FAT1 gene is localized on chromosome 4q35.2 coding a 506KDa transmembrane protein and functions as an oncogene or tumor suppressor depending on tissue types in human cancers. Our lab has identified that FAT1 has oncogenic role in glioblastoma (GBM). PDCD10 gene encodes an evolutionarily conserved protein that is widely expressed in almost all human tissues. Here in our study we are characterizing the role of FAT1 gene in primary brain tumors and in the regulation of miRNAs and its target gene in GBM.
In order to delineate molecular crosstalk among factors driving glioma progression, we used knockdown method in glioma cell lines followed by expression analysis by Q-PCR. FAT1 knockdown was done using FAT1 specific siRNA and mRNA and miRNA expression analysis by specific primers in GBM cell lines. Expression and Spearman correlation analysis of FAT1 with miR-221-3p and PDCD10 was done in GBM tumor samples (n = 30). In a sequential bioinformatics study, we analyzed TCGA GBM dataset for FAT1 and PDCD10 expression and perform spearman correlation in GBM tumors (n = 430) as compared to normal brain (n = 10).
On FAT1 knockdown, using FAT1 specific siRNA we observed significantly decreased expression of miR-221/222-3p. In-silico analysis recognized, PDCD10 as a potential targets of miR-221/222-3p. Furthermore, FAT1 knocked-down cells showed significantly increased expression of PDCD10 in all studied glioma cell lines. In order to validate our in-vitro observation and its clinical relevance, we have done expression and correlation study in GBM tumor samples. We observed significant positive spearman correlation between FAT1 and miR-221-3p (r = 0.5669, p ≤ 0.0011) and negative correlation of FAT1 with PDCD10 (r= -0.3492, p ≤ 0.0585),) and miR-221-3p with PDCD10 (r = 0.526, p ≤ 0.0028). In TCGA database we have observed negative correlation between FAT1 and PDCD10(r= -0.4209, p ≤ 0.0001). These results propose that FAT1 regulating the expression of miR-221-3p leading to downregulation of PDCD10 in GBM cell lines and GBM tumors.
Our in-vitro and GBM tumor data for the first time suggesting FAT1 to be a novel molecule regulating the expression of PDCD10 via miR221-3p in GBM.
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All authors have declared no conflicts of interest.