Studying of the effect of microRNA and natural killer cell based combined immunotherapy approach on triple negative breast cancer

Aim: In our study, we aimed to determine the effect of triple suppression on eEF2K, PD-L1 and FOXM1 with miR-3977 in triple negative breast cancer (TNBC) cells, as well as identified the natural killer cell (NK) mediated immune response effect in these cells. Materials and Method: As triple negative breast cancer cells MDA-MB-436, MDA-MB-231, BT-20 cell lines; as natural killer cells cord blood natural killer cells and NK-92 cell line will be used. To identified tumorigenesis studies colony formation assay, miRNA and siRNA mimic transfection, RNA extraction, miRNA and mRNA reverse transcription, RT-qPCR assay, protein extraction and immunoblotting assay, cell mobility, migration and invasion assays, flow cytometry, preparation of miRNA nanoparticles, orthotopic xenograft tumor model and statistical analysis methods will be used. To determination of NK mediated immunoassay studies Chromium (Cr51) analysis methods will be used. Results: In our study, we investigated the role of miR-3977 and found that miR-3977 is associated with significantly longer patient survival in TNBC and inversely correlated with levels of proto-oncogenic eEF2K, which was associated with significantly shorter overall patient survival. We showed that miR-3977 directly binds to the 3′-untranslated region (3'-UTR) of eEF2K, PD-L1 and FOXM1 mRNAs and suppresses their expression, leading to inhibition of TNBC cell proliferation, motility, and invasion. Moreover, in vivo therapeutic delivery of miR-3977 nanoparticles by systemic intraperitoneal administration delayed tumor growth of orthotopic TNBC tumor xenograft models by inhibiting eEF2K, PD-L1 and FOXM1, intratumoral proliferation and angiogenesis, and inducing apoptosis. Importantly in our study it has been shown at the first time, with either miR-3977 or siRNA treatments of each eEF2K, CD274 or FOXM1 inhibition NK-92 and CBNK cells cytotoxicity were induced. Conclusion: Our study has a high specific value due to miR-3977 gene therapy in triple negative breast cancer cells as it is the first study to both re-activate the programmed cell death (PD-L1) mechanisms and to stop the carcinogenesis process and the destruction of tumor cells with the activation of NK-mediated immune response against these cells. Furthermore, our findings provide new insights into the tumor suppressor role of miR-3977 by dual-targeting of eEF2K and FOXM1 signaling axis and suggest that miR-3977-based gene therapy may be a potential therapeutic strategy in TNBC.