Bone marrow mesenchymal stem cell-derived exosomes loaded with miR-26a through the novel immunomodulatory peptide DP7-C can promote osteogenesis.

PURPOSE: As small bioactive molecules, exosomes can deliver osteogenesis-related miRNAs to target cells and promote osteogenesis. This study aimed to investigate miR-26a as a therapeutic cargo to be loaded into bone marrow stromal cell exosomes through a novel immunomodulatory peptide (DP7-C). METHODS: After transfecting BMSCs with DP7-C as a transfection agent, exosomes were extracted by ultracentrifugation from the culture supernatant of miR-26a-modified BMSCs. We then characterized and identified the engineered exosomes. The effect of the engineered exosomes on osteogenesis was then evaluated in vitro and in vivo, including transwell, wound healing, modified alizarin red staining, western blot, real-time quantitative PCR, and experimental periodontitis assays. Bioinformatics and data analyses were conducted to investigate the role of miR-26a in bone regeneration. RESULTS: The DP7-C/miR-26a complex successfully transfected miR-26a into BMSCs and stimulated them to release more than 300 times the amount of exosomes overexpressing miR-26a compared with the ExoNC group. Furthermore, exosomes loaded with miR-26a could enhance proliferation, migration, and osteogenic differentiation of BMSCs in vitro compared with the ExoNC and blank groups. In vivo, the ExomiR-26a group inhibited the destruction of periodontitis compared with the ExoNC and blank groups, as revealed by HE staining. Micro-CT indicated that treatment of ExomiR-26a increased the percent bone volume and the bone mineral density compared with those of the ExoNC (P < 0.05) and blank groups (P < 0.001). Target gene analysis indicated that the osteogenic effect of miR-26a is related to the mTOR pathway. CONCLUSION: miR-26a can be encapsulated into exosomes through DP7-C. Exosomes loaded with miR-26a can promote osteogenesis and inhibit bone loss in experimental periodontitis and serve as the foundation for a novel treatment strategy.

miR-210-3p suppresses osteogenic differentiation of MC3T3-E1 by targeting brain derived neurotrophic factor (BDNF).

BACKGROUND AND OBJECTIVE: As an important mediator of intercellular interaction and formation of extracellular bone matrix, porous scaffolds are widely used for bone regeneration. Accumulating evidences demonstrate that microRNA are involved in the regulation of scaffolds-induced bone regeneration. Recently, we revealed that miR-210-3p was highly expressed during osteogenesis induced by HAG. In present study, we further explored the molecular mechanism underlying the effect of miR-210-3p on osteogenic differentiation. MATERIALS AND METHODS: In this study, miR-210-3p mimics and inhibitors were synthesized and transfected into MC3T3-E1 cells to explore their effects on osteogenic differentiation. The expression of osteogenic marker (Alp and Runx2) were detected by real-time quantitative PCR (qRT-PCR) and western blotting. After osteogenesis induction for 7 days, Alp staining were used to detected osteoblast differentiation of MC3T3-E1 cells. CCK8 and Transwell assays were performed to detected cell proliferation and migration. Then, top ranking list of target genes of miR-210-3p obtained from TargetScan and the expression of BDNF were detected by qRT-PCR and ELISA. The relationship between miR-210-3p and BDNF was verified by luciferase report assay. Furthermore, the effect of BDNF on osteoblast differentiation was verified by transfecting siRNA or adding BDNF to the culture medium. RESULTS: MiR-210-3p mimics markedly suppress osteogenic differentiation, cell migration and cell proliferation of MC3T3-E; nevertheless, silencing of miR-210-3p dramatically enhanced MC3T3-E1 osteogenesis, cell migration and proliferation. Furthermore, luciferase reporter assay verified that brain derived neurotrophic factor (BDNF) is a directly target of miR-210-3p. Moreover, BDNF siRNA significantly decreased the expression levels of ALP and cell migration. The addition of BDNF partially rescued the inhibition of osteogenesis by miR-210-3p. CONCLUSION: miR-210-3p inhibited the osteogenic differentiation via targeting BDNF. Our Results provide a promising target for regulating osteogenic differentiation.

Iron ion-coordinated carrier-free supramolecular co-nanoassemblies of dual DNA topoisomerase-targeting inhibitors for tumor suppression.

Overexpressed DNA topoisomerase II alpha (TOP-2A) is closely related to the invasion and metastasis of malignant breast tumors. Mitoxantrone (MTX) has been identified as a TOP-2A inhibitor with significant inhibitory activity against breast tumors. The tumor-homing ability of MTX has been further enhanced by using nanodrug delivery systems (nano-DDSs), reducing off-target side effects. However, conventional MTX nano-DDSs are still limited by low drug-loading capacity and material carrier-related toxicity. In this study, we developed metal iron-coordinated carrier-free supramolecular co-nanoassemblies of dual DNA topoisomerase-targeting inhibitors with high drug loading for superimposed DNA damage-augmented tumor regression. By introducing iron ions (Ⅲ) and another TOP-2A inhibitor quercetin (QU) onto the building blocks, Fe3+-mediated QU-MTX co-nanoassemblies are fabricated (QU-MTX-Fe) via intermolecular coordination interactions. The PEGylated co-nanoassemblies (P-QU-MTX-Fe) exhibit distinct advantages over QU/MTX solution (Sol) alone or MTX-QU mixture Sol in terms of therapeutic efficacy and systemic toxicity. Meanwhile, P-QU-MTX-Fe could efficiently suppress primary and distal breast tumor relapse by activating the CD 8+-mediated antitumor immune response. Overall, such iron-coordinated nanomedicines provide insights into the rational design of drug-likeness compounds with undesirable therapeutic performance for cancer therapy. STATEMENT OF SIGNIFICANCE: Aimed at the key target TOP-2A in the malignant breast tumor, the metal coordination-mediated supramolecular co-assemble strategy of one-target dual inhibitors was firstly proposed for superimposed DNA damage for cancer therapy. Multiple interactions involving π-π stacking interactions, hydrogen bonds and coordination forces maintained the stability of co-nanoassemblies. Meanwhile, this co-nanoassemblies not only had potentials to increase therapeutic efficacy and decrease systemic toxicity, but also activated the CD 8+-mediated antitumor immune response against distal breast tumor relapse. Such a facile and safe nanoplatform is expected to provide an important prospective for promoting the clinical transformation of drug-likeness compounds in the suppression of difficult-to-treat breast tumor.

Differential Expression Profiling of microRNAs in Human Placenta-Derived Mesenchymal Stem Cells Cocultured with Grooved Porous Hydroxyapatite Scaffolds.

Scaffold materials used for bone defect repair are often limited by osteogenic efficacy. Moreover, microRNAs (miRNAs) are involved in regulating the expression of osteogenic-related genes. In previous studies, we verified the enhancement of osteogenesis using a grooved porous hydroxyapatite scaffold (HAG). In the present study, we analyzed the contribution of HAG to the osteogenic differentiation of human placenta-derived mesenchymal stem cells (hPMSCs) from the perspective of miRNA differential expression. Furthermore, results showed that miRNAs were differentially expressed in the osteogenic differentiation of hPMSCs cocultured with HAG. In detail, 16 miRNAs were significantly upregulated and 29 miRNAs were downregulated with HAG. In addition, bioinformatics analyses showed that the differentially expressed miRNAs were enriched in a variety of biological processes, including signal transduction, cell metabolism, cell junctions, cell development and differentiation, and that they were associated with osteogenic differentiation through axon guidance, mitogen-activated protein kinase, and the transforming growth factor beta signaling pathway. Furthermore, multiple potential target genes of these miRNAs were closely related to osteogenic differentiation. Importantly, overexpression of miR-146a-5p (an upregulated miRNA) promoted the osteogenic differentiation of hPMSCs, and miR-145-5p overexpression (a downregulated miRNA) inhibited the osteogenic differentiation of hPMSCs.

Development and validation of a prognostic nomogram for colorectal cancer after surgery.

BACKGROUND: A nomogram is a diagram that aggregates various predictive factors through multivariate regression analysis, which can be used to predict patient outcomes intuitively. Lymph node (LN) metastasis and tumor deposit (TD) conditions are two critical factors that affect the prognosis of patients with colorectal cancer (CRC) after surgery. At present, few effective tools have been established to predict the overall survival (OS) of CRC patients after surgery. AIM: To screen out suitable risk factors and to develop a nomogram that predicts the postoperative OS of CRC patients. METHODS: Data from a total of 3139 patients diagnosed with CRC who underwent surgical removal of tumors and LN resection from 2010 to 2015 were collected from the Surveillance, Epidemiology, and End Results program. The data were divided into a training set (n = 2092) and a validation set (n = 1047) at random. The Harrell concordance index (C-index), Akaike information criterion (AIC), and area under the curve (AUC) were used to assess the predictive performance of the N stage from the American Joint Committee Cancer tumor-node-metastasis classification, LN ratio (LNR), and log odds of positive lymph nodes (LODDS). Univariate and multivariate analyses were utilized to screen out the risk factors significantly correlating with OS. The construction of the nomogram was based on Cox regression analysis. The C-index, receiver operating characteristic (ROC) curve, and calibration curve were employed to evaluate the discrimination and prediction abilities of the model. The likelihood ratio test was used to compare the sensitivity and specificity of the final model to the model with the N stage alone to evaluate LN metastasis. RESULTS: The predictive efficacy of the LODDS was better than that of the LNR based on the C-index, AIC values, and AUC values of the ROC curve. Seven independent predictive factors, namely, race, age at diagnosis, T stage, M stage, LODDS, TD condition, and serum carcinoembryonic antigen level, were included in the nomogram. The C-index of the nomogram for OS prediction was 0.8002 (95%CI: 0.7839-0.8165) in the training set and 0.7864 (95%CI: 0.7604-0.8124) in the validation set. The AUC values of the ROC curve predicting the 1-, 3-, and 5-year OS were 0.846, 0.841, and 0.825, respectively, in the training set and 0.823, 0.817, and 0.835, respectively, in the validation test. Great consistency between the predicted and actual observed OS for the 1-, 3-, and 5-year OS in the training set and validation set was shown in the calibration curves. The final nomogram showed a better sensitivity and specificity than the nomogram with N stage alone for evaluating LN metastasis in both the training set (-4668.0 vs -4688.3, P < 0.001) and the validation set (-1919.5 vs -1919.8, P < 0.001) through the likelihood ratio test. CONCLUSION: The nomogram incorporating LODDS, TD, and other risk factors showed great predictive accuracy and better sensitivity and specificity and represents a potential tool for therapeutic decision-making.

Structure activity relationships of chrysoeriol and analogs as dual c­Met and VEGFR2 tyrosine kinase inhibitors.

Vascular endothelial growth factor receptor 2 (VEGFR2) and c­Met are tyrosine kinases, which are involved in the tumorigenesis of various types of cancer. Previous studies have demonstrated that the elevated activation of c­Met is associated with the drug resistance of VEGFR2 inhibitors. Therefore, dual c­Met and VEGFR2 kinase inhibitors are expected to overcome VEGFR2 inhibitor resistance and subsequently lead to a superior therapeutic outcome to regular VEGFR2 inhibitors. In the present study, it was found that chrysoeriol, which can be extracted from several natural plants, was a potential dual c­Met and VEGFR2 kinase inhibitor. The results of docking experiments revealed that chrysoeriol was able to efficiently bind in the active site cavity of c­Met and VEGFR2. The results of enzymatic assays showed relatively high binding affinities of chrysoeriol to c­Met (Kd=12 µM) and VEGFR2 (Kd=11 µM). The structure activity relationships (SARs) of chrysoeriol and its analogs were investigated using pharmacological and molecular docking experiments. To the best of our best knowledge, the present study is the first to report a natural product with both c­Met and VEGFR2 inhibitory profiles, and provides insights into future dual c­Met and VEGFR2 kinase inhibitor development.