JoVE Journal > Cancer Research
Summary
Abstract
Introduction
Protocol
Resultados
Discussion
Declarações
Acknowledgements
Materials
Referências
Tadução automática
Please note that all translations are automatically generated. Click here for the English version.
Pesquisa do Câncer

TMEM200A作为泛癌生物标志物的多组学分析

Published: September 15, 2023
doi:
10.3791/65795
, , , , ,
1Department of Histology and Embryology,Youjiang Medical University for Nationalities, 2Department of Pathophysiology,Youjiang Medical University for Nationalities

Summary

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。

Abstract

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。

Introduction

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）。

Protocol

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。 转到 UCSC Xena …

Representative Results

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?…

Discussion

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，?…

Declarações

The authors have nothing to disclose.

Acknowledgements

这项工作得到了中国国家自然科学基金（82160550）的支持。

Materials

Anti-AKT antibody Proteintech Group, Inc 60203-2-Ig
Anti-E-cadherin antibody Proteintech Group, Inc 20874-1-AP
anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody Proteintech Group, Inc 10494-1-AP
Anti-N-cadherin antibody Proteintech Group, Inc 22018-1-AP
Anti-P-AKT antibody Proteintech Group, Inc 66444-1-Ig
Anti-snail antibody Proteintech Group, Inc 13099-1-AP
Anti-Vimentin antibody Proteintech Group, Inc 10366-1-AP
AxyPrepMultisourceTotalRNAMini-
prep Kit		 Suzhou Youyi Landi Biotechnology Co., Ltd UEL-UE-MN-MS-RNA-50G
BCA Protein Assay Kit Epizyme Biotech ZJ101L
CCK-8 reagent MedChemExpress HY-K0301-500T
Fetal bovine serum (FBS) CYAGEN BIOSCIENCES (GUANGZHOU) INC FBSSR-01021
GAPDH primer Sangon Biotech (Shanghai) Co., Ltd. Forward primer (5’-3’): TGACATCAAGAAGGTG
GTGAAGCAG; Reverse primer (5’-3’): GTGTCGCTGTTGAAG
TCAGAGGAG
HighGene plus Transfection reagent ABclonal RM09014P
HRP-conjugated Affinipure Goat Anti-Mouse lgG (H+L) Proteintech Group, Inc SA00001-1
HRP-conjugated Affinipure Goat Anti-Rabbit lgG (H+L) Proteintech Group, Inc SA00001-2
Human gastric mucosal epithelial GES-1 cells Guangzhou Cellcook Biotech Co.,Ltd.
Human STAD HGC-27 cells Procell Life Science&Technology Co.,Ltd
Human STAD SGC-7901 cells Procell Life Science&Technology Co.,Ltd
MonAmp SYBR Green qPCR Mix (None ROX) Mona (Suzhou) Biotechnology Co., Ltd MQ10101S
MonScript RTIII All-in-One Mix with dsDNase   Mona (Suzhou) Biotechnology Co., Ltd MR05101M
Omni-ECL Femto Light Chemiluminescence Kit Epizyme Biotech SQ201
PAGE Gel Fast Preparationb Kit  Epizyme Biotech PG111
Penicillin-streptomycin (Pen-Strep) Beijing Solarbio Science & Technology Co.,Ltd P1400-100
Polyvinylidene difluoride (PVDF) membrane Merck KGaA IPVH00010-1
Protein Free Rapid Blocking Buffer Epizyme Biotech PS108P
RIPA lysis solution Beijing Solarbio Science & Technology Co., Ltd R0010
RPMI 1640 complete medium Thermo Fisher Scientific C11875500BT
Skimmed milk Campina: Elk
TBST buffer solution Beijing Solarbio Science & Technology Co., Ltd T1082
The protein loading buffer Epizyme Biotech LT101S
TMEM200A knockdown plasmid MiaoLing Plasmid
TMEM200A primer Sangon Biotech (Shanghai) Co., Ltd. Forward primer (5’-3’): AAGGCGGTGTGGTGGTTCG; Reverse primer (5’-3’): GATTTTGGTCTCTTTGTCACGGTT
TMEM200A SiRNA1 MiaoLing Plasmid Forward primer (5’-3’): ACAACTGATGATAAGACCAG; Reverse primer (5’-3’): TGTTGACTACTATTCTGGTC
TMEM200A SiRNA2 MiaoLing Plasmid Forward primer (5’-3’): CGTGTGAATGTCAATGACTG; Reverse primer (5’-3’): GCACACTTACAGTTACTGAC
TMEM200A SiRNA3 MiaoLing Plasmid Forward primer (5’-3’): ACAACCACAACATCTGCCCG; Reverse primer (5’-3’): TGTTGGTGTTGTAGACGGGC
Transmembrane protein 200A Antibody Proteintech Group, Inc 48081-1
Equipment
CO2 cell culture incubator Haier Group PYXE-80IR
Electrophoresis instrument Bio-RAD
Fluorescence quantitative PCR instrument Bio-RAD
Gel Imaging System (Tanon 5200) Tanon Science & Technology Co., Ltd LAB-0002-0007-SHTN
Multifunctional Enzyme Labeler Berthold
DOWNLOAD MATERIALS LIST

Referências

  1. Torre, L. A., Siegel, R. L., Ward, E. M., Jemal, A. Global cancer incidence and mortality rates and trends–an update. Cancer Epidemiol Biomarkers Prev. 25 (1), 16-27 (2016).
  2. Long, X., et al. Economic burden of malignant tumors – Yichang City, Hubei Province, China, 2019. China CDC Wkly. 4 (15), 312-316 (2022).
  3. Harbeck, N., Gnant, M. Breast cancer. Lancet. 389 (10074), 1134-1150 (2017).
  4. Bagchi, S., Yuan, R., Engleman, E. G. Immune checkpoint inhibitors for the treatment of cancer: clinical impact and mechanisms of response and resistance. Annu Rev Pathol. 16, 223-249 (2021).
  5. Lam, G. T., et al. Pitfalls in cutaneous melanoma diagnosis and the need for new reliable markers. Mol Diagn Ther. 27 (1), 49-60 (2023).
  6. Gromiha, M. M., Ou, Y. Y. Bioinformatics approaches for functional annotation of membrane proteins. Brief Bioinform. 15 (2), 155-168 (2014).
  7. Schmit, K., Michiels, C. TMEM proteins in cancer: a review. Front Pharmacol. 9, 1345 (2018).
  8. Marx, S., et al. Transmembrane (TMEM) protein family members: Poorly characterized even if essential for the metastatic process. Semin Cancer Biol. 60, 96-106 (2020).
  9. Fu, K., et al. Overexpression of transmembrane protein 168 in the mouse nucleus accumbens induces anxiety and sensorimotor gating deficit. PLoS One. 12 (12), e0189006 (2017).
  10. Cuajungco, M. P., et al. Abnormal accumulation of human transmembrane (TMEM)-176A and 176B proteins is associated with cancer pathology. Acta Histochem. 114 (7), 705-712 (2012).
  11. Zhang, S., et al. TMEM116 is required for lung cancer cell motility and metastasis through PDK1 signaling pathway. Cell Death Dis. 12 (12), 1086 (2021).
  12. Zhang, N., Pan, H., Liang, X., Xie, J., Han, W. The roles of transmembrane family proteins in the regulation of store-operated Ca(2+) entry. Cell Mol Life Sci. 79 (2), 118 (2022).
  13. Zhang, X., Zheng, P., Li, Z., Gao, S., Liu, G. The somatic mutation landscape and RNA prognostic markers in stomach adenocarcinoma. Onco Targets Ther. 13, 7735-7746 (2020).
  14. Jia, D., et al. Mining TCGA database for genes of prognostic value in glioblastoma microenvironment. Aging (Albany NY). 10 (4), 592-605 (2018).
  15. Wang, S., et al. UCSCXenaShiny: an R/CRAN package for interactive analysis of UCSC Xena data. Bioinformatics. 38 (2), 527-529 (2022).
  16. Li, T., et al. TIMER2.0 for analysis of tumor-infiltrating immune cells. Nucleic Acids Res. 48 (W1), W509-W514 (2020).
  17. Thul, P. J., et al. A subcellular map of the human proteome. Science. 356 (6340), eaal3321 (2017).
  18. Li, Y., Ge, D., Lu, C. The SMART App: an interactive web application for comprehensive DNA methylation analysis and visualization. Epigenetics Chromatin. 12 (1), 71 (2019).
  19. Chandrashekar, D. S., et al. UALCAN: An update to the integrated cancer data analysis platform. Neoplasia. 25, 18-27 (2022).
  20. Tate, J. G., et al. COSMIC: the Catalogue Of Somatic Mutations In Cancer. Nucleic Acids Res. 47 (D1), D941-D947 (2019).
  21. Gao, J., et al. Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Sci Signal. 6 (269), pl1 (2013).
  22. Shen, W., et al. Sangerbox: A comprehensive, interaction-friendly clinical bioinformatics analysis platform. iMeta. 1 (3), e36 (2022).
  23. Ru, B., et al. TISIDB: an integrated repository portal for tumor-immune system interactions. Bioinformatics. 35 (20), 4200-4202 (2019).
  24. Yuan, H., et al. CancerSEA: a cancer single-cell state atlas. Nucleic Acids Res. 47 (D1), D900-D908 (2019).
  25. Sun, D., et al. TISCH: a comprehensive web resource enabling interactive single-cell transcriptome visualization of tumor microenvironment. Nucleic Acids Res. 49 (D1), D1420-D1430 (2021).
  26. Warde-Farley, D., et al. The GeneMANIA prediction server: biological network integration for gene prioritization and predicting gene function. Nucleic Acids Res. 38, W214-W220 (2010).
  27. Zhu, Y., Feng, S., Song, Z., Wang, Z., Chen, G. Identification of immunological characteristics and immune subtypes based on single-sample gene set enrichment analysis algorithm in lower-grade glioma. Front Genet. 13, 894865 (2022).
  28. Mueller Bustin, S. A., R, Real-time reverse transcription PCR (qRT-PCR) and its potential use in clinical diagnosis. Clin Sci (Lond). 109 (4), 365-379 (2005).
  29. Sun, L., Zhang, H., Gao, P. Metabolic reprogramming and epigenetic modifications on the path to cancer. Protein Cell. 13 (12), 877-919 (2022).
  30. Ntontsi, P., Photiades, A., Zervas, E., Xanthou, G., Samitas, K. Genetics and epigenetics in asthma. Int J Mol Sci. 22 (5), 2412 (2021).
  31. Martínez-Reyes, I., Chandel, N. S. Cancer metabolism: looking forward. Nat Rev Cancer. 21 (10), 669-680 (2021).
  32. Chen, Y., et al. PremPS: Predicting the impact of missense mutations on protein stability. PLoS Comput Biol. 16 (12), e1008543 (2020).
  33. Li, M., Petukh, M., Alexov, E., Panchenko, A. R. Predicting the impact of missense mutations on protein-protein binding affinity. J Chem Theory Comput. 10 (4), 1770-1780 (2014).
  34. Hirsch, D., et al. Clinical responses to PD-1 inhibition and their molecular characterization in six patients with mismatch repair-deficient metastatic cancer of the digestive system. J Cancer Res Clin Oncol. 147 (1), 263-273 (2021).
  35. Poulogiannis, G., Frayling, I. M., Arends, M. J. DNA mismatch repair deficiency in sporadic colorectal cancer and Lynch syndrome. Histopathology. 56 (2), 167-179 (2010).
  36. Chintamani, J., et al. The expression of mismatched repair genes and their correlation with clinicopathological parameters and response to neo-adjuvant chemotherapy in breast cancer. Int Semin Surg Oncol. 4, 5 (2007).
  37. Deng, H., et al. High expression of TMEM200A is associated with a poor prognosis and immune infiltration in gastric cancer. Pathol Oncol Res. 29, 1610893 (2023).
  38. Stemmler, M. P. Cadherins in development and cancer. Mol Biosyst. 4 (8), 835-850 (2008).
  39. Bill, A., et al. ANO1/TMEM16A interacts with EGFR and correlates with sensitivity to EGFR-targeting therapy in head and neck cancer. Oncotarget. 6 (11), 9173-9188 (2015).
  40. De Las Rivas, J., et al. Cancer drug resistance induced by EMT: novel therapeutic strategies. Arch Toxicol. 95 (7), 2279-2297 (2021).
  41. Tian, S., et al. SERPINH1 regulates EMT and gastric cancer metastasis via the Wnt/β-catenin signaling pathway. Aging (Albany NY). 12 (4), 3574-3593 (2020).

Tags

Keywords: TMEM200APan-cancer BiomarkerMultiomics AnalysisGastric CancerEpithelial-mesenchymal TransitionPI3K/AKT SignalingTumor Microenvironment
check_url/pt/65795?article_type=t

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Citar este artigo
Zhang, Y., Kuang, S., Qin, H., Zhao, N., Yang, Y., Xie, J. Multiomics Analysis of TMEM200A as a Pan-Cancer Biomarker. J. Vis. Exp. (199), e65795, doi:10.3791/65795 (2023).
Baixar citação
Reimpressões e Permissões

View Video

✖

To prove you're not a robot, please enter the text in the image below

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image