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. 2019 Feb 7;15(2):e1007961. doi: 10.1371/journal.pgen.1007961

Fig 5. Target gene analysis of microRNA-548ac.

Fig 5

(A) Expression of hsa-miR-548ac after transient transfection of HeLa cells with a precursor miRNA expression plasmid (mir-548ac-G). The level of mature miRNA molecules was determined relative to a reference miRNA (hsa-miR-191-5p) using real-time PCR based on TaqMan miRNA assays with specific stem-loop primers. Shown are means and standard errors of 2-ΔCt values scaled by a factor of 1000. When using a negative control vector with scrambled sequence, hsa-miR-548ac was basically not detectable. (B) Volcano plots of differential gene expression in HeLa cells transfected with the mir-548ac plasmid compared to cells transfected with the control plasmid. The transcriptome profiles were obtained by HTA 2.0 microarrays, which contain 67528 probe sets for human genes. Green dots indicate probe sets with p-value<0.05 and fold-change≤-1.5, which corresponds to RNA levels reduced by >33.3% (n = 257 at 24 h and n = 99 at 48 h post-transfection). These significantly downregulated transcripts were bioinformatically evaluated to identify the most likely direct targets of the miRNA. (C) Nine protein-coding genes, which were measured by 10 probe sets, resulted as top target gene candidates (S2 Table). RNAhybrid was used to calculate the minimum free energy (mfe) for the binding of hsa-miR-548ac at the 3' untranslated region (UTR) of each mRNA [33]. (D) Validation assays were performed in HeLa cells with luciferase reporter plasmids containing 3' UTR sequences from SDC4, SEL1L, and TNFAIP3. GLuc/SEAP ratios are given relative to cotransfections with a 3' UTR control vector, and each bar represents the mean and standard error from 3 biological replicates. For all three miRNA 3' UTR target plasmids, the relative luciferase activities were significantly decreased 24 h after transfection with the mir-548ac plasmid compared to transfection with the plasmid expressing a scrambled sequence. Welch t-test p-values are reported above the brackets. GLuc = Gaussia luciferase, HTA = Human Transcriptome Array, PCR = polymerase chain reaction, SEAP = secreted alkaline phosphatase.