Xijie Yu

MiR-335 Inhibits Small Cell Lung Cancer Bone Metastases via IGF-IR and RANKL Pathways

Small cell lung cancer (SCLC) is a rapidly progressing, incurable cancer that frequently spreads to bone. New insights are needed to identify therapeutic targets to prevent or retard SCLC metastatic progression. Human SCLC SBC-5 cells in mouse xenograft models home to skeletal and nonskeletal sites, whereas human SCLC SBC-3 cells only pervade nonskeletal sites. Because microRNAs (miRNA) often act as tumor regulators, we investigated their role in preclinical models of SCLC. miRNA expression profiling revealed selective and reduced expression of miRNA (miR)-335 and miR-29a in SBC-5 cells, compared with SBC-3 cells. In SBC-5 cells, miR-335 expression correlated with bone osteolytic lesions, whereas miR-29a expression did not. Overexpression of miR-335 in SBC-5 cells significantly reduced cell migration, invasion, proliferation, colony formation, and osteoclast induction in vitro. Importantly, in miR-335 overexpressing SBC-5 cell xenografts (n = 10), there were minimal osteolytic lesions in the majority of mice and none in three mice. Expression of RANK ligand (RANKL) and insulin-like growth factor-I receptor (IGF-IR), key mediators of bone metastases, were elevated in SBC-5 as compared with SBC-3 cells. Mechanistically, overexpression of miR-335 in SBC-5 cells reduced RANKL and IGF-IR expression. In conclusion, loss of miR-335 promoted SCLC metastatic skeletal lesions via deregulation of IGF-IR and RANKL pathways and was associated with metastatic osteolytic skeletal lesions. Implications: These preclinical findings establish a need to pursue the role of miR-335 in human SCLC with metastatic skeletal disease. Mol Cancer Res; 12(1); 101–10. ©2013 AACR.

MicroRNA-17-92 cluster regulates osteoblast proliferation and differentiation

MicroRNAs (miRNAs) have been identified to play important functions during osteoblast proliferation, differentiation, and apoptosis. The miR-17~92 cluster is highly conserved in all vertebrates. Loss-of-function of the miR-17-92 cluster results in smaller embryos and immediate postnatal death of all animals. Germline hemizygous deletions of MIR17HG are accounted for microcephaly, short stature, and digital abnormalities in a few cases of Feingold syndrome. These reports indicate that miR-17~92 may play important function in skeletal development and mature. To determine the functional roles of miR-17~92 in bone metabolism as well as osteoblast proliferation and differentiation. Murine embryonic stem cells D3 and osteoprogenitor cell line MC3T3-E1 were induced to differentiate into osteoblasts; the expression of miR-17-92 was assayed by quantitative real-time RT-PCR. The skeletal phenotypes were assayed in mice heterozygous for miR-17~92 (miR-17~92+/Δ). To determine the possibly direct function of miR-17~92 in bone cells, osteoblasts from miR-17~92+/Δ mice were investigated by ex vivo cell culture. miR-17, miR-92a, and miR-20a within miR-17-92 cluster were expressed at high level in bone tissue and osteoblasts. The expression of miR-17-92 was down-regulated along with osteoblast differentiation, the lowest level was found in mature osteoblasts. Compared to wildtype controls, miR-17-92+/Δ mice showed significantly lower trabecular and cortical bone mineral density, bone volume and trabecular number at 10xa0weeks old. mRNA expression of Runx2 and type I collagen was significantly lower in bone from miR-17-92+/Δ mice. Osteoblasts from miR-17-92+/Δ mice showed lower proliferation rate, ALP activity and less calcification. Our research suggests that the miR-17-92 cluster critically regulates bone metabolism, and this regulation is mostly through its function in osteoblasts.

MicroRNAs in Osteoclastogenesis and Function: Potential Therapeutic Targets for Osteoporosis

Abnormal osteoclast formation and resorption play a fundamental role in osteoporosis pathogenesis. Over the past two decades, much progress has been made to target osteoclasts. The existing therapeutic drugs include bisphosphonates, hormone replacement therapy, selective estrogen receptor modulators, calcitonin and receptor activator of nuclear factor NF-κB ligand (RANKL) inhibitor (denosumab), etc. Among them, bisphosphonates are most widely used due to their low price and high efficiency in reducing the risk of fracture. However, bisphosphonates still have their limitations, such as the gastrointestinal side-effects, osteonecrosis of the jaw, and atypical subtrochanteric fracture. Based on the current situation, research for new drugs to regulate bone resorption remains relevant. MicroRNAs (miRNAs) are a new group of small, noncoding RNAs of 19–25 nucleotides, which negatively regulate gene expression after transcription. Recent studies discovered miRNAs play a considerable function in bone remodeling by regulating osteoblast and osteoclast differentiation and function. An increasing number of miRNAs have been identified to participate in osteoclast formation, differentiation, apoptosis, and resorption. miRNAs show great promise to serve as biomarkers and potential therapeutic targets for osteoporosis. In this review, we will summarize our current understanding of how miRNAs regulate osteoclastogenesis and function. We will further discuss the approach to develop drugs for osteoporosis based on these miRNA networks.

Preservation of high-fat diet-induced femoral trabecular bone loss through genetic target of TNF-α

Obesity and osteoporosis are two common chronic diseases, however, the basis for the correlation between them remains largely unknown. The pro-inflammation cytokine tumor necrosis factor-alpha (TNF-α) plays important roles in lipid and bone metabolisms, which may be a good candidate in the correlation between obesity and osteoporosis. We investigated the pathological roles of TNF-α in high-fat diet (HFD)-induced bone loss. Wild-type (WT) mice and TNF-α knockout (TNF-α−/−) mice were fed with the standard diet or the HFD for 12xa0weeks. Bone marrow stromal cells (BMSCs) from both genotypes were induced to differentiate into osteoblasts and treated with palmitic acid (PA). Bone mass and microstructure of femurs were evaluated by micro-CT. Lipid and bone metabolisms were investigated by histological and plasma analyses, and real-time PCR. On the HFD, both TNF-α−/− and WT mice presented notable visceral obesity, dyslipidemia. Adipogenesis and osteoclastogenesis were enhanced, while osteoblastogenesis was reduced in both genotypes. However, the changes were significantly different between TNF-α−/− and WT mice after the HFD. The gain of body and fat-pad weight was less and adipocyte area was smaller by 22xa0% in TNF-α−/− mice. Osteoclast numbers and plasma CTX level were lower by 40xa0% and by 23xa0% in TNF-α−/− mice. There were more ALP positive cells in the PA-treated TNF-α−/− BMSCs. mRNA expression of PPAR-γ was lower while that of Runx2 was higher in the bone from TNF-α−/− HFD group and in the PA-treated TNF-α−/− BMSCs, compared to WT on the same treatment. Furthermore, femoral trabecular bone mass and trabecular bone number were significantly decreased in WT mice on the HFD, whereas they were increased by 1.56-fold and 1.53-fold, respectively, in TNF-α−/− mice on the same diet (Pxa0<xa00.05). Our results demonstrated that TNF-α gene knockout retained HFD-induced femoral trabecular bone loss mainly by suppressing adipogenesis and osteoclastogenesis, and enhancing osteoblastogenesis, which suggests that TNF-α plays a critical role in the development of HFD-related bone metabolic disorders and it may be a new potential therapeutic target for obesity-related bone loss.

Hyperactivation of mTOR critically regulates abnormal osteoclastogenesis in neurofibromatosis type 1

Individuals with nerofibromatosis Type 1 (NF1) frequently suffer a spectrum of bone pathologies, such as abnormal skeletal development (scoliosis, congenital bowing, and congenital pseudoarthroses, etc), lower bone mineral density with increased fracture risk. These skeletal problems may result, in part, from abnormal osteoclastogenesis. Enhanced RAS/PI3K activity has been reported to contribute to abnormal osteoclastogenesis in Nf1 heterozygous (Nf1+/−) mice. However, the specific downstream pathways linked to NF1 abnormal osteoclastogenesis have not been defined. Our aim was to determine whether mammalian target of rapamycin (mTOR) was a key effector responsible for abnormal osteoclastogenesis in NF1. Primary osteoclast‐like cells (OCLs) were cultured from Nf1 wild‐type (Nf1+/+) and Nf1+/− mice. Compared to Nf1+/+ controls, there were 20% more OCLs induced from Nf1+/− mice. Nf1+/− OCLs were larger and contained more nuclei. Hyperactive mTOR signaling was detected in Nf1+/− OCLs. Inhibition of mTOR signaling by rapamycin in Nf1+/− OCLs abrogated abnormalities in cellular size and number. Moreover, we found that hyperactive mTOR signaling induced abnormal osteoclastogenesis major through hyper‐proliferation. Our research suggests that neurofibromin directly regulates osteoclastogenesis through mTOR signaling pathway. Inhibiting mTOR may represent a viable strategy to treat NF1 bone diseases.

Reduced femoral bone mass in both diet-induced and genetic hyperlipidemia mice

Growing evidence argues for a relationship between lipid and bone metabolisms with inconsistent conclusions. Sphingosine-1-phosphate (S1P) has been recognized as a suitable candidate for possible link between lipid metabolism and bone metabolism. This study was designed to investigate the effects of hyperlipidemia on bone metabolism using diet-induced and genetic-induced hyperlipidemia animal models and to explore whether S1P is involved. Wild-type mice and low-density lipoprotein receptor gene deficient (LDLR-/-) mice at age of 8weeks were placed on either control diet or high-fat diet (HFD) for 12weeks. Bone structural parameters were determined using microCT. Cross-linked type I collagen (CTx) and S1P levels in plasma were measured by ELISA methods. Bone marrow cells from wild type and LDLR-/- mice were induced to differentiate into osteoblasts, osteoclasts and adipocytes respectively. Gene expressions in distal femur metaphyses and cultured cells were studied by qRT-PCR. Moderate hypercholesterolemia was found in HFD-feeding mice; severe hypercholesterolemia and moderate hypertriglyceridemia were present in LDLR-/- mice. Femoral trabecular bone mass was reduced in both diet-induced and genetic hyperlipidemia mice. Mice feeding on HFD showed higher CTx levels, and mice with hyperlipidemia had elevated S1P levels. Correlation analysis found a positive correlation between CTx and S1P levels. Lower Runx2 expression and higher TRAP expression were found in both diet-induced and genetic hyperlipidemia mice, indicating decreased osteoblastic functions and increased osteoclastic functions in these mice. Bone marrow cells from LDLR-/- mice also showed increased adipogenesis and inhibited osteogenesis accompanied by enhanced PPARγ expression. In conclusion, our study found decreased bone mass in both diet-induced and genetic hyperlipidemia mice.

Osteocalcin is inversely associated with glucose levels in middle-aged Tibetan men with different degrees of glucose tolerance.

Research on the characteristics and mechanisms of diabetes in Tibetans is scant. Especially, there is no study on the relationship between osteocalcin and glucose metabolism. The objective of this study was to investigate the associations of serum total osteocalcin (tOC) and undercarboxylated osteocalcin (ucOC) with glucose and lipid metabolism in Chinese indigenous Tibetans with different degrees of glucose tolerance.

MicroRNA-17-92 cluster regulates pancreatic beta-cell proliferation and adaptation

MiR-17-92 cluster contributes to the regulation of mammalian development, aging and tumorigenesis. The functional roles of miR-17-92 in pancreatic beta-cells are largely unknown. In this study, we found that conditional deletion of miR-17-92 in mouse pancreatic beta-cells (miR-17-92βKO) significantly reduces glucose tolerance and the first phase of insulin secretion, despite normal ad libitum fed and fasting glucose levels. Proliferation is down-regulated in pancreatic beta-cells after deleting miR-17-92. MiR-17-92βKO mice show higher phosphatase and tensin homologue (PTEN) and lower phosphorylated AKT in islets. Under high fat diet challenge for 16 weeks, miR-17-92βKO mice lose compensation and exhibit higher glucose levels, and lower insulin secretion. Collectively, these data suggest that miR-17-92 is a critical contributor to molecular mechanisms regulating glucose-stimulated insulin secretion and pancreatic beta-cell adaptation under metabolic stress.

Bone delivers its energy information to fat and islets through osteocalcin.

Bone has emerged as a novel endocrine organ for its ability to produce hormones and involvement in several regulatory feedback loops. Osteocalcin (OCN) is released into bloodstream during bone resorption and has been demonstrated to exert endocrine regulation on islets, fat and male testis to form feedback loops. We hypothesize that bone delivers its energy metabolism signals to related energy‐regulating organs through OCN based on the following evidence: First, OCN has close interactions with islets and fat, and it shows ability to stimulate islets and fat to secret insulin and adiponectin, respectively. Islets and fat are important organs involved in energy metabolism. Second, OCN undergoes physiological fluctuations during a lifetime. In children and adolescents, during the development of osteoporosis or after bone fracture, OCN level increases significantly. The elevated OCN at these stages represents enhanced bone turnover and metabolic activity, which require more energy supply. Therefore, the metabolic activity of bone and the energy‐related organs like fat and islets are closely linked by circulating OCN. Through systemic release of OCN, bone delivers its energy‐demanding information to other organs to satisfy its energy requirement.

Double suicide genes driven by kinase domain insert containing receptor promoter selectively kill human lung cancer cells

BackgroundTo investigate the selective killing efficacy of the double suicide genes driven by KDR promoter.Materials and methodsA double suicide gene system with the KDR promoter, pcDNA3-KDRp-CDglyTK, was constructed and transfected into lung cancer cell lines L9981 and NL9980, and human hepatocellular carcinoma cell line HepG2. The efficiency and specificity of the double suicide gene system were assayed by in vitro cellular proliferation and apoptosis, as well as in vivo xenograft studies.ResultsThe transgenic CD and TK genes were only expressed in L9981 and NL9980 but not in HepG2 cells. Pre-treating transfected cells with 5-Fc and GCV significantly reduced proliferation, enhanced apoptosis in L9981 and NL9980 but not in HepG2 cells. The tumor formed by L9981 and NL9980 cells with the double suicide gene system was much smaller in vivo.ConclusionTumor targeted expression of CDglyTK gene driven by KDR promotor represents a novel strategy for effective gene therapy of tumor with intrinsic KDR.