Epithelial-mesenchymal transition markers screened in a cell-based model and validated in lung adenocarcinoma

Abstract

Background: Re-capture of the differences between tumor and normal tissues observed at the patient level in cell cultures and animal models is critical for applications of these cancer-related differences. The epithelial-mesenchymal transition (EMT) process is essential for tumor migratory and invasive capabilities. Although plenty of EMT markers are revealed, molecular features during the early stages of EMT are poorly understood.

Methods: A cell-based model to induce lung cell (A549) EMT using conditioned medium of in vitro cancer activated fibroblast (WI38) was established. High-throughput sequencing methods, including RNA-seq and miRNA-seq, and advanced bioinformatics methods were used to explore the transcriptome profile transitions accompanying the progression of EMT. We validated our findings with experimental techniques including transwell and immunofluorescence assay, as well as the TCGA data.

Results: We have constructed an in vitro cell model to mimic the EMT in patients. We discovered that several new transcription factors were among the early genes (3 h) to respond to cancer micro-environmental cues which could play critical roles in triggering further EMT signals. The early EMT markers also included genes encoding membrane transporters and blood coagulation function. Three of the nine-examined early EMT hallmark genes, GALNT6, SPARC and HES7, were up-regulated specifically in the early stages of lung adenocarcinoma (LUAD) and confirmed by TCGA patient transcriptome data. In addition, we showed that miR-3613, a regulator of EGFR pathway genes, was constantly repressed during EMT progress and indicative of an epithelial miRNA marker.

Conclusions: The CAF-stimulated EMT cell model may recapture some of the molecular changes during EMT progression in clinical patients. The identified early EMT hallmark genes GALNT6, SPARC and HES7and miR-3613 provide new markers and therapeutic targets for LUAD for the further clinical diagnosis and drug screening.

Keywords: EMT; Lung adenocarcinoma; RNA-seq; WGCNA; miRNA-seq.

Fig. 1

CAF-CM induces mesenchymal phenotypes in A549 cell. a Bright-field images of CAF conditioned medium or control treated A549 cells at 3, 24and 72 h. CAF-CM treated A549 cells showed the loss of cell-cell contact and scattering phenotypes at 24 and 72 h, while control cells maintained the epithelial cobblestone shape. b DAPI staining images of CAF conditioned and control medium treated A549 cells at 3, 24 and 72 h subject to Transwell matrigel. CAF-CM treated cells showed enhanced migratory and invasive properties than control at 3, 24 and 72 h. c Quantitative analysis of Transwell assay showed enhanced migratory and invasive properties after CAF-CM treatment. * p < 0.05; ** p < 0.01; *** p < 0.001; t-test

Fig. 2

CAF-CM induces expression of mesenchymal markers in A549 cell. a Immunostaining of E-cadherin (green), vimentin (red) and DAPI (blue) of CAF treated and control cells. Beginning at 3 h, CAF treated cells showed increased vimentin and decreased E-cadherin expression compared to controls. b RT-qPCR of mRNA levels of EMT marker genes in A549 cells with CAF conditioned medium or control treatment at 3, 24 and 72 h. Error bars are SD. *, p < 0.05; **, p < 0.01; t test

Fig. 3

WGCNA analysis of the temporal mRNA expression changes of CAF-CM induced A549 EMT. a Number of DEGs at each time point between CAF-CM and control treated cells. b GO analysis of the biological process ontology of all DEGs between CAF-CM and control treated cells. Top ten terms and corrected p-values were shown. c Hierarchical cluster dendrogram of all DEG modules. Modules corresponding to branches are labeled with colors indicated by the color bands underneath the tree. d-g Eigengene bar plot of turquoise (d), red (e), yellow (f) and blue (g) modules

Fig. 4

Blue module mRNAs represent a class of early EMT markers. a Network connection of the most highly connected genes in the blue module. Blue lines indicate positive expression correlation, and red line indicates negative expression correlation. Pink circles indicate transmembrane transporters in this module. b-f RT-qPCR results of mRNA levels of key blue module genes in CAF treated and control A549 cells at different time points. Error bars are SD. *, p < 0.05; **, p < 0.01; t-test

Fig. 5

Characteristics of the yellow module mRNAs during early EMT. a Network connection of the most highly connected genes in the yellow module. Blue lines indicate a positive expression correlation, and red line indicates a negative expression correlation. Pink circles indicate transcription regulatory genes within the module. b-f RT-qPCR results of mRNA levels of key yellow module genes in CAF treated and control A549 cells at different time points. Error bars are SD. *, p < 0.05; **, p < 0.01; t-test

Fig. 6

MiR-3613 regulates EGFR pathway genes in CAF induced A549 early EMT. a Number of DEmiRNAs at each time point between CAF and control treated A549 cells. b Transcripts per million (TPM) of miR-3613-3p in CAF treated and control A549 cells. ***, p < 0.001, Fisher’s exact test. c GO analysis of the biological process ontology of miR-3613 predicted targets. Top ten terms and corrected p-values were shown. d Predicted miR-3613-3p regulatory targets in the EGFR pathway from 3 to 24 h after CAF treatment. e Barplot showing the downregulation of miR-3613 targets after miR-3613 overexpression. **, p < 0.01, ***, p < 0.001; t-test

Fig. 7

GALNT6, SPARC and HES7 are up-regulated in early stage LUAD. Violin plot showing GALNT6 (a), SPARC (b) and HES7 (c) mRNA levels in normal lung tissue and lung adenocarcinoma using RNA-seq data from TCGA database. (d) Violin plot showing the average expression level of genes with elevated expression pattern at early stages (I and II) LUAD from brown module. *, p < 0.05; **, p < 0.01; ***, p < 0.001; compared to normal tissues or as indicated