Experimental Validation of A Novel Alternative Polyadenylation Isoform of Human spp1 Gene in U87-MG Cell Line.

Objective: Osteopontin (OPN), also known as SPP1 (secreted phosphoprotein) is a secreted protein present in bodily fluids and tissues. It is also a tumour-associated protein, and elevated OPN levels are associated with tumour formation, progression and metastasis. In cancer patients expression of OPN has been associated with poor prognosis in several tumor types including breast, lung, and colorectal cancers. Research has revealed a promising role for OPN as a cancer biomarker. OPN is subject to alternative splicing, as well as post-translational modifications such as phosphorylation, glycosylation and proteolytic cleavage. Functional differences have been revealed for different isoforms and post-translational modifications. The pattern of isoform expression and post-translational modification is cell-type specific and may influence the potential role of OPN in malignancy and as a cancer biomarker. Tumor specific splice variants are being discovered at an increasing rate and their functions are also investigated in cancer progression. Materials and Methods: After culturing U87-MG cell line in proper medium, RNA extraction following by single strand cDNA synthesis has been performed. Multiple products of RT-PCR by specific primers for spp1 mRNA was isolated and cloned in pTZ57R/T vector for sequencing. Results: We found a novel transcription variant of spp1 gene in U87-MG cell line, experimentally. Our sequencing data revealed that this novel variant lacks a region that potentially is a binding site of some micro-RNAs. Conclusion: Our study revealed that this novel variant has same function with other variants of this gene but we assume that post-transcriptional control mechanisms for this variant are different from the other variants. Computational analysis reveals that shorter variant of spp1 gene has so many regions that are complementary with some micro-RNAs. Then shorter variant is more stable than longer one and has a significant association with cancer [ABSTRACT FROM AUTHOR]