馬國(guó)祥 丁茜萍 鄧海華 楊振漢

廣東深圳市寶安區(qū)福永人民醫(yī)院內(nèi)科 深圳 518103

microRNA-183通過(guò)下調(diào)Bcl-2誘導(dǎo)人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞凋亡的研究

馬國(guó)祥 丁茜萍 鄧海華 楊振漢

廣東深圳市寶安區(qū)福永人民醫(yī)院內(nèi)科 深圳 518103

目的 研究microRNA-183對(duì)神經(jīng)母細(xì)胞瘤細(xì)胞的調(diào)控作用，為神經(jīng)母細(xì)胞瘤的治療提供新策略。方法 購(gòu)買microRNA-183和對(duì)照 microRNA，轉(zhuǎn)染至人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞，再使用MTT實(shí)驗(yàn)，Caspase-3活性測(cè)定試劑盒和流式檢測(cè)細(xì)胞生長(zhǎng)和凋亡的影響。合成Bcl-2 siRNA，檢測(cè)Bcl-2對(duì)人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞凋亡的影響。轉(zhuǎn)染Bcl-2質(zhì)粒后，再轉(zhuǎn)染microRNA-183至人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞，分析Bcl-2的表達(dá)水平和人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞的凋亡。結(jié)果 轉(zhuǎn)染microRNA-183降低SK-N-SH細(xì)胞的生長(zhǎng)(P=0.005 9)，發(fā)生磷脂酰絲氨酸膜表面表達(dá)(P=0.008)和Caspase-3的激活(P=0.014)，Bcl-2的表達(dá)降低(P=0.015)。干擾SK-N-SH細(xì)胞中Bcl-2增強(qiáng)了microRNA-183誘導(dǎo)的細(xì)胞凋亡(P=0.005 8)，而過(guò)表達(dá)Bcl-2抑制了microRNA-183誘導(dǎo)的細(xì)胞凋亡(P=0.007 3)。結(jié)論 轉(zhuǎn)染microRNA-183抑制SK-N-SH細(xì)胞的生長(zhǎng)和增殖。microRNA-183通過(guò)下調(diào)Bcl-2而誘導(dǎo)SK-N-SH細(xì)胞的凋亡，提示Bcl-2可能是神經(jīng)母細(xì)胞瘤潛在的候選治療靶點(diǎn)。

細(xì)胞凋亡；microRNA-183；Bcl-2；SK-N-SH細(xì)胞

神經(jīng)母細(xì)胞瘤嚴(yán)重威脅著患者的生活和健康[1]。神經(jīng)母細(xì)胞瘤的治療主要包括放化療、手術(shù)治療、分子靶向治療等[2]，針對(duì)不同的患者，上述治療方法療效均顯著，然而，也存在毒副作用強(qiáng)、適用人群少、分子靶點(diǎn)的選擇難等缺點(diǎn)[3-4]。microRNA調(diào)控細(xì)胞生長(zhǎng)、凋亡和增殖作用[5]。在動(dòng)物實(shí)驗(yàn)中，microRNA常被作為癌癥治療的分子靶點(diǎn)[6]。然而，microRNA-183對(duì)神經(jīng)母細(xì)胞瘤細(xì)胞生長(zhǎng)和凋亡的調(diào)控作用尚不清楚。因此，本研究以人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞為細(xì)胞模型，探討microRNA-183對(duì)人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞可能的調(diào)控機(jī)制，以期為神經(jīng)母細(xì)胞瘤分子靶點(diǎn)的治療提供理論依據(jù)。

1 實(shí)驗(yàn)試劑和方法

1．1 實(shí)驗(yàn)試劑和實(shí)驗(yàn)細(xì)胞 小鼠源抗人Bcl-2的多克隆抗體，HRP標(biāo)記的羊源抗小鼠的IgG單克隆抗體，內(nèi)參抗actin內(nèi)參的多克隆抗體來(lái)源于美國(guó)Santa Cruz生物技術(shù)有限公司。細(xì)胞培養(yǎng)用血清和培養(yǎng)基來(lái)源于華蘭生物用品公司。MTT試劑盒來(lái)源于碧云天生物技術(shù)研究所。細(xì)胞凋亡試劑來(lái)源于北京鼎國(guó)科技有限責(zé)任公司公司。

microRNA如microRNA-83(5′-ACATCACA-TCGGTAGTTGCCAT-3′和5′- AAACAAAGAGGTAAAGGTGCA-3′)，對(duì)照microRNA(5′-CCAACGAGTGTCTTTCGAGC-3′和5′-TTGACGTTT-CACGATACAT-3′)和siRNA Bcl-2(5′-CTACGTGCTACCATGGATGC-3′和5′-TTCTAGAACTACGCTATGT-3′)，Bcl-2的質(zhì)粒來(lái)源于上海生物工程有限公司。Lipo2000轉(zhuǎn)染試劑來(lái)源于美國(guó)Invitrogen。

1．2 細(xì)胞培養(yǎng)的方法 復(fù)蘇人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞，將人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞重懸于DMEM培養(yǎng)基中，37 ℃，5% CO2[7]。

1．3 轉(zhuǎn)染方法 將microRNA-183和對(duì)照micro-RNA轉(zhuǎn)染至人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞。將人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞鋪板于24孔板，密度30%左右，將microRNA-183和對(duì)照microRNA加入lipo2000中，混勻，室溫放置7 min，將混合液加入細(xì)胞中，37 ℃，5% CO2[8-9]。

1．4 MTT試劑盒檢測(cè)細(xì)胞活性 按照MTT試劑盒的說(shuō)明檢測(cè)人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞的活性[10]。首先，接種人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞。按1.3的描述向人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞轉(zhuǎn)染microRNA-183或?qū)φ誱iRNA后，再加入1 mg/mL MTT反應(yīng)液，培養(yǎng)人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞24 h。最后在細(xì)胞里加入終止反應(yīng)液DMSO，終止反應(yīng)。

將6孔板人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞放入酶標(biāo)儀中，測(cè)試人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞在560 nm處的吸收值，繪制人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞生長(zhǎng)曲線[11]。

1．5 流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡 人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞的凋亡情況使用流式檢測(cè)。首先，接種人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞。按1.3的描述向人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞轉(zhuǎn)染microRNA-183或?qū)φ誱iRNA后，向200 μL體積的細(xì)胞中加入16 μL的反應(yīng)液和2 μL的染料Annexin-V-FITC，避光反應(yīng)14 min。最后上流式檢測(cè)[12]。1．6 聚丙烯酰胺凝膠電泳和Western blot免疫印跡 首先，接種人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞。按1.3的描述向人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞轉(zhuǎn)染microRNA-183或?qū)φ誱iRNA后，收集細(xì)胞，向細(xì)胞中加入細(xì)胞裂解液裂解，制備聚丙烯酰胺凝膠電泳樣品，進(jìn)行電泳，電泳結(jié)束轉(zhuǎn)膜、封閉，孵育一抗、二抗，最后進(jìn)行顯影、定影[13]。

1．7 Caspase-3活性測(cè)試 按照試劑盒的說(shuō)明檢測(cè)人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞的凋亡情況。首先，接種人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞。按1.3的描述向人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞轉(zhuǎn)染microRNA-183或?qū)φ誱iRNA后，裂解細(xì)胞，向細(xì)胞樣品中加入生色底物反應(yīng)。最后在酶標(biāo)儀上測(cè)試各組樣品的光吸收值[14]，作為細(xì)胞Caspase-3活性的相對(duì)值。

2 結(jié)果

2．1 轉(zhuǎn)染microRNA-183降低了人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞的活性 各實(shí)驗(yàn)組的人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞活性通過(guò)MTT測(cè)試，與轉(zhuǎn)染對(duì)照miRNA的人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞相比，轉(zhuǎn)染microRNA-183后，人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞的生長(zhǎng)被顯著抑制(P=0.005 9)。見(jiàn)圖1。

由于轉(zhuǎn)染對(duì)照miRNA的人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞和未轉(zhuǎn)染組人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞的活性差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)，所以，以轉(zhuǎn)染miRNA細(xì)胞組作為對(duì)照。

圖1 轉(zhuǎn)染microRNA-183降低了人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞的活性，抑制其生長(zhǎng) 與miRNA組相比，**P<0.01 圖2 轉(zhuǎn)染microRNA-183引起人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞的凋亡與miRNA組相比，**P<0.01

2．2 轉(zhuǎn)染microRNA-183引起人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞的凋亡 如圖2所示，人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞磷脂酰絲氨酸的外翻，與轉(zhuǎn)染對(duì)照miRNA的人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞相比，轉(zhuǎn)染microRNA-183后，人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞膜磷脂酰絲氨酸外翻的量顯著增加(P=0.008)。

2．3 轉(zhuǎn)染microRNA-183引起人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞中Caspase-3的活化 圖3中人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞中細(xì)胞凋亡的檢測(cè)數(shù)據(jù)提示，與轉(zhuǎn)染miRNA的人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞相比，轉(zhuǎn)染microRNA-183后，人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞凋亡增加(P=0.014)。

圖3 轉(zhuǎn)染microRNA-183引起人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞的Caspase-3的活化 與miRNA組相比，*P<0.05 圖4 轉(zhuǎn)染microRNA-183引起人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞中Bcl-2蛋白表達(dá)的下降

2．4 轉(zhuǎn)染microRNA-183引起人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞中Bcl-2蛋白表達(dá)的下降 如圖4所示，人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞中細(xì)胞凋亡的檢測(cè)數(shù)據(jù)提示，與轉(zhuǎn)染對(duì)照miRNA的人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞相比，轉(zhuǎn)染microRNA-183后，人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞細(xì)胞凋亡增強(qiáng)(P=0.015)。

2．5 敲低Bcl-2增強(qiáng)microRNA-183引起的人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞凋亡 如圖5所示，降低Bcl-2的表達(dá)后，再轉(zhuǎn)染microRNA-183后，人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞凋亡增加(P=0.007 6)。microRNA-183組和siBcl-2+microRNA-183細(xì)胞凋亡差異有統(tǒng)計(jì)學(xué)意義(P=0.005 8)。

圖5 敲低Bcl-2增強(qiáng)microRNA-183引起的人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞凋亡 與miRNA組相比，*P<0.05；microRNA-183組和siBcl-2+microRNA-183細(xì)胞凋亡比較，#P<0.05

2．6 轉(zhuǎn)染Bcl-2抑制了microRNA-183誘導(dǎo)的人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞凋亡 圖6中Western blot結(jié)果表明，Bcl-2的質(zhì)粒轉(zhuǎn)染增加了其水平。microRNA-183組和Bcl-2+microRNA-183組間Bcl-2比較差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。

圖6示，人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞中細(xì)胞凋亡的檢測(cè)結(jié)果表明，轉(zhuǎn)染Bcl-2增強(qiáng)Bcl-2水平后，再轉(zhuǎn)染microRNA-183后，人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞凋亡被明顯抑制(P=0.006)。microRNA-183組和Bcl-2+microRNA-183細(xì)胞凋亡差異有統(tǒng)計(jì)學(xué)意義(P=0.007 3)。

圖6 轉(zhuǎn)染Bcl-2抑制了microRNA-183誘導(dǎo)的人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞凋亡與miRNA組相比，*P<0.05；microRNA-183組和Bcl-2+microRNA-183比較，#P<0.05

3 討論

本研究以人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞為細(xì)胞模型，從蛋白水平上研究了microRNA-183對(duì)人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞的調(diào)節(jié)機(jī)制，結(jié)果顯示，轉(zhuǎn)染microRNA-183降低了人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞的生長(zhǎng)，引起人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞凋亡。這與前人研究結(jié)果高度一致，即microRNA調(diào)控細(xì)胞生長(zhǎng)和細(xì)胞凋亡[15]。

Bcl-2蛋白是Bcl-2蛋白家族中重要的細(xì)胞凋亡抑制蛋白之一[7-8]。據(jù)研究報(bào)道，Bcl-2可能受microRNA-183的調(diào)節(jié)，進(jìn)而調(diào)節(jié)人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞[9-10]。本研究結(jié)果證明，轉(zhuǎn)染microRNA-183降低了Bcl-2的蛋白水平。轉(zhuǎn)染microRNA-183和降低Bcl-2的表達(dá)后，人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞的凋亡增加，而轉(zhuǎn)染Bcl-2則抑制了microRNA-183誘導(dǎo)的人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞凋亡。

本文采用不同的方法證明了Bcl-2蛋白在microRNA-183誘導(dǎo)的人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞凋亡中的作用：(1)Western blot結(jié)果顯示，microRNA-183高表達(dá)時(shí)，人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞中的Bcl-2蛋白顯著降低；(2)轉(zhuǎn)染Bcl-2 siRNA后，再轉(zhuǎn)染microRNA-183至人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞后，人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞凋亡增強(qiáng)；(3)轉(zhuǎn)染Bcl-2質(zhì)粒后，再轉(zhuǎn)染microRNA-183至人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞，人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞凋亡被顯著抑制。結(jié)果表明，Bcl-2蛋白在microRNA-183誘導(dǎo)的人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞凋亡中發(fā)揮關(guān)鍵作用，Bcl-2蛋白是潛在的神經(jīng)母細(xì)胞瘤的分子靶點(diǎn)[11-12]。Bcl-2可抑制癌細(xì)胞的細(xì)胞凋亡[13-14]，這是一致性的結(jié)論。

本文的不足和缺點(diǎn)如下：(1)缺少臨床神經(jīng)母細(xì)胞瘤的樣本和對(duì)照組織中Bcl-2蛋白水平的結(jié)果；(2)缺少預(yù)后臨床神經(jīng)母細(xì)胞瘤的樣本和對(duì)照組織中Bcl-2蛋白水平的結(jié)果；(3)缺少神經(jīng)母細(xì)胞瘤的小鼠模型和以microRNA-183為靶點(diǎn)治療的神經(jīng)母細(xì)胞瘤小鼠的療效結(jié)果。

轉(zhuǎn)染microRNA-183抑制人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞的生長(zhǎng)和增殖，microRNA-183通過(guò)下調(diào)Bcl-2而誘導(dǎo)人神經(jīng)母細(xì)胞瘤細(xì)胞株SK-N-SH細(xì)胞的凋亡，提示Bcl-2可能是神經(jīng)母細(xì)胞瘤潛在的候選治療靶點(diǎn)。

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(收稿2016-10-20)

microRNA-183 induces apoptosis of human neuroblastoma cell line SK-N-SH by down regulating Bcl-2 disease rats

MaGuoxiang，DingQianping，DengHaihua，YangZhenhan

FuyongPeople'sHospitalofShenzhenBaoanDistrict，Shenzhen518103，China

Objective To study the regulation of microRNA-183 on neuroblastoma cells and to provide a new strategy for the treatment of neuroblastoma．Methods microRNA and microRNA-183 were transfected into SK-N-SH cells，and then MTT experiment and Caspase-3 activity assay and flow cytometry were employed to investigate the role of microRNA-183 in growth and apoptosis of human neuroblastoma cell line SK-N-SH．Bcl-2 siRNA was transfected into SK-N-SH cells，and effects of Bcl-2 on human neuroblastoma cell line SK-N-SH cells apoptosis was tested．After transfection of Bcl-2 plasmid，microRNA-183 was transfected into human neuroblastoma cell line SK-N-SH cells，and the expression level of Bcl-2 and apoptosis of human neuroblastoma cell line SK-N-SH were analyzed．Results The transfection of microRNA-183 reduced cell growth of the SK-N-SH cells (P=0.005 9)，and the expression of phosphatidylserine on the membrane (P=0.008) and activation of Caspase-3 were observed (P=0.014)，and the expression of Bcl-2 was decreased (P=0.015)．Bcl-2 interference of human neuroblastoma cell lines enhances apoptosis of the human neuroblastoma cell line SK-N-SH induced by microRNA-183 (P=0.005 8)，and overexpression of Bcl-2 inhibits apoptosis induced by microRNA-183 (P=0.007 3)．Conclusion The transfection of microRNA-183 inhibits the growth and proliferation of human neuroblastoma cell line SK-N-SH．MicroRNA-183 induces apoptosis of SK-N-SH by down regulating Bcl-2，which suggests that Bcl-2 may be a potential candidate therapeutic target for neuroblastoma．

Apoptosis；microRNA-183；Bcl-2；SK-N-SH cell

R730.264

A

1673-5110(2017)07-0003-04