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Objective@#To investigate the expression and potential role of heterogeneous nuclear ribonucleo-protein A2B1 (HNRNPA2B1) in mouse cerebellar development and the significance of HNRNPA2B1 in human medulloblastoma.@*Methods@#The data of HNRNPA2B1 RNA expression in mouse and human cerebella were obtained from databases. Western blot and immunohistochemical staining were performed to detect the protein level of HNRNPA2B1 in mouse cerebella at different ages. The expression level of HNRNPA2B1 in control human cerebellum and medulloblastoma was detected by immunohistochemical staining. m6A-IP-qPCR method was applied to confirm whether HNRNPA2B1 RNA in Daoy cells was modified with m6A.Western blot was used to detect the effect of MG132 treatment on the HNRNPA2B1 protein level in Daoy cells.@*Results@#The level of HNRNPA2B1 protein in postnatal mouse cerebella was higher than that in adult mouse cerebella, with weak HNRNPA2B1 staining in external granular cells while strong staining in mature Purkinje cells and molecular layer. Compared with control normal human cerebella, the RNA expression level of HNRNPA2B1 increased in medulloblastoma, while immunohistochemical staining showed that the mean intensity of HNRNPA2B1 decreased in medulloblastoma. HNRNPA2B1 RNA in medulloblastoma and Daoy cells was modified by m6A. The HNRNPA2B1 protein level in Daoy cells increased upon MG132 treatment.@*Conclusions@#HNRNPA2B1 is dynamically expressed during mouse cerebellar development. Compared with normal human cerebella, HNRNPA2B1 is significantly up-regulated at transcriptional level but obviously down-regulated at translational level in medulloblastoma. These results indicate that HNRNPA2B1 may be involved in cerebellar development process and medulloblastoma tumorigenesis. The m6A methylation in HNRNPA2B1 transcript and protein ubiquitin-proteasome pathway may account for the down-regulation of HNRNPA2B1 at protein level.
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Objective@#To investigate the biological functions of protein phosphatase 2AC(PP2AC) in the brain, and to detect its spatio-temporal expression and its involvement in neurological disorders in the brains of mice and Alzheimer′s patients.@*Methods@#Western blot was used to evaluate the expression level of PP2AC in different organs. Immunohistochemical staining was performed to detect the in situ expression levels of PP2AC in the brains of mice and patients, and the pathological changes were confirmed in the brains of patients with Alzheimer′s disease.@*Results@#Among all the tested organs in adult mouse, the expression of PP2AC protein was the highest in the brain. From embryonic day 18.5 to postnatal 2-year-old mice, PP2AC exhibited spatio-temporal specific expression in the brains. Furthermore, an age-dependent increased expression in the cerebral cortex at both protein and RNA levels was observed. Compared to control group, PP2AC protein expression was lower in the frontal cortex of Alzheimer′s patients.@*Conclusions@#The spatio-temporal specific expression profiles of PP2AC in mouse brain implicate its biological significance. Its diminished expression in the frontal cortex of Alzheimer′s disease patients implies that PP2AC plays a potential role in the pathogenesis of Alzheimer′s disease.
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Objective@#To investigate the role of Mic60 in cardiac aging.@*Methods@#Wild-type and Mic60+ /- male mice at age of 4-6 months (young group, n=6) and 18-20 months (aged group, n=9) were used. H&E and Masson staining of frozen and paraffin sections were subjected to morphologic evaluation of the cardiac tissue samples. SA-β-Gal staining was utilized to detect the activity of senescence-associated β-galactosidase. Western blot was performed to detect the expression of Mic60 and p21 in cardiac tissues.@*Results@#Expression of Mic60 in mouse cardiac tissue increased in an age-dependent manner. Haploid insufficiency of Mic60 resulted in an increased left ventricular wall thickness [(1.32±0.09) mm vs.(1.12±0.09) mm, P<0.05], cardiomyocyte hypertrophy[(474.9±27.6) μm2 vs.(358.8±48.7) μm2, P<0.05] and interstitial fibrosis [ (38.24±7.58) ×103μm2 vs.(25.81±4.12)×103μm2, P<0.05], increased activity of SA-β-Gal (2.26±0.24 vs.0.25±0.05, P<0.01) and higher expression of p21 (P<0.01) in aged mouse cardiac tissue, but not in young mice.@*Conclusion@#Haploid insufficiency of Mic60 leads to cardiac hypertrophy, interstitial fibrosis, increased activity of SA-β-Gal and higher expression of p21 in aged cardiac tissue in mice, suggesting that Mic60 may prevent cardiac aging.
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<p><b>OBJECTIVE</b>To identify novel lncRNAs involved in cerebellar neurogenesis using neuronal specific Nbs1-deficient (Nbs1(CNS-del)) mouse model.</p><p><b>METHODS</b>Microarray analysis was performed to identify differentially expressed lncRNAs between Nbs1(CNS-ctr) and Nbs1(CNS-del) mice. Expression profiles of lncRNA Gm15577 and coding gene Negr1 in mice, primary cerebellar culture and cell lines were measured using RT-qPCR. Subcellular fractionation was performed to determine the subcellular localization of Gm15577.</p><p><b>RESULTS</b>Gm15577 was specifically expressed in mice cerebellum in a developmentally regulated manner, which could be abolished upon Nbs1-deficiency. Gm15577 was located in the intronic region of Negr1 in a reversed orientation. Gm15577 modulated the RNA expression of Negr1, Shh and β-catenin. NEGR1 had a distinct expression pattern between normal and medulloblastoma patients.</p><p><b>CONCLUSION</b>Gm15577 may modulate cerebellar granule cell proliferation and differentiation by targeting Negr1, and their dysfunctions or abnormal expression may be related to tumorigenesis of medulloblastoma.</p>
Subject(s)
Animals , Humans , Mice , Cell Differentiation , Cell Proliferation , Cell Transformation, Neoplastic , Cerebellar Neoplasms , Pathology , Cerebellum , Cell Biology , Physiology , Disease Models, Animal , Introns , Medulloblastoma , Pathology , Mice, Knockout , Neurogenesis , Neurons , Physiology , RNA, Long Noncoding , MetabolismABSTRACT
Objective To assess the diagnostic value of 99Tcm-ECD SPECT for hyperacute cerebral ischemia using rats models.Methods A stable and permanent acute cerebral ischemia model using unilateral middle cerebral occlusion was tested in 24 healthy SD rats.The rats were randomly divided into 6 groups according to the time duration between imaging and induced-ischemia (1,2,3,4,5 and 6 h,respectively).The rats were sacrificed immediately after 99Tcm-ECD SPECT/CT imaging and then the brain tissue was dissected for triphenyl tetrazolium chloride (TTC) and HE staining.The count ratio of affected cortex to the contralateral cortex of ＜ 50％ was defined as ischemia on micro SPECT/CT.The volume of the ischemic area was calculated on both SPECT/CT and TTC images.Paired t test was used to determine the statistical difference between the volumes on SPECT/CT and TTC staining.Results The ischemia volume evaluated by TTC staining at 1,2,3,4,5 and 6 h after occlusion was (73.98 ± 27.76),(90.75 ±29.00),(135.00±40.83),(136.25±22.51),(158.50±32.72) and (168.00±32.75) mm3,respectively.The corresponding ischemia volume evaluated by micro SPECT/CT was (98.50 ± 27.77),(110.40±26.80),(157.00±36.82),(165.50±26.54),(175.75±31.16) and (177.25 ±34.33) mm3,respectively,which was concordant with that by TTC staining at each time point (t:-1.681 to-0.390,all P ＞0.05).The ischemic area on micro SPECT/CT imaging was consistent with the pink area by TTC staining.The volume evaluated by micro SPECT/CT tended to be constant 3 h after the occlusion.The ischemia volume showed no significant difference among 3,4,5 and 6 h (all P ＞ 0.05).Conclusions Micro SPECT/CT may have an haemodynamic value for evaluating in vivo cerebral ischemia applied in a rat model.It might have clinical value for the evaluation and decision-making of ultra acute cerebral infarctions.
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Objective To study the molecular mechanism of medulloblastoma.Methods The cell line with PARP-1 and P53 null mutation was established and characterized.Mouse medulloblastoma cell line derived from PARP-1/P53 double knockout mice was established.We analyzed cell characters after 30 passages,using neuronal cell-specific markers by immunofluorescence.The cells were transfected with pEGFP-C1-Hparp-1 and pEGFP-C1 plasmids,the expression of PARP-1 protein was detected by immunofluorescence stainning and Western blot.Results The cells showed positive immunoactivity for the neuronal-specific markers such as Vimentin,Dcx and ?Ⅲ-Tubulin,and cells were negative for PARP-1 protein.Exogenous PARP-1 expression was visualized by immunofluorescence and Western blot analysis after pEGFP-C1-Hparp-1 transfection.Conclusion Mouse medulloblastoma cell line with defective function for DNA damage recovery has been successfully established,which provides a useful tool for further dissecting the molecular mechanism and pathogenesis of medulloblastoma.