This work was supported by the NIH grant (5R01 CA178386-03) and the USC ASPIRE-1 grant to HC. We would like to thank Vrushab Gowda for his assistance with manuscript.

この研究は、全南大学校和順病院、サウスカロライナ大学の治験審査委員会によって承認されました。乳がんと一致した隣接する正常な乳房組織で構成されるTMAサンプルは全南大学校和順病院、韓国バイオバンクネットワークのメンバーのバイオバンクから提供されました。 1.溶液の調製<ol><li> DNアーゼおよびRNaseフリー水1リットルを準備します。ジエチル（DEPC）処理水を使用して、すべての化学溶液を作ります。 DEPCの500μlの水1Lを治療し、室温で3時間インキュベートします。 DEPCを不活性化するために水をオートクレーブ。 注意：それは発癌物質知られているので、DEPCを吸入しないでください。 </li><li> DEPC処理水を使用して、10×PBSから1×PBSを準備します。 </li><li> 、20×SSC（塩化ナトリウムの175.3グラムと800ミリリットルの水でクエン酸ナトリウムの88.2グラムを溶解を準備HClを14 N溶液数滴でpHを7.0に調整し、ワットで1Lにボリュームを調整ER）、4％パラホルムアルデヒド、95％エタノール、80％エタノールDEPC処理水を用いました。 </li><li> 8トリmlのDEPC処理水590ミリリットル中のHClの1.05ミリリットルを加え、アセチル化のための無水酢酸1.5ミリリットルを追加します。 </li><li>バッファ中の50μg/μlのプロテ​​イナーゼK（50 mMトリス - 塩酸、pHが8.0をDEPCで処理水を）を準備します。 </li><li>ホルムアミド500μlの、20×SSCを250μl、50×デンハルト溶液100μl、T-RNAの12.5μlを、ニシン精子DNA2.5μlの、RVCの30μlの、ブロッキング試薬及び50μlに0.02グラムで構成されるハイブリダイゼーション緩衝液を準備しますDEPC水の。新鮮な準備をします。 </li></ol> 2.乳房組織切片<ol><li>ミクロトームで5μmのホルマリン固定パラフィン包埋切片をカットし、スライド14を充電するためのセクションを適用します。 注：ここで使用される乳房組織は、韓国バイオバンクネットワークから先に調製しました。 </li></ol> 3.組織標本<ol><li>あちこちパラフィンを削除します10分間新鮮なキシレンでコプリンジャーの2倍に各々のスライドを浸漬することにより、組織をMとのddH 2 Oにエタノール（100％、95％及び80％）の濃度を減少させるで5分間のインキュベーションによって組織切片を再水和ドラフト内で、この手順を実行します。 </li><li> 5分間のDEPC処理水で組織を浸します。この時点で、疎水性バリアペンで組織切片の周りに境界線を作ります。 </li><li> PBSで洗浄した15分間、4％PFAで組織を修正しました。 </li><li> PBSで洗浄し、続いて10分間、0.3％トリトンX-100 / PBSで組織を浸します。 </li><li> 37℃で15分間の50μg/μlのプロテ​​イナーゼK溶液で試料をインキュベートします。その後PBSで洗浄し、続いて室温で5分間、トリエタノールアミンおよび無水酢酸で組織切片をインキュベートします。いいえPBS洗浄は、プロテイナーゼK処理およびトリエタノールアミン/無水酢酸処理の間に必要とされません。 </li></ol> 4.ハイブリダイゼーション<ol><li>徹底的にincu組織を洗います室温で2〜3時間のためのハイブリダイゼーション緩衝液を用いBATE組織切片。典型的には、120μlのハイブリダイゼーション緩衝液は、組織切片をカバーするのに十分です。 </li><li>希釈5&#39;-DIGは、120μlのハイブリダイゼーション溶液中の午後3時の最終濃度にLNAプローブを標識しました。ストック濃度は、通常、40 ngの/μlであるように、120μlのハイブリダイゼーション緩衝液に株式の3μlを添加し、95℃で5分間混合物をインキュベートします。 </li><li>加湿チャンバー内で54°C（Tm）がでプローブ一晩で組織切片をインキュベートします。典型的には、120μlの1部をカバーするのに十分です。 2湿ったペーパータオルを使用して湿度を作成します。室の理想的なサイズは8インチ×10インチです。 </li><li>注意：Tmは異なるmiRNAのために異なる場合があります。 54°Cは、miR-489のための言及です。 </li></ol> 5.ジェンウォッシュ<ol><li> 7分間の5倍のSSCで組織を洗浄（ハイブリダイゼーション工程の後に、RNaseを含まない条件がもはや必須ではありません）。 </li><li>組織切片のウィットを洗います二回57℃で7分間の時間1×SSC。 </li><li> 57℃で7分間、0.2×のSSCで組織切片を洗浄します。 </li><li>室温で7分間、0.2×のSSCで組織切片を洗浄します。 </li><li> 10分間、PBS中の組織切片をインキュベートします。 </li></ol> 6.ブロッキング<ol><li>バッファは、PBSの18メートルで、BSAのTween-20を3μlの10μlのを2ミリリットルのFBSを追加ブロッキングようにするには（湿潤チャンバー内で1時間室温でブロッキング緩衝液で組織切片をインキュベートします。Storeは4でブロッキング緩衝液 °C）。 </li></ol> 7.一次抗体インキュベーション開発<ol><li>ブロッキング緩衝液中に300抗DIG-AP抗体と4℃で一晩抗体を用いた組織切片をインキュベート：1に希釈します。 </li><li> 5分ごと（開発キットにより提供バッファ）のために、洗浄緩衝液で3回のセクションを洗ってください。 </li><li> CY-3蛍光光を生成するアルカリホスファターゼのための市販のキットを用いて組織切片を開発します。 </li> &lt;李&gt;は5分間の洗浄バッファーでセクションを洗ってください。洗浄緩衝液で5分間洗浄した5分間2.5 ngの/μlのヘキスト染料を100μlとステインセクション。 </li><li>製造業者のプロトコルに従ってバッファを搭載してセクションをマウントします。組織切片あたり15μLを使用して、組織拭くと、過剰なマウントソリューションを拭いてください。漏れを防止するために、明確なマニキュアでスライドを密閉します。 </li><li>トータルの倍率は100倍である10倍レンズを使用したセクションの蛍光顕微鏡を観察します。 </li></ol>

<ol>
	<li>Shah, N. R., Chen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+in+pathogenesis+of+breast+cancer:+Implications+in+diagnosis+and+treatment.">MicroRNAs in pathogenesis of breast cancer: Implications in diagnosis and treatment.</a> World J Clin Oncol. 5, 48-60 (2014).</li><li>Tang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR-185+suppresses+tumor+proliferation+by+directly+targeting+E2F6+and+DNMT1+and+indirectly+upregulating+BRCA1+in+triple-negative+breast+cancer.">miR-185 suppresses tumor proliferation by directly targeting E2F6 and DNMT1 and indirectly upregulating BRCA1 in triple-negative breast cancer.</a> Mol Cancer Ther. 13, 3185-3197 (2014).</li><li>Ye, X. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+silencing+of+miR-375+induces+trastuzumab+resistance+in+HER2-positive+breast+cancer+by+targeting+IGF1R.">Epigenetic silencing of miR-375 induces trastuzumab resistance in HER2-positive breast cancer by targeting IGF1R.</a> BMC Cancer. 14, 134 (2014).</li><li>Oneyama, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-mediated+downregulation+of+mTOR/FGFR3+controls+tumor+growth+induced+by+Src-related+oncogenic+pathways.">MicroRNA-mediated downregulation of mTOR/FGFR3 controls tumor growth induced by Src-related oncogenic pathways.</a> Oncogene. 30, 3489-3501 (2011).</li><li>McGuire, A., Brown, J. A., Kerin, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastatic+breast+cancer:+the+potential+of+miRNA+for+diagnosis+and+treatment+monitoring.">Metastatic breast cancer: the potential of miRNA for diagnosis and treatment monitoring.</a> Cancer Metastasis Rev. 34, 145-155 (2015).</li><li>Quesne, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+and+prognostic+associations+of+miR-205+and+let-7b+in+breast+cancer+revealed+by+in+situ+hybridization+analysis+of+micro-RNA+expression+in+arrays+of+archival+tumour+tissue.">Biological and prognostic associations of miR-205 and let-7b in breast cancer revealed by in situ hybridization analysis of micro-RNA expression in arrays of archival tumour tissue.</a> J Pathol. 227, 306-314 (2012).</li><li>Fernandez, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR-340+inhibits+tumor+cell+proliferation+and+induces+apoptosis+by+targeting+multiple+negative+regulators+of+p27+in+non-small+cell+lung+cancer.">miR-340 inhibits tumor cell proliferation and induces apoptosis by targeting multiple negative regulators of p27 in non-small cell lung cancer.</a> Oncogene. 34, 3240-3250 (2015).</li><li>Yu, L., Zhang, J., Guo, X., Li, Z., Zhang, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-224+upregulation+and+AKT+activation+synergistically+predict+poor+prognosis+in+patients+with+hepatocellular+carcinoma.">MicroRNA-224 upregulation and AKT activation synergistically predict poor prognosis in patients with hepatocellular carcinoma.</a> Cancer Epidemiol. 38, 408-413 (2014).</li><li>Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=microRNA-146+up-regulation+predicts+the+prognosis+of+non-small+cell+lung+cancer+by+miRNA+in+situ+hybridization.">microRNA-146 up-regulation predicts the prognosis of non-small cell lung cancer by miRNA in situ hybridization.</a> Exp Mol Pathol. 96, 195-199 (2014).</li><li>Kriegel, A. J., Liang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+in+situ+hybridization+for+formalin+fixed+kidney+tissues.">MicroRNA in situ hybridization for formalin fixed kidney tissues.</a> J Vis Exp. , e50785 (2013).</li><li>Ucar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR-212+and+miR-132+are+required+for+epithelial+stromal+interactions+necessary+for+mouse+mammary+gland+development.">miR-212 and miR-132 are required for epithelial stromal interactions necessary for mouse mammary gland development.</a> Nat Genet. 42, 1101-1108 (2010).</li><li>Nuovo, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+detection+of+microRNAs+in+paraffin+embedded,+formalin+fixed+tissues+and+the+co-localization+of+their+putative+targets.">In situ detection of microRNAs in paraffin embedded, formalin fixed tissues and the co-localization of their putative targets.</a> Methods. 52, 307-315 (2010).</li><li>Kierzek, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+locked+nucleic+acid+residues+on+the+thermodynamic+properties+of+2'-O-methyl+RNA/RNA+heteroduplexes.">The influence of locked nucleic acid residues on the thermodynamic properties of 2'-O-methyl RNA/RNA heteroduplexes.</a> Nucleic Acids Res. 33, 5082-5093 (2005).</li><li>Fischer, A. H., Jacobson, K. A., Rose, J., Zeller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paraffin+embedding+tissue+samples+for+sectioning.">Paraffin embedding tissue samples for sectioning.</a> CSH Protoc. 2008, (2008).</li><li>Li, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+expression+of+miR-182+and+miR-503+cooperatively+targets+FBXW7,+contributing+to+the+malignant+transformation+of+colon+adenoma+to+adenocarcinoma.">Sequential expression of miR-182 and miR-503 cooperatively targets FBXW7, contributing to the malignant transformation of colon adenoma to adenocarcinoma.</a> J Pathol. 234, 488-501 (2014).</li></ol>

There is nothing to disclose.

この研究の目的は、in situハイブリダイゼーションのmiRNAを用いた乳癌組織におけるmiRNA発現のレベルを決定することでした。なお、この実験では、全ての条件が乳癌組織のために最適化されたことに注意しなければなりません。さらなる最適化は、他の組織タイプのために必要になることがあります。全ての溶液は、DEPC水で行う必要があり、使用されるすべての容器はDEPC処理水ですすぎ、続いてオートクレーブ処理されるべきです。わずかなのRNaseの混入は、実験の最終的な結果を妨害することができます。 miRNAが非常に短い配列であるので、標的とのより良いハイブリダイゼーションを容易にするために、LNAプローブを使用することが必要です。また、組織固定、治療後にRNaseフリープロテイナーゼKが絶対に必要であるとそれ以外のタンパク質は、内因性のmiRNAとのプローブのハイブリダイゼーションを妨害する可能性があります。また、プローブを用いて組織切片をインキュベートする前に、後者が95にゆでする必要があります いずれかを展開するためにCを°二次構造。 一晩インキュベートした後、非特異的結合を低減するためのプロトコルで上記温度でSSCの濃度を変化させて洗浄することが重要です。蛍光の開発のために、少なくとも1時間のための基板との組織切片をインキュベートします。乱開発を防止することができるように、インキュベーション全体のセクションを観察することをお勧めします。キットに付属の取り付けメディアのセクションをマウントしてください。 1を考慮して取るべき1制限要因があります。標的miRNAの発現は、組織内の小さい場合には、この技術を用いてその発現を検出することは困難であろう。技術が確認することができ、全ての試薬が作業するようにし、彼らは自由のRNaseされているので、このようなU6などのポジティブコントロールでこのプロトコルを実行するために、常にお勧めします。 弱い符号の場合には代わりにDIG標識プローブのビオチン標識プローブを使用することが可能ですDIGとアルは、標識プローブ。信号増幅はビオチン - アビジン系を用いることにより可能です。アビジンは、ビオチンに対する強い親和性を有します。この親和性を利用して、アルカリホスファターゼで取り付けているアビジン - ビオチンデュプレックスとビオチンプローブをタグ付けすることが可能です。この戦略を用いることにより、同様に低い豊富なmiRNAのための信号を増幅することができます。 miRNAは、キー細胞プロセスにおいて重要な役割を果たすと強く癌発生にリンクされているので、miRNAの分野は急速に、浮上しています。さらに、これらのmiRNAは、癌診断におけるバイオマーカーとして使用することができます。いくつかの研究は、このような癌の予後におけるmiRNA発現の可能なアプリケーションを示唆している腫瘍段階として、miRNAの発現と臨床パラメーターとの間に強い相関を示しています。この技術はまた、他の癌タイプにおけるmiRNA発現分析のために使用することができます。例えば、のmiR-182及びmiR-503の発現は、Si中の使用結腸癌患者組織において分析しました。TUハイブリダイゼーション15。 従来の遺伝子発現解析は、組織におけるmiRNA発現レベルを明らかにすることができるが、それは、miRNAが発現されている構造の位置を明らかにすることができません。この技術を用いて、一方が容易にmiRNA発現レベルならびに場所を見つけることができます。

乳がんは世界中の女性人口に影響を与える最も一般的な悪性腫瘍の一つです。乳癌の130万以上の例は世界的に毎年1,2報告されています。腫瘍細胞は、伝統的に、生物学的に均一で高い増殖性とみなされてきたが、乳癌は遺伝的および臨床的に不均一であることが明らかになってきています。流行している抗がん剤に対する耐性は、癌の進行と距離転移3のその促進を通じ、大多数の患者の死亡率につながる、先進的な乳癌の特徴です。 マイクロRNA（miRNA）はmRNAの翻訳を遮断またはmRNA分解4を媒介することにより遺伝子発現を調節する約22ヌクレオチド長の小RNA分子のクラスです。 2,000人以上の異なるmiRNAは、これまでヒト疾患5にリンクされている人間、で同定されています。研究の増加アレイは、肝炎Eは、miRNAが癌遺伝子及び腫瘍抑制因子として機能することができ、多くの場合、腫瘍6の異常調節されていることを実証しました。 miRNAは強く腫瘍細胞の運動性、浸潤および転移7のものと一緒に、細胞周期の進行、老化、アポトーシスおよびオートファジーの主要な調節因子を制御することにより、一次腫瘍形成のすべてに影響を与えます。 異常なmiRNA発現はまた、ステージ、分化、予後およびアジュバント療法8に対する応答として臨床的意義と関連しています。具体的には、のmiR-146の発現が、 ​​肺癌患者9における予後不良と相関して増加しました。別の研究では、miRNA-let7bの失われた式は、悪性の表現型にリンクされていると、結果的に、低い生存率6されていることを実証しました。 miRNAを発現する細胞と構造の種類を理解することは、miRNAの発現に影響を​​与える細胞の変化、それを通して力学を理解することの重要な部分であり、組織は10を表現型。間質細胞で分泌されることがわかっ特定のmiRNAは、上皮細胞によって取り込まれ、具体的にその標的に作用しています。同じ静脈では、のmiR-212およびmiR-132は、間質から分泌され、乳腺発達11時の乳管伸長に必要な上皮-間質相互作用を調節しています。この論文の全体的な目的は、in situハイブリダイゼーションにおける miRNAを介して乳癌組織におけるmiRNAの発現を検出するための詳細なプロトコルを説明することです。我々は、乳癌患者サンプル中のmiRNA-489の発現のレベルをアッセイするために、すべての条件を最適化しました。この目的のために、我々は、DIGがロックされた核酸（LNA）、我々の実験でプローブを標識しました。そのヌクレオチドのいくつかは、それは、2 &#39;酸素原子と4&#39;は、ハイブリダイゼーション後の「適所にロックされた」ままになるようにヌクレオチドを​​固定リボース骨格の炭素原子を連結するメチレン架橋であり、LNA修飾を有していました。結果として、それははるかに困難ですハイブリダイズした複合体は、12,13を変性するために。

<table border="0" cellpadding="0" cellspacing="0" width="661">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">ELF-97</td>
			<td style="width:159px;">Life technology&#160;</td>
			<td style="width:119px;">E6604</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50X Denhard&#39;t&#160;</td>
			<td style="width:159px;">Life technology&#160;</td>
			<td align="right" style="width:119px;">750018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">t-RNA</td>
			<td style="width:159px;">Roche</td>
			<td align="right" style="width:119px;">109541</td>
			<td>Reconstitute in DEPC water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Herring Sperm DNA</td>
			<td style="width:159px;">Promega&#160;</td>
			<td style="width:119px;">D1815</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Roche Blocking&#160;</td>
			<td style="width:159px;">Roche</td>
			<td align="right" style="width:119px;">11096176001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RVC</td>
			<td style="width:159px;">Fisher</td>
			<td style="width:119px;">50-812-650</td>
			<td>Before use spin down it at 16.1 RCF and take supernatant&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">miR-489 probe&#160;</td>
			<td style="width:159px;">Exiqon</td>
			<td style="width:119px;">38599-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nuclease free BSA</td>
			<td style="width:159px;">Roche&#160;</td>
			<td align="right" style="width:119px;">711454</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Primary antibody-anti DIG</td>
			<td style="width:159px;">Roche&#160;</td>
			<td align="right" style="width:119px;">11093274910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Diethyl Pyrocarbonate&#160;</td>
			<td style="width:159px;">Sigma</td>
			<td align="right" style="width:119px;">1609478</td>
			<td></td>
		</tr>
	</tbody>
</table>

clinicopathological analysis of mirna expression in breast cancer tissues by using mirna in situ hybridization

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA (Locked Nucleic Acid) probe. The probe was recognized by anti-DIG alkaline phosphatase antibodies and later developed using alkaline phosphatase substrate producing fluorescence signals. Here we utilized miRNA in situ hybridization (MISH) technique to analyze expression of miR-489 in tissues from breast cancer patients. This technique can detect the localization of miRNA of interest in individual tissue samples. This technique can be used to compare the expression of desired miRNA in tumor tissue with that in adjacent normal tissue and to identify the specific structures responsible for expressing this miRNA. This technique can be very useful in answering certain clinical questions, such as role of specific miRNA in the development of cancer. Our results indicate that mammary epithelial cells express significantly higher levels of miR-489 than adjacent tumor cells.

ヒト乳癌組織のmiRNA-489の発現を決定しました。乳腺管上皮細胞は、隣接する二人の患者における腫瘍組織（ 図1Aおよび1B）よりも有意に高かったmiRNA-489レベルを発現することが見出されました。これは明らかなmiRNA-489の発現がmiRNA-489の腫瘍抑制活性を示唆し、腫瘍組織では失われていることを示しています。さらに、これらの結果を確認するために、同じ患者からの腫瘍組織と隣接する正常組織を比較しました。 図2Aおよび図2Bに観察されたように、正常組織は、その隣接する腫瘍組織よりも多くのmiRNA-489を発現することが見出されました。画像内の10μmのスケールバーを示しています。画像は、10倍レンズを使用して撮影しました。 <img alt="図1" src="/files/ftp_upload/53928/53928fig1.jpg" /> 図1：正常な乳房組織におけるmiR-489の発現両方通常のダクト構造および腫瘍細胞が同じセクションに表示されます。組織は、異なる乳癌患者からのものです。正常な乳管および乳腺不在ながら、腫瘍組織におけるmiRNA-489を発現します。スケールバー=10μmである。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53928/53928fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図2" src="/files/ftp_upload/53928/53928fig2.jpg" /> 図2：乳癌患者ペアにおけるmiR-489の発現の組織マイクロアレイは、乳癌患者におけるmiR-489の発現を研究するために使用しました。一つの代表的な患者のペアはここに示されています。スケールバー=10μmである。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53928/53928fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

Watch this Scientific Journal Video about Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization at JoVE.com

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

Here we present a protocol to detect miRNA expression in breast cancer patient samples using miRNA in situ hybridization.

miRNAを用いた乳がん組織におけるmiRNA発現の臨床病理学的解析<em&gt;その場で</em&gt;ハイブリダイゼーション

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA ...

clinicopathological-analysis-mirna-expression-breast-cancer-tissues

Nanyang Technological University

Research

JoVE Journal

Medicine

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

6.7K ビュー.  University of South Carolina. Here we present a protocol to detect miRNA expression in breast cancer patient samples using miRNA in situ hybridization.

ビデオ: Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of them being fluorescence in situ hybridization (FISH).1,2 We performed a pilot study in which oral biofilm samples collected from fixed orthodontic appliances (palatal expanders) were stained by FISH, the objective being to assess the three-dimensional organization of natural biofilm and plaque accumulation.3,4 FISH creates an opportunity to stain cells in their native biofilm environment by the use of fluorescently labeled 16S rRNA-targeting probes.4-7,19 Compared to alternative techniques like immunofluorescent labeling, this is an inexpensive, precise and straightforward labeling technique to investigate different bacterial groups in mixed biofilm consortia.18,20 General probes were used that bind to Eubacteria (EUB338 + EUB338II + EUB338III; hereafter EUBmix),8-10 Firmicutes (LGC354 A-C; hereafter LGCmix),9,10 and Bacteroidetes (Bac303).11 In addition, specific probes binding to Streptococcus mutans (MUT590)12,13 and Porphyromonas gingivalis (POGI)13,14 were used. The extreme hardness of the surface materials involved (stainless steel and acrylic resin) compelled us to find new ways of preparing the biofilm. As these surface materials could not be readily cut with a cryotome, various sampling methods were explored to obtain intact oral biofilm. The most workable of these approaches is presented in this communication. Small flakes of the biofilm-carrying acrylic resin were scraped off with a sterile scalpel, taking care not to damage the biofilm structure. Forceps were used to collect biofilm from the steel surfaces. Once collected, the samples were fixed and placed directly on polysine coated glass slides. FISH was performed directly on these slides with the probes mentioned above. Various FISH protocols were combined and modified to create a new protocol that was easy to handle.5,10,14,15 Subsequently the samples were analyzed by confocal laser scanning microscopy. Well-known configurations3,4,16,17 could be visualized, including mushroom-style formations and clusters of coccoid bacteria pervaded by channels. In addition, the bacterial composition of these typical biofilm structures were analyzed and 2D and 3D images created.

We present a protocol for structural and compositional analysis of natural oral biofilm from orthodontic appliances with in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Oral biofilm samples were collected from palatal expanders, scraping acrylic-resin flakes off their surface and referring them for molecular processing.

蛍光in situハイブリダイゼーションおよび共焦点レーザー走査顕微鏡による口蓋パンダの口腔バイオフィルムの解析

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of ...

医学

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

磁気共鳴イメージング、体積CT画像と超音波を用いた乳がんの骨転移のヌードラットモデルにおける血管新生のマルチモーダルイメージング

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

乳癌細胞におけるエストロゲン調節マイクロRNAのプロファイリング

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

3次元（3D）モデルを用いた乳癌細胞浸潤の定量化

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

マウスにおけるタキサンの薬物動態学的および薬力学的分析のための頸動脈煎茶

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that self-renew while giving rise to more differentiated progeny. Understanding and targeting these cancer stem cells in breast cancer, which may possess enhanced chemo- and radio-resistance compared to the non-stem tumor bulk, has become an important research area. Markers including CD44, CD24, and ALDH activity can be assessed using fluorescence activated cell sorting (FACS) to prospectively isolate cells that display enhanced tumorigenicity when implanted into immunocompromised mice: the mammosphere assay has also become widely used for its ability to retrospectively identify sphere-forming cells that develop from single stem cell-like clones. Here we outline approaches for the appropriate culturing of mammospheres from cell lines or primary patient samples, their passaging, and calculations to estimate sphere forming efficiency (SFE). First we discuss key considerations and pitfalls in the appropriate planning and interpretation of mammosphere experiments.

Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape.

ヒト乳癌組織および細胞株からマンモスフェア形成アッセイ

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

生体内および生体外では腹膜脂肪にミルキースポット構造の卵巣がん転移性コロニー形成を研究するためのアプローチで

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

新しい治療戦略を開発するための膵臓癌幹細胞特性を研究

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

miRNA Expression Analyses in Prostate Cancer Clinical Tissues

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, diagnosis, prognosis and treatment. In recent years, microRNAs (miRNAs) have emerged as promising alternate biomarkers for prostate cancer diagnosis and prognosis. However, the development of miRNAs as effective biomarkers for prostate cancer heavily relies on their accurate detection in clinical tissues. miRNA analyses in prostate cancer clinical specimens is often challenging owing to tumor heterogeneity, sampling errors, stromal contamination etc. The goal of this article is to describe a simplified workflow for miRNA analyses in archived FFPE or fresh frozen prostate cancer clinical specimens using a combination of quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH). Within this workflow, we optimize the existing methodologies for miRNA extraction from FFPE and frozen prostate tissues and expression analyses by Taqman-probe based miRNA RT-PCR. In addition, we describe an optimized method for ISH analyses formiRNA detection in prostate tissues using locked nucleic acid (LNA)- based probes. Our optimized miRNA ISH protocol can be applied to prostate cancer tissue slides or prostate cancer tissue microarrays (TMA).

Here we describe a simplified protocol for microRNA (miRNA) expression analyses in archived Formalin-Fixed, Paraffin-Embedded (FFPE) or fresh frozen prostate cancer (PCa) clinical tissues employing quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH).

前立腺癌臨床組織におけるmiRNA発現解析

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, ...

Systems Biology of Metabolic Regulation by Estrogen Receptor Signaling in Breast Cancer

With the advent of the -omics approaches our understanding of the chronic diseases like cancer and metabolic syndrome has improved. However, effective mining of the information in the large-scale datasets that are obtained from gene expression microarrays, deep sequencing experiments or metabolic profiling is essential to uncover and then effectively target the critical regulators of diseased cell phenotypes. Estrogen Receptor &#945; (ER&#945;) is one of the master transcription factors regulating the gene programs that are important for estrogen responsive breast cancers. In order to understand to role of ER&#945; signaling in breast cancer metabolism we utilized transcriptomic, cistromic and metabolomic data from MCF-7 cells treated with estradiol. In this report we described generation of samples for RNA-Seq, ChIP-Seq and metabolomics experiments and the integrative computational analysis of the obtained data. This approach is useful in delineating novel molecular mechanisms and gene regulatory circuits that are regulated by a particular transcription factor which impacts metabolism of normal or diseased cells.

Integration of data from genome-wide sequencing experiments and metabolomics experiments is a challenge. In this paper we report, for the first time, generation, analysis and integration of transcriptome, cistrome and metabolome data from breast cancer cells treated with estradiol.

乳癌におけるエストロゲン受容体シグナル伝達によって代謝制御のシステムバイオロジー

With the advent of the -omics approaches our understanding of the chronic diseases like cancer and metabolic syndrome has improved. However, effective ...