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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.
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.
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....
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 .......
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.......
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 .......
This work was supported by the National Natural Science Foundation of China (82160550).
....Name | Company | Catalog Number | Comments |
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 |
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