The IFT80/Hedgehog Pathway Regulates the Osteogenic-adipogenic Differentiation of Bone Marrow Mesenchymal Stem Cells.

BACKGROUND: Steroid-induced avascular necrosis of the femoral head (SANFH) is a typical refractory disease that often progresses irreversibly and has a high disability rate. Numerous studies have confirmed that abnormal osteogenic-adipogenic differentiation of bone marrow mesenchymal stem cells (BM-MSCs) is one of the major factors of SANFH. However, the mechanism remains to be elucidated. OBJECTIVES: This study aimed to investigate the mechanism and effect of the IFT80/Hedgehog-mediated osteogenic-adipogenic differentiation of BM-MSCs in SANFH. METHODS: Femoral head specimens of SANFH patients and femoral neck fractures (FNF) patients were collected to detect the expression of IFT80, Shh and osteogenic-adipogenic differentiation-related genes by immunohistochemistry (IHC), western blot (WB) and Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR). Based on the rabbit SANFH model, the mRNA expression and protein level of IFT80 and Shh were detected by RT-qPCR and WB. After the osteogenic/adipogenic differentiation based on rabbit BM-MSCs, the IFT80, Gli1, PPAR-γ, and Runx2 expression were detected. Differences in alkaline phosphodiesterase activity, calcium nodule, quantification/distribution of lipid droplets, expression of IFT80/Hedgehog axis, and the level of osteogenic- adipogenic associated factors were determined after IFT80 overexpression. RESULTS: RT-qPCR, WB and IHC revealed that IFT80 and Shh lowly expressed in the osteoblasts and intra-trabecular osteocytes at the edge of trabeculae and in the intercellular matrix of the bone marrow lumen in the SANFH specimens. The Runx2 expression was low, while the PPAR-γ expression was high in both human specimens and animal models of SANFH, suggesting that the balance of osteogenic-adipogenic differentiation was dysregulated. Rabbit BM-MSCs with stable overexpression of IFT80 showed increased alkaline phosphatase activity after induction of osteogenic differentiation, increased calcium nodule production, and decreased adipogenesis after induction of adipogenic differentiation. CONCLUSION: There is a dysregulation of the balance of osteogenic-adipogenic differentiation in SANFH. IFT80 may inhibit adipogenic differentiation while promoting osteogenic differentiation in rabbit BM-MSCs by activating the Hedgehog pathway.

Exosome-mediated miR-144-3p promotes ferroptosis to inhibit osteosarcoma proliferation, migration, and invasion through regulating ZEB1.

BACKGROUND: Osteosarcoma (OS) is the most prevalent orthopedic malignancy with a dismal prognosis. The high iron absorption rate in OS cells of patients suggests that ferroptosis may be related to the progression of OS, but its potential molecular regulatory role is still unclear. Based on the ability to couple with exosomes for targeted delivery of signals, exosome-derived micro ribonucleic acids (miRNAs) can potentially serve as diagnostic biomarkers for OS. METHODS: We identified ferroptosis-related miRNAs and messenger ribonucleic acids(mRNAs) in OS using bioinformatics analysis and performed survival analysis. Then we measured miRNA expression levels through exosome microarray sequencing, and used RT-qPCR and IHC to verify the expression level of miR-144-3p and ZEB1. Stable gene expression cell lines were fabricated for in vitro experiments. Cell viability, migration and invasion were determined by CCK-8 and transwell experiment. Use the corresponding reagent kit to detect GSH/GSSG ratio, Fe2+ level, MDA level and ROS level, and measure the expression levels of GPX4, ACSL4 and xCT through RT-qPCR and WB. We also constructed nude mice model for in vivo experiments. Finally, the stability of the miRNA/mRNA axis was verified through functional rescue experiments. RESULTS: Low expression of miR-144-3p and high expression of ZEB1 in OS cell lines and tissues was observed. Overexpression of miR-144-3p can promote ferroptosis, reduce the survival ability of OS cells, and prevent the progression of OS. In addition, overexpression of miR-144-3p can downregulate the expression of ZEB1 in cell lines and nude mice. Knockdown of miR-144-3p has the opposite effect. The functional rescue experiment validated that miR-144-3p can regulate downstream ZEB1, and participates in the occurrence and development of OS by interfering with redox homeostasis and iron metabolism. CONCLUSIONS: MiR-144-3p can induce the occurrence of ferroptosis by negatively regulating the expression of ZEB1, thereby inhibiting the proliferation, migration, and invasion of OS cells.

Batch and semi-continuous experiments examining the sludge mesophilic anaerobic digestive performance with different varieties of rice straw.

Anaerobic co-digestion of excess sludge (ES) and different varieties of rice straw including indica rice straw (IRS), japonica rice straw (JRS) and glutinous rice straw (GRS) was investigated in batch and semi-continuous experiments. The batch experiment results showed that the GRS addition presents the highest hydrolysis and methanogenesis rates, its cumulative methane yield (CMY) was 305.75 mL/g VS and its average methane content was 60.56%. After digestion, the structure of GRS was almost completely destroyed, which was beneficial to the degradation of lignocellulose. The digestive process is affected by the abundance of Actinobactereria, Proteobacteria, Methanosaetae and Methanosarcina. The results of semi-continuous digestion were similar to batch digestion. In addition, the addition of GRS increased TN concentration in biogas residue and TP concentration in biogas slurry, but was not conducive to the subsequent dehydration of sludge.