MiRNA-1271-5p regulates osteogenic differentiation of human bone marrow-derived mesenchymal stem cells by targeting forkhead box O1 (FOXO1)

Forkhead box O1 (FOXO1) is a key regulator of osteogenesis. The aim of this study was to identify the mechanisms of microRNAs (miRNAs) targeting FOXO1 in osteogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs). Three miRNA target prediction programs were used to search for potential miRNAs that target FOXO1. Quantitative real-time polymerase chain reaction was conducted to detect the expression of miR-1271-5p and FOXO1 during osteogenic differentiation. Target gene prediction and screening, luciferase reporter assay was used to verify the downstream target gene of miR-1271-5p. The expression levels of FOXO1 and Runx2 were detected by RT-qPCR and Western blot analysis. Alkaline phosphatase (ALP) activity and matrix mineralization were detected by biochemical methods. The expression levels of Runx2, ALP, and osteocalcin were detected by RT-qPCR. Our results showed that miR-1271-5p was downregulated during osteogenic induction. And the expression levels of miR-1271-5p were higher in osteoporotic tissues than that in adjacent nonosteoporotic tissues. The expression levels of FOXO1 were lower in osteoporotic tissues than that in adjacent nonosteoporotic tissues. And a negative correlation was found between miR-1271-5p and FOXO1 in osteoporotic tissues. Overexpression of miR-1271-5p downregulated FOXO1 and inhibited osteogenic differentiation in hMSCs. Overexpression of miR-1271-5p downregulated the expression of osteogenic markers and reduced ALP activity. In addition, ectopic expression of FOXO1 reversed the effect of miR-1271-5p on osteogenic differentiation. In conclusion, miR-1271-5p functioned as a therapeutic target of osteogenic differentiation in hMSCs by inhibiting FOXO1, which provides valuable insights into the use of miR-1271-5p as a target in the treatment of osteoporosis and other bone metabolic diseases.     

Antagonistic and synergistic effects of bone morphogenetic protein 2/7 and all‐trans retinoic acid on the osteogenic differentiation of rat bone marrow stromal cells

The osteogenesis of bone marrow stromal cells (BMSCs) is of paramount importance for the repair of large‐size bone defects, which may be compromised by the dietary‐accumulated all‐trans retinoic acid (ATRA). We have shown that heterodimeric bone morphogenetic protein 2/7 (BMP2/7) could induce bone regeneration in a significantly higher dose‐efficiency in comparison with homodimeric BMPs. In this study, we evaluated the effects of ATRA and BMP2/7 on the proliferation, differentiation, mineralization and osteogenic genes. ATRA and BMP2/7 exhibited both antagonistic and synergistic effects on the osteogenesis of BMSCs. ATRA significantly inhibited proliferation and expression of osteocalcin but enhanced the activity of alkaline phosphatase of BMSCs. On day 21, 50 ng/mL BMP2/7 could antagonize the inhibitive effects of ATRA and significantly enhance osteogenesis of BMSCs. These findings suggested a promising application potential of heterodimeric BMP2/7 in clinic to promote bone regeneration for the cases with dietary accumulated ATRA.     

A dual role of cholesterol in osteogenic differentiation of bone marrow stromal cells

Osteoblasts, the chief bone-forming cells, are differentiated from mesenchymal stromal/stem cells. Disruption of this differentiation process can cause osteoporosis, a bone disease characterized by low bone mass and deteriorated bone structure. Cholesterol has been implicated in pathogenesis of osteoporosis, and was recently identified as an endogenous activator of Hedgehog (Hh) signaling. However, its pathological and physiological roles in osteoblast differentiation are still poorly understood. Moreover, it is unclear whether these potential roles played by cholesterol are related to its capability to modulate Hh pathway. In this study, we investigated the role of exogenous versus endogenous cholesterol in osteogenesis and Hh pathway activation using ST2 cells, a bone marrow stromal cell line. We found that exogenous cholesterol significantly inhibited alkaline phosphatase (ALP) activity and messenger RNA expression of osteoblast markers genes (Alpl, Sp7, and Ibsp) while modestly activating expression of Gli1 (a readout of Hh signaling) under both basal osteogenic culture condition and Wnt3a treatment. Similarly, exogenous cholesterol suppressed osteogenic response of ST2 cells to sonic Hh (Shh) or purmorphamine (Purmo) treatment, which, however, was accompanied by diminished induction of Gli1, indicating the involvement of a Hh-dependent mechanism. Interestingly, depletion of endogenous cholesterol also reduced Shh-induced ALP activity and Gli1 expression. Likewise, cholesterol depletion inhibited osteogenic response to Purmo, although it did not affect Gli1 induction. Taken together, our findings have demonstrated that cholesterol plays a dual role in osteoblast differentiation likely through both Hh-dependent and -independent mechanisms.     

Circ_0005615 promotes cervical cancer cell growth and metastasis by modulating the miR-138-5p/KDM2A axis

Cervical cancer (CC) is a highly fatal gynecological malignancy due to its high metastasis and recurrence rate. Circular RNA (circRNA) has been regarded as a regulator of CC. However, the underlying molecular mechanism of circ_0005615 in CC remains unclear. The levels of circ_0005615, miR-138-5p, and lysine demethylase 2A (KDM2A) were measured using qRT-PCR or western blot. Cell proliferation was assessed by Cell Counting Kit-8, 5-ethynyl-2′-deoxyuridine, and colony formation experiments. Cell invasion and migration were tested by transwell assay and wound healing assay. Flow cytometry and Caspase-Glo 3/7 Assay kit were used to analyze cell apoptosis. The expression of proliferation-related and apoptosis-related markers was detected by western blot. The binding relationships among circ_0005615, miR-138-5p, and KDM2A were verified by dual-luciferase reporter assay or RNA immunoprecipitation assay. Xenograft assay was applied to detect the effect of circ_0005615 in vivo. Circ_0005615 and KDM2A were upregulated, while miR-138-5p was downregulated in CC tissues and cells. Circ_0005615 knockdown retarded cell proliferation, migration, and invasion, while promoting apoptosis. Besides, circ_0005615 sponged miR-138-5p, and miR-138-5p could target KDM2A. miR-138-5p inhibitor reversed the regulation of circ_0005615 knockdown on CC cell growth and metastasis, and KDM2A overexpression also abolished the inhibitory effect of miR-138-5p on CC cell growth and metastasis. In addition, we also discovered that circ_0005615 silencing inhibited CC tumor growth in vivo. Circ_0005615 acted as a tumor promoter in CC by regulating the miR-138-5p/KDM2A pathway.     

Protein expression profile in the differentiation of rat bone marrow stromal cells into Schwann cell-like cells

During the last decade, increasing evidence suggested that bone marrow stromal cells (MSCs) have the potential to differentiate into neural lineages. Many studies have reported that MSCs showed morphological changes and expressed a limited number of neural proteins under experimental conditions. However, no proteomic studies on MSCs differentiated into Schwann cell-like cells have been reported. In this study, we isolated MSCs from adult Sprague-Dawley rat femur and tibia bone marrows and induced the cells in vitro under specific conditions. By using two-dimensional gel electrophoresis (2-DE), we compared the protein profiles of MSCs before and after induced differentiation. We obtained 792 protein spots in the protein profile by 2-DE, and found that 74 spots changed significantly before and after the differentiation using PDQuest software, with 43 up-regulated and 31 down-regulated. We analyzed these 74 spots by a matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) and by database searching, and found that they could be grouped into various classes, including cytoskeleton and structure proteins, growth factors, metabolic proteins, chaperone proteins, receptor proteins, cell cycle proteins, calcium binding proteins, and other proteins. These proteins also include neural and glial proteins, such as BDNF, CNTF and GFAP. The results may provide valuable proteomic information about the differentiation of MSCs into Schwann cell-like cells. Supported by National High-Tech Research and Development Program of China (Grant No. 2006AA02A128) and National Natural Science Foundation of China (Grant No. 30670667).     

MicroRNA-218-5p relieves postmenopausal osteoporosis through promoting the osteoblast differentiation of bone marrow mesenchymal stem cells

MicroRNAs (miRs) are short noncoding RNAs that play key regulatory roles in osteoblast differentiation. In this study, the specific regulatory roles of miR-218-5p on postmenopausal osteoporosis (PMOP) were investigated. The mouse model of PMOP was established by bilateral ovariectomy, and the injection of miR-218-5p mimics significantly relieved PMOP degree. Then, bone marrow mesenchymal stem cells (BMMSCs) isolated from PMOP mice were induced into osteoblasts. When compared with normal BMMSCs , PMOP BMMSCs exhibited significantly lower alkaline phosphatase (ALP) activity and less mineralized nodules, as well as downregulated miR-218-5p, Runx2, Osterix, COL1A1, and OCN after induction (P < .05). The transfection of miR-218-5p mimics, and inhibitor significantly promoted, inhibited the osteoblast differentiation of PMOP BMMSCs, respectively. In addition, COL1A1 was a target of miR-218-5p. The transfection of miR-218-5p mimics into PMOP BMMSCs significantly upregulated COL1A1 at 14th and 21st day post-induction, but not at 7th day. Our findings suggest miR-218-5p may relieve PMOP through promoting the osteoblast differentiation of BMMSCs.     

The role of BMP‐7 in chondrogenic and osteogenic differentiation of human bone marrow multipotent mesenchymal stromal cells in vitro

This study addresses the role of bone morphogenetic protein‐7 (BMP‐7) in chondrogenic and osteogenic differentiation of human bone marrow multipotent mesenchymal stromal cells (BM MSCs) in vitro. BM MSCs were expanded and differentiated in the presence or absence of BMP‐7 in monolayer and three‐dimensional cultures. After 3 days of stimulation, BMP‐7 significantly inhibited MSC growth in expansion cultures. When supplemented in commonly used induction media for 7–21 days, BMP‐7 facilitated both chondrogenic and osteogenic differentiation of MSCs. This was evident by specific gene and protein expression analyses using real‐time PCR, Western blot, histological, and immunohistochemical staining. BMP‐7 supplementation appeared to enhance upregulation of lineage‐specific markers, such as type II and type IX collagens (COL2A1, COL9A1) in chondrogenic and secreted phosphoprotein 1 (SPP1), osteocalcin (BGLAP), and osterix (SP7) in osteogenic differentiation. BMP‐7 in the presence of TGF‐β3 induced superior chondrocytic proteoglycan accumulation, type II collagen, and SOX9 protein expression in alginate and pellet cultures compared to either factor alone. BMP‐7 increased alkaline phosphatase activity and dose‐dependently accelerated calcium mineralization of osteogenic differentiated MSCs. The potential of BMP‐7 to promote adipogenesis of MSCs was restricted under osteogenic conditions, despite upregulation of adipocyte gene expression. These data suggest that BMP‐7 is not a singular lineage determinant, rather it promotes both chondrogenic and osteogenic differentiation of MSCs by co‐ordinating with initial lineage‐specific signals to accelerate cell fate determination. BMP‐7 may be a useful enhancer of in vitro differentiation of BM MSCs for cell‐based tissue repair. J. Cell. Biochem. 109: 406–416, 2010. © 2009 Wiley‐Liss, Inc.     

MiR-138-5p Targets MACF1 to Aggravate Aging-related Bone Loss

Senile osteoporosis is one of the major health problems in an aging society. Decreased bone formation due to osteoblast dysfunction may be one of the causes of aging-related bone loss. With increasing evidence suggesting that multiple microRNAs (miRNAs) play important roles in osteoblast function, the relationship between miRNAs and senile osteoporosis has become a popular research topic. Previously, we confirmed that mechanoresponsive miR-138-5p negatively regulated bone anabolic action. In this study, the miR-138-5p level was found to be negatively correlated with BMD and osteogenic markers in bone specimens of senile osteoporotic patients by bioinformatic analysis and experimental verification. Furthermore, high miR-138-5p levels aggravated the decrease of aged osteoblast differentiation in vitro and led to worse bone loss in aged osteoblastic miR-138-5p transgenic mice in vivo. We also previously identified that the target of miR-138-5p, microtubule actin cross-linking factor 1 (MACF1), could attenuate senile osteoporosis. Here, miR-138-5p was demonstrated to regulate aged osteoblast differentiation by targeting MACF1. Finally, the therapeutic inhibition of miR-138-5p counteracted the decrease in bone formation and aging-related bone loss in aged mice. Overall, our results highlight the crucial roles and the molecular mechanism of miR-138-5p in aging-related bone loss and may provide a powerful therapeutic target for ameliorating senile osteoporosis.     

miR-138-5p targets MCU to inhibit mitochondrial biogenesis and colorectal cancer growth

miR-138-5p has been identified as a novel cancer-related miRNA molecule in a variety of malignancies. However, the functions and mechanisms underlying miR-138-5p in colorectal carcinoma (CRC) remains largely unknown. In the present study, we analysed the biological effects and clinical significance of miR-138-5p in CRC. miR-138-5p expression was analysed by quantitative real-time PCR in CRC tissues and cell lines. The effects of miR-138-5p on CRC cell growth was detected by cell proliferation, colony formation, cell cycle and cell apoptosis assays in vitro and in vivo. Our data showed that miR-138-5p was significantly downregulated in CRC. Downregulated miR-138-5p was related with poor prognosis in patients with CRC. miR-138-5p suppressed CRC growth but promoted cell death both in vitro and in vivo. Online predictions and integrated experiments identified that miR-138-5p targeted MCU, and downregulated miR-138-5p promoted mitochondrial biogenesis in CRC. In the light of the underlying mechanisms, our results indicated that downregulated miR-138-5p led to increased expression of MCU, which subsequently increased the production of ROS to promote CRC growth. Our results indicated that downregulated miR-138-5p strengthened mitochondrial biogenesis through targeting MCU, thus contributing to CRC cell growth, which may provide a potential therapeutic target for CRC.     

Silencing of RB1 and RB2/P130 during adipogenesis of bone marrow stromal cells results in dysregulated differentiation

Bone marrow adipose tissue (BMAT) is different from fat found elsewhere in the body, and only recently have some of its functions been investigated. BMAT may regulate bone marrow stem cell niche and plays a role in energy storage and thermogenesis. BMAT may be involved also in obesity and osteoporosis onset. Given the paramount functions of BMAT, we decided to better clarify the human bone marrow adipogenesis by analyzing the role of the retinoblastoma gene family, which are key players in cell cycle regulation.

Our data provide evidence that the inactivation of RB1 or RB2/P130 in uncommitted bone marrow stromal cells (BMSC) facilitates the first steps of adipogenesis. In cultures with silenced RB1 or RB2/P130, we observed an increase of clones with adipogenic potential and a higher percentage of cells accumulating lipid droplets.

Nevertheless, the absence of RB1 or RB2/P130 impaired the terminal adipocyte differentiation and gave rise to dysregulated adipose cells, with alteration in lipid uptake and release. For the first time, we evidenced that RB2/P130 plays a role in bone marrow adipogenesis.

Our data suggest that while the inactivation of retinoblastoma proteins may delay the onset of last cell division and allow more BMSC to be committed to adipocyte, it did not allow a permanent cell cycle exit, which is a prerequisite for adipocyte terminal maturation.     

miR-15a-5p和miR-16-5p在骨肉瘤组织中的表达分析

目的:使用microRNAs基因芯片及实时定量PCR法测定骨肉瘤组织中miR-15a-5p和miR-16-5p的相对表达含量,并与瘤旁组织对比,分析骨肉瘤细胞内miR-15a-5p和miR-16-5p的表达变化。方法:选取34例骨肉瘤组织蜡块样本,使用microRNAs基因芯片观察miR-15a-5p和miR-16-5p在骨肉瘤和瘤旁组织内的表达差异;实时定量PCR法测定骨肉瘤组织和瘤旁组织中miR-15a-5p和miR-16-5p的相对表达含量,并将两种结果对比分析。结果:microRNAs基因芯片结果显示,在骨肉瘤组织中,miR-15a-5p在肿瘤中的表达较瘤旁组织低1.79倍,miR-16-5p较瘤旁组织低1.62倍。实时定量PCR实验结果表明,miR-15a-5p和miR-16-5p表达较瘤旁组织降低,差异有统计学意义(P0.05)。经过统计学计算,miR-15a-5p在肿瘤中的表达较瘤旁组织低3.14倍,miR-16-5p较瘤旁组织低5.65倍。结论:在骨肉瘤中,miR-15a-5p和miR-16-5p表达含量降低,提示这两种microRNAs在骨肉瘤中可能做为抑癌因子存在。