神經(jīng)膠質(zhì)瘤中IDH2R172G突變對基因表觀修飾的研究

杜蘇萌

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神經(jīng)膠質(zhì)瘤中IDH2R172G突變對基因表觀修飾的研究

杜蘇萌

目的分析在神經(jīng)膠質(zhì)瘤細胞中異檸檬酸脫氫酶2(IDH2)突變后P15ink4b轉(zhuǎn)錄水平的變化，尋找和基因表觀遺傳修飾相關(guān)的異常表達的蛋白。重點關(guān)注P15ink4b啟動子區(qū)甲基化水平變化，進而分析IDH2突變、腫瘤抑制基因P15ink4b、2-HG和甲基胞嘧啶羥化酶(TET)之間的關(guān)系。方法pEGFP-N1、pEGFP-N1-IDH2M、pEGFP-N1-IDH2WT3個質(zhì)粒轉(zhuǎn)染C6細胞48h后，收獲細胞Western Blot檢測抑癌蛋白P15ink4b水平，qPCR檢測P15ink4bmRNA變化，5-hmC檢測試劑盒檢測P15ink4b啟動子區(qū)CpG島，CCGG位點5-hmC概率。結(jié)果IDH2突變組抑癌蛋白P15ink4b表達明顯低于對照組。IDH2突變組P15ink4bmRNA水平低于對照組，差異有統(tǒng)計學(xué)意義(P=0.043)。隨著2-羥基戊二酸濃度加大，細胞活性明顯增強，差異有統(tǒng)計學(xué)意義(P<0.05)。pEGFP-N1-IDH2WT組8個CCGG位點第二個C的5-hmC的概率都大于80%，而pEGFP-N1-IDH2M組都小于1%。結(jié)論IDH2突變后產(chǎn)生的2-羥基戊二酸可以抑制TET酶活，從而去甲基化程度降低，使得抑癌基因P15ink4b啟動子區(qū)甲基化程度增強，抑制P15ink4b基因表達，促進細胞增殖遷移。

神經(jīng)膠質(zhì)瘤；異檸檬酸脫氫酶；突變；甲基胞嘧啶羥化酶；P15ink4b

異檸檬酸脫氫酶(isocitrate dehydrogenase，IDH)，是三羧酸循環(huán)中的限速酶，在細胞中有3種異檸檬酸脫氫酶，分別是IDH1(細胞質(zhì)中、過氧化物酶體、內(nèi)質(zhì)網(wǎng))，IDH2(線粒體內(nèi))，IDH3(線粒體內(nèi))，IDH1和IDH2是煙酰胺腺嘌呤二核苷酸磷酸(NADP+)依賴型酶，IDH3煙酰胺腺嘌呤二核苷酸(NAD+)依賴型酶[1-3]。正常的異檸檬酸脫氫酶以同源二聚體形式催化異檸檬酸和NADP+為α-酮戊二酸(α-KG) 和NADPH，NADPH的產(chǎn)生避免細胞產(chǎn)生氧化性損傷。有報道IDH1，IDH2在膠質(zhì)瘤和二級多形性膠質(zhì)母細胞瘤突變的頻率大于75%，而在急性骨髓白細胞中突變的頻率約為20%[4-5]。IDH1突變經(jīng)常發(fā)生在R132，由精氨酸突變?yōu)榻M氨酸，IDH2在白血病中突變位點經(jīng)常發(fā)生在R172和R140(谷氨酰胺代替精氨酸)[6-8]。

突變以后的IDH獲得新的功能，催化α-KG+NADPH產(chǎn)生D-2-羥戊二酸 (也稱R-2-HG)和NADP+，低NADPH使膠質(zhì)母細胞瘤對放療和化療敏感，產(chǎn)生的D-2-羥戊二酸在IDH1或IDH2突變的白血病和膠質(zhì)瘤中積累。在IDH1突變的膠質(zhì)瘤中D-2-羥戊二酸可積累到驚人高濃度5～35 mmol·g-1。α-KG和2-HG結(jié)構(gòu)的相似性使得2-HG可以競爭性的抑制α-KG依賴的雙加氧酶，哺乳動物細胞中大于60種利用α-KG作為輔底物的雙加氧酶[9-11]。為探討神經(jīng)膠質(zhì)瘤細胞中IDH2突變后P15ink4b轉(zhuǎn)錄水平的變化，尋找和基因表觀遺傳修飾相關(guān)的異常表達的蛋白，進而分析IDH2突變、腫瘤抑制基因P15ink4b2-HG和甲基胞嘧啶羥化酶(translocation methylcytosine dioxygenase,TET)之間的關(guān)系，以闡明神經(jīng)膠質(zhì)瘤中IDH2突變后對基因表觀遺傳修飾的影響，現(xiàn)報道如下。

1　材料與方法

1.1材料EpiJET 5-hmC Analysis Kit(貨號：K1481，購于Thermo Scientific)；C6細胞由中科院惠贈；、pEGFP-IDH2WT、pEGFP-IDH2M由本實驗室構(gòu)建；pEGFP-N1、DNA 限制性內(nèi)切酶、RNAisoTM Plus、反轉(zhuǎn)錄試劑盒(Prime Script TMRT reagent Kit)購自TaKaRa公司；DNA膠回收試劑盒(TMGel Extraction Kit)、質(zhì)粒提取試劑盒 (TMP lasmid Mini Kit) 購于Omega公司。

1.2轉(zhuǎn)染實驗根據(jù)PEI轉(zhuǎn)染操作流程，轉(zhuǎn)染前24 h內(nèi)在含10%胎牛血清的DMEM培養(yǎng)基進行傳代培養(yǎng)，6孔板接種密度為0.5×106個細胞。按照PEI與DNA(μg)的混合比為3∶1制備轉(zhuǎn)染復(fù)合體，加入6孔板后(每孔200 μl+3 μg的DNA)立即渦旋混合。轉(zhuǎn)染后48 h內(nèi)收獲轉(zhuǎn)染細胞，進行后續(xù)實驗。

1.3Western blot實驗pEGFP-N1、pEGFP-N1-IDH2WT、pEGFP-N1-IDH2M轉(zhuǎn)染C6細胞48 h后，通過Western blot方法對細胞內(nèi)目的蛋白P15ink4b和對照蛋白β-actin進行檢測。

1.4實時熒光定量實驗pEGFP-N1、pEGFP-N1-IDH2M、pEGFP-N1-IDH2WT3個質(zhì)粒轉(zhuǎn)染C6細胞48h后，收獲細胞，利用RNAisoTM Plus提取3個樣本RNA，采用Prime ScriptTMRT reagent Kit反轉(zhuǎn)錄，SYBR Green Realtime PCR Master Mix試劑盒檢測3個樣本P15ink4bmRNA水平，擴增P15ink4bmRNA引物為Forward:5’-ATCCCAACGCCGTCAACC-3’；Reverse:5’-CGTGTCCAGGAAGCCCTCT-3’。β-actin mRNA引物為Forward:5’-AGATTACTGCCCTGGCTCCTAG-3’；Reverse:5’-CATCGTACTCCTGCTTGCTGAT-3’，每組設(shè)3個復(fù)孔。

1.5MTT實驗用1、2、3、4、6、16、33 mmol·L-1不同濃度2-HG處理C6細胞，培養(yǎng)24 h后，按照經(jīng)典MTT法檢測細胞增殖活力，每組設(shè)6個復(fù)孔，同時設(shè)置調(diào)零孔(培養(yǎng)基、MTT、二甲基亞砜)，對照孔(細胞、培養(yǎng)液、MTT、二甲基亞砜)。

1.6甲基化位點檢測上NCBI網(wǎng)站查詢P15ink4b基因，NC005104.4：107848177-107857323，截取107856323至107858323一段，即轉(zhuǎn)錄起始位點上下游各1 000 bp，登陸http://www.urogene.org/methprimer/網(wǎng)站，找出啟動子區(qū)的 CpG島從585 bp 到 1 252 bp，用藍色標出?；蚪M提取試劑盒提取3個樣本的基因組，根據(jù)CpG島每個CCGG位點序列設(shè)計8對引物，見表1。

表1　甲基化位點檢測引物

(續(xù)表)

引物名引物序列CCGG5-F5’-CCGTGGCAGAAATGGTCCT-3’CCGG5-R5’-GGTCCCCAGAGAGGAGGAG-3’CCGG6-F5’-GATTGTTACAGCCTCGTCTCTGA-3’CCGG6-R5’-TGCCTGAGGTGCGGTTCC-3’CCGG7-F5’-CACCTCAGGCACAGCTGAC-3’CCGG7-R5’-GGCGGTGCTGAACGCAG-3’CCGG8-F5’-ACTGTGCAGCTGCGTTCA-3’CCGG8-R5’-TGACGTCACCAAATGTGGGC-3’

用美國Applied Biosystems公司生產(chǎn)的熒光定量儀器，EpiJET 5-hmC Analysis KIT試劑盒檢測3個樣本P15ink4b基因啟動子區(qū)每個CCGG序列，根據(jù)公式% of 5-hmC=100/(1+E)Cq2-Cq1計算每個CCGG位點的5-hmC概率。

2　結(jié)果

2.1轉(zhuǎn)染實驗結(jié)果pEGFP-N1、pEGFP-N1-IDH2WT、pEGFP-N1-IDH2M轉(zhuǎn)染C6細胞表明pEGFP-N1空質(zhì)粒轉(zhuǎn)染效率可以達到80%左右，pEGFP-N1-IDH2R172G和pEGFP-N1-IDH2WT帶IDH2基因的質(zhì)粒轉(zhuǎn)染效率有所下降為50%左右，轉(zhuǎn)染圖見圖1。

圖1　pEGFP-N1-IDH2R172G，pEGFP-N1，pEGFP-N1-IDH2WT 3個質(zhì)粒轉(zhuǎn)染C6細胞轉(zhuǎn)染圖

2.2Western Blot實驗結(jié)果用pEGFP-N1-IDH2R172G，pEGFP-N1，pEGFP-N1-IDH2WT3個質(zhì)粒轉(zhuǎn)染C6細胞，Western Blot實驗檢測了3個樣本P15ink4b蛋白水平，表明pEGFP-N1-IDH2R172G突變的大鼠神經(jīng)膠質(zhì)瘤細胞中P15ink4b表達明顯低于pEGFP-N1、pEGFP-N1-IDH2WT對照組，Western Blot實驗結(jié)果見圖2。

1.pEGFP-N1　2.pEGFP-N1-IDH2M　3.pEGFP-N1-IDH2WT圖2　pEGFP-N1-IDH2R172G，pEGFP-N1，pEGFP-N1-IDH2WT 3個質(zhì)粒轉(zhuǎn)染C6細胞后P15ink4b 的Western Blot圖

2.3實時熒光定量實驗結(jié)果用pEGFP-N1-IDH2R172G，pEGFP-N1，pEGFP-N1-IDH2WT3個質(zhì)粒轉(zhuǎn)染C6細胞，qPCR檢測了3個樣本P15ink4bmRNA表達情況，結(jié)果表明IDH2突變組P15ink4bmRNA水平低于對照組，差異有統(tǒng)計學(xué)意義(P=0.043)

圖3　pEGFP-N1-IDH2R172G，pEGFP-N1，pEGFP-N1-IDH2WT 3個質(zhì)粒轉(zhuǎn)染C6細胞后P15ink4bmRNA的表達圖

2.42-HG處理C6細胞后細胞活性檢測結(jié)果1、2、3、4、6、16、33 mmol·L-1不同濃度2-HG處理C6細胞，MTT法檢測細胞活性實驗表明，隨著2-HG濃度增大，處理C6細胞后，細胞活性增強。33 mmol·L-1組和16 mmol·L-1組相比，差異有統(tǒng)計學(xué)意義(P=0.021)；6 mmol·L-1組和16 mmol·L-1組相比，差異有統(tǒng)計學(xué)意義(P=0.030)；4 mmol·L-1組和6 mmol·L-1組相比，差異有統(tǒng)計學(xué)意義(P=0.033)；3 mmol·L-1組和4 mmol·L-1組相比，差異有統(tǒng)計學(xué)意義(P=0.040)；MTT結(jié)果見圖4。

圖4　不同濃度的2-HG處理C6細胞后的細胞活性圖

2.5CCGG位點5-hmC概率檢測結(jié)果甲基化位點檢測實驗表明位于轉(zhuǎn)錄起始位點下游9～13 bpCCGG的第二個C上的5-hmC的概率pEGFP-N1-IDH2WT組為89%，pEGFP-N1-IDH2R172G組為0.9%；位于轉(zhuǎn)錄起始位點下游69～73 bpCCGG的第二個C上的5-hmC的概率pEGFP-N1-IDH2WT組為87%，pEGFP-N1-IDH2R172G組為1%；位于轉(zhuǎn)錄起始位點下游144～148 bpCCGG的第二個C上的5-hmC的概率pEGFP-N1-IDH2WT組為91%，pEGFP-N1-IDH2R172G組為0.75%；位于轉(zhuǎn)錄起始位點下游171～175 bpCCGG的第二個C上的5-hmC的概率pEGFP-N1-IDH2WT組為89%，pEGFP-N1-IDH2R172G組為0.9%；位于轉(zhuǎn)錄起始位點下游267～271 bpCCGG的第二個C上的5-hmC的概率pEGFP-N1-IDH2WT組為91%，pEGFP-N1-IDH2R172G組為0.6%；位于轉(zhuǎn)錄起始位點下游339～343 bpCCGG的第二個C上的5-hmC的概率pEGFP-N1-IDH2WT組為87%，pEGFP-N1-IDH2R172G組為0.83%；位于轉(zhuǎn)錄起始位點下游392～388 bpCCGG的第二個C上的5-hmC的概率pEGFP-N1-IDH2WT組為92%，pEGFP-N1-IDH2R172G組為0.76%。

圖5　P15ink4b啟動子區(qū)的CpG島圖

3　討論

神經(jīng)膠質(zhì)瘤是顱內(nèi)常見惡性腫瘤之一，約占所有顱內(nèi)腫瘤的40%，具有發(fā)病率、復(fù)發(fā)率和死亡率高，而治愈率低的特點。2008年，Parsons等[2]發(fā)現(xiàn)惡性膠質(zhì)瘤中的IDH1發(fā)生突變，以后關(guān)于IDH突變的報道相繼出現(xiàn)。通過基因組測序篩選，發(fā)現(xiàn)IDH1和IDH2這兩個基因的突變不僅在腦惡性膠質(zhì)瘤晚期出現(xiàn)，在早期樣品中也存在，證實這是腦惡性膠質(zhì)瘤最早的基因突變位點，而且IDH1或IDH2的突變只存在于膠質(zhì)瘤中，而在其他腫瘤中并未發(fā)現(xiàn)。這一發(fā)現(xiàn)對于腦惡性膠質(zhì)瘤的診斷和靶向治療意義重大，為癌癥研究打開了一扇新窗口。

本研究結(jié)果顯示IDH2突變后產(chǎn)生的副產(chǎn)物2-HG可以抑制甲基胞嘧啶酶的活性，這種抑制會隨著2-HG濃度的增大有所增強，從細胞活性實驗可以看出，4 mmol·L-1時明顯出現(xiàn)抑制，但是2-HG濃度增大到33 mmol·L-1時細胞活性抑制則不明顯。TET是一種將抑癌基因去甲基化的關(guān)鍵雙加氧酶。在正常的細胞中DNA甲基轉(zhuǎn)移酶從腺苷甲硫氨酸轉(zhuǎn)移一個甲基基團到胞嘧啶的C5上[12-14]，TET可以氧化甲基化胞嘧啶為5hmC、5fC、5caC甲基化胞嘧啶，這幾種氧化形式的甲基化胞嘧啶可以被胸腺嘧啶-DNA-糖基化酶清除，替代為未修飾的胞嘧啶。甲基胞嘧啶羥化酶被2-羥基戊二酸抑制后使得多種抑癌基因CpG島甲基化程度過高，導(dǎo)致基因沉默[15-17]。

P15ink4b則為腫瘤抑制基因之一，P15ink4b可以通過抑制TGF-P誘導(dǎo)的CDK4及CDK6的表達，從而影響細胞周期。TET酶活性被抑制后，可以使P15ink4b啟動子區(qū)甲基化升高，從而沉默P15ink4b基因的表達，促進腫瘤增殖遷移[18]。在含有IDH突變的神經(jīng)膠質(zhì)瘤中其活性被抑制后，其下游的作用蛋白P15ink4b表達異常。本研究發(fā)現(xiàn)P15ink4b啟動子區(qū)去甲基化程度降低，這從甲基化位點檢測實驗可以看出，被轉(zhuǎn)染突變IDH2的C6細胞，提取基因組后，在P15ink4b啟動子區(qū)8個CCGG位點的第二個C上的5-hmC概率普遍小于1%，這說明TET酶氧化5-mC至5-hmC這一步受到抑制，推測在IDH突變的細胞中，P15ink4b啟動子區(qū)甲基化程度增強，從而抑制了P15ink4b的表達。這和Western blot實驗，實時熒光定量實驗結(jié)果一致。Western blot實驗表明IDH突變組的P15ink4b表達量相比對照組降低，實時熒光定量實驗表明P15ink4bmRNA相對于對照組降低，差異有統(tǒng)計學(xué)意義(P=0.043)。這一發(fā)現(xiàn)提示可以從提高TET酶活上進行治療IDH突變的神經(jīng)膠質(zhì)瘤。

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Genetic Epigenetic Modification of IDH2R172G Mutation in Glioma

DU Su-meng

(Department of Environmental and Safety Engineering,Taiyuan Institute of Technology,Taiyuan 030008,China)

ObjectiveTo analyze the level of P15ink4bmRNA after isocitrate dehydrogenase 2 (IDH2) mutation in glioma cells and search for the protein abnormal expression of epigenetic modifications of gene.The changes of methylation level in P15ink4bpromoter region were focused and the relationship were analyzed among IDH2 mutations,tumor suppressor gene P15ink4b,2-HG and translocation methylcytosine dioxygenase(TET).MethodsAfter three plasmids of pEGFP-N1,pEGFP-N1-IDH2M,pEGFP-N1-IDH2WTwere transfected into C6 cells for 48h,all the cells were harvested.The level of suppression cancer protein P15ink4bwere detected by western blot,P15ink4bmRNA by qPCR,and the 5-hmC probability in CCGG sites of CpG island of P15ink4bpromoter region by 5-hmC test kits.ResultsThe 5-hmC expression of P15ink4bin IDH2 mutation group was significantly lower than that of the control group,and the difference was statistically significant (P=0.043).The cell activity was increased with the level of 2-hydroxyglutarate concentration,and the difference was statistically significant (P<0.05).All the probability of the second C in eight CCGG sites of pEGFP-N1-IDH2WTgroup was higher than 80%,but less than 1% in the pEGFP-N1-IDH2M group.ConclusionThe 2-hydroxyglutarate produced by IDH2 mutation can inhibit the activity of TET enzyme,decrease the demethylation level in anti-oncogene P15ink4bpromoter region,finally inhibit the expression of P15ink4bgene,and promote cell proliferation and migration.

glioma；isocitric dehydrogenase；mutation；methyl cytosine hydroxylase；P15ink4b

1672-688X(2016)03-0165-05DOI：10.15926/j.cnki.issn1672-688x.2016.03.002

2016-06-30

太原工業(yè)學(xué)院環(huán)境與安全工程系，山西太原 030008

杜蘇萌(1982-)，女，山西夏縣人，講師，從事生物化學(xué)教學(xué)工作。

R730.264

A