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. 2022 Nov;247(21):1907-1916.
doi: 10.1177/15353702221121631. Epub 2022 Sep 12.

Polyadenylate-binding protein cytoplasmic 1 mediates alternative splicing events of immune-related genes in gastric cancer cells

Xincai Xu  1 , Wenbin Zhang  1 , Hua Gao  1 , Yi Tan  1 , Yangchao Guo  1 , Tiehan He  1
Affiliations

Polyadenylate-binding protein cytoplasmic 1 mediates alternative splicing events of immune-related genes in gastric cancer cells

Xincai Xu et al. Exp Biol Med (Maywood). 2022 Nov.

Abstract

Polyadenylate-binding protein cytoplasmic 1 (PABPC1) is dysregulated in malignancies, which is considered as a potential therapeutic target for many cancer types. By alternative splicing (AS) for gastric cancer (GC), we described PABPC1-modulated AS events in this study. PABPC1 expression was analyzed in 408 GC tissues from The Cancer Genome Altas (TCGA) database. Human gastric adenocarcinoma (AGS) cells were transfected with PABPC1-specific small interfering RNA (siPABPC1) with siCtrl as a negative control. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was done for the determination of transcripts. To detect the differentially expressed genes (DEGs) and 10 different types of AS events, RNA sequencing (RNA-seq) was performed. DEGs were analyzed for functional categories including gene ontology, and the Kyoto encyclopedia of genes and genomes pathway were analyzed for DEGs. GC displayed an elevated expression of PABPC1. PABPC1 was efficiently knocked down in AGS cells. Here, we excavated 1234 PABPC1-regulated DEGs, among which 502 were down-regulated and 732 were up-regulated compared to the siCtrl group. A total of 94 DEGs were involved in inflammation and immune response. Results from qRT-PCR validated the up-regulation of 10 immune and inflammation-related DEGs in the siPABPC1 group. PABPC1 deficiency causes 1304 AS events differentially occurred in AGS cells. The most common type of AS events regulated by PABPC2 is alternative 5' splice sites. qRT-PCR confirmed the transcription level of five immune-related genes, in which AS events were detected in the siPABPC1 group. PABPC1 knockdown mediates AS events and thus the transcript level of immune and inflammation-related genes in AGS cells. PABPC1-regulated oncogenic AS events display potential as targets for therapeutic development.

Keywords: PABPC1; RNA sequencing; alternative splicing events; differentially expressed genes; gastric cancer; immune pathway.

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Conflict of interest statement

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.
Figure 1.
PABPC1 was down-regulated in gastric cancer tissue and PABPC1 mRNA expression was altered in AGS cells after transfection. (A) Data extracted from TCGA database suggest PABPC1 expression in gastric cancer tissues; boxplots show the median (center line), first quartile (Q1, lower quartile), third quartile (Q3, upper quartile), maximum (the highest data point in the data set excluding any outliers), and minimum (the lowest data point in the data set excluding any outliers); *P < 0.05 versus normal tissues (Student’s t-test). (B) AGS cells transfected with siRNA for PABPC1 or negative control were assayed by qRT-PCR for PABPC1 quantification. Data represent three independent technical repetitions and three independent biological repetitions; ***P < 0.001 versus siCtrl (Student’s t-test). Data are expressed as mean ± standard deviation, where the standard deviation is indicated as error bars. (A color version of this figure is available in the online journal.)
Figure 2.
Figure 2.
Identification of PABPC1-regulated DEGs by RNA-seq. (A) Volcano plots presenting differentially expressed genes in AGS cells after transfection with siPABPC1 or siCtrl. The colored dots denote significantly down-regulated (blue), up-regulated (red), or non-differentially expressed genes (gray) in siPABPC1-transfected cells compared to siCtrl-treated cells. (B) Clustering analysis of siCtrl and siPABPC1 groups based on the differentially expressed genes. The top bar values represent the relative expression of the differentially expressed genes. (A color version of this figure is available in the online journal.)
Figure 3.
Figure 3.
(A) Up-regulated and (B) down-regulated biological processes were compared between siPABPC1-treated AGS cells and siCtrl-treated cells after GO enrichment analysis. (A color version of this figure is available in the online journal.)
Figure 4.
Figure 4.
Heatmap of PABPC1-regulated genes involved in immune response and inflammation. The right bar values represent the relative expression of the indicated genes. (A color version of this figure is available in the online journal.)
Figure 5.
Figure 5.
RNA-seq quantification method for analyzing the transcript abundance of BST2, HLA-B, IFI6, IFI27, OAS1, IFIT1, IFIT3, IFITM1, ISG15, and STAT1 in siCtrl (gray) and siPABPC1 (purple) groups. ***P < 0.001 versus siCtrl (Student’s t-test). Data are expressed as mean ± standard deviation, where the standard deviation is indicated as error bars. (A color version of this figure is available in the online journal.)
Figure 6.
Figure 6.
PABPC1-regulated AS events in siCtrl and siPABPC1 groups. (A) Number of AS events in each category including A3SS&ES, A5SS&ES, 3pMXE, 5pMXE, A3SS, A5SS, cassetteExon, ES, IntronR, and MXE. (B) Number of PABP1-regulated AS events in the indicated categories. (C) Gene ontology analysis for biological processes of PABP1-regulated AS events in siPABPC1-treated AGS cells compared with siCtrl-transfected cells. (A color version of this figure is available in the online journal.)
Figure 7.
Figure 7.
A total of 57 overlapped AS events were obtained between RASG (1241) and DEGs (1177). The differentially expressed AS events and DEGs were compared between siPABPC1-treated AGS cells and siCtrl cells. (A color version of this figure is available in the online journal.)
Figure 8.
Figure 8.
Top 9 KEGG pathway items of PABPC1-regulated DEGs with alteration in AS events. (A color version of this figure is available in the online journal.)
Figure 9.
Figure 9.
Effects of PABPC1 on DEGs with changes in AS events. Relative expression of immune-related genes (C4BPB, CD55, HLA-F, IRF7, and STAT2) in siCtrl (gray) and siPABPC1 (purple) groups was determined by RNA-seq. Data represent three independent biological repetitions. ***P < 0.001 versus siCtrl (Student’s t-test). Data are expressed as mean ± standard deviation, where the standard deviation is indicated as error bars. (A color version of this figure is available in the online journal.)
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