Circ-SPATA13 regulates the osteogenic differentiation of human periodontal ligament stem cells through the miR-485-5p_R + 1/BMP7 axis.

Human periodontal ligament stem cells (PDLSCs) are widely available and have strong osteogenic differentiation ability, which makes them promising tools for bone regeneration. Circular RNAs (circRNAs) play a variety of functions in the process of cell differentiation and are potential therapeutic targets. Here, we identified a new circRNA, circ-SPATA13, and found that it was highly positively correlated with the osteogenic differentiation of PDLSCs. Therefore, in this study, we revealed the significance and mechanism of circ-SPATA13 in the osteogenic differentiation of PDLSCs. PDLSCs were isolated from third molars with incomplete apical development and induced to undergo chondrogenic, adipogenic, or osteogenic differentiation. Surface markers were detected via flow cytometry. Proliferation was assessed with EdU and CCK-8 assays. The circ-SPATA13 and miR-485-5p_R + 1-mediated control of mineral deposition was evaluated through alizarin red and alkaline phosphatase staining. Osteogenesis-related factor expression was detected through western blotting, immunofluorescence, and qRT-PCR. Fluorescence in situ hybridization was used to examine circ-SPATA13 localization within PDLSCs. The relationships among circ-SPATA13, miR-485-5p_R + 1, and BMP7 during PDLSCs osteogenesis were assessed through western blotting, qRT-PCR, dual-luciferase assay, rescue experiment, and bioinformatics approaches. Primary PDLSCs expressing mesenchymal stem cell surface markers were isolated. Circ-SPATA13 was identified and found to have no impact on PDLSC proliferation, whereas it was a positive regulator of their osteogenic differentiation, a process which was antagonized by miR-485-5p_R + 1. Dual-luciferase reporter assays revealed that circ-SPATA13 was able to function as a molecular sponge to sequester miR-485-5p_R + 1 within PDLSCs, while this miRNA was able to bind to the 3'-UTR of the target mRNA BMP7. In rescue experiments, circ-SPATA13 was confirmed to regulate the osteogenic differentiation of PDLSCs via this miR-485-5p_R + 1/BMP7 axis. Moreover, in vivo experiments in rats demonstrated that the overexpression of circ-SPATA13 in PDLSCs was associated with the promotion of bone formation in a skull defect model system. These data supported the osteogenic functions of circ-SPATA13 in PDLSCs. Mechanistically, this circRNA was found to function as a molecular sponge for miR-485-5p_R + 1, in turn targeting BMP7 to promote the osteogenic differentiation of PDLSCs. This circ-SPATA13/miR-485-5p_R + 1/BMP7 axis may be a novel target for treatments promoting PDLSCs osteogenic differentiation.

Xiao T ，Shi Y ，Ye Y ，Wang J ，Wang W ，Yu H ，Yan M ，Yu J ... -  《-》

FAM96B negatively regulates FOSL1 to modulate the osteogenic differentiation and regeneration of periodontal ligament stem cells via ferroptosis.

Periodontal ligament stem cell (PDLSC)-based therapy is one of the methods to assist bone regeneration. Understanding the functional regulation of PDLSCs and the mechanisms involved is a crucial issue in bone regeneration. This study aimed to explore the roles of the family with sequence similarity 96 member B (FAM96B) in the functional regulation of PDLSCs. To assess the osteogenic differentiation of PDLSCs, the alkaline phosphatase (ALP) activity assay, Alizarin red staining, quantitative calcium analysis, and osteogenic marker detection were conducted. Transplantation PDLSCs under the dorsum of nude mice and into the rat calvarial defects were also performed. Then, FAM96B-overexpressed PDLSCs were used for RNA-sequencing and bioinformatic analysis. To evaluate the ferroptosis of PDLSCs, cytosolic reactive oxygen species (ROS), expression of glutathione peroxidase 4 (GPX4), mitochondrial morphology and functions including the mitochondrial ROS, mitochondria membrane potential, and mitochondrial respiration were detected. The osteogenic indicators ALP activity, level of mineralization, and osteocalcin expression were decreased in PDLSCs by FAM96B, which demonstrated that FAM96B inhibited the osteogenic differentiation of PDLSCs. FAM96B knockdown promoted the new bone formation of PDLSCs subcutaneously transplanted to the dorsum of nude mice. Then, related biological functions were detected by the RNA-sequencing and the ferroptosis was focused. FAM96B enhanced the cytosolic ROS level and inhibited the expression of GPX4 and mitochondrial functions in PDLSCs. Hence, FAM96B promoted the ferroptosis of PDLSCs. Meanwhile, we found that FAM96B inhibition upregulated the target gene FOS like 1, AP-1 transcription factor subunit (FOSL1) expression and FOSL1 promoted the osteogenic differentiation of PDLSCs in vitro. FOSL1 also promoted the new bone formation of PDLSCs transplanted subcutaneously to the dorsum of nude mice and transplanted into rat calvarial defects. Then, the inhibitory effect of FOSL1 on the ferroptosis was confirmed. FAM96B depletion promoted the osteogenic differentiation and suppressed the ferroptosis of PDLSCs. FAM96B negatively regulated the downstream gene FOSL1 and FOSL1 promoted the osteogenic differentiation of PDLSCs via the ferroptosis. Hence, our findings provided a foundation for understanding the FAM96B-FOSL1 axis acting as a target for MSC mediated bone regeneration.

Qin Q ，Yang H ，Guo R ，Zheng Y ，Huang Y ，Jin L ，Fan Z ，Li W ... -  《Stem Cell Research & Therapy》

NEDD4L affects stability of the CHEK2/TP53 axis through ubiquitination modification to enhance osteogenic differentiation of periodontal ligament stem cells.

Hou W ，Sun C ，Han X ，Fan M ，Qiao W ... -  《-》

Circular RNA hsa_circ_0004650 Enhances 5-Fluorouracil Resistance in Gastric Cancer via Sponging miR-145-5p.

Chemotherapy based on 5-fluorouracil (5-Fu) is the first-line treatment for advanced gastric cancer (GC) patients. Importantly, 5-Fu resistance is recognized as a major obstacle for the successful treatment of GC. Circular RNAs (circRNAs) are non-coding RNAs involved in the pathogenesis of GC. However, their role in the mechanism of 5-Fu resistance in GC remains largely unknown. The purpose of this study was to explore and elucidate the biological function and molecular mechanism of circRNAs underlying 5-Fu resistance in GC. High-throughput sequencing results for intersection analysis were used to select a novel differentially expressed circRNA hsa_circ_0004650. The expression levels of the new circRNA between 5-Fu-sensitive and 5-Fu-resistant GC cells were evaluated using quantitative real-time polymerase chain reaction (qRT-PCR), and biological behaviors, such as proliferation and apoptosis of GC cells, were observed after silencing the hsa_circ_0004650. The mechanism of hsa_circ_0004650 sponges miR-145-5p to regulate 5-Fu resistance in GC cells was investigated by luciferase reporter assay, qRT-PCR, CCK-8 assay, Calcein AM/PI double fluorescence staining and flow cytometry. hsa_circ_0004650 was identified as a differentially expressed circRNA between 5-Fu-sensitive GC and 5-Fu-resistant GC cells. Hsa_circ_0004650 was up-regulated in 5-Fu-resistant GC cells. Silencing of hsa_circ_0004650 in 5-Fu-resistant GC cells, the survival rates of cells treated with increasing doses of 5-Fu for 24 h and 48 h were decreased (p<0.01); the mortality rates of SGC-7901-5-Fu cells were increased (17.86%±0.6 vs. 44.86%±1.52; p<0.001), and those of BGC-823-5-Fu cells were increased (8.17%±7.80 vs. 26.61%±1.12; p<0.001); and the apoptosis rates of cells treated with the same concentration of 5-Fu were increased (p<0.001). Mechanistically, miR-145-5p was confirmed as a downstream target of hsa_circ_0004650. By the down-regulation of the expression of miR-145-5p in 5-Fu-resistant GC cells, the survival rates of cells treated with increasing doses of 5-Fu for 24 h and 48 h were increased (p<0.05); the mortality rates of SGC-7901-5-Fu cells were decreased (12.86%±1.10 vs. 7.83%±0.53; p<0.01), those of BGC-823-5-Fu cells were decreased as well (16.99%±1.31 vs. 11.40%±0.72; p<0.01); and the apoptosis rates of cells treated with the same concentration of 5-Fu were decreased (p<0.001). The circRNA hsa_circ_0004650 promotes chemotherapy resistance to 5-Fu in GC cells through sponge adsorption of miR-145-5p, which offers a potential approach to overcome 5-Fu resistance in GC.

Zhou F ，Zhuo J ，Xu X ，Pan D ，Cai C ，Huang J ，Zhao X ，Mao Q ，Jiang X ，Sun XU ，Zhong L ，Gao N ，Chen J ... -  《-》

Circ-ECH1 May Compete With miR-708-5p to Regulate Ntrk2 in Bronchopulmonary Dysplasia.

Bronchopulmonary dysplasia (BPD) affects patients' quality of life. Circular RNAs participated in BPD. However, circ-ECH1's role in BPD has not been reported yet. This study aimed to explore the role and mechanism of circ-ECH1 in BPD. Hyperoxia-treated type II alveolar epithelial cells (L2 cells) were used as the in vitro BPD model. CCK-8, flow cytometry, and reactive oxygen species (ROS) were used to evaluate cell viability. Fluorescence in situ hybridization confirmed the subcellular localization. Circ-ECH1 overexpression (or inhibited) and miR-708-5p mimics were used to investigate the roles of circ-ECH1 and miR-708-5p in BPD. Quantitative reverse-transcription polymerase reaction (qRT-PCR) detected the expressions of circ-ECH1, miR-708-5p, and neurotrophic receptor tyrosine kinase 2 (Ntrk2). Ntrk2 expression was evaluated by Western blot analysis. Changes in lung tissues were evaluated by hematoxylin and eosin staining. Pulmonary fibrosis was examined by Mason staining. TUNEL staining was performed to evaluate cell apoptosis in lung tissues. RNA sequencing was performed in the lung tissues of BPD rats. The binding between circ-ECH1 and miR-708-5p was confirmed through dual luciferase activity. Hyperoxia reduced cell viability and increased cell apoptosis and ROS accumulation. In addition, hyperoxia decreased the expression levels of circ-ECH1, which is mainly located in the cytoplasm. Circ-ECH1 overexpression increased cell viability but reduced cell apoptosis and ROS accumulation. On the contrary, interference with circ-ECH1 further promoted cell apoptosis and reduced cell activity. Furthermore, circ-ECH1 overexpression reduced the incidence of pulmonary fibrosis and lung cell apoptosis. RNA sequencing, followed by qRT-PCR, confirmed that the expressions of Ntrk2 and miR-708-5p were affected by circ-ECH1. miR-708-5p mimics reversed the role of circ-ECH1 in the BPD. Mechanistically, circ-ECH1 may bind with miR-708-5p to regulate Ntrk2 expression. Circ-ECH1 may compet with miR-708-5p to regulate Ntrk2 expression in BPD. The findings provided a new target for BPD treatment.

Cheng H ，Li D ，Tang Y ，Hu T ，Wu B ... -  《-》