The COMPASS Core Member Ash2l Regulates the Cell Cycle of Neural Progenitor Cells / 中国生物化学与分子生物学报

To investigate the role of Ash2l (absent, small, or homeotic 2-like, Ash2l) on the proliferation ability and cell cycle of mouse cerebral cortical neural progenitor cells (NPCs), We examined the number and distribution of NPCs, using the radial glial cell marker PAX6 and intermediate progenitor cell marker TBR2. E16. 5 mice were labeled with EdU for 30 min to detect the proliferation ability of NPCs. Conditional knockout of Ash2l resulted in a dramatic reduction in the number of NPCs and a disordered distribution. The 30 min EdU insertion experiment showed that EdU could hardly be inserted into NPCs, indicating that the proliferation ability of NPCs was severely affected. Using the mitotic cell cycle marker pH3, the distribution of dividing NPCs was observed. We then detected the expressed level of Cyclin A by Western blotting. The distribution of cell nuclei of M-phase is disordered and the expression of G

Morphology of intervertebral disc in Chinese adults evaluated using computer tomography three-dimensional imaging / 中国组织工程研究

BACKGROUND: The artificial disc requires a height, a width, and a shape as much as possible to be similar to the original intervertebral disc in order to perfectly distribute the load. At present, most of the data are from foreign countries or measured using X-ray and magnetic resonance imaging, but there are some shortcomings. OBJECTIVE: To diagnose the spinal disease and provide data for the design of a native lumbar disc device by measuring the normal lumbar intervertebral disc using computer tomography (CT). METHODS: A total of 2 235 patients who underwent lumbar CT examination in Liuzhou Worker's Hospital from January 2012 to May 2017 were collected and analyzed. There were 62 cases, including 45 males and 17 females, after being strictly met the inclusion and exclusion criteria. From the age of 20, they were divided into four groups: 20-30, 31-40, 41-50 and more than 50 years old. The range was from L1to S1. The measurement index include intervertebral disc anteroposterior diameter, transerverse diameter, disc volume, sagittal anterior, middle and posterior height, coronal left and right height, and interverterbral angle. RESULTS AND CONCLUSION: (1) In terms of age, there was a statistically significant difference in measurement indexes between L1/2, L2/3 and L3/4(P < 0.05). Therefore, the factor of age should be taken into account. However, there was no statistically significant difference between L4/5and L5/S1segments (P > 0.05). (2) There was a statistical difference between the anterior and middle height of the sagittal position in L4/5and L5/S1(P < 0.05). (3) There were statistical differences between the angles of L2/3, L3/4, L4/5, L5/S1intervertebral space (P <0.05), but the difference of angle between L4/5and L5/S1was the most. (4) There was no statistical difference in the height between the left and right sides of the coronal position (P > 0.05). (5) There was statistical difference between the anteroposterior and transverse diameters of the disc on the adjacent cross section (P < 0.05). (6) The results showed that the lower the segment, the smaller the statistical difference between each group. It is indicated that the age difference should be considered on the L1/2, L2/3, and L3/4segments in the design of lumbar disc or interbody fusion. The lumbar artificial intervertebral disc can be placed in the middle or side. The artificial disc should be designed into the wedge shape instead of a rectangle. These will provide a good anatomical basis for the design of domestic lumbar artificial intervertebral discs.

Phenotype change of glioma stem cells is related with the RNA binding proteins in hypoxia / 基础医学与临床

Objective To explore the relationship between the change of phenotype of glioma stem cells and expres-sion of RNA binding proteins in hypoxia. Methods Glioma stem cells(U87MG-SLC and GSC5) were cultured un-der hypoxia (1% O2) and normoxia(20% O2). Cell proliferation was measured by MTS assay and self-renewal ability was determined by tumorsphere formation assay. The expression of RNA binding protein and stemness mark-ers protein were examined by Western blot and statistics was carried out. Results The proliferation of glioma stem cells was inhibited and the self-renewal ability was promoted in hypoxia. Meanwhile,hypoxia significantly promoted the expression of HIF-1α and stemness markers.Under hypoxia, the expression of RNA binding protein was changed. The expression of hnRNPF, UNRIP and HuD increased. Meanwhile the expression of PCBP2 and UNR was downregulated. But,other RNA binding proteins(hnRNPK,ADAR1,PCBP1,CIRP,EBP1,eEF1A,PTBP1,PTBP2) had no significant change. Conclusions The change of phenotype of glioma stem cells in hypoxia is relat-ed with the RNA binding proteins (hnRNPF,UNRIP,HuD,PCBP2 and UNR).

Screening alternative splicing on long noncoding RNAs regulated by PTB in glioma / 基础医学与临床

Objective To further investigate the role of PTB in regulating the alternative splicing of lncRNAs in a glioblastoma tumorigenesis,and analyze spliced lncRNAs. Methods Analyzing array data and screening a specific set of alternative spliced lncRNAs. Total RNA was isolated from PTB knockdown glioblastoma cells (U87MG) or glioblastoma and normal cell lines and tissue samples,and subjected to real-time PCR(RT-PCR) assays to detect the expression level of spliced transcripts. Alternatively spliced lncRNAs were identified as target genes that may be regulated by PTB protein by knocking down method. Nuclear and cytoplasmic isolation were performed on T98G cells to identify cellular location of lncRNA. Results Our results uncovered PTB which impact on the transcript level of several lncRNAs including linc00882. Interestingly,the lncRNA linc00882 significantly exhibited differential spli-cing patterns between two splice variants in the PTB-abundant glioblastoma cells. The alternative splicing transcripts were located in cell cytoplasm. Conclusions The results suggest that PTB may have an effect on the alternative spli-cing of linc00882 in glioma.

Advances in CRISPR/Cas9 gene-editing system / 基础医学与临床

CRISPR/Cas9 gene-editing system has been broadly used in various fields of bioscience and medicine in recent years. The system can be guided by RNA to specific DNA site thus achieving targeted gene editing.Off-target effect and editing efficiency remain to be two crucial challeges to the system. Currently, a number of researches have been focused on the optimization of the system by reducing off-target effects and increasing editing efficiency, which may enhance its safety and expand its application.

Recombinant Expression and Purification of Mouse Nectin-like 4 Glycoprotein in 293ET Cell Line / 中国医学科学杂志(英文版)

Objective To screen the transient and stable cell lines with high production of Nectin-like 4 (Necl-4) protein. Methods First, cDNA sequences encoding the extracellular domain of Necls were cloned into the modified vector pAPtag at the N terminus of alkaline phosphatase (AP) for fusion expression. Next, 293ET cells stably expressed Necls-AP fusion protein and secreted it into the culture medium which were detected by the AP activity assay and Western blot analysis. Then, by adding N-glycosylation processing inhibitor kifunensine into the medium, complex glycan was inhibited to generate. The residual glycan of purified protein was removed by endoglycosidase H. Finally, AP protein was removed by using human rhinovirus protease and size exclusion chromatography. The concentration of purified Necl-4 protein was monitored by measuring the absorbance at 280 nm and analyzed by SDS-PAGE. Result The transient and stable cell lines with high production of Necl-4 protein were screened by the color reaction with the AP-tag in the recombinant vector. The soluble and active form of purified Necl-4 protein was obtained after deglycosylation of native N-glycan protein with an expression level of 4 mg/L culture and purity of 95%. Conclusions By using modified AP mammalian protein expression system, we can easily screen the high productive stable cell lines by using AP activity assay. By adding mannosidase inhibitor kifunensine into the medium and cutting purified protein by using endoglycosidase H, we can obtain deglycosylated Necl-4 protein in milligram quantities. Our method might throw a light on the expression and purification of glycoprotein for structural and functional studies.

Astragaloside Ⅳ Protects Against Aβ1-42-induced Oxidative Stress, Neuroinflammation and Cognitive Impairment in Rats / 中国医学科学杂志(英文版)

Objective To investigate the neuroprotective action of astragaloside Ⅳ (AS-Ⅳ) on spatial learning and memory impairment induced by amyloid-beta 1-42 (Aβ1-42) in rats and elucidate its underlying molecular mechanisms. Methods Adult-male Sprague-Dawley rats (230-250 g) were divided into six groups randomly: control, Aβ1-42, AS-Ⅳ, Aβ1-42 plus 5 mg/kg·d AS-Ⅳ, Aβ1-42 plus 25 mg/kg·d AS-Ⅳ, and Aβ1-42 plus 50 mg/kg·d AS-Ⅳ groups. Aβ1-42 were delivered by intracerebroventricular injection under the guidance of a brain stereotaxic apparatus. The Morris water maze test (hidden platform test, probe trials, visible platform test) was performed one week after Aβ1-42 injection to obtain the ability of rat spatial learning and memory. AS-Ⅳ (5, 25 and 50 mg/kg·d) was administrated intraperitoneally once per day from the 8th day after Aβ1-42 injection for 5 consecutive days. Average escape latencies, distances for searching for the platform under water and the percentage of total time elapsed and distance swam in the right quadrant after removing platform were determined by behavior software system. The vision and swim speeds of rats were also determined to exclude the effect of these factors on the parameters of learning and memory. After behavioral tests, the rats were sacrificed immediately by decapitation. Hippocampus were collected. The enzyme activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-px) and catalase (CAT) in the hippocampus obtained from different-treated rat brain were measured by following the manufacturer's instructions. The levels of interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) in tissue lysates were assayed with ELISA. Results The water maze test results indicated that chronic treatments with AS-Ⅳ effectively protected the rats from Aβ1-42-induced spatial learning and memory impairment. Furthermore, the activities of SOD, GSH-px and CAT decreased by Aβ1-42 were also restored by AS-Ⅳ treatment in the hippocampus of rats. In addition, AS-Ⅳ significantly decreased the levels of IL-1β and TNF-α in the hippocampus of Aβ1-42-induced amnesia's rats. Conclusion Our findings suggest that AS-Ⅳ might be a useful chemical in improving the spatial memory and relieving the oxidative stress and neuroinflammation in Alzheimer patients.

RNA-binding Protein UNR Promotes Glioma Cell Migration and Regulates the Expression of Ribosomal Protein L9 / 中国医学科学杂志(英文版)

Objective To investigate the role of RNA binding protein─upstream-of-N-Ras (UNR) in the development of glioma and its molecular mechanism.Methods First, bioinformatics analysis of CGGA database was performed to detect UNR expression level and prognosis of patients with glioma. Western blot and real-time PCR were used to detect UNR expression level in glioma cell lines and tissues. Next, UNR siRNAs were transfected in glioma cells, and MTS assay and scratch wound-healing assay were used to detect changes in cell proliferation and migration. Then, the candidate UNR target mRNAs were identified by analyzing the sequencing data of UNR iCLIP-seq, RNA sequencing and ribosome profiling databases of human melanoma. RNA immunoprecipitation and biotin pull-down assays were used to identify the UNR target mRNAs in glioma cells. Finally, western blot was used to detect the effect of UNR knockdown on ribosomal protein L9 (RPL9) and RPL9 protein expression level in glioma cell lines. RPL9 siRNA was transfected in A172 and T98G and the expression of vimentin in the cells was detected with western blot.Results Bioinformatics analysis showed that UNR mRNA expression level was significantly higher in high-grade glioma [Grade 2 (n=126), Grade 3 (n=51), Grade 4 (n=128), P<0.001]. UNR high expression levels were associated with poor prognosis (P=0.0177). UNR had high expression level in glioma cell lines and patient samples compared with normal cell lines and normal brain samples (P<0.01). Knockdown of UNR inhibited glioma cells migration (P<0.05), but did not inhibit glioma cells growth in three glioma cell lines. UNR binded the 3' untranslated region (UTR) of PTEN and RPL9 mRNAs. RPL9 protein was significantly highly expressed in most glioma cell lines (n=9) and knockdown of UNR resulted in a downregulation of RPL9 protein expression. Epithelial-mesenchymal transition (EMT)-related marker─vimentin was positively regulated by RPL9.Conclusions UNR could bind to the 3'UTR of PTEN and RPL9 in glioma cell lines, therefore promoting glioma cell migration and regulating the expression of RPL9. Here, we establish a link between UNR and RPL9 protein, which will provide new ideas for the further study of glioma.

Functions of miR-9 and miR-9* during Aging in SAMP8 Mice and Their Possible Mechanisms / 中国医学科学院学报

<p><b>OBJECTIVE</b>To explore the functions of miR-9 and miR-9(*) in SAMP8 mice during the aging and their possible mechanisms.</p><p><b>METHODS</b>SAMP8 mice(4-,8-,12-month old,respectively)were selected,three age-matched SAMR1 mice were used as the control group with three mice in each group. The brains were collected and then sectioned for in situ hybridization of miR-9 and miR-9(*). Mimics or inhibitors of miR-9 and miR-9(*) were transfected into N2a cells,and the effects of overexpression or knockdown of the microRNAs on the cell cycle were detected by flow cytometry. Target genes were predicted by bioinformatic analysis and confirmed by dual luciferase assay.</p><p><b>RESULTS</b>Expressions of miR-9 and miR-9(*) in hippocampus of SAMP8 mice were lower than those of SAMR1 mice. Knockdown of miR-9 and miR-9(*) induced a prolonged G1 phase and a shortened S phase in N2a cells;in contrast,miR-9 and miR-9(*) overexpression showed opposite effects. The predicted target genes of miR-9 were PSEN1,SCN2B,MAP3K3,and BACE1,and that of miR-9(*) was CDKn1c. Dual luciferase reporter gene assay showed that miR-9 targeted MAP3K3 while miR-9(*) targeted CDKn1c.</p><p><b>CONCLUSION</b>miR-9 and miR-9(*) play an important role during aging via the target genes MAP3K3 and CDKn1c in the SAMP8 mice.</p>

Ribotrap analysis of proteins associated with FHL3 3'untranslated region in glioma cells / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To screen the proteins associated with four-and-a-half LIM domains 3 (FHL3) 3' untranslated region (3'UTR) in glioma cells.</p><p><b>METHODS</b>Western blot was adopted to detect the regulatory effect of poly(C)-binding protein 2 (PCBP2) on FHL3. Biotin pull-down and sliver staining were employed to screen and verify the candidate binding proteins of FHL3 3'UTR. Then liquid chromatography-tandem mass spectrometry (LC-MS/MS) and molecule annotation system were used to identify and analyze the candidate binding proteins. Immuno- precipitation was conducted to study the interaction between PCBP2 and polypyrimidine tract-binding protein 1 (PTBP1), a binding protein identified by LC-MS/MS.</p><p><b>RESULTS</b>PCBP2 could bind to FHL3 mRNA 3'UTR-A and inhibited the expression of FHL3 in T98G glioms cells. 22 candidate binding proteins were identified. Among them, there were 11 RNA binding proteins, including PCBP2. PTBP1 associated with FHL3 mRNA 3'UTR and interacted with PCBP2 protein.</p><p><b>CONCLUSIONS</b>PCBP2 and PTBP1 can both associate with FHL3 mRNA 3'UTR through forming a protein complex.</p>

Screening of substrates of protein arginine methyltransferase 1 in glioma / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To screen the asymmetric dimethyl arginines (ADMA)-containing proteins which could combine with protein arginine methyltransferase 1 (PRMT1).</p><p><b>METHODS</b>Western blot was adopted to identify the expression of PRMT1 and the proteins with ADMA in glioma cell lines and normal brain tissues, and then to detect the changes of ADMA level after knock-down of PRMT1 with RNAi transfection in U87MG cells. Co-Immunoprecipitation (Co-IP), western blot, and sliver staining were employed to screen the candidate binding proteins of PRMT1. Then liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify the binding proteins of PRMT1.</p><p><b>RESULTS</b>The expression of PRMT1 and some levels of ADMA were higher in glioma cell lines than in normal brain tissues. After knocking down PRMT1, some ADMA levels were found declined. After screening the binding proteins of PRMT1 with Co-IP and LC-MS/MS, 26 candidate binding proteins were identified. Among them, 6 candidate proteins had higher ions scores (> 38) and bioinformation analysis predicted that SEC23-IP, ANKHD1-EIF4EBP3 protein, and 1-phosphatidylinositol-3-phosphate 5-kinase isoform 2 had possible methylated aginine sites.</p><p><b>CONCLUSIONS</b>The high expression of PRMT1 in glioma may induce the change of ADMA levels. Altogether 26 candidate proteins were identified, which contain ADMA and specifically bind with PRMT1.</p>

Role of extracellular signal-regulated protein kinase 5 in the biosynthesis of follicle-stimulating hormone-stimulated progesterone in primary granulosa cells / 中国医学科学院学报

<p><b>OBJECTIVE</b>To study the role of extracellular signal-regulated protein kinase 5 (ERK5) during the biosynthesis of follicle-stimulating hormone (FSH)-mediated progesterone in primary granulosa cells.</p><p><b>METHODS</b>The expressions of phosphorylated and general forms of ERKS in primary granulosa cells after the treatment of FSH were detected by Western blot analysis. The subcellular localization of ERK5 was observed by confocal microscopy. The effect of ERK5 on FSH-mediated progesterone biosynthesis in primary granulosa cells was analyzed using recombinant adenovirus vectors.</p><p><b>RESULTS</b>ERK5 activation was induced by FSH in a time-dependent manner in primary cultured granulosa cells, although the general ERK5 protein level decreased also in a time-dependent manner. The treatment of FSH showed no remarkable effect on the subcellular distribution of endogenous ERK5, which was mainly in the cytoplasm of granulosa cells. The co-infection of Ad-caMEK5 and Ad-wtERK5 increased the progesterone production and StAR expression in primary cultured granulosa cells, whereas inhibition of ERK5 activation suppressed the FSH-stimulated progesterone production.</p><p><b>CONCLUSION</b>ERK5 may stimulate FSH-mediated progesterone production in primary cultured granulosa cells.</p>

Screening and identification of natural antisense transcript in mouse cerebral cortex / 中国医学科学院学报

<p><b>OBJECTIVE</b>To screen and identify the possible existence of natural antisense transcript (NAT) within the mouse neocortex.</p><p><b>METHODS</b>Sixty-three cerebral cortex layer-specific genes were screened by bioinformatics prediction in mice, among which 31 mice with potential NATs were screened. NAT was identified using reverse transcription polymerase chain reaction (RT-PCR) and then cloned in pGEM-T Vector System for sequencing.</p><p><b>RESULTS</b>Among 31 genes predicted using bioinformatics, 8 were proved to be NAT positive by RT-PCR.</p><p><b>CONCLUSIONS</b>NATs exist in the mouse neocortex tissue during the development of cerebral cortex. NATs may influence mouse cortical development by regulating the related coding genes.</p>

A population-based epidemiologic study of bowel habits in Guangdong province / 中华流行病学杂志

Objective To determine the bowel habits and its perceptions in the general population of Guangdong province. Methods Random clustered sampling involving permanent inhabitants aged 18-80 year was carried out under stratification of urban and suburban areas in Guangdong province. Questionnaire included the items on the characteristics of people being selected and their bowel habits. Results A total of 4103 residents (male 1878, female 2225) were investigated. Mean age among the responders was 42.81 ± 14.13 year. Among 4056 subjects (missing =47 ), 2972 subjects (73.3%) reported daily defecation, and 3951 subjects (97.4%) reported stool frequency between 3 times per week and three times per day. Two hundred and seventy subjects (6.6%) reported abnormal bowel habits by themselves. The stool frequency (OR=2.03, 95% CI:1.54-2.67) , forms of stool (OR=2.75, 95% CI: 2.35-3.22) and straining (OR=3.56, 95% CI:2.49-5.11) were significantly associated with self-reported abnormal bowel habits. Among 3949 subjects (missing= 154), 644 (16.3%) were defined as having abnormal bowel habits according to Rome Ⅱ criteria. There was poor agreement between self-reported abnormal bowel habits and that defined by Rome Ⅱ criteria (Kappa=0.312). Conclusion It seemed to be appropriate that the normal stool frequency was defined as bowel movements between 3 times per week and three times per day in the general population. The prevalence of self-reported abnormal bowel habits was lower than that defined by Rome Ⅱ criteria and the agreement between these two definitions was poor.

D-Tyr-tRNA(Tyr) deacylase, a new role in Alzheimer's-associated disease in SAMP8 mice / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To assess the expression level of D-Tyr-tRNA(Tyr) deacylase (DTD) in SAMP8 mice and speculate the function of DTD in disorders associated with Alzheimer's disease (AD).</p><p><b>METHODS</b>Altogether 12 SAMP8 mice and 12 SAMR1 mice were used in this study. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were performed to detect the mRNA and protein levels of DTD in the mice. Purified DTD protein was injected into lateral ventricle to investigate the function of DTD in SAMP mice. The behavior of the mice was tested by using a Step-through Test System.</p><p><b>RESULTS</b>Both mRNA and protein levels of DTD were found to be significantly lower in SAMP8 mice compared with those in SAMR1 mice (P<0.05). In vivo injection of DTD protein did not lead to an obvious change in behavior of SAM mice.</p><p><b>CONCLUSIONS</b>DTD might function in the process of AD-associated pathology and could possibly participate in physiology process in a long-term manner to orchestrate with other regulators in order to maintain the balance of organism.</p>

Nectin-like molecule 1 inhibits the migration and invasion of U251 glioma cells by regulating the expression of an extracellular matrix protein osteopontin / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the molecular mechanism of nectin-like molecule 1 (NECL1) inhibiting the migration and invasion of U251 glioma cells.</p><p><b>METHODS</b>We infected U251 glioma cells with adeno-nectin-like molecule 1 (Ad-NECL1) or empty adenovirus (Ad). Transwell and wound healing assays were performed to observe the migration of U251 cells incubated with the cell supernatant from Ad-NECL1 or Ad infected U251 cells. DNA microarray was applied to screen the gene expression profile after the restoration of NECL1 in U251 glioma cell lines. The differential expression of osteopontin (OPN), a gene related to migration and invasion, was further analyzed with semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunohistochemistry.</p><p><b>RESULTS</b>The restoration of NECL1 inhibited migration of U251 cells significantly (P<0.05). Altogether 195 genes were found differentially expressed by microarray, in which 175 were up-regulated and 20 down-regulated, including 9 extracellular matrix proteins involved in the migration of cells. Both mRNA and protein expressions of OPN, the most markedly reduced extracellular matrix protein, were found decreased in U251 cells after restoration of NECL1. Immunohistochemical assay also detected an increase of OPN in glioma tissues, related with the progressing of malignant grade.</p><p><b>CONCLUSION</b>A link might exist between NECL1 and the extracellular matrix protein OPN in inhibiting the migration and invasion of U251 glioma cells.</p>

Neural adhesion molecule NECL1 inhibits migration, invasion, and potentially induces differentiation of glioma cell / 中国医学科学院学报

<p><b>OBJECTIVE</b>To explore the influences of the restoration of neural adhesion molecule NECL1 on the morphology, migration, and invasion of NECL1-deficient glioma cell lines.</p><p><b>METHODS</b>Scratch and Transwell assays were used to observe the cell migration and invasion, the activities of extracellular metalloproteinases were measured, and the cell morphology was observed. Astrocytes marker glial fibrillary acidic protein was detected by Western blot after the restoration of NECL1 in glioma U251 cell line.</p><p><b>RESULTS</b>In NECL1-deficient U251 glioma cell lines, migration and invasion were inhibited. The U251 cells was differentiated potentially to astrocytes, and glial fibrillary acidic protein was up-regulated after the restoration of the NECL1 expression.</p><p><b>CONCLUSION</b>As a potential tumor repressor, the neural adhesion molecule NECL1 can inhibit the migration and invasion of glioma cell and induces its differentiation.</p>

ShcD interacts with TrkC through its PTB and SH2 domains / 中国医学科学院学报

<p><b>OBJECTIVE</b>To study the interaction between ShcD and TrkC and to reveal the molecular mechanism of the downstream signal transduction of TrkC.</p><p><b>METHODS</b>Yeast two-hybrid assay was used. The intracellular domains of TrkC and TrkC mutants were cloned into pAS2-1, and ShcD and its four domains (CH2, PTB, CH1, and SH2 domains) were cloned into pACT2 vector respectively. The constructs were separately cotransformed into yeast. beta-galactosidase activity was measured to detect their interactions. TrkC was cloned into pmRFP (carrying red fluorescent protein), and ShcD was cloned into pEGFP (carrying green fluorescent protein). pmRFP-TrkC and pEGFP-ShcD were co-transfected into 293T cells, and then the cells were fixed and subjected to confocal analysis to study their subcellular localization.</p><p><b>RESULTS</b>ShcD interacted with TrkC but not with kinase dead mutant TrkCM1(K572A). Both PTB and SH2 domains were capable of binding to TrkC, and PTB domain bound NPQY motif of TrkC. ShcD colocalized with TrkC throughout the cytoplasm and in the plasma membrane in 293T cells.</p><p><b>CONCLUSION</b>ShcD binds to TrkC in a kinase-activity-dependent manner through its PTB and SH2 domains.</p>

Dok6 promote neurite outgrowth of tropomyosin-related kinase C overexpressed PC12 cells in stimulation of neurotrophin-3 / 中国医学科学院学报

<p><b>OBJECTIVE</b>To study the role of adaptor protein Dok6 in neurite outgrowth in PC12 cells.</p><p><b>METHODS</b>Series of fusion clones were constructed by fusing different domains of Dok6 into mutant TrkC/Y516F. These constructs were transiently transfected into PC12 cells separately and the expression levels of fusion proteins were detected by Western blot. Neurite outgrowth in these PC12 cells was tested after stimulation of NT-3.</p><p><b>RESULTS</b>Each fusion clone was stably expressed in PC12 cells. The fusion clones that fused both TrkC/Y516F-Dok6 (PTB+C) and TrkC/Y516F-Dok6C rescued the loss of neurite outgrowth in PC12 cells resulting from the mutation in tyrosine 516, while fusion clones that fused with single TrkC/Y516F-Dok6PTB did not show such effect.</p><p><b>CONCLUSION</b>Dok6 can promote neurite outgrowth induced by NT-3 stimulation through its C-terminal in TrkC-positive PC12 cells.</p>

Identification of differentially expressed microRNAs by microarray: a possible role for microRNAs gene in medulloblastomas / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>MicroRNAs (miRNAs) are small noncoding regulatory RNAs whose aberrant expression may be observed in many malignancies. However, few data are yet available on human primary medulloblastomas. This work aimed to identify that whether miRNAs would be aberrantly expressed in tumor tissues compared with non-tumorous cerebellum tissues from same patients, and to explore a possible role during carcinogenesis.</p><p><b>METHODS</b>A high throughput microRNA microarray was performed in human primary medulloblastoma specimens to investigate differentially expressed miRNAs, and some miRNAs were validated using real-time quantitative RT-PCR method. In addition, the predicted target genes for the most significantly down- or up-regulated miRNAs were analyzed by using a newly modified ensemble algorithm.</p><p><b>RESULTS</b>Nine miRNA species were differentially expressed in medulloblastoma specimens versus normal non-tumorous cerebellum tissues. Of these, 4 were over expressed and 5 were under expressed. The changes ranged from 0.02-fold to 6.61-fold. These findings were confirmed using real-time quantitative RT-PCR for most significant deregulated miRNAs (miR-17, miR-100, miR-106b, and miR-218) which are novel and have not been previously published. Interestingly, most of the predicted target genes for these miRNAs were involved in medulloblastoma carcinogenesis.</p><p><b>CONCLUSIONS</b>MiRNAs are differentially expressed between human medulloblastoma and non-tumorous cerebellum tissue. MiRNAs may play a role in the tumorigenesis of medulloblastoma and maybe serve as potential targets for novel therapeutic strategies in future.</p>