SNRPD1 confers diagnostic and therapeutic values on breast cancers through cell cycle regulation

Abstract

Background: SNRPD1 is a spliceosome-associated protein and has previously been implicated with important roles in cancer development.

Methods: Through analyzing the differential expression patterns and clinical association of splicing associated genes among tumor and tumor adjacent samples across different tumors and among different breast cancer subtypes, we identify the tumor promotive role of SNRPD1 using multiple publicly available datasets. Through pathway, gene ontology enrichment analysis and network construction, we linked the onco-therapeutic role of SNRPD1 with cell cycle. Via a series of experimental studies including knockdown assay, qPCR, western blotting, cell cycle, drug response assay, we confirmed the higher expression of SNPRD1 at both gene and protein expression levels in triple negative breast cancer cells, as well as its roles in promoting cell cycle and chemotherapy response.

Results: Our study revealed that SNRPD1 over-expression was significantly associated with genes involved in cell cycle, cell mitosis and chromatin replication, and silencing SNRPD1 in breast cancer cells could lead to halted tumor cell growth and cell cycle arrest at the G₀/G₁ stage. We also found that triple negative breast cancer cells with reduced SNRPD1 expression lost certain sensitivity to doxorubicin whereas luminal cancer cells did not.

Conclusions: Our results suggested the prognostic value of SNRPD1 on breast cancer survival, its potential as the therapeutic target halting cell cycle progression for breast cancer control, and warranted special attention on the combined use of doxorubicin and drugs targeting SNRPD1.

Keywords: Breast cancer; Cell cycle arrest; Prognosis; SNRPD1; Therapeutics.

Fig. 1

Rational and workflow of this study. a Distribution of the gene expression of spliceosomal core Sm proteins across breast cancer subtypes using TCGA transcriptomic data (gene_TCGA). b Study workflow

Fig. 2

Expression profiles and clinical relevance of SNRPD1. a SNRPD1 expression across all cancer types in the gene_TCGA dataset. Expansion of abbreviations of the tumor names is listed in Additional file 1: Table S5. SNRPD1 expression across breast cancer subtypes in b gene_TCGA, c gene_CLM, and d protein_NC datasets. Overall survival (e) and relapse free survival (f) of SNRPD1 in breast cancer patients drawn using Kaplan Meier Plotter. g ROC curves of SNRPD1 and KI67

Fig. 3

Functional enrichment of SRFGs. a KEGG and b GO enrichment of SRFGs using the protein_NC dataset. c KEGG and d GO enrichment of SRFGs using the gene_TCGA dataset. e Network of enriched GO terms constructed using protein_NC data

Fig. 4

Assessment of the relationship between SNRPD1 and cell cycle. a Heatmap drawn using cell cycle related genes from SRFGs, and b GSEA of ‘GO_CELL_CYCLE’ using the protein_NC dataset. c Heatmap drawn using cell cycle related genes from SRFGs, and d GSEA of ‘BIOCARTA_CELLCYCLE_PATHWAY’ using the gene_TCGA dataset. e Forest plot showing correlations between SNRPD1 and cell cycle related genes from SRFGs across multiple datasets

Fig. 5

Cell viability upon knocking down SNRPD1. a Sequence alignment results showing the target loci of the two siRNAs on SNRPD1. Knocking down efficiency of siRNAs used for silencing SNRPD1 in MCF7 and MDAMB231 cells as assessed using b q-PCR and c western blot. ‘si-1’, ‘si-2’ and ‘si-1 + 2’ each represents siRNA-1, siRNA-2 and their pooled effects. d Normalized cell viability upon SNRPD1 silencing using pooled siRNAs

Fig. 6

Cell cycle alteration measurement upon knocking down SNRPD1. Quantified Cell cycle profiles as measured by cell flowmetry upon SNRPD1 silencing in a MCF7 cell line, b MDAMB231 cell line, c MDAMB361 cell line, d HCC1937 cell line

Fig. 7

Cell viability in response to Doxorubicin upon knocking down SNRPD1. Drug response curves with and without silencing SNRPD1 in a MCF7 cell line, b MDAMB231 cell line, c MDAMB361 cell line, d HCC1937 cell line

Fig. 8

Expression of cell cycle related genes and potential mechanism of SNRPD1 in cell cycle regulation. a Expression of cell cycle related genes CDCA5, NDC80, CCNA2, PCNA, CCNB1, CDK1, CCND1 after silencing SNRPD1 at the mRNA level in MCF7 and MDAMB231 cells. b Expression of cell cycle related gene CCND1 after silencing SNRPD1 at the (b) proteomic levels together with its quantified signal intensity in MCF7 and MDAMB231 cells. c Predicted protein–protein interaction network of SNRPD1 and cell cycle related proteins using STRING. d Immunoprecipitation of SNRPD1 and PCNA in MCF7 and MDAMB231 cells