Ruolo del miR-221 nella tumorigenesi epatica

MicroRNAs are short non-coding RNA molecules, involved in post-transcriptional gene expression regulation. Their aberrant expression is involved in several pathological conditions, including cancer. MicroRNA-221 was frequently found overexpressed in human neoplasms. This study focused on biological and molecular function of miR-221 in hepatocellular carcinoma (HCC). By analyzing miR-221 expression in 70 clinical samples from normal liver, cirrhosis and hepatocellular carcinoma we found miR-221 overexpression in 80% of cancer samples, compared to non-neoplastic tissues, suggesting that this microRNA played an important role in HCC development. Previous molecular studies identified some miR-221 target genes, that were involved in cell cycle inhibition and apoptosis. To further understand miR-221 molecular role in hepatocellular carcinoma development, gene expression profile of miR-221-transfected-SNU-398 cells (derived from human liver cancer) was analyzed through microarray assay and Sylamer algorithm. Results revealed that several down-regulated genes included sequence homology for miR-221 sequence in their 3’UTR, allowing us to identify new potential miR-221 target genes. Pathways analysis performed on the genes identified by this approach revealed that miR-221 affected cell proliferation and apoptosis. Furthermore some miR-221 target genes from Sylamer results were investigated: RB1, WEE1 (cell cycle inhibitors), APAF1 (pro-apoptotic gene), ANXA1, CTCF (transcriptional repressor) were validated as miR-221 target genes, confirming the efficacy of Sylamer analysis and the ability of microRNA to promote HCC tumorigenesis by affecting these pathways. To more directly demonstrate miR-221 oncogenic role, we created a transgenic mouse model of liver miR-221 overexpression. We proved the efficacy of this model by measuring miR-221 hepatic levels and protein expression of some miR-221 target genes (CDKN1B/p27, CDKN1C/p57, BMF), compared to wild type mice. We found that transgenic mice were prone to HCC development, since hepatic nodules were detectable in 50% of 9-12 months-old male animals. Treatment with DENA carcinogen allowed to speed tumor development and we found several liver tumors in 3 months-DENA-treated transgenic animals, while DENA-treated wild type mice developed hepatic cancer 2 months later. Tumors showed high miR-221 levels and low target proteins expression, meaning the microRNA was still present and active in cancer cells. Finally we demonstrated that anti-miR-221 injection in transgenic mice was able to reduce number and size of tumor nodules. tumorigenesis. In vivo studies allowed to demonstrate an important role of miR-221 in HCC development and growth and provided evidence of efficacy of anti-miR-221 oligonucleotide in HCC treatment.