BAY11 enhances OCT4 synthetic mRNA expression in adult human skin cells.
BAY11 enhances OCT4 synthetic mRNA expression in adult human skin cells.
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DOI：
10.1186/scrt163
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发表时间：
2013-02-06
影响因子：
7.5
通讯作者：
Byrne JA
中科院分区：
文献类型：
--
作者：
Awe JP;Crespo AV;Li Y;Kiledjian M;Byrne JA
OCT 4转录因子参与许多细胞过程，包括发育、重编程、维持多能性和分化。最近使用合成的OCT 4 mRNA（与其他重编程因子结合）来产生人诱导多能干细胞。在这里，我们发现BAY 11-7082（BAY 11），至少部分地通过基于NF-κ B抑制的机制，可以在将合成mRNA（synRNA）转染到成人皮肤细胞中后显著增加OCT 4的表达。我们测试了各种化学和分子小分子抑制合成mRNA转染后观察到的先天免疫应答的能力。三种分子-B18 R，BX 795和BAY 11-用于免疫细胞化学和基于增殖的测定。我们还利用全球转录荟萃分析结合定量PCR来鉴定OCT 4下游的相对基因表达。我们发现，在BAY 11存在下培养的人皮肤细胞导致OCT 4表达的可重复增加，而OCT 4不抑制正常细胞增殖。增加的OCT 4水平导致OCT 4下游基因的表达显著增加，包括先前鉴定的SPP 1、DUSP 4和GADD 45 G，表明表达的OCT 4是功能性的。我们还发现了一种新的OCT 4推定的下游靶基因SLC 16 A9，其在OCT 4水平升高后表现出显著增加的表达。这是我们第一次证明，使用BAY 11可以实现合成mRNA表达的基于小分子的稳定。这种基于小分子的先天免疫应答抑制和随后转染的合成mRNA的稳健表达可能在未来基于细胞的研究和治疗中具有多种应用。
The OCT4 transcription factor is involved in many cellular processes, including development, reprogramming, maintaining pluripotency and differentiation. Synthetic OCT4 mRNA was recently used (in conjunction with other reprogramming factors) to generate human induced pluripotent stem cells. Here, we discovered that BAY 11-7082 (BAY11), at least partially through an NF-κB-inhibition based mechanism, could significantly increase the expression of OCT4 following transfection of synthetic mRNA (synRNA) into adult human skin cells. We tested various chemical and molecular small molecules on their ability to suppress the innate immune response seen upon synthetic mRNA transfection. Three molecules - B18R, BX795, and BAY11 - were used in immunocytochemical and proliferation-based assays. We also utilized global transcriptional meta-analysis coupled with quantitative PCR to identify relative gene expression downstream of OCT4. We found that human skin cells cultured in the presence of BAY11 resulted in reproducible increased expression of OCT4 that did not inhibit normal cell proliferation. The increased levels of OCT4 resulted in significantly increased expression of genes downstream of OCT4, including the previously identified SPP1, DUSP4 and GADD45G, suggesting the expressed OCT4 was functional. We also discovered a novel OCT4 putative downstream target gene SLC16A9 which demonstrated significantly increased expression following elevation of OCT4 levels. For the first time we have shown that small molecule-based stabilization of synthetic mRNA expression can be achieved with use of BAY11. This small molecule-based inhibition of innate immune responses and subsequent robust expression of transfected synthetic mRNAs may have multiple applications for future cell-based research and therapeutics.
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