MicroRNAs As A Tumor Suppressor In Glioblastoma Cancer Cells

Glioblastoma and grade IV astrocytoma, is the most aggressive cancer that begins within the brain.Glioblastomas represent 15% of brain tumors. microRNA (miRNA) about 22 nucleotides functions inRNA silencing. MicroRNA-129-1 seems to behave as a tumour suppressor since its decreasedexpression in glioblastoma multiforme (GBM).Cell lines and patient sample collection: Fifteen freshGBM tissues (grade IV) and age- sex matched non-cancer postmortem brain tissues were obtainedfrom Sina Hospital (Tehran, Iran), in 2013. As well as, Human glioma cell lines U87, A172, U251 andHEK-293 T cells were purchased. Plasmids, viral vectors construction and luciferase assay: MiR-129-1 coding region was cloned into pLEX. JRed vector and for loss-of-function studies pLenti-miROff-129 construct was purchased. For luciferase assays, IGF1, IGF2BP3 and MAPK1 30-UTR,harbouring potential miR-129-1 target sites, were cloned downstream of the luciferase gene in thepSICHECK2 vector. Gene expression analysis: Total RNA was purified from cell lines and reversetranscribed to cDNA, stem-loop RT specific primers (for miR-129-1), Real-time PCR for targetmRNAs and miR-129-1 was performed. MiR-129-1 inhibits cell proliferation in GBM cell lines: Threecell lines were transduced with pLEX-miR-129-1, pLenti-miR-Off-129-1, pLEX-Ctrl and pLEX-Scr andevaluated for cell proliferation, cell cycle and apoptosis .Upregulation of miR-129-1 reduced theexpression level of IGF1, IGF2BP3, MAPK1 and CDK6 in GBM cell lines. IGF2BP3 and MAPK1 areinvolved in miR-129-1 dependent Cell cycle arrest: U251 cells were transfected with shIGF2BP3,shMAPK1, shIGF1or shCtrl vectors. IGF2BP3, MAPK1 and CDK6 expression correlate inversely withmiR-129-1 expression level in clinical GBM samples. MiR-129-1 is downregulated in GBM cancertissues compared with adjacent normal brain tissues. Our findings identify miR-129-1 as one of thetumour suppressor microRNAs that can be useful in getting a better understanding of GBM cancerpathogenicity.