Expression, potential biological behaviour and clinical significance of MCM3 in pancreatic adenocarcinoma: a comprehensive study integrating high throughput sequencing, CRISPR screening and in-house immunohistochemistry

Abstract

Background: Minichromosome maintenance complex component 3 (MCM3) plays a key role in various tumours. However, it remains largely unknown what the specific role and clinical significance of MCM3 in pancreatic adenocarcinoma (PAAD) are.

Materials and methods: We integrated high-throughput data from PAAD worldwide to analyse the expression level of MCM3 mRNA. We used immunohistochemistry to analyse MCM3 protein expression levels in 145 cases in the PAAD group and 29 cases in the non-PAAD group. We also mainly analysed the necessity of MCM3 for PAAD growth based on CRISPR screen data. In addition, we used enrichment analysis and protein-protein interaction networks to explore the molecular mechanism of MCM3 in PAAD. We also analysed the correlation between MCM3 expression, components of the immune microenvironment in PAAD tissue and clinical prognosis.

Results: In PAAD, we observed for the first time that MCM3 was significantly highly expressed at both the mRNA (SMD = 0.67, 95% CI: 0.38 ∼ 0.96) and the protein level (p < 0.05). The mRNA (AUC = 0.78, 95% CI: 0.74 ∼ 0.81; sensitivity = 0.66, 95% CI: 0.55 ∼ 0.76; specificity = 0.76, 95% CI: 0.67 ∼ 0.84) and protein (AUC = 0.929) expression levels of MCM3 had a good ability to distinguish between PAAD and non-PAAD tissue. There was heterogeneity reflected by the differential expression of MCM3 protein in PAAD cells. MCM3 played an essential role in PAAD growth, through abnormal DNA replication, p53 signalling and cell cycle checkpoints. PAAD with high MCM3 expression was sensitive to c-75, brivanib, flavopiridol and VNLG/124 drugs, with stable molecular docking models.

Conclusion: MCM3 is likely to be a critical element in promoting the initiation and growth of PAAD. Flavopiridol may exert its anti-PAAD effect through the interaction between MCM3, classic CDK1 targets in the cell cycle checkpoint and p53 pathway as well as related molecules in other pathways.

Keywords: CRISPR screening; MCM3; high-throughput sequencing; immunohistochemistry; pancreatic adenocarcinoma.

Graphical abstract

Figure 1.

The flowchart of the main design in this study.

Figure 2.

Integrated analysis of MCM3 mRNA expression in PAAD tissue worldwide. (A) The standard mean difference forest map of MCM3 mRNA expression in PAAD tissue*; (B) Funnel plot with pseudo 95% confidence limits; *Analysis based on normal pancreatic tissue.

Figure 3.

Discriminant analysis of PAAD tissue by highly expressed MCM3 map. (A) Summary receiver operating characteristic curve; (B) Sensitivity forest plots; (C) Specificity forest plot.

Figure 4.

Analysis of MCM3 protein expression in in-house PAAD tissue. (A)-(B) Expression status of MCM3 in non-PAAD tissue*; (C)-(F) Heterogeneous expression of MCM3 in PAAD tissue*; (G) Violin diagram for differential analysis of MCM3 protein expression levels between non-PAAD and PAAD groups; (H) ROC curve to evaluate the ability of MCM3 protein expression levels to discriminate between non-PAAD tissue groups and PAAD tissue groups; * Cells with positive MCM3 protein are in brown, and cells with negative MCM3 protein are in blue.

Figure 5.

Comparing the effect of knocking out MCM3 in the CRISPR system on the growth of PAAD cells. (A) Violin diagram comparing the difference in chronos scores between the MCM3 knockout group and the control group; (B) Identification of ROC curves between MCM3 knockout group and control group using chronos score.

Figure 6.

Mapping of potential biological behavioural analysis of MCM3 in PAAD based on intersection genes. (A) Bubble diagram of the biological process (BP) section for GO enrichment analysis; (B) Bubble diagram of the cellular component (CC) section for GO enrichment analysis; (C) Bubble diagram of the molecular function (MF) section for GO enrichment analysis; (D)Bubble diagram for KEGG enrichment analysis; (E) DNA replication of protein-protein interaction networks; (F) Cell cycle checkpoint signalling of protein-protein interaction networks; (G) P53 signalling pathway of protein-protein interaction networks.

Figure 7.

PAAD Tissue immune microenvironment status. (A) Single cell sequencing analysis of PAAD tissue based on UMAP and TSNE dimensionality reduction; (B) Expression of MCM3 in various cells of PAAD tissue based on UMAP dimensionality reduction; (C) Differences in immune infiltration between the high and low expression groups of MCM3.

Figure 8.

Analysis of the relationship between MCM3 expression level, PAAD tissue immune microenvironment status, and clinical prognosis. (A) Scatter plots of the correlation between the degree of infiltration of Th2 cells, eosinophils, and activated CD8+ T cells and the expression level of MCM3 in PAAD tissue; (B) Kaplan-Meier survival curves comparing overall survival in PAAD tissue with Th2 cells, eosinophils, and activated CD8+ T cells in the high infiltration and low infiltration groups*; (C) Kaplan-Meier survival curves comparing disease-free survival in PAAD tissue with Th2 cells, eosinophils, and activated CD8+ T cells in the high infiltration and low infiltration groups*; *Divided into a high infiltration group (Group 1) and a low infiltration group (Group 2) based on the median degree of infiltration of a certain cell in PAAD tissue.

Figure 9.

Analysis of the relationship between drug sensitivity and MCM3 in PAAD cell lines. (A) Cross-association analysis of MCM3 expression and drug reactivity; (B)-(E) Differences in drug sensitivity between high and low expression groups of MCM3*; (F)-(I) Relatively ideal models for drugs docking with the MCM3 protein; * Divide MCM3 into high expression groups and low expression groups based on its median expression value; The -log (IC₅₀) of these drugs is considered drug sensitivity.