Constructing a molecular genotyping assay for rs11077 based on real-time polymerase chain reaction high resolution melting (PCR HRM) technique for the prognosis of hepatocellular carcinoma (HCC) patients

Abstract

XPO5 codes for the nuclear transport factor exportin-5, which is a membrane-bound protein. This gene is responsible for the transport of pre-miRNA from the nucleus to the cytoplasmic compartments, thereby adjusting the whole miRNA expression level. The reduction of the miRNA levels was recorded when XPO5 was knocked down. rs11077 is found in the 3′UTR region of XPO5, and this SNP might affect mRNA stability and be associated with the altered expression of XPO5. This leads to the universal suppression of miRNA expression profiles, thereby mediating the HCC survival. HCC patients bearing C/C and A/C genotypes of rs11077 had a survival rate of 60% after 3 years; and this rate was reduced to 24.7% with HCC patients bearing the A/A genotype. In this study, we constructed a molecular assay based on a real-time PCR HRM technique for rs11077 genotyping. We successfully designed the primer pair for the real-time PCR HRM of rs11077. We also found the optimal concentration of MgCl2 to arrive at a clear differentiation of the three genotypes of rs11077. Thereafter, we characterised the analytical specificity and the precisions of the molecular assay. The SNP genotyping results were compared between the real-time PCR HRM and nucleotide sequencing. Finally, we evaluated the molecular assay on 123 human blood samples. The rs11077 genotyping assay in this study could be used for the prognosis of HCC patients.

Keywords

• hepatocellular carcinoma
• real-time PCR HRM
• rs11077
• SNP