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 長春, 130041)

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p53通路抑制FANCD2基因的表達來誘導(dǎo)骨肉瘤MG-63細胞凋亡

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)的通路和分子機制。方法進行范可尼貧血互補群D2(FANCD2)的siRNA構(gòu)建并轉(zhuǎn)錄至骨肉瘤細胞株MG-63細胞中。通過Western blot方法檢測MG-63細胞中FANCD2蛋白表達。結(jié)果在MG-63細胞中, FANCD2基因表達受到抑制，誘導(dǎo)細胞調(diào)亡。p53信號通路介導(dǎo)細胞凋亡。FANCD2基因表達抑制后，TP53INP1基因表達上調(diào)，促進p53的磷酸化, p21蛋白被激活，導(dǎo)致依賴半胱天冬酶介導(dǎo)的細胞凋亡。結(jié)論抑制FANCD2基因表達可以誘導(dǎo)骨肉瘤細胞凋亡。

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

骨肉瘤(OS)是骨組織中最常見的原發(fā)性惡性腫瘤，主要入侵長骨的干骺端，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。范可尼貧血互補群D2(FANCD2)是骨肉瘤的重要相關(guān)蛋白。本研究進行FANCD2的siRNA構(gòu)建并轉(zhuǎn)錄至骨肉瘤MG-63細胞，探討細胞凋亡以及凋亡相關(guān)的信號通路，揭示FANCD2在骨肉瘤發(fā)育過程中的作用，現(xiàn)報告如下。

1材料與方法

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

由圣克魯斯美國生物技術(shù)公司(Santa Cruz Biotechnology, Inc. 美國德州)設(shè)計及合成靶向FANCD2的siRNA和陰性對照iRNA(control siRNA)。利用脂質(zhì)體將其轉(zhuǎn)染到MG-63 細胞后，利用Western blot方法檢測24 h和48 h MG-63細胞中FANCD2蛋白的表達并以此評估siRNA的有效性。

1.2細胞凋亡的檢測

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

1.3細胞凋亡關(guān)聯(lián)蛋白

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

1.4統(tǒng)計學(xué)分析

所有數(shù)據(jù)采用SPSS 19.0軟件包進行分析。用平均值±標準差表示實驗數(shù)據(jù)，同質(zhì)性方差檢驗后，使用樣本獨立t檢驗的方法比較2組數(shù)據(jù)，超過2組使用方差分析，P<0.05表明差異有統(tǒng)計學(xué)意義。

2結(jié)果

2.1靶向FANCD2的siRNA對骨肉瘤MG-63細胞FANCD2表達的影響

對照組和陰性對照siRNA組FANCD2蛋白高表達，且無明顯差異，說明陰性對照對FANCD2沒有產(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，且以干擾48 h效果更明顯。見圖1。

注: 1. 對照組; 2. 陰性對照 siRNA組;3. FANCD2 siRNA干擾24 h組;4. FANCD2 siRNA干擾48 h組

圖1MG-63細胞中FANCD2蛋白表達HE染色，100倍

2.2靶向FANCD2的siRNA對骨肉瘤MG-63細胞凋亡的影響

采用流式細胞術(shù)檢測FANCD2的siRNA干擾后的MG-63細胞周期變化。與對照組比較，陰性對照siRNA組MG-63細胞正常和壞死無明顯差異，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，正常細胞百分比顯著降低，而凋亡細胞百分比顯著升高(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組正常細胞百分比顯著降低，凋亡細胞百分比顯著增加(P<0.001)。見表1、圖2。上述結(jié)果暗示靶向FANCD2的siRNA導(dǎo)致MG-63細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)成一個復(fù)雜的網(wǎng)絡(luò)，稱作FA 途徑，范可尼貧血互補群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、細胞凋亡和其他重要生命進程中的基因調(diào)控表達方面起重要作用[8-10]。范可尼貧血患者有相當高的患癌風(fēng)險[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患有頭頸、皮膚或肛門-生殖器鱗狀細胞癌[13], 但是幾乎沒有發(fā)現(xiàn)骨肉瘤與范可尼貧血途徑相關(guān)。

圖2流式細胞術(shù)檢測FANCD2的siRNA干擾后的MG-63細胞凋亡比例(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)、細胞周期停滯、重構(gòu)染色質(zhì)、DNA甲基化以及細胞凋亡等，對機體細胞生長、分化和維護至關(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)建出一個高效的靶向FANCD2基因的siRNA, 轉(zhuǎn)染至MG-63細胞后幾乎沒有觀察到FANCD2蛋白的表達。它可以抑制細胞增殖，影響細胞周期停滯和細胞凋亡。FANCD2基因受到抑制甚至缺失將嚴重改變腫瘤細胞増殖的生物進程。大量研究[17-18]表明，腫瘤細胞可以無限增殖、轉(zhuǎn)移及入侵。因此，常用的方法是通過誘導(dǎo)腫瘤細胞來控制腫瘤細胞的凋亡。FANCD2基因干擾MG-63細胞后，觀察細胞凋亡。FANCD2基因的siRNA干擾誘導(dǎo)細胞凋亡可以調(diào)節(jié)p53的信號通路, Western blot方法證實了上述結(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Western blot方法檢測。轉(zhuǎn)染到MG-63 細胞FANCD2的siRNA干擾48 h后, TP53INP1, p53, p21, caspase-9, caspase-3 mRNA的表達顯著升高; p53, p21和TP531W1蛋白產(chǎn)物也増加了。實驗結(jié)果顯示，MG-63細胞的凋亡是p53信號通路介導(dǎo)的。siRNA-FANCD2轉(zhuǎn)染到MG-63細胞后，轉(zhuǎn)錄時, FANCD2基因表達受到抑制, TP53INP1被激活。TP53INP1的表達促進p53蛋白Ser15位點的磷酸化；磷酸化的p53激活了p21, 隨后開始p53凋亡信號通路。p21是p53的下游基因，是細胞周期蛋白依賴性激酶抑制因子，與p53導(dǎo)致的細胞周期停滯相互作用?；罨膒53與的p21抑制腫瘤細胞增殖，并導(dǎo)致腫瘤細胞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，隨后將多種細胞凋亡因子釋放到細胞質(zhì)中，最終導(dǎo)致MG-63細胞凋亡。

p53也可以激活半胱天冬酶通路，半胱天冬酶的激活促進細胞凋亡。半胱天冬酶屬于半胱氨酸蛋白酶家族，在細胞凋亡的過程中起關(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，進一步裂解caspase-9和caspase-3，最后引發(fā)細胞凋亡[20-22]。而caspase-3在這個過程中是下游因子，是半胱天冬酶家族的關(guān)鍵酶，廣泛表達于各種腫瘤組織[23-24]。本研究表明，caspase-3在RNA干擾后裂解并活化，最終誘導(dǎo)細胞凋亡。

綜上所述, MG-63細胞中FANCD2的siRNA干擾導(dǎo)致FANCD2受到抑制，檢測了細胞凋亡情況。細胞凋亡可能是由p53信號通路引發(fā)的。FANCD2表達被抑制，促進TP53INP1 基因表達，進一步加強p53的磷酸化，導(dǎo)致細胞周期在G1期停滯，最后依賴半胱天冬酶誘導(dǎo)細胞凋亡。FANCD2在骨肉瘤生長發(fā)育過程中起關(guān)鍵作用，抑制FANCD2基因的表達，可以有效地促進骨肉瘤細胞的凋亡，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

中圖分類號:R 738.1

文獻標志碼:A

文章編號:1672-2353(2016)11-062-04

DOI:10.7619/jcmp.201611018