RESUMEN
BACKGROUND: A multifunctional titanium surface with osteogeneic, angiogeneic and antibacterial properties is needed to improve the osseointegration and long-term survival of dental implants. Particularly, the switch between the differentiation of mesenchymal stem cells (MSCs) into osteogenic and adipogenic lineages is regulated by numerous miRNAs. However, the association between miR-135b-5p and the Hippo signaling pathway during osteogenesis has not been elucidated. In the present study, we demonstrate that miR-135b-5p facilitates the in vitro osteogenesis of human mesenchymal stem cells (hMSCs). METHODS: hMSCs and Human Calvarial Osteoblasts (HCO) cells were cultured in vitro, followed by the transfection of the miR-135b-5p mimic or inhibitor using Lipofectamine 2000. The target of miR-135b-5p was determined by bioinformatics analysis and luciferase assay. Cell viability was tested using the MTT assay. The osteogenesis level was evaluated by alizarin red staining. miRNA and mRNA expression levels were detected by real-time PCR. The protein levels were assessed by western blotting. RESULTS: miR-135b-5p was shown to be highly expressed in osteoblasts compared with that in hMSCs. The overexpression of miR-135b-5p promotes hMSC proliferation and osteogenesis, whereas its knockdown causes the inhibition of these processes. Furthermore, aberrant expression of miR-135b-5p promotes both osteogenic and proliferation factors. We next showed that the Hippo signaling pathway was activated by miR-135b-5p transfection. Next, we found that large tumor suppressor 1 (LATS1) and MOB kinase activator 1B (MOB1B), key negative regulators of the HIPPO signaling pathway, are direct targets of miR-135b-5p. In addition, the knockdown of LATS1 or MOB1B led to an increase in TEAD activity. CONCLUSION: miR-135b-5p regulates osteogenesis by controlling LATS1 and MOB1B expression and subsequently activating the HIPPO signaling pathway.
RESUMEN
Matrine is an alkaloid extracted from a Chinese herb Sophora flavescens Ait, which has shown chemopreventive potential against various cancers. In this study, we evaluated the anticancer efficacy of a novel derivative of matrine, (6aS, 10S, 11aR, 11bR, 11cS)-10- methylamino-dodecahydro- 3a,7a-diazabenzo (de) (MASM), against human hepatocellular carcinoma (HCC) cells and their corresponding sphere cells in vitro and in vivo. Human HCC cell lines (Hep3B and Huh7) were treated with MASM. Cell proliferation was assessed using CCK8 and colony assays; cell apoptosis and cell cycle distributions were examined with flow cytometry. The expression of cell markers and signaling molecules was detected using Western blot and qRT-PCR analyses. A sphere culture technique was used to enrich cancer stem cells (CSC) in Hep3B and Huh7 cells. The in vivo antitumor efficacy of MASM was evaluated in Huh7 cell xenograft model in BALB/c nude mice, which were administered MASM (10 mg·kg-1·d-1, ig) for 3 weeks. After the treatment was completed, tumor were excised and weighed. A portion of tumor tissue was enzymatically dissociated to obtain a single cell suspension for the spheroid formation assays. MASM (2, 10, 20 µmol/L) dose-dependently inhibited the proliferation of HCC cells, and induced apoptosis, which correlated with a reduction in Bcl-2 expression and an increase in PARP cleavage. MASM also induced cell cycle arrest in G0/G1 phase, which was accompanied by increased p27 and decreased Cyclin D1 expression. Interestingly, MASM (2, 10, and 20 µmol/L) drastically reduced the EpCAM+/CD133+ cell numbers, suppressed the sphere formation, inhibited the expression of stem cell marker genes and promoted the expression of mature hepatocyte markers in the Hep3B and Huh7 spheroids. Additionally, MASM dose-dependently suppressed the PI3K/AKT/mTOR and AKT/GSK3ß/ß-catenin signaling pathways in Hep3B and Huh7 cells. In Huh7 xenograft bearing nude mice, MASM administration significantly inhibited Huh7 xenograft tumor growth and markedly reduced the number of surviving cancer stem-like cells in the tumors. MASM administration also reduced the expression of stem cell markers while increasing the expression of mature hepatocyte markers in the tumor tissues. The novel derivative of matrine, MASM, markedly suppresses HCC tumor growth through multiple mechanisms, and it may be a promising candidate drug for the treatment of hepatocellular carcinoma.