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Research Report
MiR-125b is critical for the suppression of human U251 gliomastem cell proliferation
Lei Shi a,b,1, Junxia Zhang a,1, Tianhong Pan b, Jinfang Zhou b, Weiyi Gong b, Ning Liu a,Zhen Fu a, Yongping You a,⁎aDepartment of Neurosurgery, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, PR ChinabDepartment of Neurosurgery, The First People's Hospital of Kunshan affiliated with Jiangsu University, Suzhou 215300, PR China
Article history:Accepted 20 November 2009Available online 27 November 2009
Stem cells are unique in their ability to self-renew and maintain tissue homoeostasis bydifferentiating into different cell types to replace aged or damaged cells. The keycharacteristic of the stem cell is its capacity to divide for long periods of time. MicroRNAs(miRNAs) are small noncoding RNA molecules that regulate protein expression by cleavingor repressing the translation of target mRNAs. miR-125b, one of neuronal miRNAs, recentlywas found to be necessary for stem cell fission to bypass the normal G1/S checkpoint andmake stem cells insensitive to chemotherapy signals, which normally stop the cell cycle atthe G1/S transition. Given the insensitivity of gliomas to chemotherapy and the hypothesisthat glioma stem cells cause resistance to drug therapy, exploring the functions andmechanisms of miR-125b in glioma stem cells would be valuable. In this study, we foundthat miR-125b was downregulated in human U251 glioma stem cells, therefore suggestingthat its upregulation can lead to the growth inhibition of U251 glioma stem cells in vitro.Further research on the mechanism demonstrated that inhibition of miR-125b-inducedU251 glioma stem cell proliferation was due to cell cycle arrest at the G1/S transition andinvolved the cell cycle regulated proteins CDK6 and CDC25A; miR-125b overexpressiondecreased CDK6 and CDC25A expression. These findings underscore the potential of miR-125b to regulate the proliferation of U251 glioma stem cells through the cell cycle regulatedproteins CDK6 and CDC25A.
Malignant gliomas, the most common primary malignanttumor in the brain, are aggressive, highly invasive, neuro-logically destructive, and considered the deadliest of humancancers. Despite the combination of surgery, chemotherapyand radiotherapy, the median survival time ranges from 9 to
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12 months in its most aggressive manifestation, glioblastoma(Ohgaki et al., 2004). To date, the efficacy of adjuvant therapy islimited, and no effective agents have emerged for the treatmentof glioblastoma multiforme. Therefore, there is an urgent needto develop improved conventional or novel therapeutics.
Recent findings support the existence of a stem cell-derived origin of gliomas (Vescovi et al., 2006). Glioma-derived
Fig. 1 – The expression of miR-125b based on RT-PCR isdifferent in CD133-positive and CD133-negative U251 gliomacells. CD133-positive and CD133-negative U251 cells werefirst separated by MACS. Then real-time RT-PCR was used toanalyze the expression of miR-125b in CD133-positive andCD133-negative U251 cells. The column represented therelative expression of miR-125b in CD133-positive orCD133-negative U251 cells. *P<0.05, compared withCD133-positive group.
Fig. 2 –MiR-125b inhibitsCD133-positiveU251cellsproliferationin vitro. The effects of miR-125b on CD133-positive U251 cellswere measured using the MTT assay. Cells treated withmiR-125b from 0 to 72 h were subjected to the MTT cellproliferation assay. The viability of untreated cells wasconsidered100%. *P<0.05, comparedwithnegativevectorgroup.
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stem cells (GSCs) have been isolated from both human braintumors and several glioma cell lines. GSCs are crucial to themalignancy of gliomas andmay represent the consequence ofa transformation of the normal neural stem cell compartment(Singh et al., 2004; Kondo et al., 2004; Patrawala et al., 2005).These findings are compatible with the observation thatgliomagenesis is frequently associated with adult brain stemcells, suggesting a cellular and genetic mechanism thatcontrols adult neurogenesis, which might contribute to braintumorigenesis, thereby allowing the identification of newtherapeutic strategies.
The CD133 antigen has been identified as a putative stemcell marker in normal andmalignant brain tissues. In gliomas,it is used to enrich a subpopulation of highly tumorigeniccancer cells. CD133-positive glioma cells are characterized byself-renewal and unrestricted division (Pfenninger et al., 2007).However, a critical question in the biology of GSCs is howGSCsescape cell division stop signals. MicroRNAs (miRNAs) aresmall noncoding RNAs whose function as modulators of geneexpression is crucial for the proper control of cell growth. Thenecessity of microRNA (miRNA) pathways has been found tobe one of the potential key mechanisms for proper control ofgermline GSC division. Some miRNA pathways have beenproven as necessary for GSCs to bypass the normal G1/Scheckpoint (Hatfield et al., 2005).
MiR-125b, one of neuronal miRNAs, has been found toaffect the proliferation and apoptosis of human glioma cells.Down-regulation of miR-125b decreased human glioma cellsproliferation and enhanced the sensitivity of human gliomacells to ATRA-induced apoptosis (Xia et al., 2009). In thisstudy, we found that the miR-125b could be an importantmiRNA in CD133-positive GSCs. Our study revealed that there
was lower expression of miR-125b in CD133-positive gliomacells. The upregulation of miR-125b expression specificallyresulted in a proliferation defect in the CD133-positive gliomacells and an arrest in the cell cycle G1/S transition, whichmight be due to downregulation of the cell cycle regulatedproteins CDK6 and CDC25A. CDK6 and CDC25A are twoimportant cell cycle regulators. CDK6 is one of key cyclin-dependent kinases (CDKS), participating in regulation of thecell cycle. Transition from the G0/G1 phase to S phase of thecell cycle requires sequential activation of specific complexesof cyclin and CDK. CDC25A can accelerate the G1/S transitionby dephosphorylating and activate the cyclin–CDK complexesthat are active during G1 (Blomberg and Hoffmann, 1999).Both of them were reported to be involved in the control ofthe G1/S transition in human embryonic stem cells (Zhang etal., 2009). In view of the results of this experiment, weconcluded that miR-125b was required for the proliferation ofCD133-positive glioma cells, and our goal was to furtheridentify its molecular mechanisms.
2. Results
2.1. MiR-125b expression is different in CD133-positiveand CD133-negative U251 glioma cells
In previous research, Singh et al. showed that only CD133-positive glioblastoma cells were able to form multipotentneurosphere clones or tumor xenografts, which impliedthat the functions of CD133-positive glioma cells weredifferent from CD133-negative glioma cells (Singh et al.,2003, 2004). To examine the requirement of miR-125b inCD133-positive U251 cells, we carried out RT-PCR analysisand analyzed the expression of miR-125b in CD133-positiveand CD133-negative U251 cells. Our results demonstratethat there was a significant difference in miR-125b expres-sion between the two groups. As shown in Fig. 1, theexpression of miR-125b in CD133-negative U251 cells was
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3.37-fold (P<0.05) that of CD133-positive U251 cells. Thedecreased miR-125b in CD133-positive U251 cells suggeststhat miR-125b may play an important role in CD133-positive U251 cells.
2.2. Upregulation of miR-125b expression induces growthinhibition of CD133-positive U251 cells
Given the analysis above, the functions of miR-125b inCD133-positive U251 cells were of interest. We examinedthe effect of miR-125b on the proliferation of CD133-positiveU251 cells. CD133-positive U251 cells were transfected withoverexpressed miR-125b vectors for 72 h. As shown in Fig. 2,the cell growth inhibition rates increased in a time-depen-dent manner in CD133-positive U251 cells treated withoverexpressed miR-125b vectors. The cellular proliferationof CD133-positive U251 cells significantly decreased 48 h aftertransfection with overexpressed miR-125b vectors. Theseresults suggest that the upregulation of miR-125b, at least
Fig. 3 – MiR-125b arrests the cell cycle at the G0–G1 phase with a(A) The population of G0–G1 phase in miR-125b-transduced CD1(B) The expression of CDK6 and CDC25A in miR-125b-transduced
in part, specifically leads to the inhibition of CD133-positiveU251 cell proliferation.
2.3. Upregulation of miR-125b expression reducesprogression through cell cycle arrest in CD133-positive U251 cells
Previous studies have shown that the proliferation of cellsdepends on their progression through the cell cycle. Toinvestigate the effects of miR-125b on the cell cycle, we useda flow cytometry assay to measure cell cycle progression inCD133-positive U251 cells treated with miR-125b. Our resultsshow that miR-125b caused a cell cycle defect in CD133-positive U251 cells with a significant increase in the percent-age of cells in the G0–G1 phase (70.3±1.3%) compared to thenegative and blank control groups (61.2±0.7%;60.1±0.1%) (Fig.3A). These results suggest that the effects of miR-125b on theinhibition of proliferation were due to reduced progression ofthe cell cycle, most likely due to a block or delay in the G1-Stransition.
decrease in two top cell cycle proteins of CDK6 and CDC25A.33-positive U251 cells was analyzed by cell cycle analysis.CD133-positive U251 cells was detected by Western blot.
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2.4. MiR-125b arrests the cell cycle at the G0–G1 phase ina CDK6 and CDC25A-dependent manner in CD133-positiveU251 cells
To determine the concrete mechanism by which miR-125binduced the increased percentage of cells in the G0–G1phase, the key cell cycle regulatory proteins CDK6 andCDC25A were assessed. CDK6 and CDC25A are two topscoring cell cycle regulators. Previous studies have shownthat ectopic expression of CDK6 and CDC25A accelerates theG1/S-phase transition and controls an event that is ratelimiting for entry into the S phase (Blomberg and Hoffmann,1999; Meyerson et al., 1992). CDK6 and CDC25A weredetected by Western blot analysis in CD133-positive U251cells treated with or without overexpressed miR-125b vectors
Fig. 4 –MiR-125b arresting the cell cycle at the G0–G1 phase is in awith CDK6 and CDC25A siRNAs for 48 h. Protein lysates were immcontrol). (B) The population of G0–G1 phase in miR-125b over-exCDC25A siRNA treatment were analyzed by cell cycle analysis.
at 48 h. Consistent with previous research, we found thatprotein expression levels of both CDK6 and CDC25A weredecreased with miR-125b overexpression in CD133-positiveU251 cells compared to the control (Fig. 3B).
To examine the functional interrelationship between theG0–G1 phase and CDK6 and CDC25A, CDK6 and CDC25AsiRNAs (Santa Cruz Biotechnology) were used to transfectmiR-125b-overexpressed CD133-positive U251 cells beforemiR-125b transfection (Fig. 4A). As shown in Fig. 4B, CDK6and CDC25A siRNA abrogated the G0–G1 phase differencesamong miR-125b-overexpressed CD133-positive U251 cells(69.1±0.3%), the negative control group cells (67.2±0.7%) andcontrol cells (67.7±0.3%). These results indicate that theinhibition of the G1/S transition by miR-125b depends onCDK6 and CDC25A.
CDK6 and CDC25A-dependentmanner. (A) Cells were treatedunoblotted for CDK6 and CDC25A and β-actin (for a loading
press and control CD133-positive U251 cells of CDK6 and
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3. Discussion
MiRNAs are located in introns, the noncoding regions of thegenome. Mature functional miRNAs of approximately 22nucleotides that are generated from long primary miRNA(pri-miRNA) transcripts control gene expression at the post-transcriptional level by degrading or repressing targetmRNAs.Some miRNAs aberrantly expressed in cancer have been welldocumented (Chan et al., 2005; Johnson et al., 2007; Shi et al.,2008). They were found to regulate the expression of signalingmolecules, such as cytokines, growth factors, transcriptionfactors, and proapoptotic and antiapoptotic genes.
Recently, miRNAs were found to have a role in thedifferentiation of stem cells. Proper regulation of cell differ-entiation by stem cells is crucial to normal developmentand the avoidance of cancer (Zwaka and Thomson, 2005).However, the differential expression of miRNAs on GSCs hasnot been addressed. Previous studies showed that miR-125bwas necessary for stem cell fission (Hatfield et al., 2005; Lee etal., 2005). Here we analyzed CD133-positive and CD133-negative U251 cells to identify whether the expression ofmiR-125b in GSCs was significantly different between groups.Our results show that miR-125b expression was significantlylower in CD133-positive U251 cells than CD133-negative U251cells, indicating that miR-125b might be involved in thedevelopment or replication of GSCs.
One of the key characteristics of stem cells is their capacityto divide for long periods of time when most other cells arequiescent (Hatfield et al., 2005). Because the function of miR-125b in GSCswas unknown, we identified the biological effectsof miR-125b on GSCs when miR-125b expression in CD133-positive U251 cells is up-regulated. Then the role of miR-125bon cellular growth and proliferation of transfected CD133-positive U251 cells was examined. All the results show thatoverexpression of miR-125b consistently reduces the numberof proliferating CD133-positive U251 cells. This suggests thatone or more processes related to the proliferation of CD133-positive U251 cells is affected by miR-125b. In other words,miR-125b, at least partly, controls the proliferation of CD133-positive U251 cells. To the best of our knowledge, this study isthe first to demonstrate the differential expression of miRNAson GSCs and the effect of the miRNA miR-125b on CD133-positive U251 cells, i.e., miR-125b efficiently inhibits theproliferation of CD133-positive U251 cells.
However, the underlying mechanisms of the aforemen-tioned effects are not completely understood. Previous studieshave shown that the proliferation of stem cells depends ontheir progression through the cell cycle and can be regulatedby the miRNA pathway (Hatfield et al., 2005). To investigatethe effect of miR-125b on the cell cycle, a flow cytometry assaywas used tomeasure cell cycle progression. In this study, miR-125b-induced arrest of the cell cycle at the G0–G1 phase wasobserved. Cell cycle arrest in the G0–G1 phase is thought to bea prerequisite for cell differentiation. Taken together, itstrongly suggests that miR-125b is a key regulator of cellcycle progression in CD133-positive U251 cells.
Previous research showed that some miRNAs inducedsignificant G0–G1 cell cycle arrest through the downregula-tion of cell cycle regulated proteins (Johnson et al., 2007; Sun
et al., 2008). CDK6, a key cell cycle proto-oncogene, controlscell proliferation by reducing flux through the pathways andthus promoting the G1 to S transition. CDC25A, another cellcycle regulated protein, controls an event that is rate limitingfor entry into the S phase. Activation of endogenous CDC25Aoccurs during the late G1 phase and increases in the S andG2 phases. To evaluate the function of CDK6 and CDC25A inthe control of the G1/S transition, their expression wasstudied in CD133-positive U251 cells transfected with over-expressed miR-125b vectors. In this study, we show that bothCDK6 and CDC25A decrease in miR-125b-treated CD133-positive U251 cells. To further analyze whether CDK6 andCDC25A regulated the change of G0–G1 phase in miR-125b-treated CD133-positive U251 cells, the expression of CDK6and CDC25A was silenced by siRNA. Our results show thatthe miR-125b-induced G0–G1 phase arrest was abrogated bythe CDK6 and CDC25A siRNA. Taken together, our resultsillustrate that the inhibition effect on cell proliferationinduced by the overexpression of miR-125b could be due tothe corresponding down-regulation of CDK6 and CDC25Aexpression on cell cycle progression in CD133-positive U251cells.
In summary, we have shown for the first time that miR-125b expression is low in CD133-positive U251 cells, and itsoverexpression efficiently inhibits the proliferation of CD133-positive U251 cells. Furthermore, such effects may occurthrough the down-regulation of the cell cycle regulatoryproteins CDC25A and CDK6, which block or delay the G1/Stransition of the cell cycle.
4. Experimental procedures
4.1. Cell lines and culturing conditions
Humangliomacell line, U251,was purchased from theChineseAcademy of Sciences Cell Bank. U251 glioma cells weremaintained in a 37 °C, 5% CO2 incubator in DMEM supplemen-ted with 10% fetal bovine serum (FBS) and routinely passagedat 2- to 3-day intervals.
4.2. Magnetic cell separation of CD133-positive cells
Cells were dissociated and resuspended in PBS containing 0.5%bovine serum albumin and 2 mmol/L EDTA. For magneticlabeling, CD133/1 Micro Beads were used (Miltenyi Biotech).Positivemagnetic cell separation (MACS)wasdoneusing severalMACS columns in series. Cells were stained with CD133/2-PE(Miltenyi Biotech) and analyzed on a BD FACSCalibur.
4.3. Plasmids transfection
Expression vectors formiR-125b and negative vectors (Genesil,Wuhan, China) were constructed by technical support fromWuhan Genesil. MiR-125b expressing vectors and negativevector were transfected into CD133-positive U251 glioma cellswith FuGENE HD6 (Roche) according to the manufacturer'sinstructions. The transfectedU251 CD133-positive glioma cellswith high levels of maturemiRNAwere identified by TaqMan-based real-time quantification RT-PCR.
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4.4. Real-time quantification of miRNAs by stem-loopRT-PCR
Cells were immediately frozen in liquid nitrogen for total RNAextraction after treatment. RNA was extracted using TRIzolreagent (Invitrogen). For the TaqMan-based real-time reversetranscription-polymerase chain reaction (RT-PCR) assays, theABI 7300 HT Sequence Detection system (Applied Biosystems,Foster City, CA) was used. All the primers and probes of themiR-125b (P/N: 4373148) and RNU6B endogenous controls(P/N: 4373381) for TaqMan miRNA assays were purchasedfrom Applied Biosystems. Real-time PCR was performed asdescribed in Chen et al. (2005). Relative gene expression wascalculated via a 2−ΔΔCt method (Livak and Schmittgen, 2001).
4.5. Cell growth assay
The MTT assay was used to determine relative cell growth asfollows: CD133-positive U251 cells were plated at 103 cells perwell in 96-well plateswith six replicatewells for each condition,then, transfected and assayed 72 h post-transfection. A cellgrowth assay was performed by MTT (Sigma) as described inPark et al. (1987). Cell viability was determined at 540 nmabsorbance via a scanning multiwell spectrophotometer(enzyme-linked immunosorbent assay reader, Biotek Instru-ments Inc.). All of the data points represent the mean of aminimumof sixwells. The cell growth inhibition rate formula is(AC-AT)/AC×100% (AC=Absorbance value of the blank controlgroup; AT=Absorbance value of the experimental group).
4.6. Cell cycle analysis
MiR-125b-transduced CD133-positive U251 cells were starvedwith 5% charcoal-stripped serum for 48 h, and cellular DNAsynthesis was stimulated by the addition of 10% normal FBS.Cell cycle analysis was conducted before serum stimulationand after 16 h of serum treatment. CD133-positive U251 cellstreated with miR-125b were trypsinized and fixed with ice-cold 70% ethanol for at least 1 h. After extensive washing, thecells were suspended in HBSS containing 50 μg/ml PI (Sigma-Aldrich) and 50 μg/ml RNaseA (Boehringer Mannheim, India-napolis, IN), incubated for 1 h at room temperature, andanalyzed by FACScan (Becton Dickinson, San Jose, CA). Thedata were analyzed on a BD FACS Calibur.
4.7. Cell transfection
Human CDK6 (sc-29264) and CDC25A (sc-29254) siRNAs werepurchased from Santa Cruz Biotechnology. CD133-positiveU251 cells were transfected with CDK6 and CDC25A siRNAs byFuGENE HD6 (Roche, Basel, Switzerland) according to themanufacturer's instructions at 70–90% confluence. After 48 hof transfection, the silencing effects of siRNAs were analyzedby Western blot (Fig. 4A), and then CD133-positive U251 cellswere transfected with overexpressed miR-125b vectors.
4.8. Western blot analysis
To determine the levels of protein expression, soluble proteinswere isolated by lysis buffer (137 mM NaCl, 15 mM EGTA,
0.1mMsodiumorthovanadate, 15mMMgCl2, 0.1%TritonX-100,25mMMOPS, 100 μMphenylmethylsulfonyl fluoride and 20 μMleupeptin, adjusted to pH 7.2). One-dimensional sodiumdodecyl sulfate (SDS)-polyacrylamide gel electrophoresis wasperformed with a corresponding gel concentration using thediscontinuous buffer system of Laemmli (Bio-Rad Laboratories,Richmond, CA). The electrophoresed proteins were transferredto a polyvinylidene difluoride membrane and subjected toimmunoblot analysis with antibodies to CDK6 and CDC25A(used at a 1/200 dilution, Santa Cruz Biotechnology). Thereaction was detected with enhanced chemiluminescence(Amersham Life Science, Arlington Heights, IL). The mem-branes were reblotted with a β-actin antibody (1/2000, SantaCruz Biotechnology) after washing to check for equal loadingof the gel.
4.9. Statistical analysis
All tests were done using SPSS Graduate Pack 11.0 statisticalsoftware (SPSS, Chicago, IL). Descriptive statistics includingmeanand SE in addition to one-way ANOVAs were used to determinesignificant differences. P<0.05 was considered significant.
Conflict of interest statementAll authors have declared the sources of research funding inthis manuscript and have no financial or other contractualagreements that might cause (or be perceived as causes of)conflicts of interest.
Acknowledgments
This work was supported by the China Natural ScienceFoundation (Proj. No. 30672165 and 30772231) and MedicalMajor Talent Program of Jiangsu Province (Proj. No. RC2007061).
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