CircPVT1 promotes the tumorigenesis and metastasis of osteosarcoma via mediation of miR-26b-5p/CCNB1 axis.

INTRODUCTION: Osteosarcoma (OS) is the most aggressive malignancy among the bone tumors in the world. Circular RNAs (circRNAs) have been reported to be participated in multiple cancers, including OS. Meanwhile, circPVT1 has been proved to be upregulated in OS. However, the mechanism by which circPVT1 mediates the tumorigenesis of OS remains to be further explored. MATERIALS AND METHODS: Protein and gene expressions in OS cells were measured by western blot and RT-qPCR, respectively. Cell growth was assessed by flow cytometry and colony formation, respectively. In addition, cell migration was assessed by wound healing, and invasion was evaluated by Transwell assay. Meanwhile, the correlation among circPVT1, miR-26b-5p and CCNB1 was explored by RNA pull-down and dual luciferase assay. Finally, in vivo model was established to explore the role of circPVT1 in OS in vivo. RESULTS: CircPVT1 and CCNB1 were significantly upregulated in OS cells, while miR-26b-5p was downregulated. Knockdown of circPVT1 notably inhibited proliferation and induced apoptosis of OS cells. CircPVT1 shRNA significantly suppressed the OS cell invasion and migration. Meanwhile, circPVT1 sponged miR-26b-5p and CCNB1 was found to be the direct target of miR-26b-5p. Furthermore, silencing of circPVT1 inhibited the growth and metastasis of OS in vivo. CONCLUSION: Silencing of circPVT1 notably suppressed the tumorigenesis and metastasis of OS via miR-26b-5p/CCNB1 axis. Therefore, circPVT1 might be used as a target for OS treatment.

The neurofibromatosis type I gene promotes autophagy via mTORC1 signalling pathway to enhance new bone formation after fracture.

Bone fracture is one of the most common injuries. Despite the high regenerative capacity of bones, failure of healing still occurs to near 10% of the patients. Herein, we aim to investigate the modulatory role of neurofibromatosis type I gene (NF1) to osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) and new bone formation after fracture in a rat model. We studied the NF1 gene expression in normal and non-union bone fracture models. Then, we evaluated how NF1 overexpression modulated osteogenic differentiation of BMSCs, autophagy activity, mTORC1 signalling and osteoclastic bone resorption by qRT-PCR, Western blot and immunostaining assays. Finally, we injected lentivirus-NF1 (Lv-NF1) to rat non-union bone fracture model and analysed the bone formation process. The NF1 gene expression was significantly down-regulated in non-union bone fracture group, indicating NF1 is critical in bone healing process. In the NF1 overexpressing BMSCs, autophagy activity and osteogenic differentiation were significantly enhanced. Meanwhile, the NF1 overexpression inhibited mTORC1 signalling and osteoclastic bone resorption. In rat non-union bone fracture model, the NF1 overexpression significantly promoted bone formation during fracture healing. In summary, we proved the NF1 gene is critical in non-union bone healing, and NF1 overexpression promoted new bone formation after fracture by enhancing autophagy and inhibiting mTORC1 signalling. Our results may provide a novel therapeutic clue of promoting bone fracture healing.

Downregulation of annexin A7 decreases proliferation, migration, and invasion of gastric cancer cells by reducing matrix metalloproteinase 1 and 9 expression.

High annexin A7 expression is a potential indicator of lymphatic metastasis and poor prognosis in patients with gastric cancer (GC). The mechanism underlying the effects of annexin A7 on GC cells remains unclear. In patients with GC, primary adenocarcinoma tissues had higher annexin A7 expression than adjacent non-cancerous tissues (P < 0.05). Among three human GC cell lines with high, moderate, and low levels of differentiation, respectively, the cell line with the lowest level of differentiation displayed the highest level of annexin A7 expression. We transfected cells of the human GC cell line BGC823 with short interfering RNAs (siRNAs) targeting annexin A7 and investigated the effects on signaling pathways related to cancer progression by quantitative real-time PCR and western blot. The silencing of endogenous annexin A7 suppressed the proliferation, migration, and invasion abilities of the BGC823 cells. In the cells treated with annexin A7 siRNA, the expression of p16, p21, and p27 was significantly upregulated while that of proliferating cell nuclear antigen (PCNA), cyclin A, cyclin D1, cyclin E1, matrix metalloproteinase-2 (MMP-2), MMP-9, and intercellular cell-adhesion molecule-1 (ICAM-1) was significantly downregulated compared with that in control cells. Our results suggest that the downregulation of endogenous annexin A7 inhibits GC cell proliferation, migration, and invasion by impacting cell cycle regulators and the expression of MMP-1, MMP-2, and ICAM-1. Targeting annexin A7 may represent a valuable strategy for the diagnosis and clinical treatment of GC.

Expression of annexin A7 and its clinical significance in differentiation and metastasis of gastric carcinoma.

OBJECTIVE: To investigate the expression and clinical significance of annexin A7 in the differentiation and lymphatic metastasis of gastric cancer (GC). METHODS: The clinical and pathological data were recorded for analysis. Immunohistochemical staining and Western blot were performed to analyze the expression of ANXA 7 in primary GC tissues. Logistic regression analyses were conducted to evaluate the associations between annexin A7 expression levels and differentiations of GC. Analyses of the ROC were conducted to determine the cut-off value of the ratio of pixel density of annexin A7 for predicting lymphatic metastasis of GC. RESULTS: A total of 162 GC patients were enrolled in this study, and expression rate of annexin A7 was 65.4% in GC. The survival rate of patients with positive expression of annexin A7 was lower than that in patients with negative expression (P=0.000). The results of COX regression showed that the positive expression of annexin A7, submucosal confinement and pathological stage of GC were associated with poor clinical outcomes. The ratio of pixel density value of primary GC tissues with PN 1-3 lymphatic spread was significantly higher than those in tissues with PN 0 lymphatic spread (0.56±0.09 vs. 0.42±0.07, P < 0.05). ROC analysis showed a high area under the curve for the ratio of pixel density value of annexin A7 in primary GC tissues. At a cut-off level of > 0.505, the ratio of pixel density value of annexin A7 exhibited 76.7% sensitivity and 88.3% specificity for detecting lymphatic metastasis of GC. CONCLUSION: High annexin A7 expression is associated with poor differentiation in GC patients, and it may be a predictor for lymphatic metastasis of GC.

Establishment of a chronic left ventricular aneurysm model in rabbit.

OBJECTIVES: To establish a cost-effective and reproducible procedure for induction of chronic left ventricular aneurysm (LVA) in rabbits. METHODS: Acute myocardial infarction (AMI) was induced in 35 rabbits via concomitant ligation of the left anterior descending (LAD) coronary artery and the circumflex (Cx) branch at the middle portion. Development of AMI was confirmed by ST segment elevation and akinesis of the occluded area. Echocardiography, pathological evaluation, and agar intra-chamber casting were utilized to validate the formation of LVA four weeks after the surgery. Left ventricular end systolic pressure (LVESP) and diastolic pressure (LVEDP) were measured before, immediately after and four weeks after ligation. Dimensions of the ventricular chamber, thickness of the interventricular septum (IVS) and the left ventricular posterior wall (LVPW) left ventricular end diastolic volume (LVEDV), systolic volume (LVESV), and ejection fraction (EF) were recorded by echocardiogram. RESULTS: Thirty one (88.6%) rabbits survived myocardial infarction and 26 of them developed aneurysm (83.9%). The mean area of aneurysm was 33.4% ± 2.4% of the left ventricle. LVEF markedly decreased after LVA formation, whereas LVEDV, LVESV and the thickness of IVS as well as the dimension of ventricular chamber from apex to mitral valve annulus significantly increased. LVESP immediately dropped after ligation and recovered to a small extent after LVA formation. LVEDP progressively increased after ligation till LVA formation. Areas in the LV that underwent fibrosis included the apex, anterior wall and lateral wall but not IVS. Agar intra-chamber cast showed that the bulging of LV wall was prominent in the area of aneurysm. CONCLUSIONS: Ligation of LAD and Cx at the middle portion could induce development of LVA at a mean area ratio of 33.4% ± 2.4% which involves the apex, anterior wall and lateral wall of the left ventricle.

Ameliorated stress related proteins are associated with improved cardiac function by sarcoplasmic reticulum calcium ATPase gene transfer in heart failure.

BACKGROUND: Previous studies showed that overexpression of sarco-endoplasmic reticulum calcium ATPase (SERCA2a) in a variety of heart failure (HF) models was associated with greatly enhanced cardiac performance. However, it still undefined the effect of SERCA2a overexpression on the systemic inflammatory response and neuro-hormonal factors. METHODS: A rapid right ventricular pacing model of experimental HF was used in beagles. Then the animals underwent recombinant adeno-associated virus 1 (rAAV1) mediated gene transfection by direct intra-myocardium injection. HF animals were randomized to receive the SERCA2a gene, enhanced green fluorescent protein (control) gene, or equivalent phosphate buffered saline. Thirty days after gene delivery, the cardiac function was evaluated by echocardiographic testing. The protein level of SERCA2a was measured by western blotting. The proteomic analysis of left ventricular (LV) sample was determined using two-dimensional (2-D) gel electrophoresis and MALDI-TOF-MS. The serum levels of the systemic inflammatory and neuro-hormonal factors were assayed using radioimmunoassay kits. RESULTS: The cardiac function improved after SERCA- 2a gene transfer due to the significantly increased SERCA2a protein level. Beagles treated with SERCA2a had significantly decreased serum levels of the inflammatory markers (interleukin-6 and tumor necrosis factor-α) and neuro-hormonal factors (brain natriuretic peptide, endothelin-1 and angiotensin II) compared with HF animals. The myocardial proteomic analysis showed that haptoglobin heavy chain, heat shock protein (alpha-crystallin-related, B6) were down-regulated, and galectin-1 was up-regulated in SERCA2a group compared with HF group, companied by up-regulated contractile proteins and NADH dehydrogenase. CONCLUSIONS: These findings demonstrate that regional intramyocardial injections of rAAV1-SERCA2a vectors may improve global LV function, correlating with reverse activation of the systemic inflammatory, excessive neuroendocrine factors and the stress-associated myocardial proteins, suggesting that the beneficial effects of SERCA2a gene transfer may involve the attenuation of stress-associated reaction.

Improvement in cardiac function after sarcoplasmic reticulum Ca2+-ATPase gene transfer in a beagle heart failure model.

BACKGROUND: Heart failure (HF) is a major cause of morbidity and mortality worldwide, but current treatment modalities cannot reverse the underlying pathological state of the heart. Gene-based therapies are emerging as promising therapeutic modalities in HF patients. Our previous studies have shown that recombinant adeno-associated viral (rAAV) gene transfer of Sarco-endoplasmic reticulum calcium ATPase (SERCA2a) can be effective in treating rats with chronic heart failure (CHF). The aim of this study was to examine the effects of SERCA2a gene transfer in a large HF animal model. METHODS: HF was induced in beagles by rapid right ventricular pacing (230 beats/min) for 30 days. A reduced rate ventricular pacing (180 beats/min) was continued for another 30 days. The beagles were assigned to four groups: (a) control group (n = 4); (b) HF group (n = 4); (c) enhanced green fluorescent protein group (n = 4); and (d) SERCA2a group (n = 4). rAAV1-EGFP (1 x 10(12) microg) and rAAV1-SERCA2a (1 x 10(12) microg) were delivered intramyocardially. SERCA2a expression was assessed by Western blotting and immunohistochemistry. RESULTS: Following 30 days of SERCA2a gene transfer in HF beagles its protein expression was significantly higher than in the HF group than in the control group (P < 0.05). Heart function improved along with the increase in SERCA2a expression. Left ventricular systolic function significantly improved, including the ejection fraction, left ventricular systolic pressure, maximal rate of rise of left ventricular pressure (+dp/dt(max)), and the maximal rate of decline of left ventricular pressure (-dp/dt(max)) (P < 0.05). Left ventricular end-diastole pressure significantly decreased (P < 0.05). The expression of SERCA2a in the myocardial tissue was higher in the SERCA2a group than in the HF group (P < 0.05). CONCLUSIONS: Intramyocardial injection of rAAV1-SERCA2a can improve the cardiac function in beagles induced with HF. We expect further studies on SERCA2a's long-term safety, efficacy, dosage and the optimization before using it in humans with HF.

[Overexpression of sarcoplasmic reticulum calcium ATPase induced hemodynamic and proteomic changes in a dog model of heart failure].

OBJECTIVE: Overexpression of SERCA2a could improve cardiac function in human and experimental heart failure (HF) models. We observed the proteomics changes post SERCA2a overexpression in a pacing induced HF model in dogs. METHODS: Beagles were divided into four groups: control group, HF group (230 beats/min for 4 weeks), HF + EGFP group (myocardial injection of 1 x 10(12) v.g recombinant adeno-associated virus carrying enhanced green fluorescent protein gene, rAAV2/1-EGFP) and HF + SERCA2a group (myocardial injection of 1 x 10(12) v.g recombinant adeno-associated virus carrying SERCA2a gene, rAAV2/1-SERCA2a). Thirty days after gene transduction, left ventricular systolic and diastolic functions were measured by echocardiography and invasive hemodynamics in all animals. By use of 2-dimensional gel electrophoresis (2-DE), -500 distinct protein spots were detected in myocardium of all animals. Protein spots observed to be altered between failing and SERCA2a overexpressed hearts were subjected to tryptic peptide mass fingerprinting for identification by MALDI-TOF mass spectrometry in combination with LC/MS/MS analysis. RESULTS: At 30 day after gene transfer, HF signs were significantly reduced, cardiac function [LVSP: (214.72 +/- 31.74) mm Hg (1 mm Hg = 0.133 kPa) vs. (139.32 +/- 36.79) mm Hg, +dp/dt(max): (6779.43 +/- 217.58) mm Hg/s vs. (2746.85 +/- 931.23) mm Hg/s and -dp/dt(max): (-4341.42 +/- 322.02) mm Hg/s vs. (-2531.14 +/- 616.15) mm Hg/s, LVEDP: (21.86 +/- 6.95) mm Hg vs. (59.78 +/- 6.92) mm Hg] significantly improved in HF + SERCA2a dogs than those in HF + EGFP group(all P < 0.05) and parameters were comparable between HF + SERCA2a and control groups. We identified alterations in the expression level of more than 10 proteins in myocardium. These protein changes were observed mainly in two subcellular compartments: the cardiac contractile apparatus and metabolism/energetics. CONCLUSION: These results showed that overexpression of SERCA2a could improve cardiac function accompanied with numerous alterations in protein expressions involved in calcium handling, myofibrils, and energy production in this dog model of chronic heart failure.

[Diagnostic study on the coronary artery bypass grafts lesions using 64 multi-slice computed tomography angiography].

OBJECTIVE: To evaluate the diagnostic accuracy in the assessment of coronary artery bypass grafts using 64 multi-slice computed tomography angiography (64-MSCTA) technology. METHODS: There were 228 patients post coronary artery bypass grafting (CABG) underwent 64-MSCTA from July 2005 to April 2007. Thirty-one patients with 82 bypass grafts performed coronary angiography (CAG) because of angina or grafts lesion showed by 64-MSCTA. RESULTS: All bypass grafts could be visualized by 64-MSCTA. Thirteen bypass graft occlusions and fourteen significant stenosis were detected by 64-MSCTA and confirmed by CAG. One venous grafts distal anastomosis was missed and another one was miss diagnosed as stenosis. One false negative and one false positive CT-finding resulted in a sensitivity of 93.3%, a specificity of 98.1%, a positive predictive value of 93.3%, a negative predictive value of 98.1% and an accuracy of 97.1% for grafts stenosis. As to the grafts lesion, the sensitivity, specificity, positive predictive value, negative predictive value and accuracy for grafts occlusion were 96.4%, 98.1%, 96.4%, 98.1% and 97.6%, respectively. CONCLUSION: 64-MSCTA demonstrates high diagnostic accuracy in the assessment of graft patency and suitable for the follow-up of patients post CABG.