Perturbation of Endogenous siRNA Level by DICER1 Overexpression in Adipose‑Derived Mesenchymal Stromal Cells

Background: The immunoregulatory characteristics of mesen-chymal stromal/stem cells (MSCs) raise hope for treatment of inflammatory diseases. However, the immunosuppressive po-tential of MSCs is not always achieved and manipulating the in-terplay between MSCs and immune responses is recommended to enhance their function.Despite exogenous sources for dsRNA transcripts derived from invading viruses, the vast majority of dsRNA has endogenous origin called endo-siRNA. Unlike invertebrate, mammalian cells lack a strong dsRNA processing machinery and molecular scissors such as DICER1 and RNAi pathway that could be de-tected in oocyte or embryonic stem cells. Meanwhile, differen-tiated cells elicit interferon secretion as a response to dsRNA. Furthermore, viral proteins with immunosuppressive functions has been widely used in different studies, here we used B18R, a cytokine inhibitor protein as a control in comparison with DICER1 induction state. We hypothesized that compensation of DICER1 expression in mammalian differentiated cells attenu-ate interferon response and anti-inflammatory cytokines.Materials and Methods: We cloned GFP-2A-Puromycin frag-ment inframe in pCAGGS-hsDicer vector (Addgene Plasmid #41584) and pCDH513b (GFP) constructs were utilized for transfection in HEK293T and MSCs with Lipofectamin 2000. MSCs also were treated with 200 ng/µl B18R protein. There-upon, dsRNA and interferon response genes were evaluated 48 hours post transfection using relative Real-Time PCR.Results: Our data showed that DDX58 and RNase L were over-expressed in HEK293T and MSCs. Moreover, TNFAIP6 (TSG6) and OAS2 were down-regulated in HEK293T and IFIH1 was suppressed in MSCs. It is suggested that dsRNA recognition sensors (OAS2 and DDX58) and effector enzymes (RNase L) might be increased as a feedback, because of production of en-do-siRNA following DICER1 expression. Consequently, Type I interferon and proinflammatory cytokines (IFIH1 and TSG6) significantly suppressed. In addition, we showed reduced levels of IL10 and INFβ after using B18R in MSCs which suppress the antiviral activity and interferon response.Conclusion: It might be proposed that activation of RNA inter-fering pathway in MSCs paves the way for producing immuno-compromised cells.