이 연구는 (1) 중국 국가 핵심 R&D 프로그램(2020YFE0201600)의 보조금으로 지원됩니다. (2) 국립자연과학재단(National Nature Science Foundation, 81973877 및 82174408) (3) 병원 TCM 준비의 산업 전환의 상하이 협력 혁신 센터; (4) Shanghai University of Traditional Chinese Medicine 예산 내 연구 프로젝트(2021LK047).

이 프로토콜에 사용되는 모든 재료, 시약 및 기기에 대한 자세한 내용은 재료 표를 참조하십시오. 그림 1 은 RNA FISH에 대한 전체 프로토콜을 보여줍니다. 표 1 에는 모든 용액의 조성이 포함되어 있고 표 2 에는 이 프로토콜에 사용된 프라이머 서열이 포함되어 있습니다.
1. 프로브 준비
<ol><li>예를 들어 GenBank(https://www.ncbi.nlm.nih.gov/genbank/)에서 관심 표적 lncRNA의 FASTA 염기서열을 식별하고 획득합니다. 웹 사이트의 표시에 따라 온라인으로 ISH 프로브를 설계하고19 설계 알고리즘의 제안을 검토하여 Cy3 라벨로 주문할 프로브를 나열합니다.</li>
	<li>혼성화 완충액을 37°C에서 2시간 전에 배양합니다.</li>
	<li>4 광학 밀도(OD) 프로브를 160μL의 디에틸피로카보네이트(DEPC) 처리된 ddH2O에 1mg/mL 농도로 빛에서 멀리 떨어뜨립니다. 참고: 광학 밀도(OD)는 DNA와 RNA의 측정 단위를 나타냅니다. 일반적으로 1 OD 단위 = 33 μg/mL DNA.</li>
	<li>각 웰에 대해 200μL의 프로브 혼합물(프로브 1.2μL-10μL, 혼성화 완충액 70μL, DEPC 처리된 ddH 2O 내지 200μL로 부피 구성)을 준비하고, 50μg/mL, 25μg/mL, 12.5μg/mL 및 6μg/mL 시리즈를 설정합니다. 알림: 프로브 농도는 사전에 실험적으로 탐색해야 합니다. 높은 프로브 농도는 lncRNA의 비특이적 결합으로 이어지고, 낮은 프로브 농도는 lncRNA의 둔감하거나 검출 실패로 이어집니다.</li>
	<li>프로브 혼합물을 73°C에서 5분 동안 변성시킵니다. 참고: Cy3 표지 DNA 프로브가 이중 가닥인 경우 프로브를 95°C에서 5분 동안 단일 가닥 DNA로 변성시킨 다음 얼음에서 2분 동안 빠르게 냉각합니다.</li>
</ol>2. 세포 준비
<ol><li>12웰 세포 배양 플레이트의 멸균 유리 커버슬립에 웰당 50,000개의 143B 세포를 파종하고 DMEM에서 24시간(37°C, 5% CO2) 동안 배양합니다. 참고: 여기에 파종된 특정 세포 수는 세포 크기에 따라 다르며, 이는 파종된 세포가 24시간 동안 배양된 후 50%의 합류점에 도달하는 데 적합합니다. 멸균 유리 커버슬립은 12웰 플레이트에 적합하도록 둥글게 되어 있습니다.</li>
	<li>배지를 제거하고 2x 인산염 완충 식염수(PBS)로 5 x 1분 동안 세척합니다. 참고: DEPC 처리된 ddH2O로 PBS 1개를 준비합니다. DEPC 처리가 없는 PBS는 RNase를 함유하고 있기 때문에 사용할 수 없습니다. RNase가 없는 조건에서 다음 단계를 모두 수행합니다.</li>
	<li>1x PBS를 제거하고 각 웰에 200μL의 100% 에탄올을 추가하여 실온에서 15분 동안 고정합니다.</li>
	<li>에탄올을 제거하고 각 웰에 200μL의 0.1% Triton X-100(1x PBS 기준)을 넣고 실온에서 15분 동안 배양합니다. 참고: 시간은 Triton X-100 투과성으로 엄격하게 제어해야 하며 너무 길어서는 안 됩니다.</li>
	<li>0.1% Triton X-100을 제거하고 1x PBS로 2 x 5분 동안 세척합니다. 참고: 여기서 프로토콜을 일시 중지해야 하는 경우 PBS를 70% 에탄올로 교체하고(100% 에탄올을 RNase가 없는 ddH2O로 희석) 샘플을 4°C에서 최대 3개월 동안 보관합니다.</li>
	<li>1x PBS를 제거하고, 200μL의 2x 식염수 나트륨(SSC) 완충액(RNase가 없는 ddH 2O로20xSSC 희석)을 각 웰에 넣고 37°C에서 30분 동안 배양합니다.</li>
	<li>2x SSC 버퍼를 제거하고 각 웰에 200μL의 70% 에탄올을 넣고 실온에서 3분 동안 배양합니다.</li>
	<li>70% 에탄올을 버리고 200μL의 85% 에탄올(RNase가 없는 ddH2O로 100% 에탄올 희석)을 각 웰에 넣고 실온에서 3분 동안 배양합니다.</li>
	<li>85% 에탄올을 버리고 각 웰에 200μL의 100% 에탄올을 넣고 실온에서 3분 동안 배양합니다.</li>
	<li>100% 에탄올을 흡수하고 버리십시오. 우물을 말리십시오.</li>
</ol>3. 형광 in situ hybridization (FISH)
<ol><li>200μL의 프로브 혼합물(1.5단계에서와 같이 변성됨)을 각 웰에 추가하고 37°C에서 밤새 배양합니다(16-18시간). 알림: 혼성화 온도와 프로브 농도 사이에는 강한 양의 상관 관계가 있습니다. 따라서 배경을 최적화하려면 혼성화 온도와 프로브 농도를 모두 줄여야 합니다.</li>
	<li>다음날 37 °C에서 샘플을 꺼내 프로브 혼합물을 폐기합니다. 각 웰에 0.4x SSC/0.3% Tween-20 완충액 200μL를 추가하고(65°C에서 예열) 실온에서 2분 동안 세척합니다.</li>
	<li>0.4x SSC/0.3% Tween-20 완충액을 제거하고 각 웰에 2x SSC/0.1% Tween-20 완충액 200μL를 추가한 다음 실온에서 2분 동안 세척합니다.</li>
	<li>2x SSC/0.1% 트윈-20 버퍼를 제거하고 200μL의 4',6-디아미디노-2-페닐린돌(DAPI) 염색 용액(1μg/mL)을 추가하고 빛을 피해 20분 동안 염색합니다.</li>
	<li>DAPI 염료 용액을 버리고 실온에서 1x PBS로 2분 동안 세척합니다.</li>
	<li>슬라이드에 껌이 들어 있는 장착 매체 50μL를 추가하고 유리 커버슬립을 슬라이드에 올려 고정합니다. 알림: 커버슬립은 셀 면이 아래로 향하게 하여 슬라이드에 놓아야 합니다.</li>
	<li>형광 현미경으로 관찰하십시오. 알림: 형광 현미경 또는 레이저 컨포칼 현미경을 사용하십시오. 후자는 더 높은 감도와 선명도 이미징을 생성합니다.</li>
</ol>

인간 골육종 세포에서 Long Non-coding RNA 국소화를 위한 FISH

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저자는 경쟁하는 재정적 이익이 없다고 선언합니다.

이 RNA FISH 프로토콜은 세포에서 lncRNA의 국소화를 검출할 수 있을 뿐만 아니라 세포에서 다른 RNA, DNA 또는 단백질의 공동 국소화를 검출할 수 있으며, 이는 파라핀 포매 조직에서 lncRNA의 위치를 검출하는 데에도 사용할 수 있습니다. 그러나, 이러한 경우에서의 구체적인 프로토콜은 상이한데, 그 이유는 파라핀-포매 조직이 탈왁스될 필요가 있기 때문이다21. 이 실험 절차는 48웰 또...

인간 게놈에 대한 우리의 이해는 최근 전체 게놈 기술의 발전으로 크게 확장되었습니다. 인간 게놈의 약 93%는 RNA로 전사될 수 있지만 RNA의 2%만 단백질로 번역될 수 있습니다. 단백질 번역 기능이 없는 나머지 98%의 RNA를 non-coding RNA(ncRNA)라고 합니다1. 200개 이상의 뉴클레오티드를 포함하는 긴 ncRNA(noncoding RNA(ncRNA)의 한 종류인 긴 ncRNA(lncRNA)는 분화, 주기 조절, 세포자멸사, 이동 및 침습과 같은 세포의 많은 생리학적 및 병리학적 과정에 관여하기 때문에 점점 더 주목을 받고 있습니다 3,4,5. LncRNA는 염색질 구조 및 핵 유전자 발현 조절, mRNA 접합 과정 제어, 전사 후 변형 등 다양한 메커니즘을 통해 역할을 수행합니다6. LncRNA는 전사 및 전사 후 수준 모두에서 악성 종양의 발생, 발달 및 전이를 조절합니다. 전사 조절은 염색체 구조에 대한 결합을 통해 RNA 전사에 영향을 미침으로써 핵에서 실현되는 반면, 전사 후 조절은 내인성 경쟁 RNA(ceRNA) 메커니즘을 통해 표적 유전자를 제어하여 세포질에서 실현됩니다 5,7,8. CeRNA는 RNA 상호 작용의 새로운 메커니즘, 즉 lncRNA가 miRNA를 흡착하고 관련 표적 유전자의 miRNA 매개 분해를 억제하는 스폰지 역할을 할 수 있음을 밝혔습니다9. 그러므로, lncRNAs의 subcellular 국소화에 관하여 정보는, 특정한 lncRNA가 세포질 또는 핵에서 있다는 것을, 그들의 생물학 기능을 확인하는 것을 돕도록 중요합니다.
현재 lncRNA 국소화는 주로 두 가지 방법으로 검출되는데, 하나는 핵/세포질 분획 분리 분석에 의한 것이고 다른 하나는 RNA FISH에 의한 것입니다. 전자에서는 세포질 및 핵 분획의 RNA를 각각 추출한 다음 특정 lncRNA 프라이머로 PCR 증폭을 수행하여 세포질과 핵에서 lncRNA의 비율을 검출합니다. 이 방법의 장점은 시간 효율성인 반면, 단점은 실제 lncRNA 국소화가 세포질과 핵에서 lncRNA의 상대적 비율에 의해 직접 반영되지 않는다는 것입니다. RNA FISH는 형광 염료10으로 표지한 후 lncRNA 특이적 안티센스 사슬 서열의 설계를 통해 세포에서 lncRNA 국소화를 검출할 수 있습니다. RNA FISH 분석법은 형광단 표지된 다중 올리고 프로브 세트(11), LNA 프로브(12) 및 분지형 DNA(bDNA) 프로브(13)를 포함한 프로브 기술 및 검출 방법의 발전으로 개선되었습니다. RNA FISH는 lncRNA의 국소화를 검출할 수 있을 뿐만 아니라 이중색 또는 다색 면역형광을 사용하여 다른 RNA, DNA 또는 단백질의 공동 국소화도 검출할 수 있습니다14.
이 연구에서는 RNA FISH에 의한 골육종 세포(143B)에서 lncRNA small nucleolar RNA host gene 6(SNHG6)의 상세한 세포 내 국소화 검출 프로토콜을 예로 포함했습니다. SNHG6는 성숙한 접합 형태의 600-730 뉴클레오티드 lncRNA이며 대장암, 위암, 난소 투명 세포 암종, 골육종 및 간세포 암종을 포함한 다양한 인간 암에서 새로운 종양 유전자로 확인되었습니다15,16,17,18. 연구에 따르면 SNHG6는 증식, EMT 및 자가포식과 같은 암세포의 생물학적 행동에 관여하는 것으로 확인되었으며, miRNA15,16,17에 결합(스폰지)하여 표적 유전자에 영향을 미칠 수 있는 SNHG6의 세포질 국소화를 보여주었습니다. RNA FISH에 의한 SNHG6 세포내 국소화의 상세한 검출 프로토콜이 본원에 제시되어 있다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Automatic cell counter</td>
			<td>Shanghai Simo Biological Technology Co., Ltd</td>
			<td>IC1000</td>
			<td>Counting cells</td>
		</tr>
		<tr>
			<td>Cell culture plate-12</td>
			<td>Shanghai YueNian Biotechnology Co., Ltd</td>
			<td>3513,corning</td>
			<td>Place the coverslips in the plate</td>
		</tr>
		<tr>
			<td>&#160;Cell line (143B)</td>
			<td>Cell Bank of Chinese Academy of Sciences</td>
			<td>CRL-8303</td>
			<td>osteosarcoma cancer cell line</td>
		</tr>
		<tr>
			<td>Centrifuge tube (15 mL, 50 mL)</td>
			<td>Shanghai YueNian Biotechnology Co., Ltd</td>
			<td>&#160;430790, Corning</td>
			<td>Centrifuge the cells</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Shanghai YueNian Biotechnology Co., Ltd</td>
			<td>abs7026</td>
			<td>The cells are seeded on the coverslips</td>
		</tr>
		<tr>
			<td>Cy3 label-SNHG6 DNA probe</td>
			<td>Shanghai GenePharma Co.,Ltd</td>
			<td>A10005</td>
			<td>Detect SNHG6 location</td>
		</tr>
		<tr>
			<td>DMEM media</td>
			<td>Shanghai YueNian Biotechnology Co., Ltd</td>
			<td>LM-E1141</td>
			<td>Cell culture medium</td>
		</tr>
		<tr>
			<td>Dry Bath Incubator</td>
			<td>Haimen Kylin-Bell Lab Instruments Co.,Ltd.</td>
			<td>DKT200-2&#160;</td>
			<td>Incubation at different high temperatures</td>
		</tr>
		<tr>
			<td>Ethanol 100%&#160;</td>
			<td>Sinopharm Chemical ReagentCo., Ltd</td>
			<td>10009218</td>
			<td>dehydration</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Shanghai Waihai Biotechnology Co., LTD</td>
			<td>Olympus BX43 equipped with a camera of Olympus U-TV0.5XC-3(SN:5J01719),olympus</td>
			<td>Observation and positioning</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Shanghai Yiheng Scientific Instrument Co., LTD</td>
			<td>DHP-9051</td>
			<td>The samples were incubated at 37 &#176;C.</td>
		</tr>
		<tr>
			<td>Mounting Medium</td>
			<td>Sangon Biotech (Shanghai) Co., Ltd.</td>
			<td>E675004</td>
			<td>Attach the coverslips to the slide</td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>Haimen Kylin-Bell Lab Instruments Co.,Ltd.</td>
			<td>TS-8S</td>
			<td>Washing sample</td>
		</tr>
		<tr>
			<td>Slide</td>
			<td>Shanghai YueNian Biotechnology Co., Ltd</td>
			<td>188105</td>
			<td>The coverslips is placed on the slide</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sangon Biotech (Shanghai) Co., Ltd.</td>
			<td>A600198</td>
			<td>Permeable membrane and nuclear membrane</td>
		</tr>
		<tr>
			<td>&#160;Trypsin (0.25%)</td>
			<td>Shanghai YueNian Biotechnology Co., Ltd</td>
			<td>25200056, Gibco</td>
			<td>trypsin treatment of cells</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sangon Biotech (Shanghai) Co., Ltd.</td>
			<td>A600560</td>
			<td>detergent</td>
		</tr>
	</tbody>
</table>

rna fluorescence in situ hybridization for long non-coding rna localization in human osteosarcoma cells

암세포의 증식, 상피-중간엽 전이(EMT), 이동, 침투 및 자가포식을 조절하는 것과 같은 암에서 긴 비암호화 RNA(lncRNA)의 중요한 역할이 연구되었습니다. 세포에서 lncRNA의 국소화 검출은 세포의 기능에 대한 통찰력을 제공할 수 있습니다. 형광 염료로 표지한 후 lncRNA 특이적 안티센스 사슬 서열을 설계함으로써 RNA 형광 in situ hybridization(FISH)을 적용하여 lncRNA의 세포 국소화를 검출할 수 있습니다. 현미경 검사법의 발달과 함께, RNA 물고기 기술은 지금 잘 표현되지 않는 lncRNAs의 구상 조차 허용합니다. 이 방법은 lncRNA의 국소화를 단독으로 검출할 수 있을 뿐만 아니라 이중색 또는 다색 면역형광을 사용하여 다른 RNA, DNA 또는 단백질의 공동 국소화도 검출할 수 있습니다. 여기에서는 인간 골육종 세포(143B)에서 lncRNA small nucleolar RNA host gene 6 (SNHG6)을 예로 들어 RNA FISH의 상세한 실험 조작 절차 및 주의사항을 포함하여, RNA FISH 실험, 특히 lncRNA FISH를 수행하고자 하는 연구자들에게 참고자료를 제공하였다.

인간 골육종 세포에서 SNHG6 FISH의 대표적인 이미지가 표시됩니다(그림 2). 네거티브 컨트롤은 네거티브 Ctrl 프로브로 처리됩니다. 양성 대조군은 U6 프로브 20으로 처리됩니다. SNHG6 프로브와 U6 프로브는 적색 형광을 방출하는 Cy3로 표지되어 있습니다. DAPI는 청색 형광을 방출하는 DNA를 염색하는 염료입니다. 이 결과는 SNHG6가 주로 세포질에 국한되어 있음을 ?...

Watch this Scientific Journal Video about RNA FISH for Locating lncRNA-SNHG6 in Osteosarcoma Cells at JoVE.com

저자 스포트라이트: 골육종 세포에서 lncRNA-SNHG6를 찾기 위한 RNA FISH 

본 프로토콜은 인간 골육종 세포에서 lncRNA를 국소화하기 위한 RNA 형광 in situ 교잡의 방법을 기술한다.

인간 골육종 세포에서 긴 비코딩 RNA 국소화를 위한 RNA Fluorescence In Situ Hybridization

The important roles of long non-coding RNAs (lncRNAs) in cancer have been studied, such as regulating the proliferation, epithelial-mesenchymal transition ...

우리 연구의 범위는 RNA FISH 실험을 수행하려는 과학자들에게 참조를 제공하는 상세한 RNA FISH 프로토콜을 제공하는 것입니다. 우리의 연구는 RNA FISH가 인간 골육종 세포에서 lncRNA를 국소화하는 데 사용될 수 있음을 확인했습니다. 당사의 프로토콜은 인간 골육종 세포에서 lncRNA-SNHG6를 참조로 사용하여 RNA FISH에 대한 포괄적인 실험 절차와 예방 조치를 제공했습니다.

rna-fluorescence-situ-hybridization-for-long-non-coding-rna

Research

JoVE Journal

Cancer Research

RNA Fluorescence In Situ Hybridization for Long Non-Coding RNA Localization in Human Osteosarcoma Cells

1.3K 조회수.  Shanghai University of Traditional Chinese Medicine. 우리 연구의 범위는 RNA FISH 실험을 수행하려는 과학자들에게 참조를 제공하는 상세한 RNA FISH 프로토콜을 제공하는 것입니다. 우리의 연구는 RNA FISH가 인간 골육종 세포에서 lncRNA를 국소화하는 데 사용될 수 있음을 확인했습니다. 당사의 프로토콜은 인간 골육종 세포에서 lncRNA-SNHG6를 참조로 사용하여 RNA FISH에 대한 포괄적인 실험 절차와 예방 조치를 제공했습니다.

비디오: 저자 스포트라이트: 골육종 세포에서 lncRNA-SNHG6를 찾기 위한 RNA FISH 

Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains poor when metastasis has occurred. The Cancer Stem Cell (CSC) theory holds that there is a subset of tumor cells within the tumor that have stem-like characteristics, including the capacity to maintain the tumor and to resist multidrug chemotherapy. Therefore, a better understanding of OS biology and pathogenesis is needed in order to advance the development of targeted therapies to eradicate this particular subset and to reduce morbidity and mortality among patients. Isolating CSCs, establishing cell cultures of CSCs, and studying their biology are important steps to improving our understanding of OS biology and pathogenesis. The establishment of human-derived OS-CSCs from biopsies of OS has been made possible using several methods, including the capacity to create 3-dimensional stem cell cultures under nonadherent conditions. Under these conditions, CSCs are able to create spherical floating colonies formed by daughter stem cells; these colonies are termed &#34;cellular spheres&#34;. Here, we describe a method to establish CSC cultures from primary cell cultures of conventional OS obtained from OS biopsies. We clearly describe the several passages required to isolate and characterize CSCs.

The presence of cancer stem cells (CSCs) in bone sarcomas has recently been linked to their pathogenesis. In this article, we present the isolation of CSCs from primary cell cultures obtained from human biopsies of conventional osteosarcoma (OS) using the ability of CSCs to grow under nonadherent conditions.

암의 설립은 인간 기존의 골육종에서 세포 배양 줄기

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains ...

연구

암 연구학

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance can be caused by a range of mechanisms, including increased drug elimination, decreased drug uptake, drug inactivation and alterations of drug targets. Recent data showed that other than by well-known genetic (mutation, amplification) and epigenetic (DNA hypermethylation, histone post-translational modification) modifications, drug resistance mechanisms might also be regulated by splicing aberrations. This is a rapidly growing field of investigation that deserves future attention in order to plan more effective therapeutic approaches. The protocol described in this paper is aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of several in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes. In particular, we evaluated the differential splicing of DDX5 and PKM transcripts. The aberrant splicing detected by the computational tool MATS was validated in leukemic cells, showing that different DDX5 splice variants are expressed in the parental vs. resistant cells. In these cells, we also observed a higher PKM2/PKM1 ratio, which was not detected in the Panc-1 gemcitabine-resistant counterpart compared to parental Panc-1 cells, suggesting a different mechanism of drug-resistance induced by gemcitabine exposure.

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Here we describe a protocol aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of parental and resistant in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes.

에 약물 저항 관련 소설 스플 라이스 변종을 감지하는 RNA 시퀀싱을 사용하여<em&gt; 체외</em&gt; 암 모델

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance ...

Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung colonization in osteosarcoma (OS) by fluorescence microscopy. This article provides a detailed description of the protocol, and discusses examples of obtaining image data on metastatic growth using widefield or confocal fluorescence microscopy platforms. The flexibility of the PuMA model permits researchers to study not only the growth of OS cells in the lung microenvironment, but also to assess the effects of anti-metastatic therapeutics over time. Confocal microscopy allows for unprecedented, high-resolution imaging of OS cell interactions with the lung parenchyma. Moreover, when the PuMA model is combined with fluorescent dyes or fluorescent protein genetic reporters, researchers can study the lung microenvironment, cellular and subcellular structures, gene function, and promoter activity in metastatic OS cells. The PuMA model provides a new tool for osteosarcoma researchers to discover new metastasis biology and assess the activity of novel anti-metastatic, targeted therapies.

The goal of this article is to provide a detailed description of the protocol for the pulmonary metastasis assay (PuMA). This model permits researchers to study metastatic osteosarcoma (OS) cell growth in lung tissue using a widefield fluorescence or confocal laser-scanning microscope.

폐 전이 분석 결과 사용 하 여 다리에 전이성 폐 식민지 공부에서 실용적인 고려 사항

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung ...

Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells

The in vitro RNA-pulldown is still largely used in the first steps of protocols aimed at identifying RNA-binding proteins that recognize specific RNA structures and motifs. In this RNA-pulldown protocol, commercially synthesized RNA probes are labeled with a modified form of biotin, desthiobiotin, at the 3' terminus of the RNA strand, which reversibly binds to streptavidin and thus allows elution of proteins under more physiological conditions. The RNA-desthiobiotin is immobilized through interaction with streptavidin on magnetic beads, which are used to pull down proteins that specifically interact with the RNA of interest. Non-denatured and active proteins from the cytosolic fraction of mesothelioma cells are used as the source of proteins. The method described here can be applied to detect the interaction between known RNA binding proteins and a 25-nucleotide (nt) long RNA probe containing a sequence of interest. This is useful to complete the functional characterization of stabilizing or destabilizing elements present in RNA molecules achieved using a reporter vector assay.

Desthiobiotin labeling of a synthetic 25-nucleotide RNA oligo, which contains an adenine-rich element (ARE) motif, allows specific binding of cytosolic ARE-binding protein.

Desthiobiotin Streptavidin 선호도 중재 Mesothelioma 세포에서 RNA 상호 작용 단백질의 정화

The in vitro RNA-pulldown is still largely used in the first steps of protocols aimed at identifying RNA-binding proteins that recognize specific RNA ...

Modeling Osteosarcoma Using Li-Fraumeni Syndrome Patient-derived Induced Pluripotent Stem Cells

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including osteosarcoma--one of the most frequent primary non-hematologic malignancies in the childhood and adolescence. Therefore, LFS provides an ideal model to study this malignancy. Taking advantage of iPSC methodologies, LFS-associated osteosarcoma can be successfully modeled by differentiating LFS patient iPSCs to mesenchymal stem cells (MSCs), and then to osteoblasts--the cells of origin for osteosarcomas. These LFS osteoblasts recapitulate oncogenic properties of osteosarcoma, providing an attractive model system for delineating the pathogenesis of osteosarcoma. This manuscript demonstrates a protocol for the generation of iPSCs from LFS patient fibroblasts, differentiation of iPSCs to MSCs, differentiation of MSCs to osteoblasts, and in vivo tumorigenesis using LFS osteoblasts. This iPSC disease model can be extended to identify potential biomarkers or therapeutic targets for LFS-associated osteosarcoma.

Here, we present a protocol for the generation of induced pluripotent Stem Cells (iPSCs) from Li-Fraumeni Syndrome (LFS) patient derived fibroblasts, differentiation of iPSCs via mesenchymal stem cells (MSCs) to osteoblasts, and modeling in vivo tumorigenesis using LFS patient-derived osteoblasts.

Li Fraumeni 증후군 환자에서 파생 된 유도 만능 줄기 세포를 사용 하 여 다리를 모델링

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including ...

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

다리에 대 한 3 차원 뼈 세포 외 매트릭스 모델

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

Sequencing Small Non-coding RNA from Formalin-fixed Tissues and Serum-derived Exosomes from Castration-resistant Prostate Cancer Patients

Ablation of androgen receptor (AR) signaling by androgen deprivation is the goal of the first line of therapy for prostate cancer that initially results in cancer regression. However, in a significant number of cases, the disease progresses to advanced, castration-resistant prostate cancer (CRPC), which has limited therapeutic options and is often aggressive. Distant metastasis is mostly observed at this stage of the aggressive disease. CRPC is treated by a second generation of AR pathway inhibitors that improve survival initially, followed by the emergence of therapy resistance. Neuroendocrine prostate cancer (NEPC) is a rare variant of prostate cancer (PCa) that often develops as a result of therapy resistance via a transdifferentiation process known as neuroendocrine differentiation (NED), wherein PCa cells undergo a lineage switch from adenocarcinomas and show increased expression of neuroendocrine (NE) lineage markers. In addition to the genomic alterations that drive the progression and transdifferentiation to NEPC, epigenetic factors and microenvironmental cues are considered essential players in driving disease progression. This manuscript provides a detailed protocol to identify the epigenetic drivers (i.e., small non-coding RNAs) that are associated with advanced PCa. Using purified microRNAs from formalin-fixed paraffin-embedded (FFPE) metastatic tissues and corresponding serum-derived extracellular vesicles (EVs), the protocol describes how to prepare libraries with appropriate quality control for sequencing microRNAs from these sample sources. Isolating RNA from both FFPE and EVs is often challenging because most of it is either degraded or is limited in quantity. This protocol will elaborate on different methods to optimize the RNA inputs and cDNA libraries to yield most specific reads and high-quality data upon sequencing.

Therapy resistance often develops in patients with advanced prostate cancer, and in some cases, cancer progresses to a lethal subtype called neuroendocrine prostate cancer. Assessing the small non-coding RNA-mediated molecular changes that facilitate this transition would allow better disease stratification and identification of causal mechanisms that lead to development of neuroendocrine prostate cancer.

거세 저항하는 전립선암 환자에게서 포르말린 고정 조직 및 혈청 유래 엑소좀에서 작은 비코딩 RNA를 시퀀싱

Ablation of androgen receptor (AR) signaling by androgen deprivation is the goal of the first line of therapy for prostate cancer that initially results ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

인간 유방 상피 세포에 있는 종양원성 변환의 시험관 내 평가

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

Three Differential Expression Analysis Methods for RNA Sequencing: limma, EdgeR, DESeq2

RNA sequencing (RNA-seq) is one of the most widely used technologies in transcriptomics as it can reveal the relationship between the genetic alteration and complex biological processes and has great value in diagnostics, prognostics, and therapeutics of tumors. Differential analysis of RNA-seq data is crucial to identify aberrant transcriptions, and limma, EdgeR and DESeq2 are efficient tools for differential analysis. However, RNA-seq differential analysis requires certain skills with R language and the ability to choose an appropriate method, which is lacking in the curriculum of medical education.
Herein, we provide the detailed protocol to identify differentially expressed genes (DEGs) between cholangiocarcinoma (CHOL) and normal tissues through limma, DESeq2 and EdgeR, respectively, and the results are shown in volcano plots and Venn diagrams. The three protocols of limma, DESeq2 and EdgeR are similar but have different steps among the processes of the analysis. For example, a linear model is used for statistics in limma, while the negative binomial distribution is used in edgeR and DESeq2. Additionally, the normalized RNA-seq count data is necessary for EdgeR and limma but is not necessary for DESeq2.
Here, we provide a detailed protocol for three differential analysis methods: limma, EdgeR and DESeq2. The results of the three methods are partly overlapping. All three methods have their own advantages, and the choice of method only depends on the data.

A detailed protocol of differential expression analysis methods for RNA sequencing was provided: limma, EdgeR, DESeq2.

RNA 시퀀싱을 위한 3개의 차동 발현 분석 방법: 림마, EdgeR, DESeq2

RNA sequencing (RNA-seq) is one of the most widely used technologies in transcriptomics as it can reveal the relationship between the genetic alteration ...

Fluorescence Microscopy for ATP Internalization Mediated by Macropinocytosis in Human Tumor Cells and Tumor-xenografted Mice

Adenosine triphosphate (ATP), including extracellular ATP (eATP), has been shown to play significant roles in various aspects of tumorigenesis, such as drug resistance, epithelial-mesenchymal transition (EMT), and metastasis. Intratumoral eATP is 103 to 104 times higher in concentration than in normal tissues. While eATP functions as a messenger to activate purinergic signaling for EMT induction, it is also internalized by cancer cells through upregulated macropinocytosis, a specific type of endocytosis, to perform a wide variety of biological functions. These functions include providing energy to ATP-requiring biochemical reactions, donating phosphate groups during signal transduction, and facilitating or accelerating gene expression as a transcriptional cofactor. ATP is readily available, and its study in cancer and other fields will undoubtedly increase. However, eATP study remains at an early stage, and unresolved questions remain unanswered before the important and versatile activities played by eATP and internalized intracellular ATP can be fully unraveled.
These authors&#39; laboratories&#39; contributions to these early eATP studies include microscopic imaging of non-hydrolysable fluorescent ATP, coupled with high- and low-molecular weight fluorescent dextrans, which serve as macropinocytosis and endocytosis tracers, as well as various endocytosis inhibitors, to monitor and characterize the eATP internalization process. This imaging modality was applied to tumor cell lines and to immunodeficient mice, xenografted with human cancer tumors, to study eATP internalization in vitro and in vivo. This paper describes these in vitro and in vivo protocols, with an emphasis on modifying and finetuning assay conditions so that the macropinocytosis-/endocytosis-mediated eATP internalization assays can be successfully performed in different systems.

We developed a reproducible method to visualize the internalization of nonhydrolyzable fluorescent adenosine triphosphate (ATP), an ATP surrogate, with high cellular resolution. We validated our method using independent in vitro and in vivo assays-human tumor cell lines and immunodeficient mice xenografted with human tumor tissue.

인간 종양 세포 및 종양-xenografted 마우스에 있는 매크로 피노세포증에 의해 중재된 ATP 내재화를 위한 형광 현미경 검사법

Adenosine triphosphate (ATP), including extracellular ATP (eATP), has been shown to play significant roles in various aspects of tumorigenesis, such as ...