Study Of Myocyte Enhancer Factor 2D Promotes Tumorigenicity In Malignant Glioma Cells

Background and Objective:Malignant glioma, the most frequent class of malignant primitive tumours of the central nervous system(CNS),accounts for more than 50% of all brain tumors.According to their aggressiveness,the World Health Organization(WHO) classifies them into Grades I and II or low-grade gliomas(LGG) and Grades III and IV or high-grade gliomas(HGG).(1)Grade I:pilocytic astrocytoma(PA),most of fiber astrocytomas exist in children;(2) Grade II:low-grade astrocytomas(LGA),accounting for 25% of all gliomas,most of these tumors occur in the age of 30 to 40 years;(3)Grade III:anaplastic astrocytoma(AA),easily converted to grade 4;(4)Grade IV:Glioblastoma(GBM),most of these tumors occur in the age of 45 to 60 years.Malignant astrocytoma(MA) including AA and GBM,GBM accounts for more than 50% to 80% of malignant astrocytoma.Despite advances in malignant glioma treatment including surgery,chemotherapy combined with chemotherapy and tumor immunotherapy has made tremendous progress during the past decade, the median survival is only 12-15 months.The extremely poor prognosis of patients with malignant glioma is partly due to a lack of understanding of the molecular mechanisms underlying malignant glioma formation.Because malignant glioma has Highly invasive, poor prognosis of biological characteristics,it has recurred.In order to effectively solve this clinical problem,Therefore,it is critical to identify new genes that participate in gliomagenesis and subsequently,to improve patient outcomes and develop novel strategies for the treatment of malignant glioma based on the new findings.The myocyte enhancer factor-2(MEF2) transcription factor family consists of four members, MEF2 A,B,C,and D.The MEF2 transcription factors have important roles in muscle, cardiac,neural,blood and immune system cell development.As other important factors associated with the development,MEF2 D is known to participate in human tumors.MEF2 D induce malignant transformation of normal cells in leukemia phenomenon was first reported.Chromosome translocation chromosome 1 and chromosome 19 occurs in Some patients with acute lymphoblastic leukemia,It will produces MEF2D/deleted in azoospermia-associated protein(DAZAP) 1 and DAZAP1/MEF2 D fusion proteins,which maintain the malignant phenotypes of acute lymphoblastic leukemia cells. In vitro experiments, the researchers found that overexpression MEF2 D not only given a normal cell proliferation in low serum conditions,to accelerate its rate of division,and also produce apoptosis inhibition,It can enhance the survival of transformed cells.It has been reported that MEF2 D took part in hepatocellular carcinoma.Moreover,MEF2 D is overexpressed in liver cancers,and a high level of MEF2 D is correlated with poor prognosis in hepatocellular carcinoma.MEF2 D plays an important role in the tumorigenicity of hepatocellular carcinoma by regulating G2/M transition-retarding genes,MEF2 D downregulation induces growth inhibition in liver cancer,Alternative polyadenylation isoforms of MEF2 D are differentially expressed in normal and GBM brain tissues,Furthermore, glioma-associated factor 2 is also regulated by MEF2 family proteins.MEF2 D can activate the transcription and expression of Bcl-W,so MEF2 D play a role in anti-apoptosis of tumor cells.However,the expression and biological function of MEF2 D in malignant glioma is poorly understood.Our previous study found that MEF2 D obviously overexpression in malignant gliomas and glioblastoma cell lines(U87MG and U251MG).There are evidences that MEF2 D expression may be key factors of development and progression in malignant glioma.In this study,we will try to research the function and expression of MEF2 D in malignant gliomas,to find The relationship between myocyte enhancer factor 2D and tumorigenicity in malignant glioma cells,To elucidate more about the biological function of MEF2 D in malignant gliomas, providing new reference protein molecules for the prognosis of malignant gliomas.Our findings suggest that MEF2 D may be an effective therapeutic target for malignant gliomas treatment strategies.Methods:1.Cell lines and cell cultureHuman malignant glioma cell lines,U87MG(derived from a malignant glioma patient classified as grade IV) and U251MG(derived from a malignant brain tumor containing GFAP positive cells), He La cell(were used as a MEF2D-positive control) were obtained from the American Type Culture Collection.The primary astrocyte culture was established as described previously.All cells were routinely grown in Minimum Essential Medium containing 10% fetal bovine serum at 37°C in a 5% CO2 atmosphere.2. Primary culture/ethics statementMalignant gliomas tissue(tumors and adjacent normal tissues) was obtained from patients According to protocols approved by the Ethical Review Board of the Chengdu Military General Hospital.The patients provided their written informed consent to take part in this study.All patients underwent surgical resection of primary malignant glioma at the Department of Neurological Surgery,Chengdu Military General Hospital.The malignant glioma tissues were cut into small pieces.A single cell suspension was obtained after mechanical manipulation.For primary astrocyte culture,the samples were obtained from aborted fetuses with written informed consent from pregnant women according to previously published procedures approved by the Ethical Review Board of the Chengdu Military General Hospital.Briefly, after the removal of the meninges, brain tissues from the anterior fontanelle were cut into pieces and digested with trypsin.The digested cells were filtered through a steel mesh and cultured in DMEM supplemented with 15% FBS.GFAP expression was detected by immunofluorescence staining.3. ImmunohistochemistryImmunohistological staining was conducted on formalinfixed and paraffin-embedded tissue sections using the streptavidin-biotin-peroxidase complex method.Antigens were retrieved by heating the samples in antigen retrieval solution.Antibodies against MEF2 D and Ki67 were used to detect MEF2 D and Ki67 expression. Hematoxylin was used to counterstain the nuclei. TUNEL staining was performed with In Situ Cell Death Detection Kit according to the protocols recommended by the manufacturer.4. Real-time quantitative PCRAccording to the Operation Manuals extracted Total RNA with RNA Kit.Real-time PCR was conducted as described elsewhere.Briefly,first strand c DNA synthesis and amplification were performed according to the protocols of the Prime Script TM RT reagent kit;q PCR was performed according to the instructions of SYBR premix Ex Taq with an Applied Rotor-Gene 6000 Real Time PCR System.The following primer sequences were used:MEF2D(forward)5’-AGGGAAATAACCAAAAAACTACCAAA-3’;MEF2D(reverse) 5’-GCTACATGAACACAAAAACAGAGACC-3’;GAPDH(forward) 5’-GCGAGATCGCACTCATCATCT-3’;GAPDH(reverse) 5’-TCAGTGGTGGACCTGACC-3.GAPDH m RNA levels were used for normalization. The fold change in gene expression level was calculated using the 2△△Ct method.5. Viral vectorsViral vectors were kindly provided by Dr Wang(Department of Pathology,Harvard Medical School),including Lv-GFP,Lv-MEF2 D, Lv-sh MEF2D-1,Lv-sh MEF2D-2,and Lv-CTRL.6. Western blot analysisPreparation of protein samples and immunoblotting were performed according to standard protocols. Briefly,cells were treated with lysis buffer. After centrifugation for 15 min at 12000 rpm,protein density was evaluated with Kit of BCA protein,Total proteins were then separated by SDS-PAGE on a gel.7. Proliferation assayCells were planted in 96-well plates at a concentration of 1000 per well after infected with different lentiviral vectors. MTS solution was used to detect cell proliferation rate according to the methods recommended by Promega.8. Colony formation assaysU87MG cells were plated in 3.5cm dishes at a concentration of 2000 cells per dish after infection with the indicated lentiviral vectors. U251 MG cells and astrocyte cells were plated in 24-well plates at a concentration of 100 cells per well after infection with the indicated lentiviral vectors.The cell lines were cultured for 10 days at 37°C in a 5% CO2 cell incubator.The cell lines were fixed with polyoxymethylene and then stained with crystal violet. We define 50 cells as a colony.9. Cell cycle detectionCell cycle progression was determined by FACS after PI staining as described previously.Briefly,the cells were plated in 6-well plates at a concentration of 2×105 cells per well and treated with lentiviruses.Forty-eight hours after treatment,the cells were collected with 240μl of PBS and 560μl of 100% ethanol and fixed overnight at-20°C.Cell pellets were collected by centrifugation and resuspended in 500μl of PBS containing 20 m M EDTA and 1 mg/ml RNase,and then incubated at 37°C for 1h.PI solution(50μg/ml) was used to stain the samples.The samples were then subjected to flow cytometry analysis and detected with an excitation wavelength of 536 nm and an emission wavelength of 617 nm.10. Flow cytometry analysis of cell apoptosisThe indicated cells were plated in 6-well plates at a concentration of 2×105 cells per well and treated with indicated lentiviruses.The cells were handled with lentiviral after forty-eight hours,trypsinized,and washed once with complete medium. Aliquots of cells(5×105) were collected by centrifugation and resuspended in 500μl of PBS containing and stained by fluorescein isothiocyanate(FITC)-labeled annexin V.FACS was performed immediately after staining.11. Animal studiesSix-week-old BALB/c male nude mice were used in this study. The mice(n=12) were intracranially injected with 5×106 U87 MG cells infected with 10 MOI of Lv-sh MEF2D-1 or Lv-CTRL.Their deaths were recorded every day for 40 days. All protocols of animal studies approved by the Ethical Review Board of the Chengdu Military General Hospital.Tumor volume was calculated by the formula “V=(length×width2)/2”.12. Statistical analysisExperiments were executed for three more times.The statistical significance of correlation between MEF2 D expression and survival was estimated by the log-rank test.To study the relationship between MEF2 D expression and other variables,we used either the independent sample t test.The values of quantitative real-time PCR(q RT-PCR).cell growth rate and colony formation were expressed by a two-tailed independent sample t test.The significance of difference was assessed using one-way ANOVA or two-way ANOVA where applicable.All the values were expressed as mean±SD.P<0.05(*) and P<0.01(**) mean “significant” and “very significant”, respectively.Results:1.An elevated level of MEF2 D expression predicts poor prognosis in malignant glioma patients.To examine the role of MEF2 D in the prognosis of patients with malignant glioma,we used q RT-PCR to investigate the levels of MEF2 D m RNA in malignant glioma patients.The malignant glioma samples, which were in III grade and IV grade of histological classification,were divided into a MEF2 D high group and a MEF2 D low group(We defined the groups MEF2D-high/-low by mean value).In contrast to the MEF2 D low group,the patients in the MEF2 D high group had poorer prognosis(P=0.031).Furthermore,WHO grading(2007) was positive correlated with MEF2 D m RNA level(P=0.048) of these patients’ samples.IDH1/2 mutation was also positive correlated with MEF2 D m RNA level(P=0.029).However, there was no significant association between MEF2 D m RNA level and histological classification(P=0.064).In additionally,the median overall survival period of MEF2 D low group was 12 months,which was longer than MEF2 D high group(10 months).To confirm the localization of MEF2 D in the tumor cells,we performed immunohistochemistry in malignant glioma samples.Immunohistochemical staining showed that MEF2 D protein was mainly localized to the nuclei of the cancer cells.Accordingly,the level of MEF2 D protein was higher in the malignant glioma tumor tissues compared to the adjacent tissues,as evaluated by western blotting and Immunohistochemistry.The MEF2 D m RNA levels in these samples were also measured by q RT-PCR.The m RNA level of MEF2 D in the tumor tissue was significantly higher than that of the adjacent tissues(P=0.03,0.02,and 0.02).In addition,the protein level of MEF2 D was decreased in astrocytes compared to U87 MG, U251 MG,and He La cancer cells(He La cells were used as a MEF2D-positive cell line).The m RNA level of MEF2 D in the cancer cell lines was higher than astrocytes(P<0.04).These results indicate that malignant glioma cell lines had aberrantly high expression of MEF2 D.2.MEF2 D downregulation suppresses the proliferation of malignant glioma cell lines.Because the malignant glioma cell lines had a high level of MEF2 D expression,we infected U87 MG and U251 MG cells with lentiviral vectors carrying a short hairpin RNA specifically targeting MEF2 D m RNA of(Lv-sh MEF2D-1, Lv- sh MEF2D-2) or control vector(Lv-CTRL).We examined the levels of MEF2 D protein and m RNA in U87 MG and U251 MG cells by western blotting and q RT-PCR 72 h after Infection.Lv-sh MEF2D-1 and Lv-sh MEF2D-2 resulted in an approximately 65-95% decrease in MEF2 D protein expression in the U87 MG and U251 MG malignant glioma cell lines.q RT-PCR data also showed that these sh RNAs downregulated the m RNA levels of MEF2 D in malignant glioma cell lines.MEF2 D downregulation was found to decrease the proliferation of U87 MG cells.This effect was also observed in U251 MG cells that had been infected with Lv-sh MEF2D-1 or Lv-sh MEF2D-2 but not cells infected with Lv-CTRL(P=0.02 and 0.01).Additionally, the colony numbers of U87 MG cells were affected by the changes in the levels of MEF2 D after infection with Lv-sh MEF2D-1 or Lv-sh MEF2D-2(P=0.01 and 0.01).Similar results were also obtained for U251 MG cells(P=0.01 and 0.02).The downregulation of MEF2 D reduced the colony numbers of U87 MG and U251 MG cells,indicating that MEF2 D is important for the proliferation of malignant glioma cell lines.3.MEF2 D affects cell cycle progression and apoptosis in malignant glioma cell lines.To investigate the mechanism by which MEF2 D promotes proliferation in malignant glioma cell lines, cell cycle progression was analyzed with FACS.U87 MG and U251 MG malignant glioma cells were infected with Lv-sh MEF2D-1,Lv-sh MEF2D-2,or Lv-CTRL. In accordance with the results previously reported,silencing MEF2 D expression in U87 MG and U251 MG cells led to delay of S and G2/M phases of cell cycle.MEF2 D can modulate the transcription of target genes such as CDKN1 A,GADD45A,and GADD45 B in liver cancer.To confirm whether MEF2 D regulates these genes in malignant glioma, we examined the m RNA level of MEF2 D target genes. Our data confirmed that silencing MEF2 D expression in U87 MG cells leads to increased expression of CDKN1 A, GADD45 A, and GADD45 B.We also examined whether the downregulation of MFE2 Dresulted in apoptosis in these malignant glioma cell lines.Flow cytometry analysis was performed to detect apoptosis in tumor cells after treatment with Lv-sh MEF2D-1 or Lvsh MEF2D-2.The percentage of apoptotic cells was determined by FITC-Annexin V/PI staining. Our data showed that the apoptotic rate was significantly different in the groups with different MEF2 D levels.The apoptotic rates were 39.4 and 38.2% in the Lv-sh MEF2D-1 and Lvsh MEF2D-2 infected groups,respectively.In contrast,the Lv-CTRL infected group had a lower apoptotic rate(6.1 %).These results suggested that MEF2 D suppresses malignant glioma cell proliferation by inducing delay of S and G2/M phases of cell cycle and affecting apoptosis in cancer cells.Additionally,cleaved PARP suggested that the apoptotic pathway was induced.Accordingly,the level of cleaved PARP protein was elevated in MEF2D-silenced cell lines.Therefore,our results reinforced the previous finding that MEF2 D plays an essential role in the proliferation of cancer cells by promoting cell cycle progression and suppressing apoptosis.4.The overexpression of MEF2 D promotes proliferation and cell cycle progression in astrocytes.We next examined whether MEF2 D overexpression promoted the proliferation and cell cycle progression of astrocytes of the normal brain.Astrocytes were infected with Lv-MEF2 D to elevate MEF2 D levels.Western blot analysis confirmed a significant increase in MEF2 D protein levels after infection with Lv-MEF2 D.q RT-PCR data showed that the m RNA levels of MFE2 D were higher in the Lv-MEF2 D infected astrocytes than in the control cells(P=0.01).The proliferation rates were estimated using MTS assays in astrocytes infected with Lv-MEF2 D or Lv-GFP.We found that MEF2 D overexpression increased the proliferation of astrocyt es(P=0.03).Furthermore,MEF2 D overexpression elevated the colony numbers of astrocytes,compared to a control virus(Lv-GFP)(P=0.04).Additionally,cell cycle progression was also evaluated in astrocytes overexpressing MEF2 D from a lentivirus.MEF2 D overexpression in astrocytes increased the percentage of G0/G1 phase cells and decreased the percentages of cells in the G2/M and S phases.Collectively,MEF2 D promoted the proliferation of astrocytes and accelerated the G2/M transition in astrocytes.5.The downregulation of MEF2 D abrogates tumorigenicity in malignant glioma cells.To investigate whether MEF2 D promoted malignant glioma Tumorigenicity,the effects of MEF2 D downregulation were examined in an established mouse malignant glioma model.Mice bearing MEF2 D deficient malignant glioma xenografts were observed to have a higher survival rate than the control group.Notably,the MEF2D-deficient group had 50% survival,while there was no survival in the control group 40 days after the intracranial injection of U87 MG cells.The average sizes of the tumors were significantly smaller in the Lv-sh MEF2D-1 infected groups(5.6±5.3mm3) than in the Lv-CTRL infected group(15.2±8.7mm3)(P=0.04).Western blot,q RT-PCR,and immunohistochemical staining assays all revealed extensive expression of MEF2 D in the tumors from the Lv-CTRL infected group,whereas MEF2 D expression was almost undetectable in the tumors from the Lv-sh MEF2D-1 infected groups.To clarify whether the increased survival of the mice implanted with sh MEF2 D transduced malignant glioma cells is due to a cytostatic or cytotoxic effect, we analyzed proliferation and apoptosis in tumor cells via Ki67 staining and TUNEL assays, respectively. The results showed that,compared to the Lv-CTRL infected groups,the Lv-sh MEF2D-1 infected groups had weaker Ki67 staining but stronger TUNEL staining.The cells expressing MEF2 D or Ki67 and the TUNEL positive cells were counted.The MEF2D-positive rates were 82.5 and 3.9% in the Lv-CTRL and Lv-sh MEF2D-1 treated groups,respectively.The percentages of Ki67 expressing cells in the Lv-CTRL and Lv-sh MEF2D-1 treated groups were 64.2 and 28.6%,respectively.The apoptotic rates were 8.6 and 57.8% in the Lv-CTRL and Lv-sh MEF2D-1-treated groups,respectively,as revealed by TUNEL staining.These results demonstrated that MEF2 D deficiency blocks malignant glioma tumorigenicity in vivo.Conclusions:1.We found that in malignant glioma, there is an aberrantly high expression of MEF2 D, which leads to poor prognosis of malignant glioma.2.The downregulation of MEF2 D suppresses the proliferation of malignant glioma cell lines by inducing delay of S and G2/M phases of cell cycle and promoting apoptosis.3.Furthermore, the overexpression of MEF2 D in astrocytes accelerates cell proliferation by regulating cell cycle progression.4.A mouse malignant glioma model demonstrated that MEF2 D deficiency blocks malignant glioma formation in vivo.5.We conclude that MEF2 D may act as a potential oncogene in malignant glioma and thus serve as a candidate target for malignant glioma therapy.