Abstract
Transferrin receptor 1 (TFR1) is a transmembrane glycoprotein that allows for transferrin-bound iron uptake in mammalian cells. It is overexpressed in various cancers to satisfy the high iron demand of fast proliferating cells. Here we show that in hepatocellular carcinoma (HCC) TFR1 expression is regulated by miR-148a. Within the TFR1 3′UTR we identified and experimentally validated two evolutionarily conserved miRNA response elements (MREs) for miR-148/152 family members, including miR-148a. Interestingly, analyses of RNA sequencing data from patients with liver hepatocellular carcinoma (LIHC) revealed a significant inverse correlation of TFR1 mRNA levels and miR-148a. In addition, TFR1 mRNA levels were significantly increased in the tumor compared to matched normal healthy tissue, while miR-148a levels are decreased. Functional analysis demonstrated post-transcriptional regulation of TFR1 by miR-148a in HCC cells as well as decreased HCC cell proliferation upon either miR-148a overexpression or TFR1 knockdown. We hypothesize that decreased expression of miR-148a in HCC may elevate transferrin-bound iron uptake, increasing cellular iron levels and cell proliferation.
Highlights
MicroRNAs are a class of evolutionary conserved short non-coding RNAs (~22nt) that regulate gene expression at the post-transcriptional level by binding to miRNA response elements (MREs)[1], sites with partial complementarity within the 3′ untranslated region (3′UTR) of target messenger RNA
Among miR-148a target genes are those that play a role in cell growth and proliferation, such as hematopoietic PBX-interacting protein (HPIP)[17], insulin receptor substrate 1(IRS-1)[5], insulin-like growth factor-1 receptor (IGF-IR)[5], receptor tyrosine-protein kinase erbB3 (ERBB3)[22] and mitogen-inducible gene-6 (MIG6)[23], during the cell cycle, such as cullin related protein (CAND1)[24], M-phase inducer phosphatase 2 (CDC25B)[25] and the DNA methyltransferase 1 (DNMT1)[26], as well as the anti-apoptotic protein B-cell lymphoma 2 gene (BCL-2)[27]
We selected those MREs with high evolutionary conservation and that were predicted by the following miRNA target prediction algorithms: Targetscan v.7.066, PicTar[67] and RNA22 v.2.068
Summary
The protein coding sequence (CDS) of human TFR1 was amplified from the cDNA of HepG2 cells by PCR and cloned into the BamH1-XbaI restriction sites of the pcDNA Mammalian expression vector (Invitrogen, Cat. No V79020). The complete 3′UTR sequence of the human DNMT1 and TFR1 genes were amplified from genomic DNA of HepG2 cells by PCR. 3′UTR of DNMT1 and TFR1 were cloned into the SacI-NheI restriction sites of the pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega, Cat. No E1330) to generate pMIR-DNMT1 and pMIR-TFR1, respectively. At 24 h post-transfection, cells were trypsinized and plated (5 × 103 cells/well) into a sterile 96-well culture plate with complete growth medium (Corning, Cat. No CLS3595). Two-tailed Student’s t test was applied to determine the significance for luciferase assay, qRT-PCR data and densitometric analysis.
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