Name: Unveiling Transmembrane Protein Family-Related Markers in Gastric Cancer and Implications for Targeted Therapies (Video) | JoVE
Uploaded: 2023-09-15T14:10:00
Description: Watch this Scientific Journal Video about Unveiling Transmembrane Protein Family-Related Markers in Gastric Cancer and Implications for Targeted Therapies at JoVE.com

这项工作得到了中国国家自然科学基金（82160550）的支持。

1. 癌症基因组图谱（TCGA）数据库
注：癌症基因组图谱 （TCGA） 数据库包含不同肿瘤组织中基因的测序数据14。从UCSC Xena网站15（https://xenabrowser net/datapages/）中提取TCGA中用于研究百万分之一（TPM）格式TMEM200A转录本的RNA-seq数据，并转换log2以比较样品之间的表达。
<ol><li>转到 UCSC Xena ...

TMEM200A: A Novel Oncogene Regulating EMT and PI3K/AKT in Gastric Cancer

<ol>
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TMEM200A 属于 TMEM 家族，对癌细胞增殖至关重要38。不同恶性肿瘤中 TMEM200A 的可变表达受到的关注较少，缺乏彻底的泛癌变研究。然而，越来越多的证据表明，TMEM跨膜蛋白家族可能通过与多种蛋白质的相互作用在保持癌细胞恶性方面发挥重要作用，例如，ROCK1/moesin激活TMEM16A Ca2+激活的Cl- 通道促进BRCA转移39。因此，在目前的工作中，?...

癌症已成为一个持续存在的公共卫生问题，危害全球人类健康1，因为它在世界范围内发病率和死亡率很高，给社会带来了沉重的经济和医疗负担2。近年来，由于癌症标志物3 的发现，癌症治疗取得了重大进展，研究人员开发了治疗癌症的新诊断方法和新药。然而，由于耐药性、药物副作用和化学敏感性等因素，一些癌症患者的预后仍然较差4.因此，迫切需要确定用于筛查和治疗早期癌症的新生物标志物5.
膜蛋白是可以结合并整合到细胞和细胞器膜中的蛋白质6.根据与膜结合的强度及其位置，这些可分为三类：脂质锚定蛋白、整合蛋白和外周膜蛋白 7,8。跨膜 （TMEM） 蛋白是一种完整的膜蛋白，由至少一个跨膜片段9 组成，该片段完全或部分穿过生物膜。
尽管属于TMEM家族的蛋白质的作用机制尚不清楚，但已知这些蛋白质与几种类型的癌症有关10。几种 TMEM 蛋白与迁移性、增殖性和侵袭性表型相关，它们的表达通常与患者的预后相关11。因此，TMEM家族成员成为研究的对象。对 TMEM 现有报告的全面回顾表明，它们主要与细胞间和细胞内信号转导12、免疫相关疾病和肿瘤发生10 相关。许多TMEM还具有重要的生理功能，例如质膜中的离子通道，信号转导途径的激活，以及细胞趋化性、粘附、凋亡和自噬的介导10。因此，我们假设TMEM蛋白可能是肿瘤检测和治疗的重要预后标志物。
TMEM200A 在胃癌 （GC） 中的表达显著升高。 TMEM200A13 在染色体 6q23.1 上有 8 个外显子，全长为 77.536 kb，其高表达与 GC 病例的总生存期 （OS） 预后较差有关。然而，其表达的变化在肿瘤学研究中很少报道。本文比较和分析了 TMEM200A 作为治疗靶点和肿瘤诊断标志物在各种癌症研究中的有用性，使用不同的公开数据集。我们使用来自UCSC Xena和TCGA数据库的RNA-seq数据，以及实时定量聚合酶链反应（qRT-PCR）和蛋白质印迹，评估 了TMEM200A 作为泛癌诊断和预后生物标志物的有效性及其在各种人类癌症类型中的表达水平。 
使用计算工具和数据集网站的组合进一步研究了 TMEM200A 表达水平对突变率、调控过程、肿瘤诊断和预后、免疫浸润和免疫治疗的影响。CBioPortal 和癌细胞体细胞突变目录 （COSMIC） 数据库用于检查 TMEM200A突变。利用 Sangerbox 和 TISIDB 网站来了解 TMEM200A 如何影响免疫浸润。利用肿瘤免疫单细胞中心（Tumor Immune Single Cell Center，TISCH）在线工具和CancerSEA数据库研究 TMEM200A的功能。最后，为了评估 TMEM200A 对GC细胞恶性行为和肿瘤发展功能的影响，在 体外 试验中进行了功能丧失实验。此外，还进行了蛋白质印迹分析，以评估 敲低TMEM200A 如何影响 GC 中的 PI3K/AKT 信号通路和上皮-间充质转化 （EMT）。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anti-AKT antibody</td>
			<td>Proteintech Group, Inc</td>
			<td>60203-2-Ig</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-E-cadherin antibody</td>
			<td>Proteintech Group, Inc</td>
			<td>20874-1-AP</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody</td>
			<td>Proteintech Group, Inc</td>
			<td>10494-1-AP</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-N-cadherin antibody</td>
			<td>Proteintech Group, Inc</td>
			<td>22018-1-AP</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-P-AKT antibody</td>
			<td>Proteintech Group, Inc</td>
			<td>66444-1-Ig</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-snail antibody</td>
			<td>Proteintech Group, Inc</td>
			<td>13099-1-AP</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Vimentin antibody</td>
			<td>Proteintech Group, Inc</td>
			<td>10366-1-AP</td>
			<td></td>
		</tr>
		<tr>
			<td>AxyPrepMultisourceTotalRNAMini- 
			prep Kit</td>
			<td>Suzhou Youyi Landi Biotechnology Co., Ltd</td>
			<td>UEL-UE-MN-MS-RNA-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA Protein Assay Kit</td>
			<td>Epizyme Biotech</td>
			<td>ZJ101L</td>
			<td></td>
		</tr>
		<tr>
			<td>CCK-8 reagent</td>
			<td>MedChemExpress</td>
			<td>HY-K0301-500T</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>CYAGEN BIOSCIENCES (GUANGZHOU) INC</td>
			<td>FBSSR-01021</td>
			<td></td>
		</tr>
		<tr>
			<td>GAPDH primer</td>
			<td>Sangon Biotech (Shanghai) Co., Ltd.</td>
			<td></td>
			<td>Forward primer (5&#8217;-3&#8217;): TGACATCAAGAAGGTG 
			GTGAAGCAG; Reverse primer (5&#8217;-3&#8217;): GTGTCGCTGTTGAAG 
			TCAGAGGAG</td>
		</tr>
		<tr>
			<td>HighGene plus Transfection reagent</td>
			<td>ABclonal</td>
			<td>RM09014P</td>
			<td></td>
		</tr>
		<tr>
			<td>HRP-conjugated Affinipure Goat Anti-Mouse lgG (H+L)</td>
			<td>Proteintech Group, Inc</td>
			<td>SA00001-1</td>
			<td></td>
		</tr>
		<tr>
			<td>HRP-conjugated Affinipure Goat Anti-Rabbit lgG (H+L)</td>
			<td>Proteintech Group, Inc</td>
			<td>SA00001-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Human gastric mucosal epithelial GES-1 cells</td>
			<td>Guangzhou Cellcook Biotech Co.,Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Human STAD HGC-27 cells</td>
			<td>Procell Life Science&#38;Technology Co.,Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Human STAD SGC-7901 cells</td>
			<td>Procell Life Science&#38;Technology Co.,Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MonAmp SYBR Green qPCR Mix (None ROX)</td>
			<td>Mona (Suzhou) Biotechnology Co., Ltd</td>
			<td>MQ10101S</td>
			<td></td>
		</tr>
		<tr>
			<td>MonScript RTIII All-in-One Mix with dsDNase&#160;&#160;</td>
			<td>Mona (Suzhou) Biotechnology Co., Ltd</td>
			<td>MR05101M</td>
			<td></td>
		</tr>
		<tr>
			<td>Omni-ECL Femto Light Chemiluminescence Kit</td>
			<td>Epizyme Biotech</td>
			<td>SQ201</td>
			<td></td>
		</tr>
		<tr>
			<td>PAGE Gel Fast Preparationb Kit&#160;</td>
			<td>Epizyme Biotech</td>
			<td>PG111</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin (Pen-Strep)</td>
			<td>Beijing Solarbio Science &#38; Technology Co.,Ltd</td>
			<td>P1400-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyvinylidene difluoride (PVDF) membrane</td>
			<td>Merck KGaA</td>
			<td>IPVH00010-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein Free Rapid Blocking Buffer</td>
			<td>Epizyme Biotech</td>
			<td>PS108P</td>
			<td></td>
		</tr>
		<tr>
			<td>RIPA lysis solution</td>
			<td>Beijing Solarbio Science &#38; Technology Co., Ltd</td>
			<td>R0010</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 complete medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>C11875500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>Skimmed milk</td>
			<td>Campina: Elk</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TBST buffer solution</td>
			<td>Beijing Solarbio Science &#38; Technology Co., Ltd</td>
			<td>T1082</td>
			<td></td>
		</tr>
		<tr>
			<td>The protein loading buffer</td>
			<td>Epizyme Biotech</td>
			<td>LT101S</td>
			<td></td>
		</tr>
		<tr>
			<td>TMEM200A knockdown plasmid</td>
			<td>MiaoLing Plasmid</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TMEM200A primer</td>
			<td>Sangon Biotech (Shanghai) Co., Ltd.</td>
			<td></td>
			<td>Forward primer (5&#8217;-3&#8217;): AAGGCGGTGTGGTGGTTCG; Reverse primer (5&#8217;-3&#8217;): GATTTTGGTCTCTTTGTCACGGTT</td>
		</tr>
		<tr>
			<td>TMEM200A SiRNA1</td>
			<td>MiaoLing Plasmid</td>
			<td></td>
			<td>Forward primer (5&#8217;-3&#8217;): ACAACTGATGATAAGACCAG; Reverse primer (5&#8217;-3&#8217;): TGTTGACTACTATTCTGGTC</td>
		</tr>
		<tr>
			<td>TMEM200A SiRNA2</td>
			<td>MiaoLing Plasmid</td>
			<td></td>
			<td>Forward primer (5&#8217;-3&#8217;): CGTGTGAATGTCAATGACTG; Reverse primer (5&#8217;-3&#8217;): GCACACTTACAGTTACTGAC</td>
		</tr>
		<tr>
			<td>TMEM200A SiRNA3</td>
			<td>MiaoLing Plasmid</td>
			<td></td>
			<td>Forward primer (5&#8217;-3&#8217;): ACAACCACAACATCTGCCCG; Reverse primer (5&#8217;-3&#8217;): TGTTGGTGTTGTAGACGGGC</td>
		</tr>
		<tr>
			<td>Transmembrane protein 200A Antibody</td>
			<td>Proteintech Group, Inc</td>
			<td>48081-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 cell culture incubator</td>
			<td>Haier Group</td>
			<td>PYXE-80IR</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrophoresis instrument</td>
			<td>Bio-RAD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence quantitative PCR instrument</td>
			<td>Bio-RAD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Imaging System (Tanon 5200)</td>
			<td>Tanon Science &#38; Technology Co., Ltd</td>
			<td>LAB-0002-0007-SHTN</td>
			<td></td>
		</tr>
		<tr>
			<td>Multifunctional Enzyme Labeler</td>
			<td>Berthold</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

multiomics analysis of tmem200a as a pan-cancer biomarker

TMEM200A，跨膜蛋白已知与人类癌症和免疫浸润有关。在这里，我们通过多组学分析评估了TMEM200A在常见癌症中的功能，并使用胃细胞的体外细胞培养来验证结果。使用UCSC Xena数据库中的RNA-seq数据评估了几种人类癌症类型中TMEM200A的表达。生物信息学分析揭示了TMEM200A作为诊断和预后生物标志物的潜在作用。
培养正常胃细胞系和癌细胞系的培养物，并敲除 TMEM200A 细胞系。采用实时荧光定量聚合酶链反应和蛋白质印迹法测定 TMEM200A 的表达水平。然后使用 体外 功能丧失研究来确定 TMEM200A 在胃癌 （GC） 细胞恶性行为和肿瘤形成中的作用。Western blot用于评估敲低对GC中上皮-间充质转化（EMT）和PI3K/AKT信号通路的影响。生物信息学分析表明， TMEM200A 在GC中表达水平较高。 
TMEM200A敲低抑制了GC细胞的增殖，这也降低了波形蛋白、N-钙粘蛋白和Snai蛋白，并抑制了AKT磷酸化。PI3K/AKT 信号通路似乎也参与了 TMEM200A介导的 GC 发育调节。本文提出的结果表明，TMEM200A通过影响EMT来调节肿瘤微环境。TMEM200A也可能通过PI3K/AKT信号传导影响EMT，从而影响肿瘤微环境。因此，在泛癌，尤其是GC中，TMEM200A可能是一种潜在的生物标志物和癌基因。

Multiomics Analysis of TMEM200A as a Pan-Cancer Biomarker

TMEM200A在各种癌症中的表达 如图1所示，我们首先通过不同的数据库分析了各种癌症中TMEM200A的不同表达水平。仅根据 TCGA 数据，与邻近正常组织相比，胆管癌 （CHOL）、头颈部鳞状细胞癌 （HNSC）、肾透明细胞癌 （KIRC）、肾状细胞癌 （KIRP）、肝细胞癌 （LIHC）、STAD 和甲状腺癌 （THCA） TMEM200A表达升高。然而TMEM200A，膀?...

Watch this Scientific Journal Video about Unveiling Transmembrane Protein Family-Related Markers in Gastric Cancer and Implications for Targeted Therapies at JoVE.com

Unveiling Transmembrane Protein Family-Related Markers in Gastric Cancer and Implications for Targeted Therapies (Video) | JoVE

在这里，提出了一个协议，其中结合了多种生物信息学工具来研究 TMEM200A 在癌症中的生物学功能。此外，我们还通过实验验证了生物信息学的预测。

TMEM200A作为泛癌生物标志物的多组学分析

The transmembrane protein, TMEM200A, is known to be associated with human cancers and immune infiltration. Here, we assessed the function of TMEM200A in ...

在癌症生物标志物的研究方面取得了重大进展。然而，与跨膜蛋白家族相关的肿瘤相关物质的提取功能仍未确定。因此，我们对胃癌TMEM200A的研究为相关细胞提供了新的研究方向。本研究采用在线数据库预测与实验技术验证相结合的方法，简化了相关领域的研究步骤和干细胞实验成本。未来，我们的实验室将专注于探索胃癌肿瘤的具体机制，寻找糖基化修饰与代谢记录之间的关联，并确定胃癌治疗的新靶点。