CN102965439B - miR-296-3p在制备前列腺癌发生或转移诊断试剂盒中的应用 - Google Patents
miR-296-3p在制备前列腺癌发生或转移诊断试剂盒中的应用 Download PDFInfo
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Abstract
本发明公开了miR-296-3p在制备肿瘤发生或转移诊断试剂盒中的应用。研究发现,miR-296-3p通过作用于其靶基因CDH1,使其转录受抑制或降解,使E-钙粘蛋白表达下降,从而促进前列腺癌细胞迁移侵袭。过表达miR-296-3p后,E-钙粘蛋白的表达水平降低,肿瘤细胞间的粘附减弱,迁移侵袭能力增强。因此,检测miRNA-296-3p和E-钙粘蛋白的表达,可以对前列腺癌转移进行诊断和病理分级,对其转移和预后做出评价。围绕miR-296-3p靶向E-钙粘蛋白的特点可以设计肿瘤转移干预策略,提出了anti-miR-296-3p在制备抑制前列腺癌侵袭转移药物中的应用。本发明在医学和生物制药领域有重大实际意义和巨大的潜在应用价值。
Description
技术领域
本发明涉及生物医学材料技术领域,本发明公开了一种小分子非编码RNA的用途,尤其涉及一种小分子非编码RNA即miR-296-3p在制备肿瘤发生或转移诊断试剂盒中的应用。
背景技术
前列腺癌(prostate cancer,Pca)是发生于男性前列腺组织中的恶性肿瘤,发病随年龄而增长,其发病隐匿,生长缓慢,病因复杂,给临床的早期诊断带来很大的障碍。前列腺癌容易发生淋巴结转移和骨转移,是引起前列腺癌患者死亡的主要原因,也一直是基础和临床研究的热点。前列腺癌的常用治疗手段主要有内分泌治疗、手术治疗,化疗和放疗。比较新的免疫治疗和基因治疗也有巨大应用潜力。近年来不断有文献报道microRNA在肿瘤诊断和治疗中的重要作用,前列腺癌特异性miRNA的发现为其诊断和治疗打开了一个全新的局面。microRNA是一类内源性小分子非编码RNAs,结构高度保守,具有表达时序性和组织特异性,通过与靶基因3’非编码区(3’UTR)互补序列特异性结合,导致翻译抑制或者mRNA降解,从而调节基因表达[1]。microRNA在恶性肿瘤中表达谱各异并发挥着重要的调控作用,随着大规模、高通量miRNA检测技术的推广应用,发现新的miRNA不仅为肿瘤的诊断和预后提供了颇具价值的标准,同时也为寻找新的肿瘤生物治疗靶标奠定基础[2]。
研究发现前列腺癌组织中存在miRNA表达谱差异,它们的表达水平和肿瘤细胞的分化程度密切相关,而常规的mRNA标志物不具备此特点。Kati PP,Minja JP等[3]检测了前列腺组织和细胞中319种miRNA的表达谱,发现位于染色体缺失区域的miRNA低表达,而位于扩增区的miRNA高表达,它们通过靶向一些肿瘤相关基因如RAS、BCL2等最终影响前列腺肿瘤细胞或正常细胞的生长分化。Annika Schaefer,Monika Jung等[4]采用微阵列技术和实时荧光定量PCR分析了155例前列腺癌病人组织中miRNA的表达谱,结合临床病例数据,通过ROC分析发现了可以作为诊断标志物的miR-205;而在Kaplan-Meier分析和Cox比例风险回归分析之后,发现了可以作为预后诊断标志物的miR-96。首次报道与临床前列腺癌疗效和转移相关的单个miRNA是miR-221[5]。Martin Spahn,Susanne Kneitz等在分析了不同类型的临床标本之后发现:98%的前列腺癌组织中都伴随有miR-221的特征性表达下调。根据Gleason评分或肿瘤分期和复发率,miR-221表达的进行性降低和高危肿瘤以及不良预后密切相关。而且miR-221的表达下调是前列腺癌特异性的,属于转移过程中的频发事件。进一步的研究表明其可能的作用机制与c-kit致癌基因有关。基于前列腺癌中miR表达谱差异的一系列研究为后续的功能分析提供了丰富的证据,也为临床诊断和预后评价以及寻找新的高危前列腺癌治疗靶标开辟思路。
P69和M12属于前列腺癌同源细胞系,P69来源于SV40T永生化的人非致瘤性前列腺上皮细胞[6]。随后通过裸鼠皮下成瘤实验和在体筛选,得到了具有局部侵袭和远端转移能力的M12细胞。二者的遗传背景一致,但是致瘤能力和转移潜能差异很大,是研究前列腺癌转移机制的理想模型。
有关miR-296的研究报道还比较少,Thomas等报道miR-296能够直接调控HGS mRNA有效促进肿瘤血管发生[7];miR-296能够下调p21WAF1表达,改变p53-p21WAF1通路从而促进肿瘤发生[8]。现有的文献所报道的miR-296多为miR-296-5p,对miR-296-3p的研究目前仅有6篇报道[9-14],其中一篇主要针对胶质细胞瘤的药物抵抗性[9],其他研究主要集中在miR-296-3p与其它miRNA共同调节某个或某些基因的表达[10],鲜有miR-296-3p对于肿瘤生长转移相关功能的研究。我们的研究聚焦在miR-296-3p对于前列腺癌发生与转移,对于肿瘤发生和转移的治疗具有重大意义。
我们以非转移性前列腺癌细胞系P69和高转移性前列腺癌细胞系M12两个细胞系为主要研究模型进行研究。
参考文献
1.Esquela-Kerscher A,Slack FJ.Oncomirs-microRNAs with a role in cancer[J].Nat Rev Cancer,2006,6(4):259-269。
2.Lu J,Getz G.Microrna expression profiles classify human cancers[J].Nature,2005,435:834-838。
3.Porkka KP,Pfeiffer MJ,Waltering KK,et al.Microrna expression profilingin prostate cancer[J].Cancer Res,2007,67(13):6130-5。
4.Schaefer A,Jung M,Mollenkopf HJ,et al.Diagnostic and prognosticimplications of microrna profiling in prostate carcinoma[J].Int J Cancer,2010,126(5):1166-76。
5.Spahn M,Kneitz S,Scholz CJ,et al.Expression of microrna-221 isprogressively reduced in aggressive prostate cancer and metastasis and predictsclinical recurrence[J].Int J Cancer,2010,127(2):394-403。
6.Bae VL,Jackson-Cook CK,Maygarden SJ,et al.Metastatic sublines of ansv40 large t antigen immortalized human prostate epithelial cell line[J].Prostate,1998,34(4):275-82。
7.ThomasWu¨rdinger,BakhosA.Tannous,OkaySaydam,et al.miR-296regulates growth factor receptor overexpressionin angiogenic endothelial cells[J].Cancer Cell;2008,14:382–393。
8.A-rumYoon,RanGao,ZeeniaKaul et al MicroRNA-296 is enriched in cancercells and downregulates p21WAF1 mRNA expression via interaction with its 3’untranslatedregion[J].Nucleic Acids Research,2011,1-14。
9.MiR-296-3p regulates cell growth and multi-drug resistance of humanglioblastoma by targeting ether-a`-go-go(EAG1)[J].European Journal of Cancer(In press)。
10.A TSH-CREB1-microRNA loop is required for thyroid cell growth[J].Molecular Endocrinology 2011,25(10):1819-1830。
11.MicroRNA signatures associated with immortalization of EBV-transformedlymphoblastoid cell lines and their clinical traits[J].Cell Proliferation 2011,44,59-66。
12.Differential genomic imprinting and expression of imprinted microRNAsin testes-derived male germ-line stem cells in mouse[J].Plos One 2011,6(7)e22481。
13.Expression profile of microRNAs and mRNAs in human placentas frompregnancies complicated by preeclampsia and preterm labor[J].ReproductiveSciences 2011,18(1)46-56。
14.Improving murine embryonic stem cell differentiation into cardiomyocyteswith neuregulin-1:differential expression of microRNA[J].American Journal ofPhysiology Cell Physiology 2011 7;301(1):C21–C30。
发明内容
本发明所要解决的技术问题是,提供一种与前列腺癌转移相关的小分子非编码miR-296-3p及其反义寡核苷酸的用途。
为了解决上述问题,本发明提供了miR-296-3p在制备前列腺癌发生或转移诊断试剂盒中的应用。
本发明亦提供了anti-miR-296-3p在制备抑制前列腺癌侵袭转移药物中的应用。
miR-296-3p能直接抑制E-钙粘蛋白表达,其反义寡核苷酸anti-miR-296-3p能有效抑制前列腺癌细胞侵袭和转移。所述的miRNA及其反义寡核苷酸序列通过化学合成获得或通过构建真核细胞表达载体进行表达获得。所述的miRNA及其反义寡核苷酸序列为核糖核苷酸、脱氧核糖核苷酸或二者的混合,这些序列可以经过任意修饰,如3’或5’端连接DIG或biotin等。
本发明的优点在于,本发明采用全基因组深度测序研究发现,与非转移性前列腺癌细胞系P69相比,高转移性前列腺癌细胞系M12中miR-296-3p高表达而E-钙粘蛋白低表达,Western blot和real-time PCR等方法也验证了上述结果。miR-296-3p通过作用于其靶基因E-钙粘蛋白使其降解从而促进前列腺癌细胞迁移侵袭。过表达miR-296-3p后,E-钙粘蛋白的表达水平降低,肿瘤细胞间的粘附减弱,迁移侵袭能力增强。因此,检测miRNA-296-3p和E-钙粘蛋白的表达,可以对前列腺癌转移进行诊断和病理分级,对其转移和预后做出评价,提出了miR-296-3p在制备前列腺癌发生或转移诊断试剂盒中的应用。围绕miR-296-3p靶向E-钙粘蛋白的特点可以设计肿瘤转移干预策略,提出了anti-miR-296-3p在制备抑制前列腺癌侵袭转移药物中的应用。本发明在医学和生物制药领域有重大实际意义和巨大的潜在应用价值。
附图说明
图1为前列腺癌细胞系P69和M12的迁移能力和形态学差异比较:
A为罗氏xCELLigence系统实时连续监测活细胞迁移能力时P69和M12的动力学曲线(P69-红,M12-绿);
B为光镜下3D培养基质中P69和M12的形态;
C为激光扫描共聚焦显微镜观察3D培养基质中P69和M12的形态(红-Vimentin,绿-E-cadherin,蓝-DAPI);
D为P69和M12中miR-296-3p和E-钙粘蛋白的数字基因测序结果;
E为计算机序列比对分析显示miR-296-3p与E-钙粘蛋白3’UTR区的结合;
F为实时荧光定量PCR检测P69和M12中miR-296-3p的水平;
G蛋白免疫印迹检测P69和M12中E-钙粘蛋白的表达水平(actin为内参)。
图2为miR-296-3p直接靶向E-钙粘蛋白:
A为实时荧光定量PCR验证稳定转染细胞系中miR-296-3p是否过表达;
B为实时荧光定量PCR检测miR-296-3p过表达之后E-钙粘蛋白编码基因CDH1 mRNA水平的变化;
C为蛋白免疫印迹分析miR-296-3p过表达之后E-钙粘蛋白表达水平的变化;
D为流式细胞术测定细胞系P69-Ctrl和P69-miR-296-3p中E-钙粘蛋白的表达水平;
E为琼脂糖凝胶分析M12细胞中miR-296-3p表达下调之后E-钙粘蛋白编码基因CDH1 mRNA水平的变化;
F实时荧光定量PCR分析M12细胞中miR-296-3p表达下调之后E-钙粘蛋白编码基因CDH1 mRNA水平的变化;
G E-钙粘蛋白3’UTR结构示意图(红色代表miR-296-3p可能的结合位点);
H双荧光素报告基因分析Hela细胞中转入miR-296-3p后荧光素酶活性的变化;
图3miR-296-3p通过下调E-钙粘蛋白促进前列腺癌细胞侵袭迁移:
A、B为RT-CA实时监测miR-296-3p过表达及E-钙粘蛋白回复之后P69细胞迁移侵袭能力的变化;
C:RT-CA实时监测miR-296-3p过表达之后P69细胞迁移侵袭能力的变化。
图4为anti-miR-296-3p抑制了高转移性肿瘤细胞的体内外迁移侵袭活性和克隆形成能力:
A为RT-CA实时监测miR-296-3p表达下调之后M12细胞迁移能力的变化;
B为过表达E-钙粘蛋白后RT-CA实时监测M12细胞迁移能力的变化;
C为短时(0h,48h)生物发光成像分析miR-296-3p表达下调之后高转移性细胞系M12转移能力的变化;
D为长时(4周)生物发光成像分析miR-296-3p表达下调之后高转移性细胞系M12转移能力的变化;
E裸鼠肺部肿瘤转移灶HE染色分析;
F软琼脂克隆形成实验检测miR-296-3p表达下调之后M12锚定非依赖性生长能力的变化。培养2周后克隆大小的变化;
G结晶紫染色之后定量分析克隆数目的变化并进行统计分析。
具体实施方式
下面结合具体实施例,进一步阐述本发明。下述实施例中所使用的实验方法如无特殊说明,均为常规方法。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件,例如Sambrook等人,分子克隆:实验室手册(New York:Cold SpringHarbor Laboratory Press,1989)中所述的条件,或按照制造厂商所建议的条件。
实施例1.P69和M12的迁移能力和形态学差异及miR-296-3p在两种细胞中的差异性表达
1.细胞迁移实验(Migration)
(1)组装CIM-16:lower chamber分两组,对照组每孔加入160ul SFM(serumfree medium),血清诱导组每孔加入160ul全培基(5%FBS);upper chamber每孔加入30-50ul SFM;
(2)平衡:CIM-16放入37°C培养箱平衡1h;
(3)检测背景:RT-CADP Analyzer检测CIM-16的基础值;
(4)准备细胞:细胞饥饿6-8h后,0.05%Typsin消化,细胞计数,调整密度为4×105/ml,备用;
(5)种板:upper chamber每孔加入100ul单细胞悬液,室温平衡30min;
(6)测定:CIM-16放入RT-CA DP Analyzer,启动实时测定程序,15min间隔一次,24h后停止;
(7)数据分析:RT-CA分析细胞指数CI(cell index),细胞阳性迁移结果的标准是CI≥0.2。
如图1-A所示,M12细胞的实时迁移曲线高于P69细胞,P69细胞处于基线,迁移不明显。该结果说明,M12细胞具有高迁移的特性,P69细胞具有低迁移特性。
2.三维细胞培养(3 dimensional cell culture,3D culture)
(1)解冻3D基质胶,4°C置冰盒内过夜;
(2)冰上操作,48孔板每孔加入250ul基质胶,37°C孵育30min促进胶凝;同时配制2%Assay Media(2ml matrix+98ml medium),4°C保存一周或-20°C长期保存;
(3)37°C温育Assay Media作为细胞稀释液;
(4)单层贴壁细胞消化后用24ml Assay Media重悬制备单细胞悬液,细胞计数并调整密度至1×104/ml,48孔板每孔加入500ul细胞悬液;
(5)倒置显微镜下每天观察细胞生长状况和结构变化,每隔4天换液一次;
(6)根据不同细胞系的特点,当细胞形态生长至预期的程度时,结束培养,准备后续的细胞形态和结构分析。
(7)结果分析:倒置荧光显微镜拍照保存。
如图1B\C,分别为光镜下和荧光显微镜下3D培养P69和M12细胞形态,彩色图片为荧光免疫组织化学染色。(红-Vimentin,绿-E-cadherin,蓝-DAPI)。该结果说明,M12细胞较P69细胞有更强的侵袭能力,3D培养P69细胞呈圆团状生长,而M12细胞则呈散乱的星形出芽生长,向周围迁移,具有明显的局部侵袭特性。
3.总RNA提取和实时荧光定量PCR
3.1 细胞总RNA的提取
(1)Trizol裂解细胞:1ml/Φ10皿,室温静置5min;
(2)裂解液转移至1.5mlEP管,每毫升加入200μl氯仿,用力振摇15s,室温静置2-3min,然后12000g 4°C离心15min;
(3)小心吸取上层水相(含RNA)至新的1.5ml EP管,加入500μl的异丙醇,颠倒混匀,室温静置10min,12000g 4°C离心15min;
(4)去上清,75%乙醇(RNase free水配制)洗涤沉淀,手轻弹管底使白色沉淀漂浮,重复洗涤一次,8000g 4°C离心10min;
(5)去上清,吸水纸上扣干EP管,通风橱中干燥数分钟;
(6)RNA沉淀溶于20-100μl水中,充分溶解,NanoDrop2000测定浓度,并稀释至相同浓度(1μg/μl)。
3.2 逆转录反应
(1)处理基因组DNA:10μl的处理体系。
RNA模板(1μg/μl)、10×Reaction Buffer with MgCl2、DNase Ⅰ各1μl,RNasefree水补至10μl,混匀后,37°C 30min;然后加入1μl 50mM EDTA,65°C 10min,灭活残余的DNase Ⅰ。
(2)cDNA合成:Fermentas体系
表1
上述20μl反应体系,混匀后离心,若使用Oligo(dT)18primer or Gene-specificprimer,程序设置为50°C 60min,85°C 5min;若使用Random hexamer primer,程序设置为25°C 10min,50°C 60min,85°C 5min。
3.3 实时荧光定量PCR
(1)反应体系按如下表格配制
表2
(2)根据AB公司StepOne PCR扩增仪操作说明设置程序参数,具体条件如下:
Holding Stage:95°C 10min;Cycling Stage:95°C 15s,60°C 30s,72°C 30s(40cycle);Melt Curve Stage:95°C 15s,60°C 1min,95°C 15s。
3.4 数据处理
荧光定量PCR定量检测所得miRNA的δCT值,采用Excel 2003数据分析工具,当相对标准误(STDEV)<0.5,认为结果可靠。如图1F,为实时荧光定量PCR结果,显示P69和M12细胞中miR-296-3p的表达水平差异,M12细胞较P69细胞高1.5倍。
4 免疫印迹分析
4.1 细胞总蛋白的抽提
(1)取对数生长期贴壁细胞,70%生长密度,移除旧的培养液,使用1×PBS洗涤2次;
(2)每6cm培养皿细胞加SDS裂解缓冲液(62.5mM Tris,2%SDS,pH=6.8)50ul,室温静置2-3min,细胞刮刮下,转移至1.5ml EP管,100°C水浴10min;
(3)超声波细胞粉碎机处理细胞裂解液,20kHz,30s×3;
(4)13000rpm,室温离心15min,转移上清至新的1.5ml EP管;
(5)NanoDrop2000测定蛋白浓度,立即上样或将样品保存于-20°C备用。
4.2 蛋白免疫印迹
(1)制胶
①将1.5mm厚的制胶用的玻板洗净后,晾干备用;
②将玻璃板对齐后放入夹中卡紧,卡在架子上准备灌胶(操作时要使两玻璃对齐以免漏胶);
③按下表配置12%和8%的分离胶及5%的浓缩胶;
表3 分离胶及浓缩胶配方
APS(10%)现配,加TEMED后立即混匀灌胶。
④分离胶加至距离梳齿下1cm的位置,异丙醇压线,待分离胶凝固后,倾倒掉异丙醇,再用蒸馏水清洗干净,控干水分,加入浓缩胶至短玻板顶端,迅速插入梳子,避免气泡的产生,室温下聚合约30min。
(2)SDS-PAGE电泳
①将夹子松开取出玻璃板放入电泳槽(小玻璃板面向内,大玻璃板向外;若只跑一块胶,另一边需垫塑料板)。向电泳槽注入1×电泳液,外槽亦加四分之一,拔梳子;
②根据细胞总蛋白的浓度计算蛋白的溶液体积:
样品体积(μl)=蛋白质量(μg)/蛋白浓度(μg/μl),然后再加入四分之一体积的5×上样缓冲液;
③混匀后的样品沸水浴加热5min,迅速冰上冷却,13000rpm,室温离心2min。蛋白Marker 10μl,处理方法同蛋白样品;
④上样:1.5mm板每孔上样总体积不超过40μl
按设定顺序每孔加入已经制备好的蛋白样品35μl-40μl,同时加入10μl的蛋白预染Marker。不同的样品要每次换枪头,防止交叉感染;
⑤电泳:起始70V,30min;待样品浓缩成一条线之后,改为120V,60min,根据蛋白大小和溴酚蓝的位置确定终止时间(电泳时可配制电转液)。
(3)转膜:湿法转膜
①PVDF膜(标记正反)在纯甲醇中浸泡饱和3-5s,转入电转液中,准备电转液浸泡海绵垫、滤纸;
②取下玻璃板,小心切去浓缩胶,在电转液中轻轻取下分离胶,将胶放到已铺放好的湿润滤纸上;
③夹板黑面向下,向上按层次叠放海绵、滤纸、胶、膜、滤纸、海绵。用试管擀去气泡,整个操作在电转液中进行,膜两边滤纸不得互相接触,以免短路;
④夹子放入转移槽,夹的黑面对槽的黑面,夹的白面对槽的红面(短白夹向上),槽的旁边放一块冰,转膜缓冲液要没过电转槽上的金属丝;
⑤冰上电转,260mA恒流90min或60mA稳压过夜;
⑥转膜结束后,可用1×丽春红染膜5min,然后流水冲掉参与的染液观察膜上的蛋白,或者直接进行下一步骤。
(4)免疫沉淀
①取出膜,迅速进入TBST漂洗3次,每次10min或是4次,每次5分钟;
②5%脱脂奶粉室温封闭1小时,若抗体较弱可用2%BSA封闭;
③孵一抗:一抗以2%BSA稀释,室温孵育2小时或4℃孵育过夜(第二天室温孵育30min);
④洗膜:TBST漂洗3次,每次10min;
⑤孵二抗:二抗以5%脱脂牛奶按说明书提供的参考比列稀释,室温孵育1.5h;
⑥洗膜:TBST漂洗4-5次,每次10min,充分除去非特异结合的抗体。
(5)化学发光成像
①先打开LAS4000mini化学发光成像仪,预冷至-25°C;
②打开程序,设置相关的参数,然后用镊子夹取膜于托盘上,放在预设的样品通道,调节色阶和对比度;
③采用Increment-Standard-Chemiluminescence模式扫描目的蛋白,根据目的蛋白的表达情况设置曝光的时间;切换Precision-Standard-Digital模式扫描蛋白Marker;
④16位.GIF图片格式保存所有的结果,关掉CCD程序和仪器。
(6)洗脱
如果要在同一张PVDF上对另一目的蛋白进行检测,可以对已曝光的PVDF膜进行洗脱。量取适量的洗脱缓冲液,室温中速震摇10min后,重新孵育一抗、二抗,化学发光。
(7)Adobe Photoshop CS4处理分析成像结果。
如图1G所示,E-钙粘蛋白在P69细胞中高表达,而在M12细胞中低表达,与miR-296-3p的表达模式相反。
5 慢病毒载体构建:化学合成上下游引物序列。95℃2分钟后,缓慢恢复至室温,退火后的产物通过不同的酶切位点插入到pGreenPuroTM shRNACloning and Expression Lentivector载体的多克隆位点,构建成pGreenpuro系列质粒。序列如下:
pGreenpuro-miR-296-3p上游引物序列:
5’-GATCCGGAGGGTTGGGTGGAGGCTCTCCCTTCCTGTCAGAGGAGAGCCTCGTGCCAACCCTCCTTTTTG-3’,如SEQ ID NO:1所示;
下游引物序列:
5’-AATTCAAAAAGGAGGGTTGGCACGAGGCTCTCCTCTGACAGGAAGGGAGAGCCTCCACCCAACCCTCCG-3’,如SEQ ID NO:2所示;
pGreenpuro-anti-miR-296-3p上游引物序列:
5’-GATCCGGAGGGTTGGCACGAGGCTCTCCCTTCCTGTCAGAGGAGAGCCTCCACCCAACCCTCCTTTTTG-3’,如SEQ ID NO:3所示;
下游引物序列:
5’-AATTCAAAAAGGAGGGTTGGGTGGAGGCTCTCCTCTGACAGGAAGGGAGAGCCTCGTGCCAACCCTCCG-3’,如SEQ ID NO:4所示。
6.pmiR-report CDH1-3’UTR载体构建:PCR扩增CDH1 3’UTR片段,扩增的片段通过speI和pme I克隆到pmiR-report载体上,形成pmiR-reportCDH1-3’UTR载体。序列如下:
CDH1-3’UTR上游引物序列:
5’-GGACTAGTGGGACTCGAGAGAGGCGG-3’,如SEQ ID NO:5所示;
下游引物序列:5’-GGGTTTAAACATGGTTTAACAAAATTGTT-3’,如SEQ IDNO:6所示。
实施例2.miR-296-3p直接靶向E-钙粘蛋白
1.稳定转染细胞系的构建
P69或者M12细胞种于6cm的皿中,待生长至50%-60%的汇合度时,分别用含miR-296-3p和anti-miR-296-3p的病毒上清感染细胞,所用的试剂为Lipofectamine2000(Invitrogen)和Optimal-MEM(Gibco),参照常规方法并按照说明书进行转染。24h后换液并在荧光显微镜下观察绿色荧光蛋白的表达情况以确定细胞的转染效率。48h后进行嘌呤霉素筛选。转染所用的核苷酸序列如实施例1.5和1.6所述。其中:
miR-296-3p的序列为:gagggttgggtggaggctctcc,如SEQ ID NO:7所示;
anti-miR-296-3p的序列为:ggagagcctccacccaaccctc,如SEQ ID NO:8所示。
2.生物信息学分析筛选靶基因
利用miRanda工具预测miR-296-3p的靶基因,同时查找E-钙粘蛋白编码基因(CDH1)3’-UTR潜在的miRNA结合位点,并依据基因功能进行筛选。
如图2G,miR-296-3p和CDH1的3’UTR区有很好的匹配,提示CDH1可能是miR-296-3p的靶基因。
3.总RNA提取和实时荧光定量PCR
实施方法同实施例1中3的做法。所用引物序列如下:
miR-296-3p real-time PCR茎环引物:
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGGAGAG,如SEQ ID NO:9所示。
miR-296-3p real-time PCR正向引物:CAATCGAGGGTTGGGTGGAG,如SEQ ID NO:10所示。
CDH1 real-time PCR正向引物:ATTTCTGAAAGCGGCTGATACTGAC,如SEQ ID NO:11所示。
CDH1 real-time PCR反向引物:GCCCCATTCGTTCAAGTAGTCATAG,如SEQ ID NO:12所示。
如图2A为P69细胞分别稳转Ctrl与miR-296-3p后,miR-296-3p的表达水平,显示稳转miR-296-3p后,P69-miR-296-3p细胞中miR-296-3p的表达水平显著高于P69-Ctrl细胞,细胞系构建成功。
图2B为P69-Ctrl与P69-miR-296-3p的E-钙粘蛋白编码基因CDH1 mRNA表达水平,过表达miR-296-3p后,CDH1 mRNA表达水平下调约0.5倍。
以上结果说明,P69-miR-296-3p细胞稳定过表达miR-296-3p,且能够降低CDH1 mRNA表达水平。
图2F M12细胞中miR-296-3p表达下调之后E-钙粘蛋白编码基因CDH1mRNA有所回升。
4.蛋白免疫印迹
实施方法同实施例1中4的做法。
如图2C,显示P69-miR-296-3p细胞稳定过表达miR-296-3p后能够降低E-钙粘蛋白的表达水平。
5.流式细胞术
(1)取对数生长期细胞,用0.05%胰蛋白酶消化,离心后去上清;
(2)配制Staining Buffer(1%FBS+99%PBS),重悬细胞沉淀制备单细胞悬液;
(3)1000rpm离心5min,去上清后每只流式管加Staining Buffer 50μl,然后加入流式抗体CD-3245μl,IgG为阴性对照;
(4)4°C冰箱避光孵育30min;
(5)流式细胞仪测定,筛选阳性细胞簇;
(6)数据分析。
如图2D,显示P69-miR-296-3p细胞稳定过表达miR-296-3p后能够降低E-钙粘蛋白的表达水平。与前面的免疫印迹结果互相印证。
6.琼脂糖凝胶电泳
配制2%琼脂糖凝胶,1×TAE缓冲液,电压120V,电泳25min。
如图2E,显示M12-anti-miR-296-3p细胞稳定下调miR-296-3p后能够降低CDH1基因的mRNA水平。
7.pmiR-report CDH1-3’UTR-mut载体构建:
原理同实施例1.6,所用引物序列如下
CDH1 3’UTR正向突变引物:AAATGTTGGGAGCAATCACTTTTTGGA,如SEQ ID NO:13所示。
CDH1 3’UTR反向突变引物:TTTTCTCCAAAACACTTGAGAAACAGT,如SEQ ID NO:14所示。
8.双荧光素酶报告基因分析
(1)配制细胞裂解液:取适量5×PLB,双蒸水稀释至1×,混匀备用。
(2)清洗细胞:去培养基,1×PBS清洗1-2次;
(3)细胞裂解:24孔板每孔加入150ul 1×PLB,混匀后3D摇床振摇,室温裂解15min,转移裂解液至1.5ml EP管中备用;
(4)配制发光反应液:测定前,分别使用Stop&Glo Buffer和LuciferaseAssay Buffer Ⅱ溶解Stop&Glo(液体)和Luciferase Assay Substrate(冻干粉),-80°C避光保存;
(5)BMG Novo Star化学发光测定仪设置:测定延迟设为2秒,Stop&Glo和Luciferase Assay Substrate注入体积设为100ul,读板顺序设为从左向右;
(6)自动发光测定:配制好的Stop&Glo和Luciferase Assay Substrate置于测定仪内并连接好对应管道,待测样品分别取20ul于酶标板中,启动自动测量程序,记录数据;
(7)数据分析:加入Luciferase Assay Substrate后所测的值为A,加入Stop&Glo后所测的值为B,所得比值C=A/B,计算组内均值,并至少重复3次实验,取平均值。
如图2H,当CDH1基因3’UTR转入带有荧光素酶基因的载体后,加入miR-296-3p可以抑制荧光素酶的活性,而当CDH1基因3’UTR与miR-296-3p的结合位点突变以后,荧光素酶的活性不再被抑制。说明CDH1是miR-296-3p的靶基因。
以上实验为图2的具体实施过程。
实施例3.miR-296-3p通过抑制E-钙粘蛋白的表达促进转移过程
1.细胞迁移实验(Migration)
实施方法同实施例1中1的做法。
如图3A,转入miR-296-3p后,24h即出现明显的迁移现象,迁移曲线CI值明显高于对照细胞,说明miR-296-3p能够促进细胞迁移。
如图3B,P69细胞内同时转入miR-296-3p与E-钙粘蛋白后,miR-296-3p对细胞迁移的促进作用被抑制,结合图2C,可以得到结论,miR-296-3p对细胞迁移的促进作用是通过抑制E-钙粘蛋白的表达实现的。
2.细胞侵袭实验(Invasion)
(1)铺胶:4℃冰盒内过夜融化Matrigel,可以用冷的SFM以1:20的比例稀释Matrigel,吸取50l稀释后的Matrigel加入CIM-16 upper chamber,待均匀铺展后,迅速吸出30l,每4个孔为一组,重复操作。全部孔加入Matrigel后,放入37℃培养箱孵育4h,使胶凝固。
(2)后续的操作同细胞迁移(Migration)
如图3C,P69细胞转入miR-296-3p后,其侵袭曲线在17h达到的数值,对照细胞在25h才达到,充分说明,miR-296-3p促进了肿瘤细胞的侵袭,说明miR-296-3p可以应用于制备前列腺癌发生或转移诊断试剂盒。
实施例4 沉默miR-296-3p后抑制肿瘤转移和发生
1.E-钙粘蛋白过表达稳定转染细胞的建立:
M12细胞种于6cm的皿中,待生长至50%-60%的汇合度时,分别用含E-钙粘蛋白质粒的病毒上清感染细胞,所用的试剂为Lipofectamine2000(Invitrogen)和Optimal-MEM(Gibco),参照常规方法并按照说明书进行转染。24h后换液并在荧光显微镜下观察绿色荧光蛋白的表达情况以确定细胞的转染效率。48h后进行嘌呤霉素筛选。克隆质粒所用引物序列如下:
CDH1正向引物:ATGGGCCCTTGGAGCCGCAGCCTCTCG,如SEQ IDNO:15所示;
CDH1反向引物:CTAGTCGTCCTCGCCGCCTCCGTACAT,如SEQ IDNO:16所示。
2.细胞迁移实验(Migration)
实施方法同实施例1中1的做法。
如图4A,M12细胞沉默miR-296-3p后,抑制了肿瘤细胞的转移。
如图4B,M12细胞转入E-钙粘蛋白后,同样抑制了肿瘤细胞的转移,同图4A效果相似。
3.裸鼠移植瘤模型
(1)细胞培养:细胞带有萤火虫荧光素酶基因;
(2)单层贴壁细胞消化,活细胞计数,离心去血清;
(3)无血清培养基重悬,制备单细胞悬液,置冰上;
(4)10只免疫缺陷小鼠分两组,尾静脉注射肿瘤细胞,一组注射M12-Ctrl,一组注射M12-anti-miR-296-3p;
(5)成像:小动物活体成像,检测肿瘤细胞裸鼠体内转移的情况。
如图4C,M12-anti-miR-296-3p组动物肺部48h的生物发光信号明显降低。而对照组动物肺部的生物发光信号却持续存在。这首先说明注射到裸鼠体内的肿瘤细胞短期内存活下来,其次miR-296-3p可以促进肿瘤细胞逸出肺部的血管,向远端特异性的组织器官转移。
如图4D,4周时,注射M12-anti-miR-296-3p细胞的动物体内生物荧光信号消失,而注射M12-Ctrl细胞的动物,在腹部可以看到明显的荧光信号,这说明,miR-296-3p有利于肿瘤细胞在继发灶的定植。
4.苏木精-伊红染色(HE染色)
4.1 石蜡切片的制备
(1)取成像后4周的裸鼠,颈椎脱臼法处死,取出肺组织放入固定液中(10%福尔马林)过夜;
(2)组织块采用梯度浓度酒精脱水,二甲苯透明处理;
(3)已透明的组织块置于已溶化的石蜡中,放入保温箱,包埋盒成型后迅速冷却,组织块变硬成型即可;
(4)包埋好的组织块固定于切片机上,切成5-8μm厚的薄片,热水浴中摊片至载玻片上,45°C恒温箱中烘干。
4.2 HE染色
(1)二甲苯脱去切片中的石蜡,梯度浓度酒精处理,放入ddH2O;
(2)将放入ddH2O后的切片转入苏木精水溶液中染色数分钟,酸水及氨水中分色,各数秒钟;
(3)流水冲洗1小时后入ddH2O片刻,梯度酒精中脱水各10分钟;
(4)酒精伊红染色液室温染色2-3min;
(5)染色后的切片经纯酒精脱水,再经二甲苯使切片透明;
(6)中性树胶封片,待树胶稍干后贴上标签,切片标本即可使用。
如图4E,M12-anti-miR-296-3p组没有明显的转移灶,而M12-Ctrl组动物肺部可见异型性核,存在小的转移灶定植。
5.软琼脂克隆形成实验
(1)取对数生长期细胞,用0.05%胰蛋白酶消化,制备单细胞悬液,台盼蓝细胞计数,用含10%FBS的RPMI1640培养液调整细胞密度至1×106细胞/ml。然后根据实验要求作梯度倍数稀释。
(2)用Milli Q水分别制备1.2%和0.7%两个浓度的低熔点琼脂糖胶,高温高压蒸汽灭菌后,放在37°C中培养箱备用。
(3)等体积混合1.2%的琼脂糖和2×RPMI1640培养基(含1%-5%不等的FBS),然后注入6孔板中(1.5-2ml/well),室温静置5min冷却凝固,置37°C5%CO2温箱中备用(可以在4°C冰箱保存一周,使用前室温平衡30min-60min)。
(4)等体积混合0.7%的琼脂糖和2×RPMI1640培养基(含1%-5%不等的FBS),然后根据设定的细胞密度加入一定体积的单细胞悬液,充分混匀,注入步骤(3)制备的6孔板中,室温或4°C冰箱形成双琼脂层。上层琼脂凝固后,每孔加入1ml培养基,置于37°C 5%CO2温箱中培养12-14天不等。镜下观察细胞克隆形成情况。
(5)0.005%的结晶紫染色,6孔板每孔加1ml结晶紫,室温静置1小时。弃去残留的结晶紫,通风橱中干燥后进行细胞克隆计数,统计分析。
如图4F/G,M12-anti-miR-296-3p组较M12-Ctrl组形成较小的克隆,统计分析显示,M12-anti-miR-296-3p组的克隆数相对也少于M12-Ctrl,说明anti-miR-296-3p能够抑制细胞的恶性转化,在制备抑制前列腺癌侵袭转移药物中具有广泛的应用前景。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (2)
1.miR-296-3p在制备前列腺癌转移诊断试剂盒中的应用。
2.anti-miR-296-3p在制备抑制前列腺癌侵袭转移药物中的应用。
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Asel et al.Feature of Interaction of Intergenic miRNAs with mRNA of CDH1 Gene Participating in Development of Cancer.《Moscow Conference on Computational Molecular Biology》.2011,366-367. |
Feature of Interaction of Intergenic miRNAs with mRNA of CDH1 Gene Participating in Development of Cancer;Asel et al;《Moscow Conference on Computational Molecular Biology》;20110724;366-367 * |
Riichiroh et al.Aberrant Promoter Methylation Profile of Prostate Cancers and Its Relationship to Clinicopathological Features.《Clinical Cancer Research》.2002,摘要,第518页左栏第3段. |
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