夏　鵬, 昝春芳, 李建安, 侯婷婷, 張　恒, 張　郡

(吉林大學(xué)第二醫(yī)院, 骨科, 吉林 長(zhǎng)春, 130041)

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p53通路抑制FANCD2基因的表達(dá)來(lái)誘導(dǎo)骨肉瘤MG-63細(xì)胞凋亡

夏鵬, 昝春芳, 李建安, 侯婷婷, 張恒, 張郡

(吉林大學(xué)第二醫(yī)院, 骨科, 吉林 長(zhǎng)春, 130041)

摘要:目的研究與骨肉瘤(OS)和范可尼貧血(FA)相關(guān)聯(lián)的通路和分子機(jī)制。方法進(jìn)行范可尼貧血互補(bǔ)群D2(FANCD2)的siRNA構(gòu)建并轉(zhuǎn)錄至骨肉瘤細(xì)胞株MG-63細(xì)胞中。通過(guò)Western blot方法檢測(cè)MG-63細(xì)胞中FANCD2蛋白表達(dá)。結(jié)果在MG-63細(xì)胞中, FANCD2基因表達(dá)受到抑制，誘導(dǎo)細(xì)胞調(diào)亡。p53信號(hào)通路介導(dǎo)細(xì)胞凋亡。FANCD2基因表達(dá)抑制后，TP53INP1基因表達(dá)上調(diào)，促進(jìn)p53的磷酸化, p21蛋白被激活，導(dǎo)致依賴(lài)半胱天冬酶介導(dǎo)的細(xì)胞凋亡。結(jié)論抑制FANCD2基因表達(dá)可以誘導(dǎo)骨肉瘤細(xì)胞凋亡。

關(guān)鍵詞:范可尼貧血互補(bǔ)群D2; 骨肉瘤; p53; 細(xì)胞凋亡; 范可尼貧血; p21蛋白; 半胱天冬酶

骨肉瘤(OS)是骨組織中最常見(jiàn)的原發(fā)性惡性腫瘤，主要入侵長(zhǎng)骨的干骺端，預(yù)后不良[1]。原癌基因及抑癌基因功能障礙是骨肉瘤致病機(jī)制之一。正如大多數(shù)惡性腫瘤一樣，骨肉瘤也涉及多重致癌基因激活和抑癌基因突變，原癌基因如c-myc、ras、fos等，抑癌基因如p16、p53、Rb等[2-5]。骨肉瘤是范可尼貧血的并發(fā)癥之一，骨肉瘤和范可尼貧血更容易發(fā)生在青春期。范可尼貧血互補(bǔ)群D2(FANCD2)是骨肉瘤的重要相關(guān)蛋白。本研究進(jìn)行FANCD2的siRNA構(gòu)建并轉(zhuǎn)錄至骨肉瘤MG-63細(xì)胞，探討細(xì)胞凋亡以及凋亡相關(guān)的信號(hào)通路，揭示FANCD2在骨肉瘤發(fā)育過(guò)程中的作用，現(xiàn)報(bào)告如下。

1材料與方法

1.1MG-63細(xì)胞中FANCD2 siRNA構(gòu)建及轉(zhuǎn)錄

由圣克魯斯美國(guó)生物技術(shù)公司(Santa Cruz Biotechnology, Inc. 美國(guó)德州)設(shè)計(jì)及合成靶向FANCD2的siRNA和陰性對(duì)照iRNA(control siRNA)。利用脂質(zhì)體將其轉(zhuǎn)染到MG-63 細(xì)胞后，利用Western blot方法檢測(cè)24 h和48 h MG-63細(xì)胞中FANCD2蛋白的表達(dá)并以此評(píng)估siRNA的有效性。

1.2細(xì)胞凋亡的檢測(cè)

SiRNA轉(zhuǎn)錄后24 h和48 h, 采用PI單染，流式細(xì)胞術(shù)檢測(cè)細(xì)胞周期變化，分別設(shè)立以下4組，即對(duì)照組，陰性對(duì)照siRNA組，F(xiàn)ANCD2 siRNA干擾24 h組以及FANCD2 siRNA干擾48 h組。檢測(cè)MG-63細(xì)胞G0/G1，S和G2/M期細(xì)胞百分比，采用Annexin V-FITC凋亡檢測(cè)試劑盒，流式細(xì)胞術(shù)檢測(cè)細(xì)胞周期變化。

1.3細(xì)胞凋亡關(guān)聯(lián)蛋白

通過(guò)Western blot方法檢測(cè)TP53INP1, phos-p53、p21蛋白、caspase-9和caspase-3的產(chǎn)物，如上所述，分別檢測(cè)以上4組。

1.4統(tǒng)計(jì)學(xué)分析

所有數(shù)據(jù)采用SPSS 19.0軟件包進(jìn)行分析。用平均值±標(biāo)準(zhǔn)差表示實(shí)驗(yàn)數(shù)據(jù)，同質(zhì)性方差檢驗(yàn)后，使用樣本獨(dú)立t檢驗(yàn)的方法比較2組數(shù)據(jù)，超過(guò)2組使用方差分析，P<0.05表明差異有統(tǒng)計(jì)學(xué)意義。

2結(jié)果

2.1靶向FANCD2的siRNA對(duì)骨肉瘤MG-63細(xì)胞FANCD2表達(dá)的影響

對(duì)照組和陰性對(duì)照siRNA組FANCD2蛋白高表達(dá)，且無(wú)明顯差異，說(shuō)明陰性對(duì)照對(duì)FANCD2沒(méi)有產(chǎn)生干擾效應(yīng)，而FANCD2 siRNA干擾24 h(FANCD2 siRNA 24 h)和 FANCD2 siRNA干擾48 h(FANCD2 siRNA 48 h)后，均能顯著阻斷FANCD2 蛋白的表達(dá)，且以干擾48 h效果更明顯。見(jiàn)圖1。

注: 1. 對(duì)照組; 2. 陰性對(duì)照 siRNA組;3. FANCD2 siRNA干擾24 h組;4. FANCD2 siRNA干擾48 h組

圖1MG-63細(xì)胞中FANCD2蛋白表達(dá)HE染色，100倍

2.2靶向FANCD2的siRNA對(duì)骨肉瘤MG-63細(xì)胞凋亡的影響

采用流式細(xì)胞術(shù)檢測(cè)FANCD2的siRNA干擾后的MG-63細(xì)胞周期變化。與對(duì)照組比較，陰性對(duì)照siRNA組MG-63細(xì)胞正常和壞死無(wú)明顯差異，而凋亡顯著增加(P<0.05), 可能與加入的轉(zhuǎn)染試劑產(chǎn)生的作用有關(guān); FANCD2 siRNA 干擾24 h組和FANCD2 siRNA干擾48 h組，正常細(xì)胞百分比顯著降低，而凋亡細(xì)胞百分比顯著升高(P<0.05或P<0.001); 而且與FANCD2 siRNAg干擾24 h組比較，F(xiàn)ANCD2 siRNA干擾48 h組正常細(xì)胞百分比顯著降低，凋亡細(xì)胞百分比顯著增加(P<0.001)。見(jiàn)表1、圖2。上述結(jié)果暗示靶向FANCD2的siRNA導(dǎo)致MG-63細(xì)胞死亡不是以壞死為主，而是以凋亡為主，且以48 h誘導(dǎo)的凋亡更多。

與對(duì)照組比較，*P<0.05, **P<0.01, 與FANCD2 siRNA干擾24 h組比較, ##P<0.01。

3討論

范可尼貧血(FA)是一種罕見(jiàn)的常染色體或X連鎖引發(fā)的隱性遺傳性疾病。15種FA相關(guān)聯(lián)的基因的缺失或突變均可引發(fā)范可尼貧血。這些關(guān)聯(lián)基因構(gòu)成一個(gè)復(fù)雜的網(wǎng)絡(luò)，稱(chēng)作FA 途徑，范可尼貧血互補(bǔ)群D2(FANCD2)在途徑中起關(guān)鍵作用。在轉(zhuǎn)錄后修飾(磷酸化、泛素化)等方面[6-7], FANCD2蛋白在腫瘤發(fā)生、細(xì)胞凋亡和其他重要生命進(jìn)程中的基因調(diào)控表達(dá)方面起重要作用[8-10]。范可尼貧血患者有相當(dāng)高的患癌風(fēng)險(xiǎn)[11]。研究[12]顯示, 28%的范可尼貧血患者將在40歲之前發(fā)生非造血系統(tǒng)腫瘤，表明在范可尼貧血與惡性腫瘤之間有顯著正相關(guān)性。范可尼貧血患者通常會(huì)患有頭頸、皮膚或肛門(mén)-生殖器鱗狀細(xì)胞癌[13], 但是幾乎沒(méi)有發(fā)現(xiàn)骨肉瘤與范可尼貧血途徑相關(guān)。

圖2流式細(xì)胞術(shù)檢測(cè)FANCD2的siRNA干擾后的MG-63細(xì)胞凋亡比例(n= 4)HE染色，100倍

FANCD2基因在 FA/BRCA通路上起關(guān)鍵作用。另有研究[14-16]發(fā)現(xiàn)，F(xiàn)ANCD2基因參與 DNA損傷修復(fù)、細(xì)胞周期停滯、重構(gòu)染色質(zhì)、DNA甲基化以及細(xì)胞凋亡等，對(duì)機(jī)體細(xì)胞生長(zhǎng)、分化和維護(hù)至關(guān)重要。然而，它與骨肉瘤的關(guān)聯(lián)仍不清楚。本研究成功地構(gòu)建出一個(gè)高效的靶向FANCD2基因的siRNA, 轉(zhuǎn)染至MG-63細(xì)胞后幾乎沒(méi)有觀察到FANCD2蛋白的表達(dá)。它可以抑制細(xì)胞增殖，影響細(xì)胞周期停滯和細(xì)胞凋亡。FANCD2基因受到抑制甚至缺失將嚴(yán)重改變腫瘤細(xì)胞増殖的生物進(jìn)程。大量研究[17-18]表明，腫瘤細(xì)胞可以無(wú)限增殖、轉(zhuǎn)移及入侵。因此，常用的方法是通過(guò)誘導(dǎo)腫瘤細(xì)胞來(lái)控制腫瘤細(xì)胞的凋亡。FANCD2基因干擾MG-63細(xì)胞后，觀察細(xì)胞凋亡。FANCD2基因的siRNA干擾誘導(dǎo)細(xì)胞凋亡可以調(diào)節(jié)p53的信號(hào)通路, Western blot方法證實(shí)了上述結(jié)果。

TP53INP1(p53的上游蛋白), p53的磷酸化(phos-p53), p21蛋白 (參與調(diào)節(jié)G1期的下游蛋白)，激活caspase-9和caspase-3(凋亡相關(guān)蛋白)通過(guò)Western blot方法檢測(cè)。轉(zhuǎn)染到MG-63 細(xì)胞FANCD2的siRNA干擾48 h后, TP53INP1, p53, p21, caspase-9, caspase-3 mRNA的表達(dá)顯著升高; p53, p21和TP531W1蛋白產(chǎn)物也増加了。實(shí)驗(yàn)結(jié)果顯示，MG-63細(xì)胞的凋亡是p53信號(hào)通路介導(dǎo)的。siRNA-FANCD2轉(zhuǎn)染到MG-63細(xì)胞后，轉(zhuǎn)錄時(shí), FANCD2基因表達(dá)受到抑制, TP53INP1被激活。TP53INP1的表達(dá)促進(jìn)p53蛋白Ser15位點(diǎn)的磷酸化；磷酸化的p53激活了p21, 隨后開(kāi)始p53凋亡信號(hào)通路。p21是p53的下游基因，是細(xì)胞周期蛋白依賴(lài)性激酶抑制因子，與p53導(dǎo)致的細(xì)胞周期停滯相互作用?；罨膒53與的p21抑制腫瘤細(xì)胞增殖，并導(dǎo)致腫瘤細(xì)胞G1期的停滯。此外, p53可介導(dǎo)磷酸化線粒體凋亡途徑。它導(dǎo)致線粒體通透性的改變，隨后將多種細(xì)胞凋亡因子釋放到細(xì)胞質(zhì)中，最終導(dǎo)致MG-63細(xì)胞凋亡。

p53也可以激活半胱天冬酶通路，半胱天冬酶的激活促進(jìn)細(xì)胞凋亡。半胱天冬酶屬于半胱氨酸蛋白酶家族，在細(xì)胞凋亡的過(guò)程中起關(guān)鍵作用[19]。在半胱天冬酶途徑中，首先激活caspase-7, 然后激活caspase-12，進(jìn)一步裂解caspase-9和caspase-3，最后引發(fā)細(xì)胞凋亡[20-22]。而caspase-3在這個(gè)過(guò)程中是下游因子，是半胱天冬酶家族的關(guān)鍵酶，廣泛表達(dá)于各種腫瘤組織[23-24]。本研究表明，caspase-3在RNA干擾后裂解并活化，最終誘導(dǎo)細(xì)胞凋亡。

綜上所述, MG-63細(xì)胞中FANCD2的siRNA干擾導(dǎo)致FANCD2受到抑制，檢測(cè)了細(xì)胞凋亡情況。細(xì)胞凋亡可能是由p53信號(hào)通路引發(fā)的。FANCD2表達(dá)被抑制，促進(jìn)TP53INP1 基因表達(dá)，進(jìn)一步加強(qiáng)p53的磷酸化，導(dǎo)致細(xì)胞周期在G1期停滯，最后依賴(lài)半胱天冬酶誘導(dǎo)細(xì)胞凋亡。FANCD2在骨肉瘤生長(zhǎng)發(fā)育過(guò)程中起關(guān)鍵作用，抑制FANCD2基因的表達(dá)，可以有效地促進(jìn)骨肉瘤細(xì)胞的凋亡，為治療骨肉瘤提供新的研究方向。

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The p53 signaling pathway induces osteosarcoma MG-63 cell apoptosis by inhibiting expression of gene FANCD2

XIA Peng, ZAN Chunfang, LI Jian′an, HOU Tingting, ZHANG Heng, ZHANG Jun

(DepartmentofOrthopedics,TheSecondHospitalofJilinUniversity,Changchun,Jilin130041)

KEYWORDS:fanconi anemia complementation group D2; osteosarcoma; p53; cell apoptosis; fanconi anemia; p21 protein; caspase

ABSTRACT:ObjectiveTo investigate the associated pathway between osteosarcoma (OS) and fanconi anemia (FA) and its molecular mechanism. MethodsThe siRNA for fanconi anemia complementation group D2 (FANCD2) was constructed and transfected into the osteosarcoma cell line MG-63 cells. The FANCD2 protein expression of MG-63 cells was detected by Western blot. ResultsIn MG-63 cells, expression of gene FANCD2 was inhibited, and cell apoptosis was induced. The apoptosis was mediated by the p53 signaling pathway. After FANCD2 expression was inhibited, TP53INP1 gene expression was up-regulated, phosphorylation of p53 was promoted and the p21 protein was activated, leading to cell cycle arrested in G1, and finally resulted in caspase-dependent cell apoptosis. ConclusionInhibition of gene FANCD2 expression can induce apoptosis of osteosarcoma cells.

收稿日期:2015-12-09

通信作者:張郡, E-mail: 861916323@qq. com

中圖分類(lèi)號(hào):R 738.1

文獻(xiàn)標(biāo)志碼:A

文章編號(hào):1672-2353(2016)11-062-04

DOI:10.7619/jcmp.201611018