EFFECT OF miR-21, miR-182 AND let-7i ON TSP-1 EXPRESSION IN COLON CANCER CELL LINE

Background:MicroRNAs (miRNAs) are small non-coding RNAsthat regulate the expression of different genes, including genesinvolved in cancer progression, angiogenesis and metastasis.Thrombospondin-1 (TSP-1) has been shown to contrastangiogenesis in vivo. TSP-1 expression levels are inverselycorrelated with tumor vascularity and metastasis in coloncancer. Bio-informatic statistical analysis indicated that TSP-1 ishypothetical target of miR-21, miR-182, overexpressed in CRC, andlet-7i which expression is down-regulated in this tumor. In thiswork we investigated whether TSP-1 expression could be regulatedby miR-21, mir-182 and let-7i in HT29 colon cancer cell line.Methods:To investigated whether miR-21, mir-182 and let-7idirectly modulates TSP-1 expression, we transfected HT29 cell linewith pre-mir21, pre-mir182 and pre-let7i by using siPortNeo FXtranfection agent and after 48h we evaluated TSP-1 mRNA, usingQuantitative Real Time-PCR, and intracellular and secreted proteinlevel performed by Western blotting and ELISA. To confirm themodulation of TSP-1 by miRNAs we transfected HT29 cell line withanti-mir to target the mature form of miR-21, miR182 and let-7i.Results:Using Real-Time PCR we did not find any variation ofTSP-1 mRNA expression levels after transfection with pre-mir21 inHT29 cell line, but we observed a down-regulation of cytosolic andsecreted protein by Western blot and ELISA. In cells transfectedwith pre-mir182 we did not observe any down-regulation both TSP-1 mRNA and cytosolic and secreted protein. Finally, we didnot find any variation of TSP-1 level in cells transfected with let-7i. Results were confirmed by transfection with anti-mir21, anti-mir182 and anti-let7i and, using the same method, we evaluatedTSP-1 expression.Conclusions:Data suggest that mir-182 induces degradation ofTSP-1 mRNA in HT29 cell line, whereas mir-21 affects probablyby blockage of TSP-1 translation. Let-7i does not seem involved inregulation of TSP-1 expression in HT29 cells. Understanding themolecular mechanism by which miRNAs regulate TSP-1 expressioncould be used to restore TSP-1 expression to contrast angiogenicevents in colon cancer.