determining the cell viability of tmem200a sirna transfected hgc-27 cell lines using cck-8 assays

TMEM200A: Um novo oncogene que regula EMT e PI3K/AKT no câncer gástrico

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. Therefore, there is an urgent need for identifying new biomarkers for screening and treating early-stage cancers5.
Membrane proteins are proteins that can bind and integrate into cells and organelle membranes6. These can be grouped into three categories depending on the strength of binding to the membrane and their location: lipid-anchored proteins, integral proteins, and peripheral membrane proteins7,8. A transmembrane (TMEM) protein is an integral membrane protein that consists of at least one transmembrane segment9, which passes either completely or partially through the biological membrane. 
Although the mechanisms of action of proteins belonging to the TMEM family are not well understood, these proteins are known to be involved in several types of cancers10. Several TMEM proteins are associated with migratory, proliferative, and invasive phenotypes, and their expression is often associated with a patient's prognosis11. Therefore, TMEM family members have become the target of research. A comprehensive review of existing reports on TMEM revealed that they are mostly associated with inter- and intracellular signaling12, immune-related diseases, and tumorigenesis10. Many TMEMs also possess important physiological functions, for example, ion channels in the plasma membrane, activation of signal transduction pathways, as well as the mediation of cell chemotaxis, adhesion, apoptosis, and autophagy10. Therefore, we hypothesized that TMEM proteins may be important prognostic markers in the detection and treatment of tumors.
TMEM200A expression is significantly elevated in gastric cancer (GC). Higher expression of TMEM200A13, which has eight exons and a full length of 77.536 kb on chromosome 6q23.1, has been linked to a poor prognosis for overall survival (OS) in cases of GC. Yet the changes in its expression have rarely been reported in oncology studies. This article compares and analyzes the usefulness of TMEM200A as a therapeutic target and tumor diagnostic marker in various cancer studies using different publicly available datasets. We assessed the effectiveness of TMEM200A as a pan-cancer diagnostic and prognostic biomarker as well as its expression levels in various human cancer types using RNA-seq data from the UCSC Xena and TCGA databases, as well as by real-time quantitative polymerase chain reaction (qRT-PCR) and western blotting. 
The effect of TMEM200A expression levels on mutation rates, regulatory processes, tumor diagnosis and prognosis, immune infiltration, and immunotherapy was further investigated using a mix of computational tools and dataset websites. CBioPortal and the Catalog of Somatic Mutations in Cancer Cells (COSMIC) databases were used to examine mutations in TMEM200A. Sangerbox and TISIDB websites were utilized to understand how TMEM200A influences immune infiltration. The Tumor Immune Single Cell Center (TISCH) online tool and CancerSEA database were used to investigate the function of TMEM200A. Finally, to assess the impact of TMEM200A on the malignant behavior and tumor development function of GC cells, a loss-of-function experiment was conducted in an in vitro assay. Additionally, western blotting was performed to assess how TMEM200A knockdown affected the PI3K/AKT signaling pathway and the epithelial-mesenchymal transition (EMT) in GC.

Autor em destaque: Revelando marcadores relacionados à família de proteínas transmembranares no câncer gástrico e implicações para terapias direcionadas 

Análise Multiômica de TMEM200A como Biomarcador Pan-Câncer

Watch this Scientific Journal Video about Determining the Cell Viability of TMEM200A siRNA Transfected HGC-27 Cell Lines Using CCK-8 Assays at JoVE.com

Determining the Cell Viability of TMEM200A siRNA Transfected HGC-27 Cell Lines Using CCK-8 Assays  

Determinação da Viabilidade Celular de TMEM200A Linhagens Celulares de HGC-27 Transfectadas com SiRNA Usando Ensaios CCK-8

Comece semeando células HGC-27 em boas condições de crescimento em placas de 96 poços. Semeando as células transfectadas em placas de 96 poços divididas em grupos controle negativo e TMEM200A siRNA. Incubar as células a 37 graus Celsius.Após intervalos de tempo específicos, adicione o reagente CCK-8 a cada poço e incube a 37 graus Celsius por duas horas. Finalmente, meça a absorbância a 450 nanômetros usando um rotulador enzimático multifuncional. Os ensaios CCK-8 mostraram que TMEM200A grupos knockdown apresentaram uma queda notável na viabilidade celular em comparação com o grupo controle negativo.

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Research

JoVE Journal

Cancer Research

Determining the Cell Viability of TMEM200A siRNA Transfected HGC-27 Cell Lines Using CCK-8 Assays

271 visualizações. Comece semeando células HGC-27 em boas condições de crescimento em placas de 96 poços. Semeando as células transfectadas em placas de 96 poços divididas em grupos controle negativo e TMEM200A siRNA. Incubar as células a 37 graus Celsius.Após intervalos de tempo específicos, adicione o reagente CCK-8 a cada poço e incube a 37 graus Celsius por duas horas. Finalmente, meça a absorbância a 450 nanômetros usando um rotulador enzimático multifuncional. Os ensaios CCK-8 mostrar...

Vídeo: Determining the Cell Viability of TMEM200A siRNA Transfected HGC-27 Cell Lines Using CCK-8 Assays  

Multiomics Analysis of TMEM200A as a Pan-Cancer Biomarker

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.

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 ...