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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Here, a protocol is presented in which multiple bioinformatic tools are combined to study the biological functions of TMEM200A in cancer. In addition, we also experimentally validate the bioinformatics predictions.

Abstract

The transmembrane protein, TMEM200A, is known to be associated with human cancers and immune infiltration. Here, we assessed the function of TMEM200A in common cancers by multiomics analysis and used in vitro cell cultures of gastric cells to verify the results. The expression of TMEM200A in several human cancer types was assessed using the RNA-seq data from the UCSC Xena database. Bioinformatic analysis revealed a potential role of TMEM200A as a diagnostic and prognostic biomarker.

Cultures of normal gastric and cancer cell lines were grown and TMEM200A was knocked down. The expression levels of TMEM200A were measured by using quantitative real-time polymerase chain reaction and western blotting. In vitro loss-of-function studies were then used to determine the roles of TMEM200A in the malignant behavior and tumor formation of gastric cancer (GC) cells. Western blots were used to assess the effect of the knockdown on epithelial-mesenchymal transition (EMT) and PI3K/AKT signaling pathway in GC. Bioinformatic analysis showed that TMEM200A was expressed at high levels in GC.

The proliferation of GC cells was inhibited by TMEM200A knockdown, which also decreased vimentin, N-cadherin, and Snai proteins, and inhibited AKT phosphorylation. The PI3K/AKT signaling pathway also appeared to be involved in TMEM200A-mediated regulation of GC development. The results presented here suggest that TMEM200A regulates the tumor microenvironment by affecting the EMT. TMEM200A may also affect EMT through PI3K/AKT signaling, thus influencing the tumor microenvironment. Therefore, in pan-cancers, especially GC, TMEM200A may be a potential biomarker and oncogene.

Introduction

Cancer has emerged as a persistent public health issue endangering human health globally1due to its high morbidity and mortality rates worldwide, posing a heavy financial and medical burden to society2. Significant advancements in cancer therapy have been achieved in recent years thanks to the discovery of cancer markers3, and researchers have developed novel diagnostic methods and new drugs to treat cancer. However, some patients with cancer still have poor prognoses because of factors such as medication resistance, side effects of drugs, and chemical sensitivity4. Therefo....

Protocol

1. The Cancer Genome Atlas (TCGA) database

NOTE: The Cancer Genome Atlas (TCGA) database contains the sequencing data of genes in different tumor tissues14. RNA-seq data in TCGA for the study of TMEM200A transcripts per part per million (TPM) formats were extracted from the UCSC Xena website15 (https://xenabrowser. net/datapages/) and log2 transformed for comparing the expressions between .......

Representative Results

Expression of TMEM200A in various cancers
As illustrated in Figure 1, we first analyzed the differential expression levels of TMEM200A in various cancers through different databases. TMEM200A expression was elevated in cholangiocarcinoma (CHOL), head and neck squamous cell carcinoma (HNSC), renal clear cell carcinoma (KIRC), renal papillary cell carcinoma (KIRP), hepatocellular carcinoma (LIHC), STAD, and thyroid carcinoma (T.......

Discussion

TMEM200A belongs to a family of TMEMs that is essential for cancer cells to proliferate38. The variable expression of TMEM200A in different malignancies has received less attention, and a thorough pan-cancer investigation is lacking. However, evidence continues to accumulate, showing that the TMEM transmembrane protein family may be important in keeping cancer cells malignant through interactions with several proteins, for example, activation of TMEM16A Ca2+-activated .......

Acknowledgements

This work was supported by the National Natural Science Foundation of China (82160550).

....

Materials

NameCompanyCatalog NumberComments
Anti-AKT antibodyProteintech Group, Inc60203-2-Ig
Anti-E-cadherin antibodyProteintech Group, Inc20874-1-AP
anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodyProteintech Group, Inc10494-1-AP
Anti-N-cadherin antibodyProteintech Group, Inc22018-1-AP
Anti-P-AKT antibodyProteintech Group, Inc66444-1-Ig
Anti-snail antibodyProteintech Group, Inc13099-1-AP
Anti-Vimentin antibodyProteintech Group, Inc10366-1-AP
AxyPrepMultisourceTotalRNAMini-
prep Kit
Suzhou Youyi Landi Biotechnology Co., LtdUEL-UE-MN-MS-RNA-50G
BCA Protein Assay KitEpizyme BiotechZJ101L
CCK-8 reagentMedChemExpressHY-K0301-500T
Fetal bovine serum (FBS)CYAGEN BIOSCIENCES (GUANGZHOU) INCFBSSR-01021
GAPDH primerSangon Biotech (Shanghai) Co., Ltd.Forward primer (5’-3’): TGACATCAAGAAGGTG
GTGAAGCAG; Reverse primer (5’-3’): GTGTCGCTGTTGAAG
TCAGAGGAG
HighGene plus Transfection reagentABclonalRM09014P
HRP-conjugated Affinipure Goat Anti-Mouse lgG (H+L)Proteintech Group, IncSA00001-1
HRP-conjugated Affinipure Goat Anti-Rabbit lgG (H+L)Proteintech Group, IncSA00001-2
Human gastric mucosal epithelial GES-1 cellsGuangzhou Cellcook Biotech Co.,Ltd.
Human STAD HGC-27 cellsProcell Life Science&Technology Co.,Ltd
Human STAD SGC-7901 cellsProcell Life Science&Technology Co.,Ltd
MonAmp SYBR Green qPCR Mix (None ROX)Mona (Suzhou) Biotechnology Co., LtdMQ10101S
MonScript RTIII All-in-One Mix with dsDNase  Mona (Suzhou) Biotechnology Co., LtdMR05101M
Omni-ECL Femto Light Chemiluminescence KitEpizyme BiotechSQ201
PAGE Gel Fast Preparationb Kit Epizyme BiotechPG111
Penicillin-streptomycin (Pen-Strep)Beijing Solarbio Science & Technology Co.,LtdP1400-100
Polyvinylidene difluoride (PVDF) membraneMerck KGaAIPVH00010-1
Protein Free Rapid Blocking BufferEpizyme BiotechPS108P
RIPA lysis solutionBeijing Solarbio Science & Technology Co., LtdR0010
RPMI 1640 complete mediumThermo Fisher ScientificC11875500BT
Skimmed milkCampina: Elk
TBST buffer solutionBeijing Solarbio Science & Technology Co., LtdT1082
The protein loading bufferEpizyme BiotechLT101S
TMEM200A knockdown plasmidMiaoLing Plasmid
TMEM200A primerSangon Biotech (Shanghai) Co., Ltd.Forward primer (5’-3’): AAGGCGGTGTGGTGGTTCG; Reverse primer (5’-3’): GATTTTGGTCTCTTTGTCACGGTT
TMEM200A SiRNA1MiaoLing PlasmidForward primer (5’-3’): ACAACTGATGATAAGACCAG; Reverse primer (5’-3’): TGTTGACTACTATTCTGGTC
TMEM200A SiRNA2MiaoLing PlasmidForward primer (5’-3’): CGTGTGAATGTCAATGACTG; Reverse primer (5’-3’): GCACACTTACAGTTACTGAC
TMEM200A SiRNA3MiaoLing PlasmidForward primer (5’-3’): ACAACCACAACATCTGCCCG; Reverse primer (5’-3’): TGTTGGTGTTGTAGACGGGC
Transmembrane protein 200A AntibodyProteintech Group, Inc48081-1
Equipment
CO2 cell culture incubatorHaier GroupPYXE-80IR
Electrophoresis instrumentBio-RAD
Fluorescence quantitative PCR instrumentBio-RAD
Gel Imaging System (Tanon 5200)Tanon Science & Technology Co., LtdLAB-0002-0007-SHTN
Multifunctional Enzyme LabelerBerthold

References

  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....

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