MicroRNA expression patterns in osteosarcomas and their potential role in tumour progression

Abstract

There is an increasing evidence that microRNAs are involved in control of developmental timing, cell proliferation, apoptosis, morphogenesis, fat metabolism. In many studies, performed to investigate the genes and gene products that drive the metastatic process, it has become evident that, in addition to alterations in protein-encoding genes, abnormalities in non-coding genes can also contribute to cancer pathogenesis. Changes in miRNA levels may be related to dysregulated growth in some cancer cells and in this field the differential expression of miRNA may have substantial diagnostic and prognostic value. In the context of microRNAs in tumours, our aim was to evaluate whether pro-metastatic and non-metastatic sarcoma cells, may differ in their microRNA expression, in the effort to identify single pro-metastatic microRNAs in bone and soft-tissue sarcomas. microRNAs were separated from total RNA of MG-63 and 143B osteosarcoma cells with different intravasation behaviour and malignancy degree. Specific libraries were constructed using TopoTA cloning system supported by 5’- and 3’ adaptors. Differentially expressed microRNA were identified by sequencing followed by bioinformatic analysis. A number of microRNAs reported in the data base miRNA registry were found to be differential expressed in the two osteosarcoma model cell lines. Of the over 19 microRNAs identified, the majority correspond to “oncomiR”, i.e. microRNAs involved in neoplastic transformation and progression. One of the analysed sequences, localized on chromosome 7 of the human genome, showed miRNA configuration and studies are in progress to define its precise characteristics. Two of the differential expressed microRNA, miR-93 and miR-210, for which no functional data were available, were analyzed in different cell lines and tissues and investigated for their potential involvement in motility phenomena by their mis-expression through transduction in to osteosarcoma cells. To identify their molecular targets, different approaches were performed using bioinformatic softwares, through PCR-based strategies and DNA microarray analyses. The expression of another set of four miRNAs (miR-9, miR-183, miR-196a, miR-484) differentially expressed, was identified through a global expression analysis, and analyzed in surgical specimens of low- and high-grade osteosarcoma patients. To better define the significance of the expression pattern of these microRNAs, studies on a wider patient cohort are in progress.