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. 2019 Jul 18;21(9):849–862. doi: 10.1016/j.neo.2019.06.004

Figure 6.

Figure 6

Putative mRNA targets of miR-125a-5p are associated with locoregional recurrence, are upregulated in advanced squamous cell carcinoma of the head and neck, compared to normal tissue and directly targets TXNRD1 and EIF4EBP1 mRNA to downregulate protein expression. (A) Messenger RNA microarray analysis from patient tumor samples collected from a prospective trial of postoperative radiation therapy was performed, revealing an association of local recurrence with increased mRNA expression of putative target mRNAs, TXNRD1 and EIF4EBP1, compared to patients with no evidence of disease. The data were analyzed with the Student's t-test and is presented as the mean ± standard error of the mean. (B) mRNA expression levels for EIF4EBP1, which encodes for the eukaryotic translation initiation factor 4E-binding protein 1, and TXNRD1, which encodes for thioredoxin reductase 1, from The Cancer Genome Atlas were analyzed according to mRNA expression levels, determined by RNA-seq, and analyzed based on cancer stage. UALCAN (ualcan.path.uab.edu) [10] was utilized for data acquisition and analysis, which combines mRNA expression data with clinical data. (C) Predicted binding sites of miR-125a-5p on the 3′ untranslated region of TXNRD1 and EIF4EBP1 mRNA. The TargetScan (www.targetscan.org) [14] was utilized to predict for the binding site based on conservation of the mRNA binding site to the miRNA seed sequence. Vertical lines show Watson-Crick base pairing between the mRNA and miRNA. (D) In vitro pulldown assay with a synthetically biotinylated miR-125a-5p were transfected into HN5, using biotinylated miR-551a as a negative control, and then recovered using streptavidin-agarose beads, which were then washed. Recovered RNAs were analyzed using quantitative RT-PCR, which revealed that TXNRD1 and EIF4EBP1 mRNAs are directly bound to miR-125a-5p, compared to the RNA species bound to miR-551a. (E) Transfection of miR-125a-5p results in decreased TXNRD1 and EIF4EBP1 protein expression, compared to control miRNA, in HN5 and UM-SCC-22B cell lines. (F) Transfection of miR-125a-5p results in decreased TXNRD1 mRNA expression, compared to control miRNA. (G) Transfection of miR-125a-5p does not change the levels of the EIF4EBP1 transcript, suggesting that translational repression is main mechanism of gene regulation for EIF4EBP1. Data were analyzed with the Student's t-test and are presented as the mean ± standard deviation. Experiment was carried out in triplicate.