多形性膠質(zhì)母細(xì)胞瘤(Glioblastoma multiforme,GBM)是一種神經(jīng)上皮來源的惡性程度最高、最為常見的原發(fā)性膠質(zhì)瘤亞型(WHOⅣ級(jí))。約占腦膠質(zhì)瘤的25%~50%,被世界公認(rèn)的一種致命的人類癌癥。GBM常見于20歲以上的成年人,其中40歲-70歲最為常見,男性略多于女性,發(fā)病率約1/10萬。約60%的GBM為原發(fā)性新生膠質(zhì)瘤,由低級(jí)別膠質(zhì)瘤的惡性演變而成的GBM約占總GBM患者的40%。最近文獻(xiàn)報(bào)道指出被新診斷的GBM患者一般存活時(shí)間約為8~15個(gè)月,復(fù)發(fā)的GBM患者存活時(shí)間也只有3~9個(gè)月。即使手術(shù)后經(jīng)過系統(tǒng)的放化療,GBM患者2年生存率不超過10%,5年生存率更不到5%。目前GBM的基因治療及靶向治療是研究的熱點(diǎn),到目前為止已發(fā)現(xiàn)不少蛋白的異常表達(dá)與GBM的發(fā)生和進(jìn)展有關(guān),但至今尚未發(fā)現(xiàn)一種與之發(fā)生有關(guān)的特異蛋白,雖然對(duì)GBM的研究已有重大進(jìn)展,但GBM發(fā)生的原因仍不明了。本課題組查閱大量文獻(xiàn),發(fā)現(xiàn)一種潛在的抗癌因子,發(fā)現(xiàn)了含MYND結(jié)構(gòu)的鋅指蛋白11(Zinc finger MYND domain-containing protein11,ZMYND11)。ZMYND11又稱為BS69,分子量為69kDa,由ZMYND11基因合成,基因定位于細(xì)胞核內(nèi)染色體10p15.3,包含15個(gè)外顯子,跨度120kb。在人體和動(dòng)物的多種組織器官都有不同水平的表達(dá)。最早于1995年被Hateboer等發(fā)現(xiàn)可與人腺病毒早期區(qū)域蛋白1A(Early region1A,E1A)特異性結(jié)合并抑制E1A蛋白的反式激活作用從而發(fā)揮其抗癌作用。有研究發(fā)現(xiàn)ZMYND11可通過其特有的結(jié)構(gòu)域特異性識(shí)別H3.3K36me3中的H3K36me3結(jié)構(gòu),作為共因子抑制RNA聚合酶Ⅱ的活性進(jìn)而抑制相關(guān)致癌基因的轉(zhuǎn)錄。也有學(xué)者發(fā)現(xiàn)ZMYND11可抑制乳腺癌的發(fā)生及進(jìn)展。直到目前,有關(guān)ZMYND11與GBM的研究目前尚未報(bào)道。為探討ZMYND11對(duì)GBM的作用,我們收集GBM腫瘤標(biāo)本經(jīng)western檢測(cè)發(fā)現(xiàn)ZMYND11在GBM中低表達(dá),并通過GBM細(xì)胞系U87細(xì)胞的慢病毒轉(zhuǎn)染、細(xì)胞功能學(xué)實(shí)驗(yàn)及體內(nèi)試驗(yàn)發(fā)現(xiàn)ZMYND11可抑制GBM的發(fā)生及進(jìn)展。但ZMYND11在GBM中低表達(dá)的原因目前尚不為人知。本課題組查閱文獻(xiàn)及生物信息網(wǎng)站最終發(fā)現(xiàn)miR-196a-5p與GBM的發(fā)生與患者預(yù)后密切相關(guān),并且極可能靶向調(diào)節(jié)ZMYND11的表達(dá),利用雙熒光素酶報(bào)道基因等實(shí)驗(yàn)證實(shí)ZMYND11是miR-196a-5p下游靶點(diǎn)。經(jīng)過拯救試驗(yàn)我們明確miR-196a-5p可通過靶向抑制ZMYND11發(fā)揮其促腫瘤的作用。第一部分ZMYND11在多形性膠質(zhì)母細(xì)胞瘤的表達(dá)及臨床意義目的:對(duì)比ZMYND11在GBM腫瘤與非腫瘤腦組織中表達(dá)的差別并探討ZMYND11表達(dá)水平與GBM患者生存時(shí)間的關(guān)系。方法:收集河北醫(yī)科大學(xué)第二醫(yī)院神經(jīng)外科2015年1月-2016年6月多形性膠質(zhì)母細(xì)胞瘤標(biāo)本20例,重度顱腦外傷患者術(shù)中無腫瘤腦組織標(biāo)本20例,分為腫瘤組及對(duì)照組。并對(duì)上述GBM患者術(shù)后進(jìn)行隨訪。病理證實(shí)所取標(biāo)本均為多形性膠質(zhì)母細(xì)胞瘤,取Western Blot及q RT-PCR方法檢測(cè)兩組標(biāo)本中ZMYND11的表達(dá)情況并進(jìn)行比較。根據(jù)每個(gè)GBM標(biāo)本ZMYND11表達(dá)水平與腫瘤組ZMYND11平均表達(dá)水平的比較情況,將上述腫瘤組的GBM患者又分為ZMYND11高表達(dá)組與低表達(dá)組,利用統(tǒng)計(jì)學(xué)探討ZMYND11的表達(dá)水平與GBM患者生存時(shí)間的關(guān)系。結(jié)果:1.GBM患者臨床信息及資料歸納患者臨床資料:GBM患者(腫瘤組)20例,男12例,女8例,年齡44-68歲,平均年齡54.9;重度顱腦外傷患者20例(對(duì)照組)男性14例,女性6例,年齡21-65歲,平均35.78歲。GBM患者術(shù)后隨訪3.6-16.1個(gè)月,平均9.74個(gè)月。所有GBM患者術(shù)前均未進(jìn)行任何放化療。術(shù)后GBM患者中2例未進(jìn)行放化療,16例術(shù)后化療,術(shù)后18例行放療,對(duì)照組中的20例顱腦外傷的患者均無其他系統(tǒng)腫瘤的發(fā)生。2.ZMYND11在GBM腫瘤組織中表達(dá)顯著低于正常腦組織所有納入本研究的GBM患者均為我院病理科所證實(shí),本課題組利用western blot及q RT-PCR檢測(cè)兩組ZMYND11的表達(dá),結(jié)果顯示:GBM治療中的ZMYND11的表達(dá)水平顯著低于無腫瘤腦組織。3.ZMYND11的表達(dá)水平與GBM患者的中位生存時(shí)間有關(guān)為進(jìn)一步分析ZMYND11表達(dá)的臨床意義,我們隨訪了上述20例GBM患者并得到其生存時(shí)間。將所收集的GBM標(biāo)本根據(jù)其ZMYND11表達(dá)水平與腫瘤組ZMYND11平均表達(dá)水平比較,高于平均水平的標(biāo)本稱為高表達(dá)組,低于平均水平的稱為低表達(dá)組,統(tǒng)計(jì)兩組GBM患者平均生存時(shí)間及中位生存時(shí)間。結(jié)果發(fā)現(xiàn)ZMYND11高表達(dá)組的患者的中位生存時(shí)間長(zhǎng)于低表達(dá)組,ZMYND11高表達(dá)組與低表達(dá)組平均生存生存時(shí)間無明顯統(tǒng)計(jì)學(xué)差異(P0.05)。小結(jié):1.ZMYND11在GBM中呈明顯低表達(dá)。2.ZMYND11表達(dá)水平與GBM患者中位生存時(shí)間存在明顯正相關(guān)性,提示ZMYND11對(duì)GBM的發(fā)生及進(jìn)展可能存在一定的抑制作用。第二部分ZMYND11對(duì)多形性膠質(zhì)母細(xì)胞瘤的增殖、侵襲及凋亡等生物學(xué)行為的影響目的:通過建立ZMYND11過表達(dá)的U87細(xì)胞系,探討ZMYND11對(duì)GBM細(xì)胞增殖,遷移,凋亡等生物學(xué)行為的影響。方法:向人多形性膠質(zhì)母細(xì)胞瘤細(xì)胞系U87細(xì)胞通過慢病轉(zhuǎn)染使ZMYND11過表達(dá),經(jīng)Western blot檢測(cè)轉(zhuǎn)染效果后并通過CCK-8試驗(yàn)檢測(cè)細(xì)胞增殖能力,流式細(xì)胞術(shù)檢測(cè)細(xì)胞周期變化,AnnexinⅤ-PE/7-AAD雙染色法檢測(cè)細(xì)胞凋亡比例,transwell實(shí)驗(yàn)檢測(cè)細(xì)胞侵襲力,并通過裸鼠成瘤實(shí)驗(yàn)驗(yàn)證ZMYND11在體內(nèi)對(duì)GBM生長(zhǎng)的影響。結(jié)果:1.ZMYND11過表達(dá)的U87細(xì)胞模型建立應(yīng)用ZMYND11過表達(dá)的慢病毒及空載慢病毒分別轉(zhuǎn)染GBM細(xì)胞系U87細(xì)胞建立ZMYND11過表達(dá)的U87細(xì)胞系及對(duì)照U87細(xì)胞系,經(jīng)Western blot實(shí)驗(yàn)檢測(cè)兩組細(xì)胞的ZMYND11表達(dá)水平,結(jié)果發(fā)現(xiàn)ZMYND11過表達(dá)組的ZMYND11表達(dá)水平顯著高于對(duì)照組,可以進(jìn)行后續(xù)的細(xì)胞功能學(xué)實(shí)驗(yàn)。2.ZMYND11可抑制GBM細(xì)胞的增殖經(jīng)CCK-8實(shí)驗(yàn)檢測(cè)ZMYND11過表達(dá)的U87細(xì)胞及對(duì)照細(xì)胞的不同時(shí)期的細(xì)胞活力,并繪制兩組細(xì)胞增殖曲線。結(jié)果發(fā)現(xiàn)ZYMND11過表達(dá)的U87細(xì)胞不同時(shí)期的活力均明顯低于對(duì)照組。由此推斷ZMYND11可抑制GBM細(xì)胞的增殖。3.ZMYND11可抑制GBM細(xì)胞有絲分裂并促進(jìn)其細(xì)胞凋亡經(jīng)流式細(xì)胞術(shù)及Ⅴ-PE/7-AAD雙染染色檢測(cè)ZMYND11過表達(dá)的U87細(xì)胞及對(duì)照組細(xì)胞的周期變化及細(xì)胞凋亡情況,結(jié)果發(fā)現(xiàn)ZMYND11過表達(dá)組G0/G1期的細(xì)胞比例明顯高于對(duì)照組,而且G2/M期的細(xì)胞比例顯著低于對(duì)照組;在ZMYND11過表達(dá)組中的早期及中期凋亡細(xì)胞比例明顯高于對(duì)照組。表明ZMYND11可抑制GBM細(xì)胞的有絲分裂并促進(jìn)GBM細(xì)胞凋亡4.ZMYND11可抑制GBM細(xì)胞的侵襲力通過transwell實(shí)驗(yàn)檢測(cè)ZMYND11過表達(dá)的U87細(xì)胞及對(duì)照細(xì)胞的侵襲力,結(jié)果顯示ZMYND11過表達(dá)的U87細(xì)胞透過膜的細(xì)胞數(shù)明顯少于對(duì)照組細(xì)胞,由此推斷ZMYND11能抑制GBM細(xì)胞的侵襲。5.ZMYND11可有效抑制GBM的生長(zhǎng)上述體外實(shí)驗(yàn)后,我們將ZMYND11過表達(dá)的U87細(xì)胞系及對(duì)照細(xì)胞置入4周大的雌性裸鼠皮下進(jìn)行裸鼠成瘤實(shí)驗(yàn),同樣分為ZMYND11過表達(dá)組與對(duì)照組,對(duì)ZMYND11對(duì)GBM在體內(nèi)生長(zhǎng)的影響進(jìn)行研究,結(jié)果發(fā)現(xiàn)ZMYND11過表達(dá)組的裸鼠所形成的腫瘤生長(zhǎng)速度、腫瘤體積及腫瘤重量均小于對(duì)照組的裸鼠。此結(jié)果表明ZMYND11可在體內(nèi)有效抑制GBM的生長(zhǎng)。小結(jié):1.ZMYND11過表達(dá)可有效抑制GBM細(xì)胞的增殖及有絲分裂。2.ZMYND11過表達(dá)可明顯抑制GBM細(xì)胞的侵襲力。3.ZMYND11過表達(dá)可顯著促進(jìn)GBM細(xì)胞的凋亡進(jìn)程。4.ZMYND11過表達(dá)可有效抑制GBM腫瘤的生長(zhǎng)。第三部分miR-196a-5p靶向抑制ZMYND11并促進(jìn)GBM細(xì)胞增殖、侵襲及抗凋亡目的:探討miR-196a-5p在GBM中可靶向抑制ZMYND11的表達(dá)并可通過此機(jī)制促進(jìn)GBM細(xì)胞增殖、侵襲及抗凋亡的研究。方法:利用Western blot及q RT-PCR方法檢測(cè)所收集的GBM標(biāo)本中ZMYND11與miR-196a-5p的表達(dá)水平,分析兩者表達(dá)水平的相關(guān)性。取慢病毒及質(zhì)粒分別轉(zhuǎn)染U87細(xì)胞系以抬高或敲低miR-196a-5p的表達(dá),并利用Western blot檢測(cè)miR-196a-5p上調(diào)或下調(diào)后的ZMYND11的表達(dá)水平,驗(yàn)證兩者表達(dá)的相互關(guān)系。為進(jìn)一步證實(shí)miR-196a-5p和ZMYND11的靶向關(guān)系,本課題組設(shè)計(jì)并進(jìn)行了雙熒光素酶報(bào)告基因?qū)嶒?yàn)。此外,我們還通過慢病毒及siRNA轉(zhuǎn)染U87細(xì)胞成功的建立miR-196a-5p和ZMYND11均下調(diào)的U87細(xì)胞及對(duì)照細(xì)胞系,完成了拯救試驗(yàn),驗(yàn)證miR-196a-5p可通過抑制GBM中ZMYND11的表達(dá)而發(fā)揮其對(duì)GBM發(fā)生與進(jìn)展的促進(jìn)作用。結(jié)果:1.在GBM中miR-196a-5p和ZMYND11的表達(dá)呈明顯負(fù)相關(guān)性。我們對(duì)利用Western blot及qRT-PCR方法分別檢測(cè)所收集的20例GBM標(biāo)本中ZMYND11與miR-196a-5p的表達(dá)水平,發(fā)現(xiàn)兩者表達(dá)呈明顯負(fù)相關(guān);此外通過轉(zhuǎn)染U87細(xì)胞改變miR-196a-5p表達(dá)后再檢測(cè)ZMYND11的表達(dá)改變情況,結(jié)果發(fā)現(xiàn)miR-196a-5p與ZMYND11表達(dá)呈負(fù)相關(guān)。由此推斷miR-196a-5p與ZMYND11在GBM中表達(dá)呈負(fù)相關(guān)性。2.在GBM中miR-196a-5p可靶向抑制ZMYND11的表達(dá)并通過此機(jī)制促進(jìn)GBM的發(fā)生與進(jìn)展。通過雙熒光素酶報(bào)告基因?qū)嶒?yàn)本課題組發(fā)現(xiàn)ZMYND11野生型組的U87細(xì)胞熒光素酶活性顯著低于ZMYND11突變型組,提示miR-196a-5p可特異性結(jié)合于ZMYND11的mRNA中3’UTR區(qū)并抑制ZMYND11的表達(dá)。此外,我們經(jīng)慢病毒及siRNA轉(zhuǎn)染U87細(xì)胞成功建立miR-196a-5p和ZMYND11均低表達(dá)的U87細(xì)胞及僅miR-196a-5p下調(diào)的對(duì)照細(xì)胞系以完成拯救試驗(yàn),經(jīng)Western blot檢測(cè)轉(zhuǎn)染效果后將兩組細(xì)胞進(jìn)行細(xì)胞功能學(xué)實(shí)驗(yàn)并發(fā)現(xiàn)miR-196a-5p和ZMYND11均低表達(dá)的U87細(xì)胞的增殖、侵襲與抗凋亡均高于對(duì)照細(xì)胞系,此實(shí)驗(yàn)證實(shí)miR-196a-5p在GBM中通過抑制ZMYND11表達(dá)從而促進(jìn)腫瘤增殖、侵襲及抗凋亡。小結(jié):1.在GBM中miR-196a-5p可靶向抑制其下游的ZMYND11的表達(dá),導(dǎo)致miR-196a-5p與ZMYND11在GBM細(xì)胞中的表達(dá)呈明顯負(fù)相關(guān)。2.在GBM中miR-196a-5p通過特異性的靶向抑制ZMYND11的表達(dá)從而促進(jìn)GBM細(xì)胞的增殖、侵襲及抗凋亡能力。結(jié)論:1.ZMYND11在GBM中呈明顯低表達(dá),ZMYND11高表達(dá)的GBM患者中位生存時(shí)間明顯長(zhǎng)于ZMYND11低表達(dá)的GBM患者。2.ZMYND11過表達(dá)可有效抑制GBM細(xì)胞的增殖、侵襲并促進(jìn)其細(xì)胞凋亡。3.在GBM中miR-196a-5p可靶向抑制下游的ZMYND11的表達(dá)從而促進(jìn)GBM的細(xì)胞增殖、侵襲及抗細(xì)胞凋亡。
【學(xué)位單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位年份】：2018
【中圖分類】：R739.4
【部分圖文】：

圖 1 GBM 中 ZMYND11 的表達(dá)水平明顯低于正常腦組織Fig.1 The expression level of ZMYND11 was significantly lower in GBMtumor than that in non-neoplasm brain tissueA: The protein expression level of ZMYND11 was significantly decreased inGBM samples than that in non-neoplasm brain samples measured by WestBlot assay (***P<0.001). B: The mRNA level of ZMYND11 wasremarkably lower in GBM samples than that in normal brain tissue detectedby qRT-PCR assay (***P < 0.001)

圖 1 GBM 中 ZMYND11 的表達(dá)水平明顯低于正常腦組織Fig.1 The expression level of ZMYND11 was significantly lower in GBMtumor than that in non-neoplasm brain tissue: The protein expression level of ZMYND11 was significantly decreased inBM samples than that in non-neoplasm brain samples measured by Westlot assay (***P<0.001). B: The mRNA level of ZMYND11 wasmarkably lower in GBM samples than that in normal brain tissue detectedy qRT-PCR assay (***P < 0.001)

圖 1 轉(zhuǎn)染后成功抬高 U87 細(xì)胞系的 ZMYND11 表達(dá)Fig.1 ZMYND11was successfully upregulated in U87 cell line aftertransfectione: 96 hours after transfection withZMYND11-overexpression lentiv control lentivirus, ZMYND11 expression was determined by meantern blot in U87 cell line. GAPDH was used as a normalization conults were represented as means ±SD from at least three independent P<0.001

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相關(guān)期刊論文 前2條

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