Knockdown of FAM83A to Verify Its Role in Cervical Cancer Cell Growth and Cisplatin Sensitivity

The scope of our research and focus are the investigating the role of FAM83A in cervical cancer cell growth and cisplatin sensitivity. By addressing this question, we hope to contribute to the scientific understanding of the cervical cancer biology and potentially identify new therapeutic targets for this disease. We found lysing FAM83A expressions desensitizes cervical cancer cells to cisplatin treatment, enhancing the drug's cytotoxic effect and inhibiting the cell viability and migration.

Our laboratory will focus on the foreign research question in the future, illustrating the medical mycolysis of FAM83A in cervical cancer, developing FAM83A inhibitors or activators therapies, starting the correlation between FAM83A and the patient prophylaxis and investigating the role of FAM83A in other types of cancer. To begin cell transfection, seed human cervical tumor cells in six-well plates. Prepare a mixture of 50 nanomolar cypham 83A and eight microliters of transfection agent in serum-free DMEM, then add 500 microliters of the mixture to the plate.

To the negative control, add only the serum-free DMEM. Now, replace the medium with one milliliter of fetal calf serum-supplemented DMEM. After 48 hours, treat the cells with one milliliter of five micromolar cisplatin for 24 hours, then harvest the cells for total RNA extraction and qRT-PCR analysis.

Wash the above treated cells with ice cold PBS to remove any residual media and add 50 microliters of the RIPA lysis buffer containing PMSF to lyse the cells. Collect the cells in a tube. Incubate the cells on ice for a few minutes to ensure complete lysis, then centrifuge at four degrees Celsius at 12, 000 G for 15 minutes.

Then, mix the cell lysate with a loading buffer and heat the mixture at 95 to 100 degrees Celsius for five to 10 minutes on a water bath to denature the proteins. Load 10 microliters of total protein from each sample onto an SDS-PAGE gel. Run the gel under a constant voltage of 80 volts.

Stop the electrophoresis when the bromophenol blue reaches the bottom of the separation gel, then use a wet transfer system in an ice bath to transfer the separated proteins from the gel onto a polyvinylidene difluoride membrane. After the completion of the transfer, remove the membrane and incubate the membrane in a blocking buffer at room temperature for about one hour. Incubate the membrane with a secondary antibody conjugated to HRP.

Visualize the protein bands on the membrane using a chemiluminescent substrate and capture the signal using a chemiluminescence imaging system. Western blot analysis showed that the suppressed expression of FAM83A increased the BAX and cleaved caspase-three proteins, decreased the expression of BCL2, and increased cytochrome c release.