Reversine suppresses osteosarcoma cell growth through targeting BMP-Smad1/5/8-mediated angiogenesis.

Reversine, or 2-(4-morpholinoanilino)-6cyclohexylaminopurine, is a 2,6-disubstituted purine derivative. This small molecule exhibits tumor-suppressive activities through different molecular mechanisms. In this study, in vitro and in vivo angiogenic models were used to elucidate the effect of Reversine on angiogenesis in the tumor suppression. Firstly, we grafted osteosarcoma-derived MNNG/HOS cell aggregates onto chick embryonic chorioallantoic membrane (CAM) to examine the vascularization of these grafts following Reversine treatment. Following culture, it was determined that Reversine inhibited MNNG/HOS grafts growth, and decreased the density of blood vessels in the chick CAM. We then used CAM and chick embryonic yolk-sac membrane (YSM) to investigate the effects of Reversine on angiogenesis. The results revealed Reversine inhibited the proliferation of endothelial cells, where cells were mainly arrested at G1/S phase of the cell cycle. Scratch-wound assay with HUVECs revealed that Reversine suppressed cell migration in vitro. Furthermore, endothelial cells tube formation assay and chick aortic arch sprouting assay demonstrated Reversine inhibited the sprouting, migration of endothelial cells. Lastly, qPCR and western blot analyses showed BMP-associated Smad1/5/8 signaling expressions were up-regulated by Reversine treatment. Our results showed that Reversine could suppress tumor growth by inhibiting angiogenesis through BMP signaling, and suggests a potential use of Reversine as an anti-tumor therapy.

Dysbacteriosis-induced LPS elevation disturbs the development of muscle progenitor cells by interfering with retinoic acid signaling.

Whether myogenesis is affected by the maternal gut dysbacteriosis still remains ambiguous. In this study, first we show the elevated level of lipopolysaccharides (LPS) in a gut microbiota dysbiosis mouse model. Second, we demonstrate that the diameter of muscle fibers, limb development, and somitogenesis were inhibited in both the gut microbiota dysbiosis and LPS exposed mice and chicken embryos. These might be due to LPS disturbed the cell survival and key genes which regulate the somitogenesis and myogenesis. RNA sequencing and subsequent validation experiments verified that retinoic acid (RA) signaling perturbation was mainly responsible for the aberrant somite formation and differentiation. Subsequently, we found that LPS-induced reactive oxygen species (ROS generation and antioxidant genes such as Nrf2, AKR1B10) contributed to the above -mentioned interference with RA signaling. These findings highlight that the gut microbiota homeostasis is also involved in regulating the development of muscle progenitor cells during pregnancy.

Dysbacteriosis induces abnormal neurogenesis via LPS in a pathway requiring NF-κB/IL-6.

In this study, we identified elevated levels of LPS and suppressed neurogenesis in a successfully established mouse model of gut microbiota dysbiosis. We mimicked these phenotypes using mouse and chicken embryos exposed to LPS and found that dramatic variation in gene expression was due to changes in the dorsal-ventral patterning of the neural tube. Cell survival and excess ROS were also involved in this process. Antioxidant administration alleviated LPS-activated NF-κB signaling, while directly blocking NF-κB signaling altered the LPS-induced inhibition of neurogenesis. Furthermore, IL-6 was proven to play a vital role in the expression of crucial neurogenesis-related genes and NF-κB. In summary, we found that the suppression of neurogenesis induced by dysbacteriosis-derived LPS was significantly reversed in mice with fecal microbiota transplantation. This study reveals that gut dysbacteriosis-derived LPS impairs embryonic neurogenesis, and that the NF-κB/IL-6 pathway could be one of the main factors triggering the downstream signaling cascade.

Dysbacteriosis-Derived Lipopolysaccharide Causes Embryonic Osteopenia through Retinoic-Acid-Regulated DLX5 Expression.

Growing evidence suggests an adverse impact of gut microbiota dysbiosis on human health. However, it remains unclear whether embryonic osteogenesis is affected by maternal gut dysbacteriosis. In this study, we observed that elevated lipopolysaccharide (LPS) levels led to skeletal developmental retardation in an established mouse model of gut microbiota dysbiosis. Using chick embryos exposed to dysbacteriosis-derived LPS, we found restriction in the development of long bones as demonstrated by Alcian blue and alizarin red staining. Micro-CT and histological analysis exhibited decreased trabecular volume, bone mineral density, and collagen production, as well as suppressed osteoblastic gene expression (Ocn, Runx2, Osx, and Dlx5) in chick embryonic phalanges following LPS treatment. Atomic force microscopy manifested decreased roughness of MC3T3-E1 cells and poorly developed matrix vesicles (MVs) in presence of LPS. The expression of the aforementioned osteoblastic genes was suppressed in MC3T3-E1 cells as well. High-throughput RNA sequencing indicated that retinoic acid (RA) may play an important role in LPS-induced osteopenia. The addition of RA suppressed Dlx5 expression in MC3T3-E1 cells, as was also seen when exposed to LPS. Quantitative PCR, Western blot, and immunofluorescent staining showed that retinoic acid receptor α (RARα) was upregulated by LPS or RA treatment, while the expression of DLX5 was downregulated. CYP1B1 expression was increased by LPS treatment in MC3T3-E1 cells, which might be attributed to the increased inflammatory factors and subsequently activated NF-κB signaling. Eventually, blocking RA signals with AGN193109 successfully restored LPS-inhibited osteoblastic gene expression. Taken together, our data reveals that maternal gut microbiota dysbiosis can interfere with bone ossification, in which Dlx5 expression regulated by RA signaling plays an important role.

A feedforward circuit between KLF5 and lncRNA KPRT4 contributes to basal-like breast cancer.

Basal-like breast cancer (BLBC) is the most aggressive subtype of breast cancer with a poor prognosis. Long noncoding RNAs (lncRNAs) play critical roles in human cancers. Krüppel-like Factor 5 (KLF5) is a key oncogenic transcription factor in BLBC. However, the underlying mechanism of mutual regulation between KLF5 and lncRNA remains largely unknown. Here, we demonstrate that lncRNA KPRT4 promotes BLBC cell proliferation in vitro and in vivo. Mechanistically, KLF5 directly binds to the promoter of KPRT4 to promote KPRT4 transcription. Reciprocally, KPRT4 recruits the YB-1 transcription factor to the KLF5 promoter by interacting with YB-1 at its 5' domain and forming an RNA-DNA-DNA triplex structure at its 3' domain, resulting in enhanced transcription of KLF5 and ultimately establishing a feedforward circuit to promote cell proliferation. Moreover, the antisense oligonucleotide (ASO)-based therapy targeting KPRT4 substantially attenuated tumor growth in vivo. Clinically, the expression levels of YB-1, KLF5 and KPRT4 are positively correlated in clinical breast specimens. Together, our data suggest that KPRT4 is a major molecule for BLBC progression and that the feedforward circuit between KLF5 and KPRT4 may represent a potential therapeutic target in BLBC.

Interaction between retinoic acid and FGF/ERK signals are involved in Dexamethasone-induced abnormal myogenesis during embryonic development.

Despite the common application in pregnancy at clinical practice, it remains ambiguous whether dexamethasone (Dex) exposure can affect embryonic myogenesis. In this study, firstly we showed that 10-6 M Dex (Cheng et al., 2016; 2017) treatment resulted in abnormal myogenesis in chicken embryos. Secondly, we demonstrated that 10-6 M Dex-induced abnormality of myogenesis resulted from aberrant cell proliferation, as well as from alteration of the differentiation process from the early stage of somitogenesis up to the late stage of myogenesis. The above-mentioned results caused by Dex exposure might be due to the aberrant gene expressions of somite formation (Raldh2, Fgf8, Wnt3a, ß-catenin, Slug, Paraxis, N-cadherin) and differentiation (Pax3, MyoD, Wnt3a, Msx1, Shh). Thirdly, RNA sequencing implied the statistically significant differential gene expressions in regulating the myofibril and systemic development, as well as a dramatical alteration of retinoic acid (RA) signaling during somite development in the chicken embryos exposed to Dex. The subsequent validation experiments verified that Dex treatment indeed led to a metabolic change of RA signaling, which was up-regulated and principally mediated by FGF-ERK signaling revealed by means of the combination of chicken embryos and in vitro C2C12 cells. These findings highlight that 10-6 M Dex exposure enhances the risk of abnormal myogenesis through interfering with RA signaling during development.

Baicalin rescues hyperglycemia-induced neural tube defects via targeting on retinoic acid signaling.

We, in this study, studied whether or not antioxidant activities of Baicalin could reduce the incidence of neural tube defects (NTDs) in the presence of hyperglycemia. Using early chick embryos, we demonstrated that Baicalin at 6 µM dramatically reduced NTDs rate and impaired neurogenesis in E4.5-day and HH10 chick embryo neural tubes induced by high glucose (HG). Likewise, immunofluorescent staining showed that Baicalin mitigated the HG-induced regression of Pax7 expression in neural tubes of both HH10 and E4.5-day chick embryos. Additionally, PHIS3 immunofluorescent staining in neural tubes of both HH10 and E4.5-day chick embryos manifested that cell proliferation inhibited by HG was significantly reversed by the administration of Baicalin, and similar result could also be observed in neurosphere assay in vitro. c-Caspase3 or γH2AX immunofluorescent staining and quantitative PCR showed that Baicalin administration alleviated HG-induced cell apoptosis and DNA damage. Bioinformatics results indicated that retinoic acid (RA) was likely to be the signaling pathway that Baicalin targeted on, and this was confirmed by whole-mount RALDH2 in situ hybridization and quantitative PCR of HH10 chick embryos in the absence/presence of Baicalin. In addition, blocking RA with an inhibitor abolished Baicalin's protective role in HG-induced NTDs, suppression of neurogenesis and cell proliferation, and induction of apoptosis, which further verified the centrality of RA in the process of Baicalin confronting HG-induced abnormal neurodevelopment.