INTRODUCTION AND OBJECTIVES: Osteopontin (OPN) is an extracellular matrix protein overexpressed in several tumor types, including colorectal cancer (CRC). The primary OPN transcript undergoes alternative splicing, generating the splicing variants (OPN-SV), called OPNa, OPNb and OPNc, and the more recently described OPN4 and OPN5. Although total OPN (tOPN) roles in CRC progression and as a biomarker are already known, the contribution of each OPN-SV in CRC has not been investigated. Therefore, this study aims to characterize the expression profile and the possible functional roles of OPNa, OPNb and OPNc in CRC cell lines.
MATERIAL AND METHODS: The CRC tumor cell lines Caco-2 (differentiated phenotype), HT-29 (moderate phenotype of differentiation) and HCT-116 (undifferentiated phenotype) have been used to evaluate the transcriptional profile of OPNa, OPNb and OPNc by means of qualitative and quantitative RT-PCR (reverse transcription polymerase chain reaction). RT-PCR assays were performing using OPN-SV specific oligonucleotides and β-ACTIN as housekeeping gene control. The transcriptional expression of each investigated OPN-SV and the overall survival were also examined by in silico analysis using GEPIA 2 (http://gepia2.cancer-pku.cn) and TVSdb database (http://www.tsvdb.com). Functional evaluation of these OPN-SV was investigated by transfecting Caco-2 cell line with pCR3.1 expression vector containing the complete cDNA of each OPN-SV or the empty vector control plasmid. Cell proliferation rates was evaluated by trypan blue staining assays.
RESULTS AND CONCLUSION: Among the OPN-SVs evaluated in Caco-2, HT-29 and HCT-116 cells lines, OPNa is the most expressed variant (p<0.05). Moreover, we also observed a higher expression of OPNa, OPNb and OPNc isoforms in HCT-116 lines and HT-29 in relation to Caco-2 cells. These data suggest that the highest OPNa expression levels may be associated with the most undifferentiated phenotype of CRC cell lines. By using in silico expression analysis, we found that the three isoforms OPNa, OPNb and OPN are overexpressed in CRC tumors when compared to non-tumor samples. This analysis also showed that OPNa is overexpressed in tumor samples in relation to the other two tested splice variants. When compared to OPNb and OPNc, OPNa is also upregulated in primary, recurrent and metastatic colorectal tumors, as well as in CRC more advanced pathological stages. Moreover, higher expression levels of these three OPN-SV are associated to poor overall survival in CRC patients. Accordingly, Caco-2 cell lines stably overexpressing OPNa displayed higher proliferative rates than those cell clones overexpressing OPNb or OPNc. Further functional studies will be performed using these cell clones ectopically overexpressing these OPN-SVs to better comprehend their roles in CRC biology.