Identification of New Key Genes and Their Association with Breast Cancer Occurrence and Poor Survival Using In Silico and In Vitro Methods

Abstract

Breast cancer is one of the most prevalent types of cancer diagnosed globally and continues to have a significant impact on the global number of cancer deaths. Despite all efforts of epidemiological and experimental research, therapeutic concepts in cancer are still unsatisfactory. Gene expression datasets are widely used to discover the new biomarkers and molecular therapeutic targets in diseases. In the present study, we analyzed four datasets using R packages with accession number GSE29044, GSE42568, GSE89116, and GSE109169 retrieved from NCBI-GEO and differential expressed genes (DEGs) were identified. Protein-protein interaction (PPI) network was constructed to screen the key genes. Subsequently, the GO function and KEGG pathways were analyzed to determine the biological function of key genes. Expression profile of key genes was validated in MCF-7 and MDA-MB-231 human breast cancer cell lines using qRT-PCR. Overall expression level and stage wise expression pattern of key genes was determined by GEPIA. The bc-GenExMiner was used to compare expression level of genes among groups of patients with respect to age factor. OncoLnc was used to analyze the effect of expression levels of LAMA2, TIMP4, and TMTC1 on the survival of breast cancer patients. We identified nine key genes, of which COL11A1, MMP11, and COL10A1 were found up-regulated and PCOLCE2, LAMA2, TMTC1, ADAMTS5, TIMP4, and RSPO3 were found down-regulated. Similar expression pattern of seven among nine genes (except ADAMTS5 and RSPO3) was observed in MCF-7 and MDA-MB-231 cells. Further, we found that LAMA2, TMTC1, and TIMP4 were significantly expressed among different age groups of patients. LAMA2 and TIMP4 were found significantly associated and TMTC1 was found less correlated with breast cancer occurrence. We found that the expression level of LAMA2, TIMP4, and TMTC1 was abnormal in all TCGA tumors and significantly associated with poor survival.

Keywords: breast cancer; differentially expressed genes; down regulated genes; poor survival; up regulated genes.

Figure 1

DEGs in breast cancer tissues. Volcano plot presenting DEGs in breast cancer tissues in datasets. (a) GSE29044 (b) GSE42568 (c) GSE89116 (d) GSE109169 (e)Venn diagram represented the down regulated overlapping DEGs among GSE29044, GSE42568, GSE89116, and GSE109169 datasets. (f) Venn diagram represented the up-regulated overlapping DEGs among GSE29044, GSE42568, GSE89116, and GSE109169 datasets.

Figure 2

Protein-protein interaction network showing down-regulated (green) DEGs and up-regulated (red) DEGs. Blue colour is presenting supporting genes.

Figure 3

Network/modules/sub-modules at different levels of network.

Figure 4

Comparisons of the expression of the nine genes between breast cancer and normal breast tissues in TCGA and GTEx based on GEPIA. The Y axis represents the log2 (TPM + 1) for gene expression. The Gray bar indicates the normal tissues, and the red bar shows the breast cancer tissues. These figures were derived from GEPIA. TPM: transcripts per kilobase million. The box plots (a–i) of all nine hub genes demonstrate that the genes were abnormally expressed in breast cancer as compared to normal breast tissue. (a) ADAMTS5—down-regulated, (b) COL11A1—up-regulated, (c) PCOLCE2—down-regulated, (d) RSPO3—down-reguloated, (e) LAMA2—down-regulated, (f) MMP11—up-regulated, (g) COL10A1—up-regulated, (h) TIMP4—down-regulated, and (i) TMTC1—down-regulated. * p < 0.05.

Figure 5

The expression-stage plot of three genes associated with breast cancer. The plots were achieved by the GEPIA web server. The expression-stage plot analysis (violin plots a–c) revealed that three genes namely (a) LAMA2, (b) TMTC1, and (c) TIMP4 among these nine genes were found significantly associated (p < 0.05) with different stages of breast cancer.

Figure 6

Violin plot showing gene expression among groups of patients categorized according to age (a–c). We found that the genes namely (a) LAMA2, (b) TMTC1, and (c) TIMP4 were significantly expressed among different age groups of patients, i.e., lower 21 age to higher 97 age groups as indicated by the violin plots.

Figure 7

Asociation of genes with breast cancer occurrence (a–c). (a) LAMA2 and (b) TIMP4 were found significantly associated and (c) TMTC1 gene was found less correlated with breast cancer occurrence.

Figure 8

Pan-cancer view of LAMA2, TIMP4, and TMTC1 expression level (a–c). We found that the expression level of (a) LAMA2, (b) TIMP4, and (c) TMTC1 was higher in all TCGA tumors.

Figure 9

Analysis of the prognostic value of three differentially expressed genes in breast cancer patients using The Cancer Genome Atlas data. All the three genes (a) LAMA2, (b) TIMP4, and (c) TMTC1were found significantly (p < 0.05) associated with poor survival.

Figure 10

Validation of key regulatory genes in human breast cancer cell lines using qRT-PCR. In the figure, (a–c) presents up-regulated and (d–g) presents down-regulated genes, ** p < 0.01, *** p < 0.001.