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miRNA Characterization from the Extracellular Vesicles
细胞外囊泡中miRNA的鉴定   

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Abstract

Cancer cells and cancer associated stromal cells co-evolve secrete extracelluar vesicles to the surrounding regions and regulate several processes involved in cancer metastasis. miRNAs have been known to be mediators of cancer progression and metastasis. miRNAs consist of short noncoding RNA. miRNAs are stable in extracellular fluids such as serum, plasma and urine. miRNAs are secreted by cells in normal and diseased conditions. miRNAs signatures have been identified specific to certain disease conditions. Therefore they are valuable biomarkers for different diseases. In our study we identified certain miRNAs, miR-409-3p and miR-409-5p, which were secreted by activated stromal fibroblast cells and were taken up by cancer cells to induce explosive tumor growth, through activation of epithelial to mesenchymal transition of cancer cells. Here we describe a procedure to determine miRNAs (miR-409-3p and miR-409-5p) in extracellular vesicles, which were secreted by prostate cancer stromal cells expressing miR-409. In this procedure, conditioned media from the stromal fibroblasts was used to extract the vesicular fraction. RNA was purified from the vesicular fraction, and specific miRNA was reverse transcribed and quantitated using real-time PCR assay.

Keywords: miRNA(miRNA), Extracellular vesicles(细胞外囊泡), Quantitaitive real-time PCR(定量实时PCR), Cancer(癌症), Cancer associated stroma(癌症相关基质), Biofluids(生物流体), Biomarkers(生物标志物), Exosomes(外来体)

Background

miRNA are short non-coding RNA of 20-23 nucleotides in size. miRNAs have been detected in tissues and body fluids. miRNA expression levels have been determined using Northern blotting and quantitative real-time PCR. miRNA are powerful biomarkers for disease conditions. Under frozen conditions, they are stable in biofluids. Recent emphasis is on the miRNA in the vesicular fractions of cells and extracellular fluids (Josson et al., 2015). Extracellular vesicles (EV) contain different proteins, lipids, DNA, RNA and miRNA. EVs range from sizes of 30 nm to a few µm. EVs has been isolated using kits or differential centrifugation (Morello et al., 2013). In this study we describe the detection of miRNA in the vesicular fraction of activated stromal fibroblast cells. miRNAs are typically detected by Northern blot or quantitative real-time PCR assay (Josson et al., 2015). Using the recently available exosome purification kits we isolated exosomes and purified the miRNA from this fraction. The specific miRNA was reverse transcribed and quantitated using real-time PCR assay.

Materials and Reagents

  1. 6 well plates
  2. 6-well dish
  3. MicroAmp® optical 96-well reaction plate (Thermo Fisher Scientific, Applied BiosystemsTM)
  4. MicroAmp® optical adhesive tape (Thermo Fisher Scientific, Applied BiosystemsTM)
  5. SON normal human stromal fibroblast cells expressing miR-409 miRNA
  6. Pencillin-streptomycin (1%, stock: 10,000 U/ml)
  7. Exosome depleted FBS media supplement (System Biosciences, catalog number: EXO-FBS-50A-1 )
  8. Exo-Quick-TC (System Biosciences, catalog number: EXOTC10A-1 )
  9. SeraMir exosome RNA purification kit (System Biosciences, catalog number: RA808A-1 )
  10. Heat inactivated fetal bovine serum (Bio-Whittaker)
  11. DMEM
  12. F-12K
  13. Sodium bicarbonate
  14. Insulin
  15. Triiodothyronine
  16. Transferrin
  17. Biotin
  18. Adenine
  19. dNTP mixture (Thermo Fisher Scientific, Applied BiosystemsTM)
  20. M_MLV reverse transcriptase (Thermo Fisher Scientific, Applied BiosystemsTM)
  21. 10x RT buffer (Thermo Fisher Scientific, Applied BiosystemsTM)
  22. RNase inhibitor (Thermo Fisher Scientific, Applied BiosystemsTM)
  23. dH2O (Thermo Fisher Scientific, Applied BiosystemsTM)
  24. Taqman miRNA primer sets for hsa-miR-409-3p, hsa-miR-409-5p and RNU6B (Thermo Fisher Scientific, Applied BiosystemsTM)
  25. Taqman universal PCR master mix, No AmpErase® UNG (Thermo Fisher Scientific, Applied BiosystemsTM)
  26. T-medium (see Recipes)
  27. Reverse transcription mix for 1 reaction (see Recipes)
  28. Real Time PCR reaction mixture for a single reaction (see Recipes)

Equipment

  1. Centrifuge
  2. Nanodrop 2000/2000c spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: NanodropTM 2000 / 2000c Spectrophotometer )
  3. Applied Biosystems 7500 Real-Time PCR machine (Thermo Fisher Scientific, model: 7500 Real-Time PCR System )
  4. PCR machine

Software

  1. Taqman software
  2. Graphpad Prism software

Procedure

  1. Cell culture
    Prostate normal human stromal fibroblast cells, SON was stably transduced with miR-409 lentiviral particles. Stromal cells were derived from a patient. Stromal cells were cultured at a concentration of 3 x 105 cells in 6 well plates, and maintained in T-medium. The stromal cells were maintained in T-medium with exosome depleted FBS media supplement for 72 h. The conditioned media was collected, and used to extract EVs.

  2. Extraction of exosomes
    1. The conditioned media (CM) from the cells was centrifuged for 15 min. at 3,000 x g to remove cellular debris.
    2. The supernatant (5 ml) was used for extracting EVs using the Exo-Quick-TC for tissue culture media. Exo-Quick-TC (1 ml) was mixed with 5 ml of CM, and the tube was inverted 3 times and kept at 4 °C overnight.
    3. The next day the solution was centrifuged at 13,000 rpm for 2 min. The supernatant was removed and the exosome pellet was used.
    4. Lysis buffer (350 μl) from SeraMir exosome RNA purification kit was added to the exosome pellet and vortexed for 15 sec. The solution was placed at RT for 5 min.

  3. miRNA extraction
    SeraMir exosome RNA purification kit was used to extract miRNA from EVs as per manufacturer’s instructions. The RNA concentration and purity was measured using a Nanodrop spectrophotometer.

  4. miRNA detection
    1. MiRNA was detected separately for hsa-miR-409-5p, hsa-miR-409-3p and RNU6B. Equal amounts of miRNA (25 ng) were added to the reaction. Procedure was followed as per the manufacturer’s instructions.
    2. Reverse transcription: The reverse transcription mix was made as described below (see Recipes). The RT reaction was as follows: 16 °C-30 min, 42 °C- 30 min, 85 °C-5 min.
    3. Quantitative real-time PCR (RT-PCR): The RT-PCR reaction was performed as described below (see Recipes). Assays were run in duplicates. The reaction mixtures were put into the MicroAmp Optical 96 well reaction plate and sealed with a MicroAmp® optical adhesive tape. The assay was run in an Applied Biosystems 7500 Real-Time PCR machine, using the Taqman software, under standard run speed and using a reaction volume of 10 µl. The Ct values were used to determine the relative fold change and the results were normalized to RNU6B. The differential miRNA expression from the vesicular fraction was determined and plotted in a graph.

Data analysis

Analysis of data is performed as mentioned in the Applied Biosystems manual. Real-time PCR assays are performed in duplicates. The Taqman software determines the Ct value of the specific miRNA (miR-409-3p for example). RNU6B is used as the endogenous control. The difference between the target miRNA and RNU6B is determined by subtraction. The normalized value (x), is then calculated as 2x. Values were expressed as means ± standard deviation. Different groups are compared, and miRNA concentration is plotted graphically, as relative change of miRNA normalized to RNU6B using Graphpad Prism software. If 2 groups are being compared, Student’s t-test is used for statistical analysis. If more than 2 groups are compared, One-Way Anova test is used. All experiments were done in duplicates at least two to three independent times.

Notes

miRNA concentrations vary in cells verses extracellular vesicles. For miRNA detection from conditioned media of stromal fibroblasts we used slightly higher RNA concentration for reverse transcription (25 ng). When determining certain miRNA, which are present in low concentration from EVs from conditioned media or body fluids, higher concentration of RNA will be needed to detect the miRNA of interest. Concentrations of 25 ng to 100 ng will be required for a pilot test. Since different miRNA are present in different levels, an initial dose response curve will be useful to determine an appropriate RNA starting concentration.

Recipes

  1. T-medium
    DMEM:F-12K, 4:1, supplemented with:
    5% FBS
    3 g/L sodium bicarbonate
    5 µg/ml insulin
    13.6 pg/ml triiodothyronine
    5 µg/ml transferrin
    0.25 µg/ml biotin
    25 µg/ml adenine
  2. Reverse transcription mix for 1 reaction

  3. Real Time PCR reaction mixture for a single reaction 

Acknowledgments

These experiemntal procedures has been published Josson et al., 2015. Grant support for this work is from P01-CA98912, DAMD-17-03-02-0033, RO1-CA122602 (L.W.K. Chung).

References

  1. Josson, S., Gururajan, M., Sung, S. Y., Hu, P., Shao, C., Zhau, H. E., Liu, C., Lichterman, J., Duan, P., Li, Q., Rogatko, A., Posadas, E. M., Haga, C. L. and Chung, L. W. (2015). Stromal fibroblast-derived miR-409 promotes epithelial-to-mesenchymal transition and prostate tumorigenesis. Oncogene 34(21): 2690-2699.
  2. Morello, M., Minciacchi, V. R., de Candia, P., Yang, J., Posadas, E., Kim, H., Griffiths, D., Bhowmick, N., Chung, L. W., Gandellini, P., Freeman, M. R., Demichelis, F. and Di Vizio, D. (2013). Large oncosomes mediate intercellular transfer of functional microRNA. Cell Cycle 12(22): 3526-3536.

简介

癌细胞和癌症相关的基质细胞将分泌的细胞外囊泡共同进入周围区域并调节参与癌症转移的几个过程。已知miRNA是癌症进展和转移的介质。 miRNA由短的非编码RNA组成。 miRNA在细胞外液体如血清,血浆和尿液中是稳定的。 miRNA在正常和患病条件下由细胞分泌。已经鉴定了特定疾病条件的miRNAs特征。因此,它们是不同疾病的宝贵生物标志物。在我们的研究中,我们确定了某些miRNAs,miR-409-3p和miR-409-5p,其由激活的基质成纤维细胞分泌并被癌细胞摄取以诱导爆发性肿瘤生长,通过激活癌细胞的上皮至间质转化细胞。在这里,我们描述了一种在细胞外囊泡中测定miRNA(miR-409-3p和miR-409-5p)的方法,其由表达miR-409的前列腺癌基质细胞分泌。在该方法中,使用来自间质成纤维细胞的条件培养基来提取囊泡部分。从囊泡部分纯化RNA,并使用实时PCR测定将特异性miRNA逆转录并定量。

背景 miRNA是大小为20-23个核苷酸的短非编码RNA。已经在组织和体液中检测到miRNA。已经使用Northern印迹和定量实时PCR测定了miRNA表达水平。 miRNA是疾病状况的强大生物标志物。在冷冻条件下,它们在生物流体中是稳定的。最近的重点是细胞和细胞外液的水泡部分中的miRNA(Josson等人,2015)。细胞外囊泡(EV)含有不同的蛋白质,脂质,DNA,RNA和miRNA。 EV范围从30nm到几μm。使用试剂盒或差速离心法分离EV(Morello等人,2013)。在本研究中,我们描述了激活的基质成纤维细胞囊泡部分中miRNA的检测。 miRNA通常通过Northern印迹或定量实时PCR测定(Josson等人,2015)检测。使用最近可用的外来体纯化试剂盒,我们分离出外来体,并从该级分纯化miRNA。使用实时PCR测定将特异性miRNA逆转录并定量。

关键字:miRNA, 细胞外囊泡, 定量实时PCR, 癌症, 癌症相关基质, 生物流体, 生物标志物, 外来体

材料和试剂

  1. 6孔板
  2. 6孔碟
  3. MicroAmp ®光学96孔反应板(Thermo Fisher Scientific,Applied Biosystems TM
  4. MicroAmp ®光学胶带(Thermo Fisher Scientific,Applied Biosystems TM
  5. SON正常人间质成纤维细胞表达miR-409 miRNA
  6. 青霉素 - 链霉素(1%,存量:10,000 U/ml)
  7. 外来体消耗的FBS培养基补充(系统生物科学,目录号:EXO-FBS-50A-1)
  8. Exo-Quick-TC(系统生物科学,目录号:EXOTC10A-1)
  9. SeraMir外源体RNA纯化试剂盒(系统生物科学,目录号:RA808A-1)
  10. 热灭活的胎牛血清(Bio-Whittaker)
  11. DMEM
  12. F-12K
  13. 碳酸氢钠
  14. 胰岛素
  15. 三碘甲腺原氨酸
  16. 转铁蛋白
  17. 生物素
  18. Adenine
  19. dNTP混合物(Thermo Fisher Scientific,Applied Biosystems TM
  20. M_MLV逆转录酶(Thermo Fisher Scientific,Applied Biosystems TM
  21. 10x RT缓冲液(Thermo Fisher Scientific,Applied Biosystems TM
  22. RNase抑制剂(Thermo Fisher Scientific,Applied Biosystems TM
  23. dH 2 O(Thermo Fisher Scientific,Applied Biosystems TM
  24. 用于hsa-miR-409-3p,hsa-miR-409-5p和RNU6B的Taqman miRNA引物组(Thermo Fisher Scientific,Applied Biosystems TM
  25. Taqman通用PCR主混合物,无AmpErase UNG(Thermo Fisher Scientific,Applied Biosystems TM
  26. T介质(见配方)
  27. 逆转录混合1次反应(见配方)
  28. 单次反应的实时PCR反应混合物(参见食谱)

设备

  1. 离心机
  2. Nanodrop 2000/2000c分光光度计(Thermo Fisher Scientific,Thermo Scientific TM,型号:Nanodrop TM 2000/2000c分光光度计)
  3. Applied Biosystems 7500实时PCR机(Thermo Fisher Scientific,型号:7500实时PCR系统)
  4. PCR机器

软件

  1. Taqman软件
  2. Graphpad Prism软件

程序

  1. 细胞培养
    前列腺正常人间质成纤维细胞,SON稳定转导与miR-409慢病毒颗粒。基质细胞来自患者。在6孔板中以3×10 5个细胞的浓度培养基质细胞,并保持在T培养基中。将基质细胞维持在具有外来体消耗的FBS培养基补充物的T介质中72小时。收集条件培养基,用于提取EV。

  2. exosomes的提取
    1. 将来自细胞的条件培养基(CM)离心15分钟。在3,000×g处除去细胞碎片。
    2. 使用Exo-Quick-TC用于组织培养基的上清液(5ml)用于提取EV。将Exo-Quick-TC(1ml)与5ml CM混合,将管倒置3次,并在4℃保持过夜。
    3. 第二天,溶液以13,000rpm离心2分钟。除去上清液,使用外来体颗粒。
    4. 将来自SeraMir外源体RNA纯化试剂盒的裂解缓冲液(350μl)加入到外来体颗粒中并涡旋15秒。将溶液置于室温下5分钟
  3. miRNA提取
    根据制造商的说明书,使用SeraMir外源RNA纯化试剂盒从EV中提取miRNA。使用Nanodrop分光光度计测量RNA浓度和纯度
  4. miRNA检测
    1. 分别检测hsa-miR-409-5p,hsa-miR-409-3p和RNU6B的MiRNA。将相等量的miRNA(25ng)加入到反应中。按照制造商的说明遵循程序。
    2. 逆转录:逆转录混合物如下所述进行(参见食谱)。 RT反应如下:16℃-30分钟,42℃-30分钟,85℃-5分钟。
    3. 定量实时PCR(RT-PCR):如下所述进行RT-PCR反应(参见食谱)。测试以重复的方式运行。将反应混合物放入MicroAmp Optical 96孔反应板中,并用MicroAmp 光学胶带密封。使用Taqman软件,在标准运行速度下,使用10μl的反应体积,在Applied Biosystems 7500实时PCR仪中进行测定。使用C 值来确定相对折叠变化,并将结果归一化为RNU6B。确定来自囊泡部分的差异miRNA表达并绘制在图中。

数据分析

数据分析按照Applied Biosystems手册中的说明进行。实时PCR测定以重复的方式进行。 Taqman软件确定特异性miRNA(例如,miR-409-3p)的C 值。 RNU6B用作内源对照。通过减法确定靶miRNA和RNU6B之间的差异。然后将归一化值(x)计算为2 x 。值表示为平均值±标准偏差。比较不同的组,并且使用Graphpad Prism软件将miRNA浓度图形化为miRNA的相对变化,使其归因于RNU6B。如果比较2组,则使用Student's -test进行统计分析。如果比较超过2组,则使用单向Anova测试。所有的实验都是以至少两到三次独立的时间重复进行的。

笔记

细胞中的miRNA浓度在胞外囊泡中变化。对于从基质成纤维细胞的条件培养基进行miRNA检测,我们使用稍高的RNA浓度用于逆转录(25ng)。当确定来自条件培养基或体液的EV的低浓度的某些miRNA时,需要更高浓度的RNA来检测感兴趣的miRNA。中试测试需要25 ng至100 ng的浓度。由于不同的miRNA存在于不同的水平,初始剂量反应曲线将有助于确定合适的RNA起始浓度。

食谱

  1. T-medium
    DMEM:F-12K,4:1,补充:
    5%FBS
    3克/升碳酸氢钠
    5μg/ml胰岛素 13.6 pg/ml三碘甲状腺原氨酸 5μg/ml转铁蛋白
    0.25μg/ml生物素
    25μg/ml腺嘌呤
  2. 反转录混合1次反应

  3. 实时PCR反应混合物,用于单次反应

致谢

这些实验程序已经出版了Josson等人,2015年。这项工作的授权来自P01-CA98912,DAMD-17-03-02-0033,RO1-CA122602(L.W.K.Chung)。

参考文献

  1. Josson,S.,Gururajan,M.,Sung,SY,Hu,P.,Shao,C.,Zhau,HE,Liu,C.,Lichterman,J.,Duan,P.,Li,Q.,Rogatko, A.,Posadas,EM,Haga,CL和Chung,LW(2015)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/25065597"基质成纤维细胞衍生的miR-409促进上皮 - 间质转化和前列腺肿瘤发生。 34(21):2690-2699。
  2. Morello,M.,Minciacchi,VR,de Candia,P.,Yang,J.,Posadas,E.,Kim,H.,Griffiths,D.,Bhowmick,N.,Chung,LW,Gandellini,P.,Freeman ,MR,Demichelis,F.和Di Vizio,D.(2013)。  大型癌基因介导功能性微小RNA的细胞间转移。细胞周期 12(22):3526-3536。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Josson, S., Gururajan, M. and Chung, L. W. (2017). miRNA Characterization from the Extracellular Vesicles. Bio-protocol 7(4): e2139. DOI: 10.21769/BioProtoc.2139.
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