Long non-coding RNA taurine upregulated gene 1 is downregulated in osteoporosis and influences the osteogenic differentiation of bone marrow mesenchymal stem cells.

BACKGROUND: With aging, an imbalance in bone remodeling leading to increased bone resorption and decreased bone formation is thought to contribute to osteoporosis. Osteoblastic differentiation of bone marrow mesenchymal stem cells (BMMSCs) plays a vital role in the pathogenesis of osteoporosis. However, the detailed molecular mechanisms of osteoporosis remain incompletely understood. Given that long non-coding RNA taurine upregulated gene 1 (lnc TUG1) plays a critical role in the osteogenic differentiation, and microRNA-23b (miR-23b) as a putative sponge for lnc TUG1 has upregulated expression in osteoporosis. Therefore, this study investigated the roles of TUG1/miR-23b in osteoporotic pathology. MATERIAL AND METHODS: TUG1 and miR-23b expression in the plasma of osteoporotic patients were evaluated by quantitative real-time PCR (qRT-PCR). The osteogenic differentiation in human BMMSCs was evaluated by qRT-PCR, western blot, Alizarin red staining after knockdown of TUG1 by small interfering RNA (siRNA) treatment. RESULTS: Decreased expression of TUG1 and increased expression of miR-23b evident in the plasma of patients with osteoporosis than in that of age- and sex-matched healthy controls. Additionally, increased miR-23b expression inhibited runt-related transcription factor 2 (RUNX2), osteocalcin, and osteopontin expression and reduced calcified nodule formation based on the results of qRT-PCR, western blot, and Alizarin Red S staining. CONCLUSION: The study for the first time reported that silence of lncRNA TUG1 significantly suppressed the osteogenic differentiation of BMMSCs possibly by targeting the miR-23b/RUNX2 signaling pathway. This mechanism of TUG1/miR-23b/RUNX2 signaling within the osteogenic differentiation of BMMSCs might provide new insight for the development of lncRNA-directed diagnostic and therapeutic strategies for osteoporosis.

New Therapeutic Approach for Targeting Hippo Signalling Pathway.

Nuclear localization signals are short amino acid sequences that target proteins for nuclear import. In this manuscript, we have generated a chimeric tri-functional peptide composed of a cell penetrating peptide (CPP), a nuclear localization sequence and an interfering peptide blocking the interaction between TEAD and YAP, two transcription factors involved in the Hippo signalling pathway, whose deregulation is related to several types of cancer. We have validated the cell penetration and nuclear localization by flow cytometry and fluorescence microscopy and shown that the new generated peptide displays an apoptotic effect in tumor cell lines thanks to the specific nuclear delivery of the cargo, which targets a protein/protein interaction in the nucleus. In addition, the peptide has an anti-tumoral effect in vivo in xenograft models of breast cancer. The chimeric peptide designed in the current study shows encouraging prospects for developing nuclear anti- neoplastic drugs.

SEMA3B-AS1-inhibited osteogenic differentiation of human mesenchymal stem cells revealed by quantitative proteomics analysis.

Human mesenchymal stem cells (hMSCs) are fibroblastoid multipotent adult stem cells with capacities of differentiation into osteoblasts and chondrocytes and show great potential in new bone formation and bone repair-related clinical settings, such as osteoporosis. Long noncoding RNAs (lncRNAs) have been demonstrated to play important roles in various biological processes. Here, we report an antisense lncRNA SEMA3B-AS1 regulating hMSCs osteogenesis. SEMA3B-AS1 is proximal to a member of the semaphorin family Sema3b. Overexpression of SEMA3B-AS1 using the lentivirus system markedly inhibits the proliferation of hMSCs and meanwhile reduces osteogenic differentiation. Using a comprehensive proteomic technique named isobaric tag for relative and absolute quantitation, we found that SEMA3B-AS1 significantly alters the process of osteogenesis through downregulating the expression of proteins involved in actin cytoskeleton, focal adhesion, and extracellular matrix-receptor interaction, while increasing the expression of proteins in the spliceosome. Collectively, we find that SEMA3B-AS1 is a target for controlling osteogenesis of hMSCs.

miR-142-5p in Bone Marrow-Derived Mesenchymal Stem Cells Promotes Osteoporosis Involving Targeting Adhesion Molecule VCAM-1 and Inhibiting Cell Migration.

Osteoporosis is a systemic bone metabolic disease that is highly prevalent in the elderly population, particularly in postmenopausal women, which results in enhanced bone fragility and an increased susceptibility to fractures. However, the underlying molecular pathogenesis mechanisms still remain to be further elucidated. In this study, in a rat ovariectomy- (OVX-) induced postmenopausal osteoporosis model, aberrant expression of a microRNA miR-142-5p and vascular cell adhesion molecule 1 (VCAM-1) was found by RNA sequencing analysis and qRT-PCR. Using a dual-luciferase reporter assay, we found that miR-142-5p can bind to and decrease expression of VCAM-1 mRNA. Such reduction was prohibited when the miR-142-5p binding site in VCAM-1 3'UTR was deleted, and Western blotting analyses validated the fact that miR-142-5p inhibited the expression of VCAM-1 protein. Bone marrow-derived mesenchymal stem cells (BMMSCs) transfected with miR-142-5p showed a significantly decreased migration ability in a Transwell migration assay. Collectively, these data indicated the important role of miR-142-5p in osteoporosis development involving targeting VCAM-1 and inhibiting BMMSC migration.

Identification and characterization of novel enhanced cell penetrating peptides for anti-cancer cargo delivery.

Cell penetrating peptides (CPP) are able cross the membrane and to transport cargos, presenting a great potential in drug delivery and diagnosis. In this paper, we have identified novel natural or synthetic CPPs. We have validated their rapid and efficient time and dose-dependent penetration, the absence of toxicity, the intracellular localization and the stability to proteases degradation, one of the main bottlenecks of peptides. Moreover, we have associate a cargo (an interfering peptide blocking the association of the serine/threonine phosphatase PP2A to its inhibitor, the oncogene SET) to the new generated shuttles and showed that they new bi-functional peptides keep the original properties of the shuttle and, in addition, are able to induce apoptosis due to the properties of the cargo. The CPPs identified in this study have promising perspectives for future anti-cancer drug delivery.

miR-542-3p prevents ovariectomy-induced osteoporosis in rats via targeting SFRP1.

Secreted frizzled-related protein-1 (SFRP1) is a negative regulatory molecule of the WNT signaling pathway and serves as a therapeutic target for bone formation in osteoporosis. In this study, we first established an ovariectomized (OVX) rat model to simulate postmenopausal osteoporosis and found significant changes in miR-542-3p and sFRP1 expression by RNA sequencing and qRT-PCR. In addition, there was a significant negative correlation between miR-542-3p and sFRP1 mRNA levels in postmenopausal women with osteoporosis. We found that miR-542-3p inhibited the expression of sFRP1 mRNA by luciferase reporter assay. When the miR-542-3p binding site in sFRP1 3'UTR was deleted, it did not affect its expression. Western blot results showed that miR-542-3p inhibited the expression of SFRP1 protein. The expression of SFRP1 was significantly increased in osteoblast-induced mesenchymal stem cells (MSC), whereas the expression of miR-542-3p was significantly decreased. And miR-542-3p transfected MSCs showed a significant increase in osteoblast-specific marker expression, indicating that miR-542-3p is necessary for MSC differentiation. Inhibition of miR-542-3p reduced bone formation, confirmed miR-542-3p play a role in bone formation in vivo. In general, these data suggest that miR-542-3p play an important role in bone formation via inhibiting SFRP1 expression and inducing osteoblast differentiation.

Correlations between the levels of acute infection markers and serum albumin in elderly patients with hip fracture.

OBJECTIVE: The aim of this study was to explore a clinical index that could predict the decline of serum albumin (ALB) in elderly patients (over 60 years old) with hip fractures in 2014. METHODS: All the data came from the retrospective survey, and the correlations between the ALB changes and acute infection markers were then analyzed using correlation analysis. The changes of infection markers and ALB before and after surgery were compared using the t test. RESULTS: There was no correlation of the serum ALB blood with interleukin-6 (IL-6) (r = 0.072, P = 0.588), C-reactive protein (CRP) (r = -0.249, P = 0.057), or calcitonin (PCT) (r = -0.038, P = 0.775) when patients were admitted, but it was negatively correlated with the total amount of infection markers (TAIMs) (r = -0.301, P = 0.020). The postoperative levels of IL-6 (154.23 ± 177.14 pg/mL) (P < 0.001), CRP (69.52 ± 39.84 mg/L) (P < 0.001), and PCT (1.27 ± 2.4 ng/mL) (P < 0.001) were significantly increased than those before surgery [IL-6 (44.96 ± 54.58 pg/mL), CRP (31.78 ± 29.90 mg/L), and PCT (0.42 ± 1.06 ng/mL)]. The postoperative level of serum ALB (29.93 ± 3.02 g/L) was significantly reduced than that before surgery (33.95 ± 3.69 g/L) (P < 0.001). The serum ALB level was negatively correlated with IL-6 (r = -0.333, P = 0.015) before surgery, but not correlated with TAIMs (r = -0.256, P = 0.061). The serum ALB level was negatively correlated with IL-6 (r = -0.292, P = 0.034) and TAIMs (r = -0.271, P = 0.050) after surgery. CONCLUSIONS: The serum IL-6 level could predict the changes of ALB during the disease process.

Evaluation of Caspase-9b and PP2Acα2 as potential biomarkers for chronic lymphocytic leukemia.

BACKGROUND: Disruption of alternative splicing in apoptotic factors has been associated to chronic lymphocytic leukemia among other cancers and hematological malignancies. The proapoptotic proteins Caspase-9 and PP2Acα are functionally related in a direct interaction, which constitutes a promising target for cancer therapy. Both proteins present aberrant mRNA splicing variants that are antiapoptotic (Caspase-9b) and catalytically inactive (PP2Acα2), respectively. RESULTS: In this work we have analyzed the relative abundance of the aberrant spliced forms Caspase-9b and PP2Acα2 in several cell lines and chronic lymphocytic leukemia patients and correlated it with several parameters of the disease. Despite 40 % of the patients presented Caspase-9b dysregulation, there was no direct association between alterations in Caspase-9b relative abundance and the parameters analyzed in medical records. More importantly, PP2Acα2 dysregulation was observed in 88 % of CLL patients and was related with advanced stages of the malignancy. CONCLUSIONS: Caspase-9b dysregulation seemed to be associated with the disease, although the differences between healthy donors and CLL patients were not statistically significant. However, PP2Acα2 dysregulation was significantly different between healthy donors and CLL patients and correlated with Binet B and C stages; therefore, we propose the use of PP2Acα2 dysregulation as a potential biomarker for advanced stages of chronic lymphocytic leukemia.

Restrictive blood transfusion strategies and associated infection in orthopedic patients: a meta-analysis of 8 randomized controlled trials.

This study sought to evaluate whether restrictive blood transfusion strategies are associated with a risk of infection in orthopedic patients by conducting a meta-analysis of randomized controlled trials (RCTs). RCTs with restrictive versus liberal red blood cell (RBC) transfusion strategies were identified by searching Medline, Embase, the Cochrane Central Register of Controlled Trials and the Cochrane Database of Systematic Reviews from their inception to December 2014. Eight RCTs with infections as outcomes were included in the final analysis. According to the Jadad scale, all studies were considered to be of high quality. The pooled risk ratio [RR] for the association between transfusion strategy and infection was 0.65 (95% CI, 0.47-0.91; p = 0.012), and the number of patients needed to treat to avoid an infection using a restrictive transfusion strategy was 62. No heterogeneity was observed. The sensitivity analysis indicated unstable results, and no significant publication bias was observed. This meta-analysis of RCTs demonstrates that restrictive transfusion strategies in orthopedic patients result in a significant reduction in infections compared with more liberal strategies.

Opioids contribute to fracture risk: a meta-analysis of 8 cohort studies.

OBJECTIVE: To evaluate the association between chronic opioid use for non-cancer pain and fracture risk by conducting a meta-analysis of cohort studies. METHODS: Cohort studies were identified by searching PubMed and EMBASE from their inception to July 2014. A fracture was considered an endpoint. The information was extracted by two authors independently. When the heterogeneity was significant, a random-effects model was used to calculate the overall pooled risk estimates. RESULTS: Eight cohort studies were included in the final meta-analysis. On the basis of the Newcastle-Ottawa Scale (NOS), six studies were considered to be of high quality. The overall combined relative risk for the use of opioids and fractures was 1.88 (95% confidence interval [CI] 1.51-2.34). A subgroup analysis revealed the sources of heterogeneity. The sensitivity analysis indicated stable results, and no publication bias was observed. CONCLUSIONS: This meta-analysis of cohort studies demonstrates that opioids significantly increase the risk of fractures.

Identification of luteolin as enterovirus 71 and coxsackievirus A16 inhibitors through reporter viruses and cell viability-based screening.

Hand, foot and mouth disease (HFMD) is a common pediatric illness mainly caused by infection with enterovirus 71 (EV71) and coxsackievirus A16 (CA16). The frequent HFMD outbreaks have become a serious public health problem. Currently, no vaccine or antiviral drug for EV71/CA16 infections has been approved. In this study, a two-step screening platform consisting of reporter virus-based assays and cell viability­based assays was developed to identify potential inhibitors of EV71/CA16 infection. Two types of reporter viruses, a pseudovirus containing luciferase-encoding RNA replicons encapsidated by viral capsid proteins and a full-length reporter virus containing enhanced green fluorescent protein, were used for primary screening of 400 highly purified natural compounds. Thereafter, a cell viability-based secondary screen was performed for the identified hits to confirm their antiviral activities. Three compounds (luteolin, galangin, and quercetin) were identified, among which luteolin exhibited the most potent inhibition of viral infection. In the cell viability assay and plaque reduction assay, luteolin showed similar 50% effective concentration (EC50) values of about 10 µM. Luteolin targeted the post-attachment stage of EV71 and CA16 infection by inhibiting viral RNA replication. This study suggests that luteolin may serve as a lead compound to develop potent anti-EV71 and CA16 drugs.

High-level expression, purification, and characterization of bifunctional ScFv-9R fusion protein.

Fibroblast growth factor receptor 3 (FGFR3) is a noted proto-oncogene involved in the pathogenesis of many tumors, so more and more studies focus on the potential use of receptor kinase inhibitor and therapeutic antibodies against FGFR3. In this study, we designed a novel fusion protein containing the single-chain Fv (ScFv) against FGFR3 and 9-arginine, denoted as ScFv-9R. To achieve the high-level production and soluble expression, ScFv and ScFv-9R were fused with small ubiquitin-related modifier (Sumo) by polymerase chain reaction and expressed in Escherichia coli BL21 (DE3). The recombinant bacteria was induced by 0.5 mM isopropyl-ß-D-thiogalactopyranoside for 20 h at 20 °C; supernatants of Sumo-ScFv was harvested and purified by DEAE Sepharose FF and Ni-NTA orderly, and supernatants of Sumo-ScFv-9R was harvested and purified by Ni-NTA. After cleaved by the Sumo protease, the recombinant ScFv or ScFv-9R was released from the fusion protein, respectively. The purity of ScFv or ScFV-9R was shown to be higher than 90 %, and their yield reached 3-5 mg per liter of bacterial culture. In vitro data showed that ScFV-9R can attenuate the phosphorylation of FGFR3 and ERK in the absence or presence of FGF9. Gel retardation assay showed that 1 µg of ScFv-9R could efficiently bind to about 4 pmol siRNA. Fluorescent microscope analysis showed that ScFv-9R can efficiently bind and deliver siRNA into RT112 cells. In conclusion, we use Sumo fusion system to acquire high-level production, soluble expression, and bifunctional activity of ScFv-9R in E. coli. Our results also revealed that ScFv-9R, as a novel carrier, may have potential applications in antitumor studies and pharmaceutical development.