Role of EFNA1 SNP (rs12904) in Tumorigenesis and Metastasis of Colorectal Cancer: A Bioinformatic Analysis and HRM SNP Genotyping Verification

Abstract

Objective: Colorectal cancer is a prevalent disease with a poor prognosis and is known as a heterogeneous disease with many differences in clinical Symptoms and molecular profiles. The present study aimed to systematically evaluate the association of SNPs in miRNA binding sites of target genes that are involved in CRC angiogenesis, epithelial to mesenchymal transition, and cytoskeleton organization with tumorigenesis and metastasis of CRC.

Methods: A case-control study was performed on 146 samples of CRC patients and 132 healthy samples. After that, the DNA of all samples was isolated by the salting-out method. Finally, the genotypes for EFNA1 SNP (rs12904) were identified by HRM (High-resolution melting analysis) method. In order to evaluate the results of genotyping, two samples from each genotype were sequenced using the sanger sequencing method.

Result: The frequency of AA genotype and the frequency of GG for rs12904 in satge4 and other stages are different from each other (P-value <0.0001) (P-value = 0.008). Also, the frequency of AA genotype in patients with different grades is different from each other (P-value = 0.035), while the frequency of AG genotype and the frequency of GG genotype is not significantly different in patients with different grades (P-value = 0.377) (P-value = 0.284).

Conclusion: Results of this study indicated that patients carrying the GA and GG genotypes reduced the risk of disease progression compared to the AA genotype. As a result, this polymorphism plays a key role in CRC pathogenesis and metastasis and could be used as a biomarker in molecular diagnosis and metastatic state prediction in the near future after further study of its signaling pathways and molecular mechanism.

Keywords: Colorectal Neoplasms; EFNA1; Metastasis; Polymorphism; rs12904.

Figure 1

Schematic Diagram of Bioinformatics Analysis Workflow

Figure 2

Venn Diagram of Identified Genes for GO Terms Angiogenesis, Cytoskeleton Organization and EMT. The three genes SFRP2, TGFBR2, and EFNA1 are involved in both the processes of angiogenesis and EMT, while the SRF gene is involved in the processes of angiogenesis and cytoskeleton organization

Figure 3

Genotyping the rs12904 on EFNA1 via HRM. (a) Normalized melting curve, (b) Normalized Melting Peaks, C) Difference Plot. In all plots, samples were categorized similarly to 3 genotypes including AA, GG, and AG

Figure 4

Sanger Sequencing Analysis for Samples. six samples which their geneotypes were identified with HRM method evaluated with sanger sequencing including 2 GA genotype , 2 AA genotype and 2 GG genotype