Sequencing of high-frequency mutated genes in breast cancer (BRCA) and associated-functions analysis

Mutations or aberrant expression of genes in an organism tend not to be completely random and this cumulative effect predisposes to the development of malignant tumours. This study aims to reveal the possible aberrant expression of high frequency mutated genes, and then to investigate their role in development, prognosis, signalling pathway function and drug resistance in breast cancer.

The mutated genes in breast cancer (BRCA) clinical samples were identified and detected by high-throughput sequencing. High-frequency mutant genes were counted. Gene expression profiles and the relationship with prognosis were analysed throughout TCGA database. qRT-PCR was used to analyse the mRNA levels of the six high-frequency mutant genes in BRCA tissues and cell lines. IHC was used to analyse the protein levels of the six high-frequency mutant genes in BRCA tissues. The linear interaction, single-cell layer clustering status and the influence in immune cell infiltration degree among these six high-frequency mutant genes were analysed by bioinformatics analysis. The STITCH and cMAP datasets were used for high-frequency mutant gene interaction networks, association signalling pathway enrichment and drug-transcriptome analyses. The effects of trastuzumab on the proliferative capacity of breast cancer cells, as well as on the expression of six high-frequency mutated genes were determined by CCK8 assay.

The genes that were statistically found to have high-frequency mutations in the samples recruited in the present study by high-throughput sequencing analysis included TP53, PIK3CA, NF1, TBX3, BRCA1 and BRCA2. The expression profiles of these genes and the correlation with prognosis were further demonstrated using the TCGA database: the trend in this study was similar to that of BRCA in TCGA. The mRNA and protein expression of these genes showed that the expression of TP53, NF1, TBX3, BRCA1 and BRCA2 was higher in tumor samples than that in normal samples, with an opposite trend for PIK3CA, a similar trend was observed in BRCA cell lines. The protein expressions of TP53, NF1, TBX3, BRCA1 and BRCA2 displayed the same trend by IHC. Other correlation results include 1) the single cell layer clustering of these six genes resulted in significant clustering with few overlapping regions; 2) these six genes showed different degrees of influence on BRCA immune cell infiltration; 3) these six genes showed a significant correlation between each other; 4) the network of each gene had partially overlapping molecules; and 5) the PI3K pathway was a key association pathway in BRCA. Finally, the cell proliferation ability results confirmed the optimal concentration of trastuzumab and its effect on mRNA expression of these six genes.