Significant advancements have been made in the identification of cancer biomarkers. Nonetheless, the transfunctional tumor-related matters associated with transmembrane protein family remains unidentified. As a result, our research on the role TMEM200A in gastric cancer has generated no research directions for related valves.
In this study, we used a combination of online database prediction and experimental technique validation, which simplifies large research time and stems the cost of experiments in related fields. In future, our laboratory, we are focused on exploring the specific mechanism of tumor metaphysis in gastric cancer, searching for association twin glycosylation, modification, and major body recording, and I didn't find new targets for gastric cancer treatment. To begin, navigate to the TIMER 2.0 database website interface.
Select the cancer exploration tab, input the gene ID into the search box, and click submit to produce the differential expression images of TMEM200A in various tumor types. For transfection, label three microcentrifuge tubes as siRNA-1, two, and three, then add transfection reagent followed by the basal medium in each tube. Add four micrograms of siRNA to the respective tubes and mix thoroughly.
Now, distribute the mixture into the three wells of the six-well plate containing the adenocarcinoma cells. Gently shake the plate to ensure even distribution of the mixture. Label the three wells where the mixture was added as siRNA-1, siRNA-2, and siRNA-3.
After incubating the cells for four to six hours, replace half of the medium in the labeled wells with fresh, complete medium. Incubate the cells for 24 to 48 hours before using them for quantitative RTPCR and western blotting. To begin, transfect the stomach adenocarcinoma cells with TMEM200A siRNA and incubate for two days, then, discard the cell culture medium from the wells and gently wash the cells two times with one milliliter of PBS.
Using an RNA isolation kit, isolate the total RNA of the cells. Estimate the RNA concentration and synthesize CDNA with one microgram of RNA using a CDNA synthesis kit, following the manufacturer's instructions. Now, set up quantitative RTPCR on tenfold dilutions of CDNA in a 10 microliter reaction mix.
Add primers in quantitative PCR master mix using the real-time PCR assay system. Use the quantitative PCR reaction conditions shown here and determine the relative expression of each gene using the double delta CT technique. The HGC-27 cell line dramatically overexpressed TMEM200A protein and mRNA transcript compared to the GES-1 cell line.
The differential mRNA expression of TMEM200A in human gastric cancer cells, SGC-7901, is higher than in GES-1 cells. To begin, transfect the stomach adenocarcinoma cells with TMEM200A siRNA and incubate for two days, then discard the cell culture medium from the wells and gently wash the cells two times with one milliliter of PBS. Place the cells on ice and add 150 microliters of pre-cooled radioimmunoprecipitation assay lysis buffer.
Using a plastic cell scraper, carefully detach the adhering cells from the dish and transfer the cell solution gently into a pre-cooled microcentrifuge tube. Then, centrifuge the cell lysate at 1.5 by 10 raised to the power of 4G for 10 minutes at four degrees Celsius and transfer the supernatant to a new 1.5 milliliter microcentrifuge tube. Using a BCA protein assay kit, determine the protein content following the manufacturer's instructions.
Load 30 grams of protein from each sample onto a 10%SDS-PAGE gel. Run the gel at 80 volts for 0.5 hours, followed by 1.5 hours at 120 volts. Next, transfer the proteins from the gel to a 45 micrometer PVDF membrane at a constant current of 300 milliamperes for one to 1.5 hours.
Place the PVDF membrane on a shaker for three cycles of five minutes each. After shaking, add TBST to the container with the protein side facing up. Place the membrane in the blocking buffer for 30 minutes at room temperature.
Wash the membrane with TBST for three cycles of 10 minutes each. Incubate the membrane with primary antibodies overnight at four degrees Celsius. After washing the membrane three times with TBST, add a rabbit or mouse secondary antibody and incubate for one hour at room temperature.
Then, add ECL substrate to the PVDF membrane for 30 seconds and detect the signal using an imaging system. The efficiency of TMEM200A was significantly reduced in gastric cancer cell HGC-27 transfected with siRNA compared to the non-transfected cells. The TMEM200A siRNA significantly inhibited the related proteins in epithelial mesenchymal transition and affected the phosphorylation of AKT in the phosphoinositide 3-kinase signaling pathway.
Begin by seeding HGC-27 cells in good growth condition in 96-well plates. Seed the transfected cells into 96-well plates divided into negative control and TMEM200A siRNA groups. Incubate the cells at 37 degrees Celsius.
After specific time intervals, add CCK reagent to each well and incubate at 37 degrees Celsius for two hours. Finally, measure the absorbance at 450 nanometers using a multifunctional enzyme labeler. The CCK-8 assays showed that TMEM200A knockdown groups showed a notable drop in cell viability as compared to the negative control group.
