Effects of anti-miR-182 on Thrombospondin-1 expression in human colon cancer cells

ABSTRACT Background: MicroRNAs are small non-coding RNAs that regulate the expression of different genes, involved in cancer progression, angiogenesis and metastasis. Bio-informatic statistical analysis indicated that miR-182, over-expressed in colorectal cancer (CRC), has like predictive target Thrombospondin-1 (TSP-1), a protein inversely correlated with tumor vascularity and metastasis that results downregulated in different types of cancer including CRC. Early Growth Response 1 (Erg-1) and Specificity Protein 1 (Sp-1) are transcriptional factors that bind consensus sequence on TSP-1 gene promoter and are putative target of miR-96/182/183 cluster. MiR-182 could target SMAD4, which expression influences angiogenesis, increasing expression of TSP-1. Aims: Considered that we investigated whether and how the silencing of miR-182 could restore TSP-1 expression levels in HT-29 and HCT-116 cells. Materials and Methods: We determined the expression levels of TSP-1 and miR-182 in HT-29 and HCT-116 by qRT-PCR. Then, cells were transfected with synthetic oligonucleotides targeting miR-182 (anti-miR-182) and we evaluated TSP-1 mRNA, using qRT-PCR, and intracellular and secreted protein levels by Western blotting (WB) and ELISA. Next, we evaluated Erg-1 and Sp-1 total and fractionated protein levels by WB and we assessed their function by ChIP. Finally we evaluate intracellular SMAD4 levels by WB. Results: We found that TSP-1 expression increased after transfection with anti-miR-182. Moreover, we observed that anti-miR-182 induced Egr-1 expression, nuclear traslocation and its binding on TSP-1 promoter in HCT- 116. Instead Sp-1 was up-regulated both as total and as nuclear protein in HT-29 tranfescted and then recruited on the TSP-1 promoter consensus sequence. Finally, we found increased SMAD4 levels in transfected cells. Conclusions: Our data suggest that anti-miR-182 could determine an upregulation TSP-1 expression, modulating Egr-1 and Sp-1 function. Moreover, the restoration of SMAD4 expression induced by silencing of miR-182 could be another mechanism by which anti-miR-182 could restore the antiangiogenic phenotype. Understanding the molecular mechanism by which miRNAs regulate gene expression could be used to restore TSP-1 expression to contrast angiogenic and invasive events in colon cancer.