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miRNA-149靶向IGFBP5抑制膀胱癌细胞侵袭的分子机制研究

2018-02-26饶大庞虞海峰王帅彬孙来芳

中国现代医生 2018年35期
关键词:膀胱癌靶向活性

饶大庞 虞海峰 王帅彬 孙来芳

[摘要] 目的 探讨miRNA-149靶向IGFBP5抑制膀胱癌细胞侵袭的分子机制。 方法 采用双萤光报告基因系统分别检测miRNA-149对目的基因IGFBP5和PDGFRA的3'UTR的影响。在T24膀胱癌细胞中瞬时转染miRNA-149模拟物(mimic)和阴性对照miRNA(mimic NC),并且在该细胞中分别转染IGFBP5 过表达质粒(H10495)、阴性对照(H155)和PDGFRA过表达质粒(H10496),利用Transwell法检测细胞的迁移和侵袭能力。 结果 miRNA-149显著抑制IGFBP5的 3UTR 活性,差异具有统计学意义(P<0.0001)。Transwell实验显示T24-H155-miRNA149-mimics组较T24-H155-mimic-NC对照组(control)侵袭能力降低,差异具有统计学意义(P<0.0001),而在T24-H10495-miRNA-149-mimics组较 T24-H155-miRNA-149-mimics组侵袭能力明显增强(P<0.01)。另一方面,T24-H10496-miRNA-149-mimics组较 T24-H155-miRNA-149-mimics组侵袭能力无明显变化(P>0.05)。 结论 该研究表明miRNA-149通过抑制其下游基因IGFBP5从而抑制膀胱癌侵袭,为临床膀胱癌转移的分子治疗研究提供了新的靶点和理论依据。

[关键词] miRNA-149;膀胱癌;IGFBP5;PDGFRA;侵袭

[中图分类号] R737.9          [文献标识码] A          [文章编号] 1673-9701(2018)35-0001-04

[Abstract] Objective To investigate the molecular mechanism of miRNA-149 targeting IGFBP5 to inhibit the invasion of bladder cancer cells. Methods The effect of miRNA-149 on the 3'UTR of the target genes IGFBP5 and PDGFRA was examined using a dual fluorescent reporter system. miRNA-149 mimics and negative control miRNAs(mimic NC) were transiently transfected into T24 bladder cancer cells, and IGFBP5 overexpression plasmid(H10495), negative control (H155) and PDGFRA overexpression plasmid (H10496) were transfected into the cells, respectively. The migration and invasion ability of the cells were detected by Transwell method. Results miRNA-149 significantly inhibited the 3'UTR activity of IGFBP5 with a statistically significant difference(P<0.0001). Transwell experiment showed that the T24-H155-miRNA149-mimics group had a lower invasive ability than the T24-H155-mimic-NC control group, and the difference was significant(P<0.0001). While the invasive ability of the T24-H10495-miRNA-149-mimics group was significantly enhanced compared with that of the T24-H155-miRNA-149-mimics group(P<0.0001). On the other hand, there was no significant change in the invasive ability of the T24-H10496-miRNA-149-mimics group compared with that of the T24-H155-miRNA-149-mimics group(P>0.05). Conclusion This study demonstrates that miRNA-149 inhibits the invasion of bladder cancer through inhibiting its downstream gene IGFBP5, which provides a new target and theoretical basis for the molecular treatment of clinical bladder cancer metastasis.

[Key words] miRNA-149; Bladder cancer; IGFBP5; PDGFRA; Invasion

膀胱癌是世界范圍内最常见的泌尿系统恶性肿瘤之一,具有较高的发病率和死亡率,给患者造成了极大威胁[1,2]。2017年估计有79 030例膀胱癌新发病例和16 870例膀胱癌死亡[3]。膀胱癌可分为两类:非肌肉浸润性膀胱癌和肌肉浸润性膀胱癌。研究显示,1/3的非肌肉浸润性膀胱癌(NMIBC)患者可随后进展为肌肉浸润性(MIBC)或转移,并且约70%的患者进行根治性膀胱切除术和淋巴结切除术后同样发展为转移性疾病,降低膀胱癌患者的预后水平[4-7]。因此,阐明膀胱癌转移机制已成为改善膀胱癌患者预后的必然趋势。

MicroRNA(miRNA)是真核生物内源性的非编码小单链RNA,长度约为19~24个核苷酸[8]。研究显示miRNA通过降低mRNA的表达或者通过结合到目标mRNA的3UTR位点来调节编码蛋白的基因表达从而参与多种生命过程,包括细胞生长、增殖、分化和凋亡[9,10]。我们前期研究发现miRNA-149在膀胱癌中显著降低,miRNA-149抑制膀胱癌细胞迁移和侵袭,且抑制其下游靶基因Insulin-like growth factor binding protein 5(IGFBP5)和platelet—derived growth factor receptor alpha(PDGFRA)蛋白的表达。本研究将在前期研究的基础上,进一步探究miRNA-149是否通过靶向IGFBP5和(或)PDGFRA抑制膀胱癌细胞的迁移和侵袭,为临床膀胱癌的转移防治提供了新的靶点和理论依据。

1 材料与方法

1.1 材料与试剂

人膀胱癌细胞株T24由本实验室保存,293T细胞株来源于中科院细胞库。胎牛血清、DMEM/Ham's F-12(1:1 volume)培养基购于美国GIBCO公司,pMIR-REPORT Luciferase-IGFBP5 3UTR(Wt)(H10046);pMIR-REPORT Luciferase-PDGFRA 3UTR(Wt)(H10047);pLenti-CMV-MCS-3FLAG(H155)、pLenti-CMV-IGFBP5-3FLAG(H10495)、pLenti-CMV-PDGFRA-3FLAG(H10496)均购自上海和元生物技术有限公司;pRL-CMV(H321)购自美国Promega公司;lipofectamine 2000购自美国invitrogen公司;mimics NC、hsa-miRNA-149-5p mimics均购自上海吉玛制药技术有限公司,Transwell和invision小室购自美国BD公司。

1.2 细胞株的培养、转染和分组

实验用所有细胞株均使用含5%胎牛血清的DMEM/Hams F-12(1:1 volume)培养基,并置于37℃含体积分数5%的CO2培养箱中培养。将293T细胞按 70%的汇合度接种到96孔板,24 h后转染萤光素酶报告基因质粒和 RNA,每个样品设置6个复孔。并按照萤火虫萤光素酶载体(Firefly):海腎萤光素酶载体(Renilla):转染试剂=0.2 μg:0.004 μg:0.25 μL的比例,采用脂质体LipofectamineTM 2000的说明书要求对细胞进行转染。另一方面,使用慢病毒H155、H10495、H10496感染T24细胞,然后采用LipofectamineTM 2000 转染试剂,将miRNA-149 mimic及其对照转染至T24-H155,T24-H10495和T24-H10496细胞。

1.3 双荧光素酶报告基因实验

将IGFBP5-3UTR(Wt)(H10046);PDGFRA-3UTR(Wt)(H10047),双荧光素酶报告载体转染细胞后,放入细胞培养箱内24 h。提前将仪器开机并预热,此后步骤尽量冰上操作。将细胞移至室温,弃掉培养基,用PBS洗涤细胞1次。加入Lysis Buffer(1x PLB)100 μL,在室温轻缓晃动培养板15 min,把裂解液转移到检测试管中。瞬间离心,吸取上清 20 μL加入检测板。加入20 μL底物。测量 luciferase(firefly)。加入stop buffer与nenal底物混合物(50∶1)50 μL。立即再次测量,检测结束后,进行数据分析。

1.4 Transwell侵袭实验

将转染24 h后的膀胱癌T24细胞进行消化,用无血清的培养基重悬细胞,并取400 μL重悬液分别至普通小室和带基质胶的小室中,细胞数为104个,在下室中加入700 μL的完全培养基,继续培养24 h后,将小室取出,放入新的24孔中,小室上下同时用3.7%的甲醛固定2 min,PBS洗2次,再用100%甲醇通透20 min,PBS洗2次后,用吉姆萨室温避光染色15 min后,在显微镜下观察细胞迁移和侵袭的数目,并拍照。

1.5 评价指标

经慢病毒转染48 h后,采用荧光定量PCR检测miRNA-149是否过表达水平,采用WB检测PDGFRA和IGFBP5蛋白水平是否过表达。观察Transwell侵袭实验中,在显微镜下观察细胞迁移和侵袭的数目,检测PDGFRA和IGFBP5过表达后是否逆转miRNA-149过表达抑制膀胱癌侵袭的作用。

1.6 统计学方法

所有实验数据均以均数±标准差(x±s)表示,采用 SPSS18.0 统计软件进行分析,利用t检验方法检测两组之间的差异,P<0.05为差异有统计学意义。

2 结果

2.1 miRNA-149对PDGFRA和IGFBP5的3UTR的影响

本研究使用TargetScan在线软件预测miRNA-149的靶基因,发现PDGFRA和IGFBP5的3UTR区域分别包含一个保守的miRNA-149的靶位点,如封三图1A、封三图1B所示。接着通过双荧光素酶报告基因实验检测 miRNA-149是否影响PDGFRA和IGFBP5的3UTR活性。结果显示miRNA-149可抑制PDGFRA 和IGFBP5 3UTR活性,下调比例分别为 38.83%和30.51%,差异具有统计学意义(P<0.0001)。

2.2 IGFBP5对miRNA-149抑制膀胱癌细胞侵袭的影响

为进一步探究PDGFRA和IGFBP5对miRNA-149诱导的膀胱癌细胞侵袭抑制的影响,本研究单独或联合使用miRNA-149mimic和IGFBP5过表达载体转染T24细胞。转染后,T24-H155-miRNA-149-mimic组较 T24-H155-mimics-NC组侵袭能力降低,差异具有统计学意义(P<0.001);而T24-H10495-miRNA-149-mimic组较T24-H155-miRNA-149-mimic组侵袭能力明显增强(P<0.01)。IGFBP5过表达载体对miRNA-149抑制膀胱癌细胞侵袭产生逆转作用。

2.3 PDGFRA对miRNA-149抑制膀胱癌细胞侵袭的影响

另一方面,本研究同样单独或联合使用miRNA-149mimic和PDGFRA过表达载体转染T24细胞。同样的,转染后,T24-H155-miRNA-149-mimic组较 T24-H155-mimics-NC组侵袭能力降低,差异具有统计学意义(P<0.001);然而T24-H10496-miRNA-149-mimic组较T24-H155-miRNA-149-mimic组侵袭能力未发生明显变化(P>0.05)。PDGFRA过表达载体对miRNA-149抑制膀胱癌细胞侵袭未产生逆转作用。

3 讨论

越来越多的研究表明,miRNA作为致癌基因或抑癌基因在肿瘤发生发展过程中发挥重要作用[11-13]。miRNA在多种恶性肿瘤中异常表达,包括膀胱癌[14]。大量的功能性研究已表明膀胱癌中多种miRNA具有潜在的新的预后价值[15,16]。本研究前期miRNA-149在膀胱癌中表达降低,过表达miRNA-149可显著抑制膀胱癌细胞侵袭,且miRNA-149下调其潜在的靶基因PDGFRA和IGFBP5蛋白水平的表达。以往研究表明,miRNA-149在不同类型的肿瘤(包括结肠癌、骨髓性白血病、肝细胞癌和肾细胞癌)进展中发挥不同的功能[17-20]。miRNA-149异位表达可通过靶向肌动调节蛋白PPM1F抑制肝细胞癌的转移;miRNA-149可通过直接靶向FOXM1从而显著抑制结肠癌细胞的迁移和侵袭[21,22]。因此本研究在前期研究的基础上,进一步明确miRNA-149是否通过靶向PDGFRA和IGFBP5抑制膀胱癌细胞侵袭。我们的结果表明miRNA-149可显著抑制PDGFRA和IGFBP5的3UTR活性,miRNA-149可能靶向作用于IGFBP5和PDGFRA。

血小板源性生长因子受体A(PDGFRA),是一种细胞表面酪氨酸激酶受体。与其他许多受体酪氨酸激酶(RTKs)一样,PDGFRA通过表达升高或由于突变和染色体重排导致的活性增加从而参与各种肿瘤的进展,如白血病、胃肠道间质瘤(GISTs)、胶质母细胞瘤和肝细胞癌等[23-25]。研究显示,PDGFRA在炎性乳腺癌中过度表达且具有独特的活性,可能是IBC治疗的理想靶点[26]。活化的PDGFRA通过激活AKT通路刺激纤维原细胞增殖,并诱导心肌成纤维细胞激活,可作为心肌纤维化治疗的潜在靶点[27]。此外,PDGFRA参与间充质细胞增殖和雄性特异性中肾细胞迁移[28]。本研究在前期发现miRNA-149降低PDGFRA蛋白表达的基础上,进一步探究miRNA-149是否通过靶向作用于PDGFRA抑制膀胱癌细胞侵袭。结果显示miRNA-149抑制PDGFRA 3UTR的活性,然而PDGFRA过表达未能逆转miRNA-149抑制膀胱癌细胞侵袭的作用。因此miRNA-149抑制膀胱癌细胞侵袭可能与PDGFRA没有相关作用。

另一方面,胰岛素样生长因子结合蛋白5(IGFBP5)是IGFBPs同源蛋白家族之一,且在人类肿瘤和转移组织中异常表达[29]。研究表明IGFBP5在肿瘤发生和发展过程中具有多种生物学功能,调控细胞的存活、生长、迁移和侵袭[29]。IGFBP5通过IGF依赖和不依赖的机制调节细胞功能,可抑制各种组织或环境下的肿瘤生长和转移,也可促进肿瘤转移从而作为致癌基因发挥作用[30]。其中,IGFBP5在肿瘤中表达升高,被认为具有促进转移的能力;外源性IGFBP5表达已被证实对神经酰胺诱导的细胞凋亡具有保护作用;且IGFBP5的过表达被发现与乳腺癌患者预后不良有关[31-33]。而最近发表的一项研究表明IGFBP5在尿路上皮癌進展中发挥重要作用并且IGFBP5过表达与肿瘤晚期、频繁有丝分裂和较差的临床预后紧密相关[34]。此外,IGFBP5不同的结构域对人类不同肿瘤的致瘤性和转移产生不同的影响[35]。然而未见IGFBP5在膀胱癌转移中的报道。本研究在前期工作的基础上进一步探讨了miRNA-149是否通过靶向作用于IGFBP5抑制膀胱癌细胞的侵袭。结果显示miRNA-149抑制IGFBP5 3UTR的活性,并且IGFBP5过表达明显逆转miRNA-149抑制膀胱癌细胞侵袭的作用。因此该结果表明miRNA-149通过靶向作用于IGFBP5抑制膀胱癌细胞侵袭,为临床膀胱癌转移防治提供了新的靶点和理论依据。

综上所述,miRNA-149通过靶向IGFBP5基因抑制膀胱癌细胞侵袭,具有重要的临床意义。然而miRNA-149是否在体内同样通过该调控机制抑制膀胱癌转移尚需进一步的探究。

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(收稿日期:2018-08-03)

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