Alterations in Gene Expression and Alternative Splicing Induced by Plasmid-Mediated Overexpression of GFP and P2RY12 Within the A549 Cell Line

Abstract

Phenotypic modifications and their effects on cellular functions through the up-regulation of target gene expression have frequently been observed in genetic studies, but the unique roles of cell lines and their introduced plasmids in influencing these functions have not been fully revealed. In this research, we developed two distinct cell lines derived from the A549 cell line: one that stably overexpresses GFP and another that is a polyclonal stable line overexpressing both GFP and P2RY12. We then utilized transcriptome sequencing (RNA-seq) technology to screen out differentially expressed genes (DEGs) and genes with differential transcript usage (gDTUs) after GFP overexpression (GFP-OE) and P2RY12 overexpression (P2RY12-OE). We found that, compared with A549, there were more than 1700 differentially expressed genes (DEGs) in both GFP-OE and P2RY12-OE cells, while only 866 DEGs were identified in GFP-OE and P2RY12-OE cells. Notably, the differences in transcript usage were relatively minor, with only over 400 genes exhibiting changes across all three groups. The functional analysis of DEGs and gDTUs showed that they were both highly enriched in the pathways associated with cell proliferation and migration. In summary, we performed an extensive analysis of the transcriptome profile of gene expression and alternative splicing with GFP-OE and P2RY12-OE, enhancing our comprehension of how genes function within cells and the processes that control gene expression.

Keywords: A549; DEGs; GFP; P2RY12; gDTUs; overexpression cell line.

Figure 1

Validation of GFP-OE and P2RY12-OE: (A) a pictorial representation illustrating the process of plasmid transfection aimed at creating GFP-OE and P2RY12-OE; (B) the fluorescence of A549, GFP-OE, and P2RY12-OE; and (C) a histogram illustrating the qRT-PCR results for samples that either were within the normal range or exhibited increased levels of P2RY12. Each sample was tested with three biological replicates. ****: p< 0.0001, ns means not significant, one-way ANOVA test.

Figure 2

Translational differences between A549, GFP-OE, and P2RY12-OE: (A) principal component analysis (PCA) based on TPM values of three biological replicates in each group; volcano plots of the DEGs from the comparisons between GFP-OE and A549 (B), P2RY12-OE and A549 (C), and P2RY12-OE and GFP-OE (D), Blue dots show genes with down-regulated expression and red dots show genes with up-regulated expression; the top ten enriched GO terms of DEGs from the comparisons between GFP-OE and A549 (E), P2RY12-OE and A549 (F), and P2RY12-OE and GFP-OE (G); and the top ten enriched KEGG pathways of DEGs from the comparisons between GFP-OE and A549 (H), P2RY12-OE and A549 (I), and P2RY12-OE and GFP-OE (J).

Figure 3

Common DEGs and qRT-PCR validation: (A) a Venn diagram of shared DEGs in three comparisons; (B) an UpSet diagram showing the intersection of molecular function in three comparisons, the dots with lines show genes were simultaneously enriched in multiple molecular functions; (C) an UpSet diagram showing the intersection of KEGG pathways in three comparisons, the dots with lines show genes were enriched across different pathways; and (D) bar plots showing the TPM values and qRT-PCR results for six selected DEGs. All experiments were performed in triplicate.

Figure 4

Alternative splicing events in GFP-OE and P2RY12-OE: (A) the number of genes with different isoform counts; (B) the number of genes with different types of alternative splicing events. AL, alternative last exon; AF, alternative first exon; A5, alternative 5′ splice site; RI, retained intron; SE, skipping exon; ME, mutually exclusive exons; A3, alternative 3′ splice site, the grey boxes show constitutive exon, the red boxes show alternatively splice exon; the top ten enriched GO terms of gDTUs from the comparisons between GFP-OE and A549 (C), P2RY12-OE and A549 (D), and P2RY12-OE and GFP-OE (E); and the top ten enriched KEGG pathways of gDTUs from the comparisons between GFP-OE and A549 (F), P2RY12-OE and A549 (G), and P2RY12-OE and GFP-OE (H).

Figure 5

Overlap between DEGs and gDTUs. Venn plots depicting the intersection of DEGs and gDTUs in the comparison between GFP–OE against A549 (A), P2RY12–OE against A549 (B), and P2RY12–OE against GFP–OE (C); and (D) the visualization of FBL expression alterations in GFP-OE compared to A549 using IGV.