Pression (p 0.01; Figure 6B). inflammatory Ziritaxestat Metabolic Enzyme/Protease makers, which includes the upregulation of NLRP
Pression (p 0.01; Figure 6B). inflammatory makers, such as the upregulation of NLRP3 (3.60 vs. 0.55, p 0.01), IL-1 The IL-1 mRNA 3-UTR area was predicted to have an 8 mer pairing web page seed (2.24 vs. 0.49, p 0.01), TNF (1.64 vs. 0.83, p 0.01), CCL2 (3.62 vs. 1.94, p 0.01), and match with miR-26A, 0.001) binding sequence on IL-1 (Figure 5F). CCL5 (7.56 vs. 3.40, p and theinduced by LPS in myotubes3-UTR was 276-A, 277-G, 278A, 279-A, 280-C, 281-A, 282-G, and 283-A by Targetscan (Figure 6C). Then, we cloned the sequences containing target genes IL-1 3-UTR (WT) into the pGL3 Expression ReporterInt. J. Mol. Sci. 2021, 22, 12435 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW9 of 16 9 of2.six. miR-26A Targeting the encodes the reporter gene firefly luciferase. To confirm Vector Method, which alsoIL-1 mRNA 3 -UTR Alleviated Myotube Inflammation no matter if IL1 The results of Targetscan prediction and inflammation-related pathway evaluation reis the target gene of miR-26A, we chose cotransfection into human renal epithelial cells (293Tcandidate targetmiR-26A miR-26A, such as IL-1, of its higher transfection effivealed the cells) with the genes of mimic. This was simply because PIK3R3, and SMAD2. qPCR ciency and mature transfection conditions, whichtarget genes wereinfluence of the operaresults showed that mRNA levels of all screening can exclude the drastically elevated bility LPS treatment, even though the overexpression of miR-26A considerably inhibited them soon after on the experiment itself around the benefits. miR-26A’s mimicked cotransfection caused a marked6A). Finally, miR-26A expression and targeting partnership among miR-26A and (Figure enhance in we intended to verify the a lower in luciferase transcription. DeleIL-1, the predicted miR-26A final binding sequence on the IL-1 3-UTR formed a mution of as a result of its high targeted score (Targetscan score = 94). Their correlation analysis also verified (MUT), which inhibited the was drastically lower in IL-4 Protein Epigenetic Reader Domain transcriptional tant sequencethat miR-26A overexpression miR-26A-induced negatively correlated with IL-1 mRNA expression activity (Figure 6B ). (p 0.01; Figure 6B).Figure six. miR-26A targeting IL-1 mRNA 3-UTR alleviated myotube inflammation. (A) qPCR val6. miR-26A targeting IL-1 mRNA 3 -UTR alleviated myotube inflammation. (A) qPCR idation of candidate target genes (IL-1, Pik3R3, Smad2) of miR-26A. (B) Correlation analysis of validation of candidate target genes (IL-1, Pik3R3, Smad2) of miR-26A. (B) Correlation miR-26A with IL-1 mRNA. (C) The binding region of miR-26A using the IL-1 mRNA 3 -UTR was miR-26A with IL-1 mRNA. (C) The binding region of miR-26A together with the IL-1 mRNA 3-UTR was predicted by Targetscan. (D) The dual luciferase assay verified the targeting relationship amongst predicted by Targetscan. (D) The dual luciferase assay verified the targeting relationship involving miR-26A and IL-1 in the 293T cells. The data shown would be the means SEM, n = three. p 0.05, p miR-26A and IL-1 in the 293T cells. The information shown would be the implies SEM, n = 3. p 0.05, 0.01, p 0.001 vs. manage cells, and ### p 0.001 vs. LPS treated cells. Unmarked graphs show no p 0.01, p 0.001 vs. manage cells, and ### p 0.001 vs. LPS treated cells. Unmarked graphs significant distinction. show no considerable difference.3. Discussion The IL-1 mRNA 3 -UTR area was predicted to have an eight mer pairing site seed In the miR-26A, and we’re the first to report that -UTR was 276-A, 277-G, 278-A, match with present study,the binding sequence on IL-1.