Downregulation of the LncRNA MEG3 Promotes Osteogenic Differentiation of BMSCs and Bone Repairing by Activating Wnt/ß-Catenin Signaling Pathway.

BACKGROUND: Previous studies have demonstrated that long non-coding RNA maternally expressed gene 3 (MEG3) emerged as a key regulator in development and tumorigenesis. This study aims to investigate the function and mechanism of MEG3 in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and explores the use of MEG3 in skull defects bone repairing. METHODS: Endogenous expression of MEG3 during BMSCs osteogenic differentiation was detected by quantitative real-time polymerase chain reaction (qPCR). MEG3 was knockdown in BMSCs by lentiviral transduction. The proliferation, osteogenic-related genes and proteins expression of MEG3 knockdown BMSCs were assessed by Cell Counting Kit-8 (CCK-8) assay, qPCR, alizarin red and alkaline phosphatase staining. Western blot was used to detect ß-catenin expression in MEG3 knockdown BMSCs. Dickkopf 1 (DKK1) was used to block wnt/ß-catenin pathway. The osteogenic-related genes and proteins expression of MEG3 knockdown BMSCs after wnt/ß-catenin inhibition were assessed by qPCR, alizarin red and alkaline phosphatase staining. MEG3 knockdown BMSCs scaffold with PHMG were implanted in a critical-sized skull defects of rat model. Micro-computed tomography(micro-CT), hematoxylin and eosin staining and immunohistochemistry were performed to evaluate the bone repairing. RESULTS: Endogenous expression of MEG3 was increased during osteogenic differentiation of BMSCs. Downregulation of MEG3 could promote osteogenic differentiation of BMSCs in vitro. Notably, a further mechanism study revealed that MEG3 knockdown could activate Wnt/ß-catenin signaling pathway in BMSCs. Wnt/ß-catenin inhibition would impair MEG3-induced osteogenic differentiation of BMSCs. By using poly (3-hydroxybutyrate-co-3-hydroxyhexanoate, PHBHHx)-mesoporous bioactive glass (PHMG) scaffold with MEG3 knockdown BMSCs, we found that downregulation of MEG3 in BMSCs could accelerate bone repairing in a critical-sized skull defects rat model. CONCLUSIONS: Our study reveals the important role of MEG3 during osteogenic differentiation and bone regeneration. Thus, MEG3 engineered BMSCs may be effective potential therapeutic targets for skull defects.

Hypoxia-induced USP22-BMI1 axis promotes the stemness and malignancy of glioma stem cells via regulation of HIF-1α.

AIMS: This study intends to investigate the mechanisms of ubiqutin-specific protease 22 (USP22)/B cell-specific Moloney murine leukemia virus integration site 1 (BMI1) on the biological phenotypes of glioma stem cells (GSCs) under hypoxia. MAIN METHODS: Western blot, Cell Counting Kit-8, colony formation and flow cytometry assays were preformed to evaluate cells biological behaviors. Luciferase assay was utilized to identify the associations among USP22, HIF-1α and BMI1. KEY FINDINGS: Silencing USP22 reduced the stemness and proliferation of GSCs, and increased its apoptosis in response to hypoxia. Whilst, overexpression of BMI1 reversed these phenomena. Whilst, a significant decrease in proliferation and stemness of GSCs caused by HIF-1α exhaustion were inversed by overexpression of USP22 or BMI1. SIGNIFICANCE: Function of USP22-BMI1 on biological behaviors of GSCs was regulated by HIF-1α in response to hypoxia.

Activation of DRD5 (dopamine receptor D5) inhibits tumor growth by autophagic cell death.

Dopamine agonists such as bromocriptine and cabergoline have been successfully used in the treatment of pituitary prolactinomas and other neuroendocrine tumors. However, their therapeutic mechanisms are not fully understood. In this study we demonstrated that DRD5 (dopamine receptor D5) agonists were potent inhibitors of pituitary tumor growth. We further found that DRD5 activation increased production of reactive oxygen species (ROS), inhibited the MTOR pathway, induced macroautophagy/autophagy, and led to autophagic cell death (ACD) in vitro and in vivo. In addition, DRD5 protein was highly expressed in the majority of human pituitary adenomas, and treatment of different human pituitary tumor cell cultures with the DRD5 agonist SKF83959 resulted in growth suppression, and the efficacy was correlated with the expression levels of DRD5 in the tumors. Furthermore, we found that DRD5 was expressed in other human cancer cells such as glioblastomas, colon cancer, and gastric cancer. DRD5 activation in these cell lines suppressed their growth, inhibited MTOR activity, and induced autophagy. Finally, in vivo SKF83959 also inhibited human gastric cancer cell growth in nude mice. Our studies revealed novel mechanisms for the tumor suppressive effects of DRD5 agonists, and suggested a potential use of DRD5 agonists as a novel therapeutic approach in the treatment of different human tumors and cancers.

[Microsurgical strategies of glioma located in lateral fissure area].

OBJECTIVE: To investigate the microsurgical strategies of glioma located in lateral fissure area. METHODS: The clinical data of 123 patients with glioma located in lateral fissure area confirmed by pathology, 76 males and 47 females, aged 46.2 (4-75), were retrospectively analyzed. The optimal surgical approach and comprehensive therapeutic strategies were selected according to the imaging features and pathological properties of tumors. Resections were performed by the pterion approach in all cases to remove the tumors, navigational orientation was used in 17 cases, and supervision by B mode ultrasonography was used in 12 cases. The branches of middle cerebral artery and fissure vein were protected carefully. The patients with tumors above grade U, confirmed pathologically after resection, underwent chemotherapy ( teniposide + semustine or temozolomide) and radiotherapy that was designated individually according to the pathological grade and distribution of the tumors. Follow-up was conducted by telephone, mail or outpatient department visit on 102 of the 133 patients (82.9%). RESULTS: 82 patients (66.7%) underwent total resection, 18 (14.6%) underwent subtotal resection, 16 (13.0%) underwent major resection, and 7 (5.7%) underwent partial resection. Postoperatively cerebral vasospasm in 8 cases, brief aphasia and reaction clumsily in 4 cases, muscle strength decline in 3 cases, and epilepsy in 1 case, these patients were submitted to symptomatic treatment with progressive improvement of the above-mentioned signs and symptoms. One patient died of malignant intracranial hypertension. The follow-up showed that 97 patients survived over 1 year, the 5-year survival rate was 25.6%, and the average survival time was 21.7 months. CONCLUSION: The lateral fissure area glioma can be treated through proficient microsurgical technique after the anatomic training. It is the key in the surgery on the lateral fissure glioma to protect the main branches of middle cerebral artery, trunk of middle cerebral vein, and important brain functional areas.

[Establishment of animal model of chronic optic nerve injury and pathological changes thereof: experiment with cats].

OBJECTIVE: To establish an animal model of chronic optic nerve injury which is suitable for experimental research. METHODS: Dil, a tracer, was injected through the bone windows into the brain of 48 cats so as to mark the retinal ganglion cells (RGCs). Two weeks later the 48 cats were randomly divided into 6 equal groups. The normal group did not receive any other treatment. Other 8 cats underwent sham operation. Imitating the clinical pterional approach, a balloon was implanted into the place under the optic nerve and chiasm in the other 32 cats, then the volume of the balloons were increased by injecting contrast agent at different times to cause the optic nerve and chiasm compressed chronically for 1, 2, 4, or 6 weeks. Flash-visual evoked potential (F-VEP) was measured before operation and at the corresponding observation times in different groups. By the end of the experiment the cats were killed with the specimens of retina and optic nerve taken out to undergo light microscopy and electron microscopy to observe the pathological changes. Eight eyes were taken out from each group to calculate the number of RCGs 1, 2, 4, and 8 weeks after operation respectively. RESULTS: Microscopy showed retina showed profound morphological changes 8 weeks after compression; Demyelination of optic nerve began to occur 2 weeks after compression and progressed later. Axonal degeneration was found 4 week after compression and became more significant 8 weeks later. Under electron microscopy, pathological changes of retina was found 4 weeks and more prominent 8 weeks after compression; Slight demyelination and disorganized of cytoskeleton in the optic nerve were shown 2 weeks after compression, and became more profound later; Myelin regeneration was found 8 weeks after compression. The number of RGCs was reduced significantly by 37% (293/465) since 8 weeks after compression. F-VEP recording showed an extension of latency and depression of amplitude 4 weeks after compression, and the changes were more significant 8 weeks later. CONCLUSION: An animal model of chronic optic nerve injury by compression has been established which is stable and well repeatable. The pathological changes of compressed optic nerve are aggravated gradually as the compression lasts and the volume increases. Degeneration of RGCs occurs secondarily and obviously later than the axonal degeneration.

[Regional specificity in proliferation capacity of neural stem cells from hippocampus and epithalamus: study with rats].

OBJECTIVE: To compare the regional specificity in proliferation capacity of neural stem cells derived from hippocampus and epithalamus: study with rats. METHODS: Neural stem cells (NSCs) were separated mechanically from the hippocampus and the epithalamus of an embryo of SD rat respectively and cultured in serum-free medium to observe the growth status dynamically and their in vitro growth curves were drawn. Single cell clones were established and identified by immunofluorescence staining. The surface morphology of the clone balls were observed by using scanning electron microscope. The ultrastructure was observed by scanning electron microscopy. The 3D morphology was observed under a laser confocal microscope. NSCs were co-incubated with 5-bromodeoxyuridine (Brdu) and immunofluorescence staining was used to observe their in vitro proliferation potentials. Studies on. RESULTS: The in vitro growth of the hippocampus-derived NSCs was basically the same as that of the epithalamus-derived NSCs, however, the culture fluid of the epithalamus-derived NSCs became yellowish 18 hours after the inoculation, significantly earlier than that of the hippocampus-derived NSCs. Showing a faster multiplication rate of the latter. Scanning electron microscopy showed that these two types of NSCs had a similar ultrastructural feature. Phase contrast microscopy and laser confocal microscopy showed similar form in the NSCs from two different sources. Co-incubation with found that the Brdu positive rate of the epithalamus-derived NSCs was 74.87%, significantly higher than that of the hippocampus-derived NSCs (63.07%, P < 0.05). The cell growth curves of the NSCs from both sources displayed the same multiplication trend, however, the NSC count of the epithalamus-derived NSCs at the 8th, 12th, 16th, 20th, and 24th days after culture were all higher than those of the corresponding hippocampus-derived NSCs at the same time-points (all P < 0.05). CONCLUSION: Both embryonic hippocampus-derived and epithalamus-derived NSCs can be acquired by using mechanical separation and serum-free culture method. The two types of cells have a similar morphological structure and possess the in vitro multiplication capacity. However, the multiplication rate of the epithalamus-derived NSCs is higher than that of the hippocampus-derived NSCs.

[Experience of resection large clinoidal meningiomas].

OBJECTIVE: To investigate the clinical applied anatomy in the region of anterior clinoid process, and to improve the therapeutic efficacy of clinoidal tumors. METHODS: Twelve patients with large meningiomas located in clinoid were surgically treated via the extended anterior and middle fossa combined with epidural approach between January 1998 and August 2004. The surgical outcome and follow-up results were reviewed retrospectively. Supraorbital-posterional approach and cranioorbital zygomatic approach were used when tumors involved cavernous sinus. Anterior clinoid process was grinded with high-speed drilling. Supply of tumors were blocked extradurally. Tumors were resected intradurally. RESULTS: Of the 12 cases in large meningiomas located in clinoid, 8 cases had total removal of tumors, 3 patients had subtotal removal. Of the 10 patients with pre-operative severe visual deterioration, 6 patients was markedly improved, one patient unchanged and one patient worsened post-operatively. No death was found in this group. CONCLUSIONS: Using epidural approach for clinoidal meningiomas and grinding anterior clinoid process was advantageous to block tumors base blood supply and detach infraclinoidal tumors from internal carotid artery. Supraorbital-pterional approach could minimize brain retraction and was advantageous to expose superior pole of giant tumors.

[Cluster analysis and identify significance of novel genes related to glioma in molecule classification].

OBJECTIVE: To screen differentially expressed genes in the development of human glioma and establish molecular classification of glioma preliminary based on gene expression using cDNA microarray. METHODS: Brain specimens were obtained from 18 patients with glioma, 10 males and 8 females, aged 14 approximately 62 with an average age of 44.4. The total RNAs of these glioma specimens and 2 specimens of donated brain of normal adults were extracted. BioStarH140S microarray (including 8347 old genes and 5592 novel genes) were adopted and hybridized with probes which were prepared from the total RNAs. Differentially expressed genes between the normal tissues and glioma tissues were assayed after scanning cDNA microarray with ScanArray 4000. Northern hybridization, and in situ hybridization (ISH) were used to identify the functions of novel genes. Those differentially expressed genes were studied with Hierarchical method and molecular classification of glioma was preliminarily carried out. RESULTS: Among the 13 939 target genes, there were 1200 (8.61%) differentially expressed genes and 395 (2.83%) novel genes. 348 genes were up-regulated and 852 genes were down-regulated in glioma. The results of bioinformatical analysis, Northern hybridization and ISH revealed that those novel genes were highly associated with glioma. There were multiple genes which were relevance to classification by Hierarchical method, such as MAP gene, cytoskeleton and matrix motility genes, etc. Molecular classification of glioma with Hierarchical cluster was in accordance with pathology and revealed internal essence in tumorigenesis and development. CONCLUSION: Multiple genes play important roles in development of glioma. cDNA microarray technology is a powerful technique in screening for differentially expressed genes between two different kinds of tissues.