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 RNAs that regulate the expression of different genes, including genes involved in cancer progression, angiogenesis and metastasis. Thrombospondin-1 (TSP-1) has been shown to contrast angiogenesis in vivo. TSP-1 expression levels are inversely correlated with tumor vascularity and metastasis in colon cancer. Bio-informatic statistical analysis indicated that TSP-1 is hypothetical target of miR-21, miR-182, overexpressed in CRC, and let-7i which expression is down-regulated in this tumor. In this work we investigated whether TSP-1 expression could be regulated by miR-21, mir-182 and let-7i in HT29 colon cancer cell line. Methods: To investigated whether miR-21, mir-182 and let-7i directly modulates TSP-1 expression, we transfected HT29 cell line with pre-mir21, pre-mir182 and pre-let7i by using siPortNeo FX tranfection agent and after 48h we evaluated TSP-1 mRNA, using Quantitative Real Time-PCR, and intracellular and secreted protein level performed by Western blotting and ELISA. To confirm the modulation of TSP-1 by miRNAs we transfected HT29 cell line with anti-mir to target the mature form of miR-21, miR182 and let-7i. Results: Using Real-Time PCR we did not find any variation of TSP-1 mRNA expression levels after transfection with pre-mir21 in HT29 cell line, but we observed a down-regulation of cytosolic and secreted protein by Western blot and ELISA. In cells transfected with pre-mir182 we did not observe any down-regulation both TSP-1 mRNA and cytosolic and secreted protein. Finally, we did not 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 evaluated TSP-1 expression. Conclusions: Data suggest that mir-182 induces degradation of TSP-1 mRNA in HT29 cell line, whereas mir-21 affects probably by blockage of TSP-1 translation. Let-7i does not seem involved in regulation of TSP-1 expression in HT29 cells. Understanding the molecular mechanism by which miRNAs regulate TSP-1 expression could be used to restore TSP-1 expression to contrast angiogenic events in colon cancer.