Background: Bone marrow mesenchymal stem cells (BMSCs) exhibit the capacity to self-renew and differentiate into multi-lineage cell types, including osteoblasts, which are crucial regulators of fracture healing. Thus, this study aims to investigate the effect of microRNA (miR)-22-3p from BMSC-derived EVs on osteogenic differentiation and its underlying mechanism. Methods: Extracellular vesicles (EVs) were isolated from BMSCs and taken up with BMSCs. Dual-luciferase reporter gene assay was used to verify the binding relationship between miR-22-3p and FTO. Loss-and gain-of-function experiments were performed to determine the roles of EV-delivered miR-22-3p and FTO in osteogenic differentiation as well as their regulatory role in the MYC/PI3K/AKT axis. To determine the osteogenic differentiation, ALP and ARS stainings were conducted, and the levels of RUNX2, OCN, and OPN level were determined. In vivo experiment was conducted to determine the function of EV-delivered miR-22-3p and FTO in osteogenic differentiation, followed by ALP and ARS staining. Results: miR-22-3p expression was repressed, while FTO expression was elevated in the ovariectomized mouse model. Overexpression of miR-22-3p, EV-delivered miR-22-3p, increased ALP activity and matrix mineralization of BMSCs and promoted RUNX2, OCN, and OPN expressions in BMSCs. miR-22-3p negatively targeted FTO expression. FTO silencing rescued the suppressed osteogenic differentiation by EV-delivered miR-22-3p inhibitor. FTO repression inactivated the MYC/PI3K/AKT pathway, thereby enhancing osteogenic differentiation both in vivo and in vitro. Conclusion: In summary, miR-22-3p delivered by BMSC-derived EVs could result in the inhibition of the MYC/PI3K/ AKT pathway, thereby promoting osteogenic differentiation via FTO repression.

We recently showed the ability of lovastatin to inhibit the function of the epidermal growth factor receptor (EGFR) and its downstream signaling of the phosphatidylinositol-3 kinase/ AKT pathway. Combining lovastatin with gefitinib, a potent EGFR inhibitor, induced synergistic cytotoxicity in various tumor-derived cell lines. In this study, lovastatin treatment was found to inhibit ligand-induced EGFR dimerization in squamous cell carcinoma cells and its activation of AKT and its downstream targets 4E-binding protein 1 and S6 kinase 1. This inhibition was associated with global protein translational inhibition shown by a decrease in RNA associated polysome fractions. The effects of lovastatin on EGFR function were reversed by the addition of geranylgeranyl pyrophosphate, which functions as a protein membrane anchor. Lovastatin treatment induced actin cytoskeletal disorganization and the expression of geranylgeranylated rho family proteins that regulate the actin cytoskeleton, including rhoA. Lovastatin-induced rhoA was inactive as EGF stimulation failed to activate rhoA and inhibition of the rho-associated kinase, a target and mediator of rhoA function, with Y-27632 also showed inhibitory effects on EGFR dimerization. The ability of lovastatin to inhibit EGFR dimerization is a novel exploitable mechanism regulating this therapeutically relevant target. To explore the potential clinical significance of this combination, we evaluated the effect of statin on the overall survival (OS) and disease-specific survival (DSS) of patients with advanced nonsmall-cell lung cancer enrolled in the NCIC Clinical Trials Group phase III clinical trials BR21 (EGFR tyrosine kinase inhibitor erlotinib versus placebo) and BR18 (carboplatin and paclitaxel with or without the metalloproteinase inhibitor BMS275291). In BR18, use of statin did not affect OS or DSS. In BR21, patients showed a trend for improvement in OS (HR: 0.69, P ¼ 0.098) and DSS (HR: 0.62, P ¼ 0.048), but there was no statin  treatment interaction effect (P ¼ 0.34 and P ¼ 0.51 for OS and DSS, respectively).

The present study investigated the effects of social defeat stress on the behaviours and expressions of 78-kDa glucose-regulated protein (Grp78), CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) and choline acetyltransferase (Chat) in the brains of adolescent mice. Adolescent male C57BL/6J mice were divided into two groups (susceptible and unsusceptible) after 10 d social defeat stress. In expt 1, behavioural tests were conducted and brains were processed for Western blotting on day 21 after stress. In expt 2, social avoidance tests were conducted and brains were subsequently processed for Western blotting on day 12 after stress. Chronic social defeat stress produced more pronounced depression-like behaviours such as decreased locomotion and social interaction, increased anxiety-like behaviours and immobility, and impaired memory performance in susceptible mice. Moreover, susceptible mice showed greater expression of Grp78 and CHOP in the amygdala (Amyg) on days 12 and 21 compared with the other groups. Susceptible and unsusceptible groups showed significant increases in Grp78 and CHOP expression in the prefrontal cortex (PFC) and hippocampus (Hipp) on day 12 compared with the control group; this persisted until day 21. The levels of Chat measured on days 12 and 21 were significantly lower in the PFC, Amyg and Hipp of all defeated mice compared with controls. The findings of the behavioural tests indicate that chronic social defeat in adolescents produces anxiety-like behaviours, social withdrawal, despair-like behaviours and cognitive impairment. The Grp78, CHOP and Chat results suggest that the selective response of endoplasmic reticulum stress proteins in the Amyg plays an important role in the vulnerability-stress model of depression.

The epidermal growth factor receptor (EGFR) is commonly amplified and mutated in glioblastoma, making it a compelling therapeutic target. Recent reports have demonstrated clinical activity of the EGFR inhibitors gefitinib and erlotinib in a subset of glioblastoma patients. Co-expression of EGFRvIII, a constitutively active mutant receptor expressed in 50% of tumours, and PTEN, an inhibitor of PI3K activity, by glioblastoma cells is associated with clinical response to these EGFR kinase inhibitors. PTEN loss and resulting increased PI3K pathway activity appears to act as a resistance factor. A critical therapeutic challenge is to identify agents that enhance the anti-cancer effects of these agents and promote responsiveness to EGFR kinase inhibitors in a broader spectrum of glioblastoma patients. For example, combining gefitinib with inhibitors of the PI3K/AKT pathway show enhanced cytotoxicity in glioblastoma derived cell lines. Here, we show that targeting HMG-CoA reductase with lovastatin, that can affect the activity of multiple cell signaling pathways, significantly enhanced the sensitivity of glioblastoma cells to the EGFR kinase inhibitor gefitinib in the five cell lines tested. In an isogenic model system, U87MG glioblastoma cells expressing EGFRvIII and PTEN in relevant combinations, we show that combined gefitinib and lovastatin treatments induce potent synergistic cytotoxicity irrespective of EGFRvIII and PTEN status. These studies demonstrate the potential of lovastatin to augment the cytotoxic effects of gefitinib and provide a rationale for combined statin/EGFR targeted therapies in glioblastoma patients.

AIM:To investigate the expression of three types of mucin (MUC1, MUC2, MUC5AC) and E-cadherin in human gastric carcinomas and their clinical significance. METHODS:Ninety-four gastric cancer specimens were classified according to WHO criteria and detected by immunohistochemical assay of expression of mucins and E-cadherin. RESULTS:The positive expression rates of MUC1, MUC2, MUC5AC and E-cadherin were 82% (77/94), 84% (79/94), 40% (38/94) and 56% (53/94) respectively. MUC1 expression was significantly correlated with the types of cancer (the positive rates of MUC1 in well and moderately differentiated tubular adenocarcinoma, poorly differentiated adenocarcinoma, signet-ring cell carcinoma and mucinous carcinoma were 91%, 87%, 71%, 71%, respectively, P<0.05), age of patients (the positive rates of it among the people who are younger than 40 years, between 40-60 years and over 60 year were 74%, 81%, 89%, P<0.05), lymph nodes involvement (the positive rates in the non-interfered group and the interfered group were 78%, 85%, P<0.05) and tumor size (the positive rates in the tumors with the size less than 3 cm, 3-6 cm and larger than 6 cm were 69%, 92%, 69%, P<0.05); MUC2 expression was significantly associated with types of cancers and had the strongest expression in mucinous carcinomas (the positive rates of MUC2 in well and moderately differentiated tubular adenocarcinoma, poorly differentiated adenocarcinoma, signet-ring cell carcinoma and mucinous carcinoma were 94%, 70%, 81%, 100%, P<0.05), but it had no obvious relation to age, gender, tumor location, lymph nodes involvement, depth of invasion and metastasis to extra-gastric organs (P>0.05); MUC5AC expression was not related to any of the characteristics investigated except that it had relation to gender, whereas MUC5AC showed the tendency to higher expression in less invasive lesions and lower expression in advanced stage cancers (P>0.05); No significant difference was found for E-cadherin expression. There were strong positive relationships between the expression of MUC1 and E-cadherin, MUC2 and E-cadherin, MUC1 and MUC2 (R = 0.33, R = 0.22, R = 0.32, respectively, P<0.05). According to the COX proportional hazards model, older patients, involvement of lymph nodes, different types of gastric cancer and MUC2 expression were significantly associated with poorer outcome of gastric carcinoma patients (β = 0.08, β = 3.94, β = 1.33, β = 0.75, respectively, P<0.05).CONCLUSION: MUC1 and MUC2 are good markers of different types of gastric cancer. MUC2 is especially a good marker of mucinous carcinoma. MUC1, MUC2 may interfere with the function of E-cadherin in gastric carcinomas, and have synergic effect on progression of gastric cancers.Zhang HK, Zhang QM, Zhao TH, Li YY, Yi YF. Expression of mucins and E-cadherin in gastric carcinoma and their clinical significance.

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ObjectiveMesenchymal stem cells (MSCs) confer therapeutic benefits in various pathologies and cancers by releasing extracellular vesicles (EVs) loaded with bioactive compounds. Herein, we identified bone marrow MSC (BMSC)-derived EVs harboring microRNA (miR)-29b-3p to regulate osteogenic differentiation through effects on the suppressor of cytokine signaling 1 (SOCS1)/nuclear factor (NF)-κB pathway via targeting of lysine demethylase 5A (KDM5A) in osteoporosis.MethodsWe quantified the miR-29b-3p in BMSC-derived EVs from bone marrow specimens of osteoporotic patients and non-osteoporotic patients during total hip arthroplasty (THA). miR-29b-3p targeting KDM5A was confirmed by promoter luciferase assay, and enrichment of KDM5A in the promoter region of SOCS1 was analyzed by chromatin immunoprecipitation (ChIP). The expression and translocation of NF-κB to the nucleus were detected by western blot analysis and immunofluorescence staining, respectively. An ovariectomized (OVX) osteoporosis mouse model was established to further confirm the in vitro findings.ResultsBMSC-derived EVs of osteoporotic patients exhibited downregulated miR-29b-3p. EV-encapsulated miR-29b-3p from BMSCs potentiated osteogenic differentiation by specifically inhibiting KDM5A. KDM5A inhibited osteogenic differentiation by the regulation of H3K4me3 and H3K27ac of SOCS1. SOCS1 potentiated osteogenic differentiation by inhibiting NF-κB pathway.ConclusionEV-encapsulated miR-29b-3p derived from BMSCs potentiated osteogenic differentiation through blockade of the SOCS1/NF-κB pathway by inhibition of KDM5A.

BackgroundIn a recent study, we demonstrated the ability of lovastatin, a potent inhibitor of mevalonate synthesis, to inhibit the function of the epidermal growth factor receptor (EGFR). Lovastatin attenuated ligand-induced receptor activation and downstream signaling through the PI3K/AKT pathway. Combining lovastatin with gefitinib, a potent EGFR inhibitor, induced synergistic cytotoxicity in a variety of tumor derived cell lines. The vascular endothelial growth factor receptor (VEGFR) and EGFR share similar activation, internalization and downstream signaling characteristics.Methodology/Principal FindingsThe VEGFRs, particularly VEGFR-2 (KDR, Flt-1), play important roles in regulating tumor angiogenesis by promoting endothelial cell proliferation, survival and migration. Certain tumors, such as malignant mesothelioma (MM), also express both the VEGF ligand and VEGFRs that act in an autocrine loop to directly stimulate tumor cell growth and survival. In this study, we have shown that lovastatin inhibits ligand-induced VEGFR-2 activation through inhibition of receptor internalization and also inhibits VEGF activation of AKT in human umbilical vein endothelial cells (HUVEC) and H28 MM cells employing immunofluorescence and Western blotting. Combinations of lovastatin and a VEGFR-2 inhibitor showed more robust AKT inhibition than either agent alone in the H28 MM cell line. Furthermore, combining 5 µM lovastatin treatment, a therapeutically relevant dose, with two different VEGFR-2 inhibitors in HUVEC and the H28 and H2052 mesothelioma derived cell lines demonstrated synergistic cytotoxicity as demonstrated by MTT cell viability and flow cytometric analyses.Conclusions/SignificanceThese results highlight a novel mechanism by which lovastatin can regulate VEGFR-2 function and a potential therapeutic approach for MM through combining statins with VEGFR-2 inhibitors.