Effects Of SelS Gene Silence On The Expression Of ER Localized Selenoproteins MRNA In HepG2Cells And Off-Target

The endoplasmic reticulum (ER) is one of the important organelle of cells ineukaryotic organism. The ER lumen is involved in the synthesis, modification, folding,assembly and translocation of proteins, as also is an important Ca2+reservoir within thecell. Research shows that ER stress is associated with a variety of diseases. ER biologicalfunction plays an important role in cell survival. Selenium is an essential trace element inmammals. Seven out of25mammalian selenoproteins have been identified as residents ofthe ER. They are type2iodothyronine deiodinase (D2),15-kDaselenoprotein (Sep15) andselenoproteins S, K, M, N and T. Many of their biological functions have been less-wellstudied except D2, but some studies reveal that these selenoproteins play important rolesof in ER.In this paper, to study the relationship between these ER localized selenoproteins, theeffects of SelS gene silence on the mRNA expression of the other ER localizedselenoproteins in HepG2cells were investigated. The main results are as follows:(1) Two kinds of SelS gene siRNA sequences were tested. Cell viability wasevaluated by MTT assay using different concentrations of SelS siRNA. Results indicatedhigh concentration of SelS siRNA was toxic to the HepG2cells and caused cell death.Optimal concentration of SelS siRNA was selected. The effects of SelS and SelK genesilence on cell apoptosis were studied by flow cytometry. The results showed that cellapoptosis was not influenced by SelS or SelK gene silence in24h.(2) The effects of SelS, SelK and Derlin-1gene silence on the mRNA expression ofER localized selenoproteins in HepG2cells were examined by RT-PCR. The resultsindicated that SelS gene silence for24h could down-regulate the mRNA level of other ERlocalized selenoproteins, Sep15, SelK, SelN and SelT, in a different extent. In addition,the similar results were got when the cells were treated using two kinds of SelS genesiRNA sequences. However, the mRNA expression of these selenoproteins in ER had nosignificant change when the SelK or Derlin-1gene was silenced for24h. Meanwhile, thespecificity of SelS siRNA sequence was identified using BLAST analysis and the results showed that no other selenoprotein was homologous with SelS siRNA sequence,suggesting that SelS has a certain role with the other ER selenoproteins. Its mechanismremains to be further determined.