Differential Expression of E-cadherin Gene by Real Time RT-PCR and Exon 1 Sequence Analysis in Invasive Breast Carcinoma

Abstract

Introduction: Genomic alterations in key genes such as tumour suppressor genes have been reported to contribute to human cancers like breast cancer. Loss of E-cadherin (CDH1) mediated adhesion characterises the transition from benign lesions to invasive, metastatic cancer. Genetic changes occurring in the CDH1 gene has not yet been completely studied despite the remarkable biological function of the signal peptide of CDH1. Many of these genomic alterations have altered messenger Ribonucleic Acid (mRNA) and protein expression that play a role in the pathophysiology of cancers and warrant further studies. Aim: To identify mutations in CDH1 gene (Exon 1) in invasive breast carcinoma and evaluate CDH1 expression by real time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). Materials and Methods: The present study was a retrospective cross-sectional study in which tissue samples were collected from Madras Medical College and VS Hospitals, Chennai, Tamil Nadu, India between March 2010 to August 2011 and molecular biology work and analysis was carried out at Sri Ramasamy Memorial Institute of Science and Technology-Department of Biotechnology (SRM-DBT) Platform for Advanced Life technologies, SRM Institute of Science and Technology (SRMIST) between June 2017 to September 2017. The Exon 1 region of the human E-cadherin gene of normal and breast cancer (Infiltrating Ductal Carcinoma) patients were sequenced by Sanger method. The sequences were compared using the National Centre for Biotechnology Information-Basic Local Alignment Search Tool (NCBI-BLAST) utility. The change in the sequence was identified between the normal and tumour samples. The fold change of E-cadherin gene expression in tumour was calculated by comparing with control non neoplastic breast tissue. The fold change in the relative expression level between tumour and normal sample was determined using real time RT-PCR. Results: In the present study upon comparing the Exon 1 sequences of normal gene with that of tumour, two deletion mutation and one CT transition was observed. In present investigation also 0.08-fold down regulation of CDH1 mRNA was observed in the tumour tissue when compared with the normal tissue. Conclusion: The present study has attempted to study the alterations at genome level (CDH1 gene, Exon 1, encoding for the biologically active signal peptide region), transcriptional and translational level with respect to CDH1. The effect of the mutations detected including two loci with deletion mutations and one single nucleotide change could affect the structural conformation of the protein and functional impact including aberrant expression. Molecular docking studies and in-vitro studies with cell lines and animal studies could be done to confirm these findings.

Keywords

• expression level
• fold change
• reverse transcriptase polymerase chain reaction
• sequence change
• signal peptide