Analyzing the Molecular Mechanism of Fam26f Through Knockdown and Overexpression

Abstract

FAM26F (family with sequence similarity 26, member F) is a tetraspanin-like membrane glycoprotein involved in calcium homeostasis and in immune modulation. There is substantial evidence supporting FAM26F's potential role in immunity, but the protein's complete modulatory signalling pathways have yet to be investigated. The current study aims to explore the expression of FAM26F in vitro in human embryonic kidney cell line (HEK 293), and how its inhibition or overexpression affects the downstream signalling cascade proteins. This was investigated through two ways: 1) siRNA based knockdown study carried out by transfecting cells with FAM26F siRNA to assess the expression of FAM26F. 2) Overexpression achieved in the HEK293 cell line through plasmid-mediated delivery of the FAM26F gene. The transfection was confirmed through one-step qPCR and western blotting of transfected cells, and then MS/MS analysis was performed to determine the proteins affected by inhibition and overexpression of FAM26F. MS/MS analysis revealed that among the overexpressed cells, there were 780 proteins, 85 of which were up-regulated and 695 were down-regulated. In the knockdown group, however, only two proteins were up regulated and twenty were down-regulated. Out of these, all of knockdown proteins, and top 50 hits from overexpressed category were selected and analysed using STRING software to predict their protein-protein interaction, and Reactome pathway browser for pathway analysis. STRING detected no interactions between FAM26F and any of the other proteins. However, it identified interactions among other various proteins. Reactome pathway analysis revealed proteins to be primarily involved in gene expression, immune system, protein metabolism (primarily translation), infectious disease (HIV and SARS CoV2), RNA metabolism, and vesicle-mediated transport pathways. Validating the findings of previous research, this study demonstrates that FAM26F plays a role in immunity and various anomalies by either regulating differential gene expression or protein metabolism. The exact relation of FAM26F with these proteins can be validated in future by employing real time PCR technique for gene expression analysis and western blotting for proteomics analysis.