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RNA干扰沉默MSI1表达对人脑胶质瘤细胞化疗敏感度的影响

刘细国, 付锴, 江普查, 宫睿

刘细国, 付锴, 江普查, 宫睿. RNA干扰沉默MSI1表达对人脑胶质瘤细胞化疗敏感度的影响[J]. 肿瘤防治研究, 2016, 43(9): 754-757. DOI: 10.3971/j.issn.1000-8578.2016.09.005
引用本文: 刘细国, 付锴, 江普查, 宫睿. RNA干扰沉默MSI1表达对人脑胶质瘤细胞化疗敏感度的影响[J]. 肿瘤防治研究, 2016, 43(9): 754-757. DOI: 10.3971/j.issn.1000-8578.2016.09.005
LIU Xiguo, FU Kai, JIANG Pucha, GONG Rui. Effect of RNA Interference Decreasing MSI1 Expression on Chemosensitivity of Human Glioma Cells[J]. Cancer Research on Prevention and Treatment, 2016, 43(9): 754-757. DOI: 10.3971/j.issn.1000-8578.2016.09.005
Citation: LIU Xiguo, FU Kai, JIANG Pucha, GONG Rui. Effect of RNA Interference Decreasing MSI1 Expression on Chemosensitivity of Human Glioma Cells[J]. Cancer Research on Prevention and Treatment, 2016, 43(9): 754-757. DOI: 10.3971/j.issn.1000-8578.2016.09.005

RNA干扰沉默MSI1表达对人脑胶质瘤细胞化疗敏感度的影响

详细信息
    作者简介:

    刘细国(1980-),男,博士,主治医师,主要从事脑肿瘤的基础与临床研究

    通讯作者:

    付锴.E-mail: kaizige@hotmail.com

  • 中图分类号: R739.41

Effect of RNA Interference Decreasing MSI1 Expression on Chemosensitivity of Human Glioma Cells

More Information
  • 摘要:
    目的 

    探讨RNA干扰沉默MSI1表达对人脑胶质瘤细胞化疗药物敏感度的影响。

    方法 

    构建针对MSI1 mRNA的小分子RNA干扰(siRNA)重组质粒pSIREN-MSI1,转染人脑胶质瘤U-87MG细胞;Western blot检测U-87MG细胞内MSI1蛋白的表达;CCK-8法观察U-87MG细胞对化疗药物替莫唑胺(TMZ)敏感度的改变;流式细胞仪(FCM)检测pSIREN-MSI1转染后对U-87MG细胞凋亡的影响。

    结果 

    成功构建了重组质粒pSIREN-MSI1,并成功转染U-87MG细胞;Western blot结果显示pSIREN-MSI1抑制U-87MG细胞中MSI1蛋白的表达(P<0.01);CCK-8结果显示在替莫唑胺作用下,pSIREN-MSI1组U-87MG细胞的存活率明显下降(P<0.01);FCM结果表明pSIREN-MSI1转染后明显提高U-87MG细胞的凋亡率(P<0.01)。

    结论 

    MSI1 siRNA能特异性抑制人脑胶质瘤细胞U-87MG细胞中MSI1的表达,增强人脑胶质瘤U-87MG细胞对化疗药物的敏感度,并促进细胞的凋亡。

     

    Abstract:
    Objective 

    To explore the effect of RNA interference decreasing MSI1 gene expression on the chemosensitivity of human glioma cells U-87MG.

    Methods 

    The design and synthesis of Msi1-specific siRNA and the transfection of U-87MG cells were conducted. The expression of MSI1 in U-87MG cells was detected by Western blot. CCK-8 was employed to observe the sensitivity of U-87MG cells to temozolomide(TMZ). FCM was used to observe the apoptosis rate of U-87MG cells after pSIREN-MSI1 transfection.

    Results 

    Lentiviral vector-mediated MSI1-siRNA was successfully constructed. Immunofluorescence assay demonstrated that the transfection efficiency was above 60%. Western blot analyses demonstrated that pSIREN-MSI1 could significantly inhibit the expression of MSI1 in U-87MG cells (P<0.01); CCK-8 results showed that when U-87MG cells were exposed to temozolomide, the growth inhibiting rate of pSIREN-MSI1 transfected cells was significantly increased, compared with those of blank control group and negative control group (P<0.01). FCM results showed that the apoptosis rate of U-87MG cells were increased after pSIREN-MSI1 transfection(P<0.01).

    Conclusion 

    MSI1-siRNA could significantly inhibit the expression of MSI1 in human glioma cells U-87MG. It could induce the apoptosis and enhance the chemosensitivity of U-87MG cells.

     

  • 胶质瘤是人类中枢神经系统最常见的恶性肿瘤。胶质瘤细胞能激活相关调控的信号通路,从而产生对肿瘤细胞凋亡的抵抗作用,使常规放化疗效果不佳[1]。MSI1(musashi 1)是一种进化保守的RNA结合蛋白,在干细胞功能的维持以及自我更新能力中起重要作用,被认为是干细胞或祖细胞的一种重要标志物[2]。近年来有研究发现MSI1不仅在成体干细胞中高表达,在胃癌、结肠癌等实体细胞肿瘤中也高表达,且与肿瘤的转移和预后不佳关系密切[3-4]。本研究通过RNA干扰技术沉默人脑胶质瘤U-87MG细胞中MSI1的表达,观察其在人脑胶质瘤U-87MG细胞中的表达水平及对化疗药物敏感度的影响,并探索其作用机制。

    人脑胶质瘤U-87MG细胞(上海中国科学院细胞所);RPMI 1640培养液(美国Gibco公司);胎牛血清(美国Hyclone公司);pSIREN载体、DH5α大肠杆菌菌株(上海吉凯基因技术有限公司);RT-PCR试剂盒(日本Takara公司);Lipofectamine2000(美国Invitrogen公司);Western blot检测试剂盒(上海碧云天生物技术有限公司);MSI1单克隆抗体(美国Santa Cruz Biotechnology公司);FITC-AnnexinV凋亡检测试剂盒(美国BioVision公司);替莫唑胺(江苏天士力帝益药业公司)。

    将人脑胶质瘤U-87MG细胞置于含10%新生小牛血清的RPMI 1640培养液中,内加青霉素1×105 u/ml,链霉素100 mg/ml,置于5%CO2、饱和湿度、37℃恒温培养箱内培养。

    合成具有互补序列的能够编码短发卡RNA(shRNA)的双链寡核苷酸模板DNA。其转录产物所形成的siRNA的作用靶点为人MSI1 mRNA 498-516位核苷酸(GenBank ID:44440)。双链寡核苷酸的序列为:5’-GATCCACGCCATGCTGATGTTTGATTCAAGAGATCAAACATCAGCATGGCGTTTTTTTG-3’和5’-AATTCAAAAAAACGCCATGCTGATGTTTGATCTCTTGAATCAAACATCAGCATGGCGTG-3’。

    实验分为三组。空白对照组:未转染;阴性对照组:转染液中仅加入空质粒pSIREN-negative;pSIREN-MSI1组:转染液加入重组pSIREN-MSI1质粒。将人脑胶质瘤U-87MG细胞分三组用Lipofectamine2000TM介导进行转染。

    收集细胞,用细胞裂解液处理细胞,离心后取上清液,考马斯亮蓝法测定蛋白质浓度,聚丙烯酞胺凝胶电泳,将蛋白电转至硝酸纤维膜上,与MSI1抗体结合,之后用辣根过氧化物酶结合的二抗结合,ECL法显色后照相。最后以Image-Pro Plus 4.5图像分析系统测定各条带灰度值,以此反映MSI1蛋白的表达程度。

    取转染48 h后的3组细胞,按2×103/孔(0.1 ml)种植于96孔板中。24 h后加入不同浓度的替莫唑胺(1、4、8 μg/ml),72 h后吸出原培养液,加入含10%CCK-8的培养液10 μl,37℃继续培养 2 h。在全自动酶标仪上测450 nm波长的吸光度(OD)值。细胞存活率=(OD实验组-OD空白对照组)/(OD阴性对照组-OD空白对照组)×100%。实验重复3次。

    将空白对照组、阴性对照组、pSIREN-MSI1组三组细胞分别用替莫唑胺10 μg/ml处理48 h后,用胰酶消化并用预冷磷酸缓冲液(PBS)将细胞定量为(1~2)×106/毫升,预冷PBS洗涤、低速(900 r/min)、4℃离心5 min,共3次后尽量去除PBS,用预冷结合缓冲液200 μl重新悬浮细胞,先加入FITC-AnnexinV 5 μl和PI 5 μl孵育,15 min后再加入300 μl结合缓冲液,上机检测前5 min加入5 μl碘化丙啶。

    应用SPSS15.0统计学软件进行数据分析,用t检验进行两组间差异比较,P<0.05为差异有统计学意义。

    Western blot结果显示pSIREN-MSI1组转染U-87MG细胞后MSI1蛋白的表达明显减少,与空白对照组和阴性对照组比较差异均有统计学意义(均P<0.01),见图 1,而在空白对照组和阴性对照组细胞中表达无明显变化(P>0.05)。说明MSI1 siRNA转染U-87MG细胞后特异性地抑制了MSI1蛋白的表达。

    图  1  Western blot检测pSIREN- MSI1转染U-87MG细胞后MSI1蛋白的表达
    Figure  1  Expression of MSI1 protein in U-87MG cells after pSIREN-MSI1 transfection detected by Western blot
    The expression of MSI protein was significantly inhibited,compared with Blank control group and Negative control group (all P<0.01)

    CCK-8法结果显示加入不同浓度替莫唑胺处理后,pSIREN-MSI1组的U-87MG细胞存活率明显下降,分别为47.63%(1 μg/ml),38.16%(4 μg/ml),27.52%(8 μg/ml),与空白组及阴性对照组比较差异均有统计学意义(P=0.0079,P=0.0057),见图 2;而加入不同浓度替莫唑胺后空白对照组与阴性对照组相比U-87MG细胞存活率差异均无统计学意义[P=0.361,P=0.279(1 μg/ml); P=0.478,P=0.519(4 μg/ml); P=0.611,P=0.905(8 μg/ml)]。结果说明MSI1 siRNA转染U-87MG细胞后明显提高了细胞对化疗药物的敏感度。

    图  2  CCK-8法检测不同浓度替莫唑胺作用于各组转染细胞后的细胞存活率
    Figure  2  Survival rates of U-87MG cells treated with TMZ in each group detected by CCK-8
    The survival ratio of pSIREN-MSI1 was significantly inhibited,compared with Blank control group and Negative control group [P=0.0046,P=0.0039 (8μg/ml)]

    FCM检测结果显示,pSIREN-MSI1组的细胞凋亡率(36.79±2.38)%明显高于空白对照组(6.20±2.62)%和阴性对照组(8.77±1.35)%(P=0.0069,P=0.0076),见图 3;而空白对照组和阴性对照组细胞凋亡率相比差异无统计学意义(P=0.307)。结果说明MSI1 siRNA转染U-87MG细胞后能明显促进细胞的凋亡。

    图  3  流式细胞仪检测pSIREN-MSI1转染U-87MG细胞后的细胞凋亡率
    Figure  3  Apoptosis rate of U-87MG cells after pSIREN-MSI1 transfection detected by FCM
    The apoptosis rate of pSIREN-MSI1 group was significantly inhibited,compared with Blank control group and Negative Control group (P=0.0069,P=0.0076)

    近年提出的肿瘤干细胞学说认为,肿瘤组织中存在极少量充当着干细胞角色的瘤细胞,它们具有自我更新和多向分化潜能,在启动肿瘤形成和生长中起着重要作用,是肿瘤生长、转移、复发以及产生极强耐药性的根源[5-7]。MSI1作为一种RNA结合蛋白能够通过两个特异性RNA识别模序识别结合(G/A)UnAGU(n=1-3)位点,阻止如CDKN2A、Numb、p21WAF-1等mRNA的翻译,而这些基因在肿瘤的发生发展中起重要作用,主要影响细胞周期、增殖、细胞分化和凋亡以及蛋白修饰[8]。同时MSI1还通过一条独特的自分泌信号通路激活Wnt和Notch等肿瘤生长的相关信号通路,导致肿瘤的发生[9]。MSI1在多种肿瘤干细胞中表达,与肿瘤和肿瘤干细胞的关系密切,参与了肿瘤的发生、发展、浸润和转移,是肿瘤潜在的诊断标志物、预后指标和治疗靶点,是肿瘤干细胞的新型标志物。

    本研究成功构建了针对MSI1基因的siRNA真核表达载体转染人脑胶质瘤U-87MG细胞中。通过Western blot的检测显示转染后U-87MG细胞内的MSI1蛋白表达明显减少。提示本研究构建的siRNA真核表达载体的确能够在细胞内持续地表达siRNA,高效、特异地抑制MSI1基因的表达。

    有研究表明,在结肠癌等肿瘤中Wnt和Notch相关信号通路可通过调节一系列的下游基因表达介导癌细胞药物耐药过程[10]。本研究通过CCK-8法检测转染后在化疗药物作用下细胞成活率的情况,结果表明,稳定转染MSI1 siRNA真核表达载体后替莫唑胺对细胞生长抑制作用增加,U-87MG细胞的存活率明显下降,细胞存活率最低达到27.52%,明显低于空白对照组和阴性对照组(P<0.01)。同时本研究还通过FCM检测发现MSI1 siRNA转染U-87MG细胞后细胞凋亡率显著增加,最高达(36.79±2.38)%,明显高于空白对照组和阴性对照组(P<0.01)。以上研究结果显示:用RNA干扰技术抑制MSI1表达极有可能通过抑制 Wnt和Notch等通路的激活,诱导细胞周期停滞,从而导致肿瘤干细胞分化或凋亡,降低对化疗药物的耐受。

    综上所述,通过RNA干扰技术沉默MSI1的表达可诱导人脑胶质瘤U-87MG细胞凋亡,并提高肿瘤细胞化疗敏感度。MSI1基因可作为重要的肿瘤干细胞标志物成为治疗肿瘤的有效靶点。

  • 图  1   Western blot检测pSIREN- MSI1转染U-87MG细胞后MSI1蛋白的表达

    Figure  1   Expression of MSI1 protein in U-87MG cells after pSIREN-MSI1 transfection detected by Western blot

    图  2   CCK-8法检测不同浓度替莫唑胺作用于各组转染细胞后的细胞存活率

    Figure  2   Survival rates of U-87MG cells treated with TMZ in each group detected by CCK-8

    图  3   流式细胞仪检测pSIREN-MSI1转染U-87MG细胞后的细胞凋亡率

    Figure  3   Apoptosis rate of U-87MG cells after pSIREN-MSI1 transfection detected by FCM

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出版历程
  • 收稿日期:  2015-08-04
  • 修回日期:  2015-11-26
  • 网络出版日期:  2024-02-04
  • 刊出日期:  2016-08-31

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