MicroRNA-1Targets Fibronecin1in Human Laryngeal Carcinoma Cell Line HEp2

Aims MicroRNAs (miRNAs), involved in many physiological and pathological progresses, are a class of small non-coding RNAs that regulate gene expression post-transcriptionally. According to recent researches, miRNAs regulate the gene expression by specially binding to the3’-untranslated regions (3’-UTRs) of the target gene. Some miRNAs can function as oncogene or tumor suppressor in the process of the tumor generation and development by regulating the expression of target gene negatively. Using a miRNA array, we found that the expression of microRNA-1(miR-1) was significantly downregulated in the human laryngeal squamous cancer tissues compared with the matched non-neoplastic controls. To explore the molecular mechnisms of the function of miR-1, we studied the effect of miR-1on the growth of HEp2cell and identified a new target gene for miR-1.Methods HEp2cells were firstly transfected with miR-1overexpressing plasmid or miR-1antisense oligonucleotides(ASO-1), and the MTT and colony formation experiment were used to detect the viability and proliferation of HEp2cells. Then, to explore new target genes of miR-1, target prediction from bioinformatics was combined with cDNA expresson profiling. The direct effct of miR-1on predicted target gene was validated by a fluorescent reporter assay and the suppression of miR-1on target gene expression was measured by real-time PCR, Western blot and immunofluorescence. Meantime, the cell gowth phenotype was also checked when the target gene was knockdown by specific siRNA.Results Overexpression of miR-1inhibited the proliferation potential of HEp2cells, whereas knockdown of miR-1by specific ASO elevated growth of the cells. Fibronectin1(FN1) was pridicted to be one of the target gene of miR-1, whose3’-untranslated region (3’-UTR) contain a putative binding site of miR-1. An EGFP reporter assay was used to demonstrate that miR-1regulates FN1expression directly through the UTR binding region. What’s more, the mRNA and protein level of FN1was negatively regulated by miR-1. Meanwhile, the viability and proliferation of HEp2cells was decreased when endogenous FN1was knockdown by siRNA, which is in coincidence with the effect of miR-1overexpression. Conclusions In HEp2cells, miR-1can act as a tumor suppressor by inhibiting cell growth; miR-1could regulate the HEp2cell growth by targeting the3’-UTR of FN1; FN1may function as a oncogene to promote the cell proliferation. The identification of tumor suppressor miR-1and its target gene FN1in laryngeal carcinoma is potentially valuable for us to understand the mechanisma of tumor generation and development, and it also may have diagnostic and therapeutic value in the future.