Purpose. To investigate the DNMT3A/miR-149/NOTCH1/Hedgehog axis regulating the development of osteosarcoma. Methods. First, microRNA and mRNA expression microarrays were downloaded from the GEO database for osteosarcoma and differentially expressed microRNAs were analyzed. Subsequently, we collected cancerous tissues and corresponding paracancerous tissues from 42 osteosarcoma patients and examined the expression levels of miR-149, DNMT3A, and NOTCH1 in the samples. Subsequently, miR-149 was overexpressed in osteosarcoma cells to detect cell proliferation and metastatic ability changes. We then queried the methylation level of the miR-149 promoter on the bioinformatics website and verified it by experiment. We further demonstrated the expression level of miR-149 with NOTCH1 using a dual luciferase assay and confirmed the role of NOTCH1 on osteosarcoma cell growth and metastasis by functional rescue assay. Finally, we detected the activation level of the Hedgehog/catenin signaling pathway by WB and immunofluorescence. Results. miR-149 was significantly low expressed in osteosarcoma tissues and cells, while DNMT3A and NOTCH1 were highly expressed in osteosarcoma tissues and cells, and negatively correlated with miR-149 expression levels. Overexpression of miR-149 significantly inhibited the growth and metastasis of osteosarcoma cells in vitro and in vivo, and we found that DNMT3A could promote the methylation modification of the miR-149 promoter, thereby inhibiting the expression of miR-149. Subsequently, the experimental results showed that miR-149 could target negative regulation of NOTCH1, and further overexpression of NOTCH1 in cells with high miR-149 expression could promote the growth and metastasis of osteosarcoma cells in vitro. Conclusion. The methyltransferase DNMT3A suppresses miR-149 expression by promoting methylation modification of the miR-149 promoter, resulting in elevated expression levels of NOTCH1 in cells, therefore exacerbating activation of the Hedgehog signaling pathway and therefore exacerbating the development and progression of osteosarcoma.

To investigate the regulatory mechanisms and effects of LIM and SH3 protein 1 (LASP1) on osteoarthritis (OA). IL‐1β was used to induce OA in cell models. Viability and apoptosis of chondrocytes were assessed. The expressions of tumor necrsis factor‐α (TNF‐α) and IL‐6 were measured by ELISA kit, and Quantitative reverse transcription polymerase chain reaction (qRT‐PCR) and Western blot were performed to test the expression of related proteins. The STRING database was used to predict the relationship between LASP1 and DNA methyltransferase 1 (DNMT1). The tight junction protein 2 (TJP2) and Gene Expression Omnibus data were analyzed for differential OA genes. Methylation‐specific PCR detected methylation of the TJP2 promoter region, and chromatin immunoprecipitation detected the enrichment of DNMT1 in the TJP2 promoter region. Safranin O‐Fast Green staining and hematoxylin and eosin staining were used to determine the OARSI score and evaluate the pathological conditions of the joint tissues. LASP1 was highly expressed in IL‐1β‐induced cell models. Silencing of LASP1 promoted chondrocyte proliferation and expression of Collagen II and Aggrecan and inhibited chondrocyte apoptosis, inflammatory factors, and matrix metalloprotein expression. TJP2 is weakly expressed in OA models, and LASP1 promotes methylation of the TJP2 promoter region by interacting with DNMT1. Silencing of LASP1 attenuated IL‐1β‐induced chondrocyte degeneration by promoting TJP2 expression. Similarly, silencing LASP1 promotes TJP2 expression to alleviate articular cartilage injury in mice with OA. Silencing of LASP1 inhibited the methylation of the TJP2 promoter region by interacting with DNMT1, thereby alleviating articular cartilage damage in OA mice.

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