Long noncoding RNA and mRNA profiling in MDA-MB-231 cells following RNAi-mediated knockdown of SIRT7

Abstract

Breast cancer is one of the most common malignant cancers among women and a major clinical obstacle. Although studies have reported the abnormal expression of SIRT7 in breast cancer, whether the function of SIRT7 regulates the expression of long noncoding RNAs (lncRNAs) in breast cancer remains unknown. We aimed to determine the differential expressions of mRNAs and lncRNAs associated with SIRT7 and understand the regulatory mechanism of SIRT7 in breast cancer. RNA sequencing was performed to explore the transcriptome in MDA-MB-231 cells after SIRT7 depletion, and a total of 50,634 different transcripts were identified. In comparison with the negative control, siSIRT7 groups showed 240 differentially expressed mRNAs and 26 differentially expressed lncRNAs. Gene ontology analysis revealed that the differentially expressed mRNAs mainly regulated DNA replication, CXCR chemokine receptor binding, and maturation of large subunit rRNA from tricistronic rRNA transcript, nucleoplasm, mitochondrion, and NAD⁺ ADP-ribosyltransferase activity. Kyoto Encyclopedia of Genes and Genomes analysis showed that the differentially expressed mRNAs were mainly involved in pathways associated with MAPK signaling pathway, tumor necrosis factor signaling pathway, hepatitis B, and cancer. Moreover, the target genes of the differentially expressed lncRNAs mainly regulated the carboxylic acid metabolic processes and were involved in glycolysis pathway. The mRNA-lncRNA coexpression network comprised 186 mRNAs and 23 lncRNAs. Our results provide essential data regarding differentially expressed lncRNAs and mRNAs after the depletion of SIRT7 in breast cancer cells, which may be useful to elucidate the role of SIRT7 in breast cancer development.

Keywords: RNA-Seq; SIRT7; breast cancer cell; lncRNA; mRNA.

Figure 1

The mRNA expression and protein levels were detected by qRT-PCR (A), agarose gel electrophoresis (B), and Western blot (C, D) after transfection of MDA-MB-231 breast cancer cells with three different pairs of siRNA. si-1, si-2, and si-3 represent different sequences of siSIRT7. **p<0.01 and ***p<0.001. Abbreviation: NC, negative control.

Figure 2

Differentially expressed mRNAs (A) and lncRNAs (B) were analyzed by hierarchical clustering within NC and siSIRT7 groups in MDA-MB-231 cells; red indicates high relative expression, and green indicates low relative expression. (C, D) The number of mRNAs and lncRNAs differentially expressed. Abbreviation: NC, negative control.

Figure 3

qRT-PCR was performed to confirm the validity of the RNA-Seq data in MDA-MB-231 and MCF-7 cells. The mRNAs (A, C) and lncRNAs (B, D) were expressed as log2 values (RNA-Seq) and −ΔΔCt values (qRT-PCR). Abbreviation: RNA-Seq, RNA sequencing.

Figure 4

GO analysis of the differentially expressed genes after depletion of SIRT7. (A) GO analysis results showed that these mRNAs were enriched in the biological functions related to cellular processes, biological regulation, metabolic process, and signal-organism process. (B) The cellular component of these genes included cell, cell part, membrane, and organelle. (C) The molecular function of these genes composed of binding, catalytic activity, transporter activity, and molecular transducer activity. Abbreviation: GO, gene ontology.

Figure 5

The top 10 GO enrichment analysis of the upregulated mRNAs: (A) biological process, (B) cell component, and (C) molecular function. Abbreviations: DE, differentially expressed; GO, gene ontology.

Figure 6

The top 10 GO enrichment analysis of the downregulated mRNAs: (A) biological process, (B) cell component, and (C) molecular function. Abbreviations: DE, differentially expressed; GO, gene ontology.

Figure 7

KEGG analysis of the differentially expressed mRNAs. (A) The KEGG analysis results showed that the most enriched pathways included MAPK signaling pathway; those involved in cancer, HTLV-I, and hepatitis B infection; and TNF signaling pathway. (B, C) The top 10 pathway corresponding to the upregulated mRNAs and downregulated mRNAs. Abbreviations: DE, differentially expressed; HTLV-I, human T-cell lymphotropic virus type I; KEGG, Kyoto Encyclopedia of Genes and Genomes; TCA, tricarboxylic acid; TNF, tumor necrosis factor.

Figure 8

GO and KEGG analyses of the target genes of these differentially expressed lncRNAs. (A) Top 10 GO terms of biological processes for lncRNA-targeted genes. (B) Top 10 pathways corresponding to the lncRNA-targeted genes. Abbreviations: DE, differentially expressed; GO, gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 9

The differentially expressed mRNA-lncRNA coexpression network. Box nodes represent lncRNAs, while circular nodes represent mRNAs. Red represents upregulation, and blue represents downregulation.