo-inductive activity as functional particles. Subsequently compounding them into L-polylactic acid (PLLA) biomaterial ink to further explore 3D printing processes of the composite scaffold, and reveal their potential as biomimetic 3D scaffolds for bone tissue repair. The research results of this project provide a new idea for the construction of a 3D scaffold with growth-factor-free biomimetic structure, personalized customization ability and osteo-inductive activity.

Articular cartilage reconstruction with TGF-ß1-simulating self-assembling peptide hydrogel-based composite scaffold.

Transforming growth factor-ß (TGF-ß) is an important inducing factor for the differentiation of mesenchymal stem cells and the secretion of collagen II, but the inaccessibility and instability limit its application in clinical practice. In this study, the TGF-ß1-simulating peptide LIANAK (CM) was connected with the self-assembling peptide Ac-(RADA)4-CONH2 (RAD) to obtain the functionalized self-assembling peptide Ac-(RADA)4-GG-LIANAK-CONH2 (RAD-CM). The results indicated that the CM-functionalized RAD hydrogel contributed to the enhanced expressions of chondrogenic genes and extracellular matrix deposition. The self-assembling peptides were then combined with decellularized cartilage extracellular matrix (DCM) to construct a composite scaffold for articular cartilage repair. The CM-functionalized composite scaffold RAD/RAD-CM/DCM (R/C/D) exhibited good bioactivity and structural stability and exhibited satisfactory performance in promoting neocartilage restoration and the reconstruction of the osteochondral unit. This study provides a promising strategy for in situ cartilage regeneration via the stable presentation of TGF-ß1-simulating peptide. STATEMENT OF SIGNIFICANCE: Deficiency of effective chondrogenic inducers (especially, the TGF-ß family) significantly limits the self-regeneration of cartilage in osteochondral defect cases. Oligopeptide LIANAK, named CM, could simulate TGF-ß1's bioactivity with particular structure, but traditional chemical crosslinking to polymer scaffolds resulted in risks of safety and complication, which is unfavorable for clinical applications. Here, self-assembling peptide RAD was used to load CM, to obtain a TGF-ß1 mimetic peptide hydrogel. Depending on the homology (amino acids) of RAD and CM, the synthesis of the whole peptide only needs simply extended sequences of CM following that of RAD by automated solid-phase peptide synthesis. The modified peptide effectively demonstrated osteochondrogenic bioactivity, ensured the convenience, safety, and mass production, which displayed great potential in tissue engineering research and translational medicine.

Stem cell-homing hydrogel-based miR-29b-5p delivery promotes cartilage regeneration by suppressing senescence in an osteoarthritis rat model.

Osteoarthritis (OA) is a common joint disease characterized by progressive loss of cartilage and reduction in lubricating synovial fluid, which lacks effective treatments currently. Here, we propose a hydrogel-based miRNA delivery strategy to rejuvenate impaired cartilage by creating a regenerative microenvironment to mitigate chondrocyte senescence that mainly contributes to cartilage breakdown during OA development. An aging-related miRNA, miR-29b-5p, was first found to be markedly down-regulated in OA cartilage, and their up-regulation suppressed the expression of matrix metalloproteinases and senescence-associated genes (P16INK4a/P21) via ten-eleven-translocation enzyme 1 (TET1). An injectable bioactive self-assembling peptide nanofiber hydrogel was applied to deliver agomir-29b-5p, which was functionalized by conjugating a stem cell-homing peptide SKPPGTSS for endogenous synovial stem cell recruitment simultaneously. Sustained miR-29b-5p delivery and recruitment of synovial stem cells and their subsequent differentiation into chondrocytes led to successful cartilage repair and chondrocyte rejuvenation. This strategy enables miRNA-based therapeutic modality to become a viable alternative for surgery in OA treatment.

LncRNA LINC01303 Promotes the Progression of Oral Squamous Cell Carcinomas via the miR-429/ZEB1/EMT Axis.

OBJECTIVES: The aim of this research was to uncover the biological role and mechanisms of LINC01303 in oral squamous cell carcinoma (OSCC). MATERIALS AND METHODS: Real-time quantitative PCR (qRT-PCR) was used to determine LINC01303 expression in OSCC tissues. Subcellular distribution of LINC01303 was examined by nuclear/cytoplasmic RNA fractionation and FISH experiments. The role of LINC01303 in the growth of TSCCA and SCC-25 was examined by CCK-8 assay, colony formation, transwell invasion assay in vitro, and xenograft tumor experiment in vivo. Dual-luciferase reporter assay was used to verify the interaction between LINC01303 and miR-429. RNA pull-down assay was used to discover miR-429-interacted protein, which was further examined by qRT-PCR, western blot, and rescue experiments. RESULTS: LINC01303 expression was higher in OSCC tissues compared with adjacent nontumor tissues. LINC01303 was found to be localized in the cytoplasm of OSCC cells. Knockdown of LINC01303 inhibited OSCC cell proliferation and invasion, whereas increasing the expression of LINC01303 showed the opposite effects. Furthermore, LINC01303 served as a miR-429 "sponge" and positively regulated ZEB1 expression. Moreover, LINC01303 promoted OSCC through miR-429/ZEB1 axis both in vivo and in vitro. CONCLUSIONS: LINC01303 plays an oncogenic role in OSCC and is a promising biomarker for OSCC patients.

Hierarchically electrospraying a PLGA@chitosan sphere-in-sphere composite microsphere for multi-drug-controlled release.

Sequential administration and controlled release of different drugs are of vital importance for regulating cellular behaviors and tissue regeneration, which usually demands appropriate carriers like microspheres (MS) to control drugs releases. Electrospray has been proven an effective technique to prepare MS with uniform particle size and high drug-loading rate. In this study, we applied electrospray to simply and hierarchically fabricate sphere-in-sphere composite microspheres, with smaller poly(lactic-co-glycolic acid) MS (∼8-10 µm in diameter) embedded in a larger chitosan MS (∼250-300 µm in diameter). The scanning electron microscopy images revealed highly uniform MS that can be accurately controlled by adjusting the nozzle diameter or voltage. Two kinds of model drugs, bovine serum albumin and chlorhexidine acetate, were encapsulated in the microspheres. The fluorescence-labeled rhodamine-fluoresceine isothiocyanate (Rho-FITC) and ultraviolet (UV) spectrophotometry results suggested that loaded drugs got excellent distribution in microspheres, as well as sustained, slow release in vitro. In addition, far-UV circular dichroism and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) results indicated original secondary structure and molecular weight of drugs after electrospraying. Generally speaking, our research proposed a modified hierarchically electrospraying technique to prepare sphere-in-sphere composite MS with two different drugs loaded, which could be applied in sequential, multi-modality therapy.

Corrigendum to: Hierarchically electrospraying a PLGA@chitosan sphere-in-sphere composite microsphere for multi-drug-controlled release.

[This corrects the article DOI: 10.1093/rb/rbaa009.][This corrects the article DOI: 10.1093/rb/rbaa009.].