Hongqing Cao

miR-214 targets ATF4 to inhibit bone formation

Emerging evidence indicates that microRNAs (miRNAs) have important roles in regulating osteogenic differentiation and bone formation. Thus far, no study has established the pathophysiological role for miRNAs identified in human osteoporotic bone specimens. Here we found that elevated miR-214 levels correlated with a lower degree of bone formation in bone specimens from aged patients with fractures. We also found that osteoblast-specific manipulation of miR-214 levels by miR-214 antagomir treatment in miR-214 transgenic, ovariectomized, or hindlimb-unloaded mice revealed an inhibitory role of miR-214 in regulating bone formation. Further, in vitro osteoblast activity and matrix mineralization were promoted by antagomir-214 and decreased by agomir-214, and miR-214 directly targeted ATF4 to inhibit osteoblast activity. These data suggest that miR-214 has a crucial role in suppressing bone formation and that miR-214 inhibition in osteoblasts may be a potential anabolic strategy for ameliorating osteoporosis.

CKIP-1 Inhibits Cardiac Hypertrophy by Regulating Class II Histone Deacetylase Phosphorylation Through Recruiting PP2A

Background— Sustained cardiac pressure overload–induced hypertrophy and pathological remodeling frequently leads to heart failure. Casein kinase-2 interacting protein-1 (CKIP-1) has been identified to be an important regulator of cell proliferation, differentiation, and apoptosis. However, the physiological role of CKIP-1 in the heart is unknown. Methods and Results— The results of echocardiography and histology demonstrate that CKIP-1–deficient mice exhibit spontaneous cardiac hypertrophy with aging and hypersensitivity to pressure overload–induced pathological cardiac hypertrophy, as well. Transgenic mice with cardiac-specific overexpression of CKIP-1 showed resistance to cardiac hypertrophy in response to pressure overload. The results of GST pull-down and coimmunoprecipitation assays showed the interaction between CKIP-1 and histone deacetylase 4 (HDAC4), through which they synergistically inhibited transcriptional activity of myocyte-specific enhancer factor 2C. By directly interacting with the catalytic subunit of phosphatase 2A, CKIP-1 overexpression enhanced the binding of catalytic subunit of phosphatase-2A to HDAC4 and promoted HDAC4 dephosphorylation. Conclusions— CKIP-1 was found to be an inhibitor of cardiac hypertrophy by upregulating the dephosphorylation of HDAC4 through the recruitment of protein phosphatase 2A. These results demonstrated a unique function of CKIP-1, by which it suppresses cardiac hypertrophy through its capacity to regulate HDAC4 dephosphorylation and fetal cardiac genes expression.

Pretreatment of rat bone marrow mesenchymal stem cells with a combination of hypergravity and 5-azacytidine enhances therapeutic efficacy for myocardial infarction.

Background and Purpose: The in vivo cardiac differentiation and functional effects of unmodified adult bone marrow mesenchymal stem cells (BMSCs) after myocardial infarction (MI) is controversial. Our previous results suggested that hypergravity promoted the cardiomyogenic differentiation of BMSCs, and thus we postulated that ex vivo pretreatment of BMSCs using hypergravity and 5‐azacytidine (5‐Aza) would lead to cardiomyogenic differentiation and result in superior biological and functional effects on cardiac regeneration of infarcted myocardium. Methods: We used a rat MI model generated by ligation of the coronary artery. Homogeneous rat BMSCs were isolated, culture expanded, and differentiated into a cardiac lineage by adding hypergravity (2G) for 3 days and 5‐Aza (50 lmol/L, 24 h). Rats underwent BMSCs (labeled with DAPI) injection after the infarction and were randomized into five groups. Group A rats received the control medium, Group B rats received unmodified BMSCs, Group C rats received BMSCs treated with hypergravity, Group D rats received BMSCs treated with 5‐Aza, and Group E rats received BMSCs treated with 5‐Aza and hypergravity (n = 6). Results: After hypergravity and 5‐Aza treatment, BMSCs showed positive for the early muscle and cardiac markers GATA‐4, MEF‐2, and Nkx2‐5 with RT‐PCR. We also found that hypergravity could enhance the activities of MEF‐2 via promoting the nuclear export of HDAC5. The frozen section showed that the implanted BMSCs labeled with DAPI survived and angiogenesis was identified at the implantation site. In Groups B, C, D, and E rats, pre‐treated BMSCs colocalized with α‐actinin, and Group E rats showed a significantly larger increase in left ventricular function. Conclusions: The biological ex vivo cardiomyogenic differentiation of adult BMSCs with hypergravity and 5‐Aza prior to their transplantation is feasible and appears to improve their in vivo cardiac differentiation as well as the functional recovery in a rat model of the infarcted myocardium.

Identification of suitable reference gene and biomarkers of serum miRNAs for osteoporosis

Our objective was to identify suitable reference genes in serum miRNA for normalization and screen potential new biomarkers for osteoporosis diagnosis by a systematic study. Two types of osteoporosis models were used like as mechanical unloading and estrogen deficiency. Through a large-scale screening using microarray, qPCR validation and statistical algorithms, we first identified miR-25-3p as a suitable reference gene for both type of osteoporosis, which also showed stability during the differentiation processes of osteoblast and osteoclast. Then 15 serum miRNAs with differential expression in OVX rats were identified by microarray and qPCR validation. We further detected these 15 miRNAs in postmenopausal women and bedrest rhesus monkeys and evaluated their diagnostic value by ROC analysis. Among these miRNAs, miR-30b-5p was significantly down-regulated in postmenopausal women with osteopenia or osteoporosis; miR-103-3p, miR-142-3p, miR-328-3p were only significantly decreased in osteoporosis. They all showed positive correlations with BMD. Except miR328-3p, the other three miRNAs were also declined in the rhesus monkeys after long-duration bedrest. Their AUC values (all >0.75) proved the diagnostic potential. Our results provided a reliable normalization reference gene and verified a group of circulating miRNAs as non-invasive biomarkers in the detection of postmenopausal- and mechanical unloading- osteoporosis.

Involvement of Cholinergic Dysfunction and Oxidative Damage in the Effects of Simulated Weightlessness on Learning and Memory in Rats

The present study aimed to determine how the learning and memory gradually change with the prolonged hindlimb unloading (HU) treatment in rats. Different HU durations (7 d, 14 d, 21 d, and 28 d) in Sprague-Dawley (SD) rats were implemented. Cognitive function was assessed using the Morris water maze (MWM) and the shuttle box test. Additionally, parameters about cholinergic activity and oxidative stress were tested. Results showed that longer-than-14 d HU led to the inferior performances in the behavioral tasks. Besides, acetylcholine esterase (AChE) activity, malondialdehyde (MDA) level in brain, reactive oxygen species (ROS), and 8-hydroxy-2-deoxyguanosine (8-OHdG) concentrations of HU rats were significantly increased. Furthermore, choline acetyltransferase (ChAT), superoxide dismutase (SOD), and catalase (CAT) activity in brain were notably attenuated. Most of these effects were more pronounced after longer exposure (21 d and 28 d) to HU, although some indicators had their own characteristics of change. These results indicate that cholinergic dysfunction and oxidative damage were involved in the learning and memory impairments induced by longer-than-14 d HU. Moreover, the negative effects of HU tend to be augmented as the HU duration becomes longer. The results may be helpful to present possible biochemical targets for countermeasures development regarding the memory deficits under extreme environmental conditions.

Expression profiles and functional annotation analysis of mRNAs in suprachiasmatic nucleus of Clock mutant mice

The core circadian clock gene, Clock, is a positive component of the transcription/translation feedback loop in the master pacemaker suprachiasmatic nucleus (SCN) in mammals. The robust daytime peak of some clock genes in the wild-type SCN is absent in Clock mutant mice. However, very little is known about the impact of Clock mutation on the expression of other functional genes in SCN. Here, we performed cDNA microarray and found 799 differentially expressed genes (DEGs) at zeitgeber time 2 (ZT2) and 1289 DEGs at ZT14 in SCN of Clock△19/△19 mutant mice. KEGG pathway analysis showed that the changed mRNAs were highly associated with hedgehog signaling pathway, retinol metabolism, allograft rejection, drug metabolism, hematopoietic cell lineage and neuroactive ligand-receptor interaction. The top 14 and 71 hub genes were identified from the protein-protein interaction (PPI) network at ZT2 and ZT14, respectively. The sub-networks revealed hub genes were involved in olfactory transduction and neuroactive ligand-receptor interaction pathways. These results demonstrate the Clock△19/△19 mutation alters the expression of various genes involved in a wide spectrum of biological function in mouse SCN, which are helpful for better understanding the function of Clock and potential regulatory mechanisms.

Activation of HIF-1α and its downstream targets in rat hippocampus after long-term simulated microgravity exposure

Microgravity has many detrimental impact on brain functions, however the underlying mechanism remain unclear. In present study, 28 days of tail-suspension (30°) was used to simulate microgravity in rats. We showed that oxidative stress in hippocampus was increased after 28 days of simulated microgravity in consideration of the decreased expression of NF-E2-related factor 2 (Nrf2) and the declined activities of total superoxide dismutase (T-SOD), CuZn-SOD, glutathione peroxidase (GSH-PX) and total antioxidant capacity (T-AOC). Using RNA-seq, we further investigated the effect of simulated microgravity on the expression of genes in hippocampus, and 849 genes were found to be differentially expressed. According to pathway analysis, the differentially expressed genes involved in cytoskeleton, metabolism, immunity, transcription regulation, etc. It is interesting to note that the differentially expressed genes were involved in hypoxia-associated pathway. In agreement with this, the expression of hypoxia induced factor-1α (HIF-1α), the master regulator of oxygen homeostasis, was significantly increased. Meanwhile, HIF-2α, a HIF-1α paralog, was elevated compared with the control group. The expression of pyruvate dehydrogenase kinase 1 (PDK1), lactate dehydrogenase A (LDHA) and vascular endothelial growth factor (VEGF), three well-defined downstream targets of HIF-1α, were up-regulated in hippocampus after 28 days of simulated microgravity exposure. Additionally, brain oxygen saturation (SO2) and blood flow analyzed by the tissue oxygen analysis system were also significantly reduced. These findings indicate that simulated microgravity might cause an alteration in oxygen homeostasis, providing novel insight into better understanding of how simulated microgravity affects the function of hippocampus and a new direction to the development of countermeasure for brain dysfunction during spaceflight (actual microgravity).