Noncoding RNAs in skeletal development and disorders.

Protein-encoding genes only constitute less than 2% of total human genomic sequences, and 98% of genetic information was previously referred to as "junk DNA". Meanwhile, non-coding RNAs (ncRNAs) consist of approximately 60% of the transcriptional output of human cells. Thousands of ncRNAs have been identified in recent decades, and their essential roles in the regulation of gene expression in diverse cellular pathways associated with fundamental cell processes, including proliferation, differentiation, apoptosis, and metabolism, have been extensively investigated. Furthermore, the gene regulation networks they form modulate gene expression in normal development and under pathological conditions. In this review, we integrate current information about the classification, biogenesis, and function of ncRNAs and how these ncRNAs support skeletal development through their regulation of critical genes and signaling pathways in vivo. We also summarize the updated knowledge of ncRNAs involved in common skeletal diseases and disorders, including but not limited to osteoporosis, osteoarthritis, rheumatoid arthritis, scoliosis, and intervertebral disc degeneration, by highlighting their roles established from in vivo, in vitro, and ex vivo studies.

Epigenetic regulations of cellular senescence in osteoporosis.

Osteoporosis (OP) is a prevalent age-related disease that is characterized by a decrease in bone mineral density (BMD) and systemic bone microarchitectural disorders. With age, senescent cells accumulate and exhibit the senescence-associated secretory phenotype (SASP) in bone tissue, leading to the imbalance of bone homeostasis, osteopenia, changes in trabecular bone structure, and increased bone fragility. Cellular senescence in the bone microenvironment involves osteoblasts, osteoclasts, and bone marrow mesenchymal stem cells (BMSCs), whose effects on bone homeostasis are regulated by epigenetics. Therefore, the epigenetic regulatory mechanisms of cellular senescence have received considerable attention as potential targets for preventing and treating osteoporosis. In this paper, we systematically review the mechanisms of aging-associated epigenetic regulation in osteoporosis, emphasizing the impact of epigenetics on cellular senescence, and summarize three current methods of targeting cellular senescence, which is helpful better to understand the pathogenic mechanisms of cellular senescence in osteoporosis and provides strategies for the development of epigenetic drugs for the treatment of osteoporosis.

Cirsiliol induces autophagy and mitochondrial apoptosis through the AKT/FOXO1 axis and influences methotrexate resistance in osteosarcoma.

BACKGROUND: Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents, with poor outcomes for patients with metastatic disease or chemotherapy resistance. Cirsiliol is a recently found flavonoid with anti-tumor effects in various tumors. However, the effects of cirsiliol in the regulation of aggressive behaviors of OS remain unknown. METHODS: The effect of cirsiliol on the proliferation of OS cells was detected using a cell counting kit-8 (CCK-8) assay and 5-ethynyl-2'-deoxyuridine (EdU) staining, while cell apoptosis was detected using flow cytometry. Immunofluorescence was applied to visualize the expression level of the mitochondria, lysosomes and microtubule-associated protein light chain 3 (LC3). A computational molecular docking technique was used to predict the interaction between cirsiliol and the AKT protein. The impact of cirsiliol on resistance was investigated by comparing it between a methotrexate (MTX)-sensitive OS cell line, U2OS, and a MTX-resistant OS cell line, U2OS/MTX. Finally, in situ xenogeneic tumor models were used to validate the anti-tumor effect of cirsiliol in OS. RESULTS: Cirsiliol inhibited cell proliferation and induced apoptosis in both U2OS and U2OS/MTX300 OS cells. In addition, treatment with cirsiliol resulted in G2 phase arrest in U2OS/MTX300 and U2OS cells. Cell fluorescence probe staining results showed impaired mitochondria and increased autophagy in OS cells after treatment with cirsiliol. Mechanistically, it was found that cirsiliol targeted AKT by reducing the phosphorylation of AKT, which further activated the transcriptional activity of forkhead Box O transcription factor 1 (FOXO1), ultimately affecting the function of OS cells. Moreover, in situ tumorigenesis experiments showed that cirsiliol inhibited the tumorigenesis and progression of OS in vivo. CONCLUSIONS: Cirsiliol inhibits OS cell growth and induces cell apoptosis by reducing AKT phosphorylation and further promotes FOXO1 expression. These phenomena indicate that cirsiliol is a promising treatment option for OS.

Brief research report: Effects of Pinch deficiency on cartilage homeostasis in adult mice.

Pinch1 and Pinch2 are LIM domain-containing proteins with crucial functions in mediating focal adhesion formation. Our previous studies have demonstrated that Pinch1/2 expression is essential for cartilage and bone formation during skeletal development in mice. Loss of Pinch expression (Prx1Cre; Pinch1flox/flox; Pinch2-/-) inhibits chondrocyte proliferation and promotes chondrocyte apoptosis, resulting in severe chondrodysplasia and limb shortening. Based on these observations, we wonder if Pinch proteins have a role in adult cartilage and whether Pinch deficiency will compromise cartilage homeostasis and promote osteoarthritis (OA)-related defects in adult mice. To this end, we generated the AggrecanCreERT2; Pinch1flox/flox; Pinch2-/- mice, in which the Pinch1 gene can be inducibly deleted in aggrecan-expressing chondrocytes by tamoxifen and the Pinch2 gene is globally inactivated. Immunofluorescent staining confirmed that the expression of Pinch proteins was significantly decreased in articular cartilage in tamoxifen-treated adult AggrecanCreERT2; Pinch1flox/flox; Pinch2-/- mice. Unexpectedly, our results showed that Pinch loss did not induce marked abnormalities in articular cartilage and other joint tissues in the knee joints of either adult (10-month-old) mice or aged (17-month-old) mice. In a destabilization of the medial meniscus (DMM)-induced OA model, the surgically-induced OA lesions were comparable between Pinch-deficient mice and control mice. Given the fact that Pinch proteins are essential for chondrogenesis and cartilage formation during skeletal development, these findings suggest that Pinch expression is seemingly not indispensable for adult cartilage homeostasis in mice.

Effective Skill Transfer From Fundamentals of Arthroscopic Surgery Training to Shoulder Arthroscopic Simulator in Novices.

PURPOSE: To investigate whether novices could improve performance on a shoulder arthroscopic simulator (high-fidelity) through short-term training on a Fundamentals of Arthroscopic Surgery Training (FAST) simulator (low-fidelity). METHODS: Twenty-eight novices with no experience in arthroscopy were recruited to perform a pre-test on a shoulder arthroscopic simulator. Then they were randomized into two groups: the experimental group practiced five modules on the FAST simulator three times, and the control group did nothing. The experimental group performed a post-test immediately after FAST simulator practice. Control group rested for 70 minutes after experiencing pre-test before performing post-test. All parameters were recorded by the simulator. RESULTS: The experimental group outperformed the control group in terms of total score, procedure time, camera path length, and grasper path length. However, there was no statistical difference in scratching of humerus cartilage or glenoid cartilage. Significant differences were found in the improvement of both groups in total score, procedure time, and camera path length. CONCLUSIONS: Arthroscopic skills gained after short-term training on FAST simulator could be transferred to the shoulder arthroscopic simulator. This research provides important evidence of the benefits of FAST simulator in shoulder arthroscopy training program.

Interleukin-1 induces receptor activator of nuclear factor-κB ligand-independent osteoclast differentiation in RAW264.7 cells.

Interleukin-1 (IL-1) is a pro-inflammatory cytokine which induces bone destruction in various diseases, such as osteoporosis and rheumatoid arthritis. RAW264.7 cells are frequently used in studies as osteoclast precursors, however it remains unclear whether IL-1 can induce osteoclast differentiation from RAW264.7 cells without the stimulation of receptor activator of nuclear factor-κB ligand (RANKL). Hence, the present study aimed to investigate the effects of IL-1 on the formation of osteoclasts from RAW264.7 cells. The cell viability was determined via the Cell Counting Kit-8 (CCK-8) assay. Protein and gene expression were measured by western blotting and reverse transcription-quantitative PCR, respectively. Tartrate-resistant acid phosphatase (TRAP) staining and the resorption pit assay were performed to determine the formation and activity of osteoclasts. A significantly increased quantity of osteoclasts were found in the IL-1 group compared with the control group, and also in the RANKL+IL-1 group compared with the RANKL group. In addition IL-1 significantly increased both the protein and mRNA expression of specific genes associated with osteoclastogenesis, including nuclear factor of activated T cells cytoplasmic 1, matrix metalloprotein-9, cathepsin K and TRAP. The findings of the present study suggested that IL-1 can induce osteoclast differentiation and upregulate the quantity of osteoclasts differentiated from RAW264.7 cells. These results may lay a foundation for further study of diseases involving inflammation-associated bone loss. The combined blockade of IL-1 and RANKL may be effective for the prevention of inflammatory bone loss.