Name: Quantification of mRNA in TMEM200A siRNA Transfected Stomach Adenocarcinoma Cells
Uploaded: 2023-09-15T14:10:00
Description: Watch this Scientific Journal Video about Quantification of mRNA in TMEM200A siRNA Transfected Stomach Adenocarcinoma Cells at JoVE.com

quantification of mrna in tmem200a sirna transfected stomach adenocarcinoma cells

TMEM200A:胃がんにおけるEMTおよびPI3K/AKTを調節する新規がん遺伝子

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.

著者スポットライト:胃がんにおける膜貫通タンパク質ファミリー関連マーカーの発表と標的療法への影響 

汎がんバイオマーカーとしての TMEM200A のマルチオミクス解析

Significant advancements have been made in the investigation of cancer biomarkers. Nevertheless, the extract function of tumor-related matters associated with transmembrane protein family remains unidentified. As a result, our research on the TMEM200A in gastric cancer has generated novel research directions for related cells.In this study, we used the combination of online database prediction and experimental technique validation, which simplifies the research steps and the stem cell cost of experiment in related field. In future, our laboratory will focus on exploring the specific mechanism of tumor in gastric cancer, searching for the association between glycosylation modification and the metabolic recording, and identifying find new targets for gastric cancer treatment.

Watch this Scientific Journal Video about Quantification of mRNA in TMEM200A siRNA Transfected Stomach Adenocarcinoma Cells at JoVE.com

Quantification of mRNA in TMEM200A siRNA Transfected Stomach Adenocarcinoma Cells  

TMEM200A siRNAトランスフェクション胃腺癌細胞におけるmRNAの定量

まず、胃腺癌細胞にTMEM200A siRNAをトランスフェクションし、2日間インキュベートします。次に、ウェルから細胞培養培地を廃棄し、1ミリリットルのPBSで細胞を2回静かに洗浄します。RNA単離キットを使用して、細胞のトータルRNAを単離します。製造元の指示に従い、cDNA合成キットを使用してRNA濃度を推定し、1マイクログラムのRNAでcDNAを合成します。次に、10 マイクロリットルの反応混合物中の cDNA を 10 倍希釈した定量的 RTPCR をセットアップします。リアルタイムPCRアッセイシステムを使用して、プライマーと定量PCRマスターミックスを添加します。ここに示す定量的PCR反応条件を使用し、ダブルデルタCT技術を使用して各遺伝子の相対発現を決定します。HGC-27細胞株は、GES-1細胞株と比較してTMEM200Aタンパク質およびmRNA転写産物を劇的に過剰に発現しました。ヒト胃がん細胞SGC-7901におけるTMEM200AのmRNA発現差は、GES-1細胞よりも高い。

Research

JoVE Journal

Cancer Research

Quantification of mRNA in TMEM200A siRNA Transfected Stomach Adenocarcinoma Cells

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 ...

がん研究

Transfection of TMEM200A siRNA in Stomach Adenocarcinoma Cell Lines

Transfection of TMEM200A siRNA in Stomach Adenocarcinoma Cell Lines  

胃腺癌細胞株における TMEM200A siRNAのトランスフェクション

Protein Detection in TMEM200A siRNA Transfected Stomach Adenocarcinoma Cells Using Western Blotting

Protein Detection in TMEM200A siRNA Transfected Stomach Adenocarcinoma Cells Using Western Blotting  

ウェスタンブロッティングを用いた TMEM200A siRNAトランスフェクション胃腺癌細胞におけるタンパク質検出(英語)

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

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

CCK-8アッセイを用いた TMEM200A siRNAトランスフェクションHGC-27細胞株の細胞生存率の決定