PABPC1 promotes cell proliferation and metastasis in pancreatic adenocarcinoma by regulating COL12A1 expression

Abstract

Background: The expression of cytoplasmic poly (A) binding protein-1 (PABPC1) has been reported in multiple cancer types. This protein is known to modulate cancer progression. However, the effects of PABPC1 expression in pancreatic adenocarcinoma (PAAD) have not been investigated. Here, we investigate the regulatory targets and molecular mechanisms of PABPC1 in PAAD.

Methods: PABPC1 and collagen type XII α1 chain (COL12A1) expression in PAAD and their role in tumor prognosis and tumor stage were investigated using The Cancer Genome Atlas database analysis. After silencing PABPC1, messenger RNA sequencing and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed. The expression of differentially expressed genes (DEGs), cell viability, apoptosis, and cell migration and invasion were explored using reverse transcription-quantitative polymerase chain reaction, Cell Counting Kit-8 assay, flow cytometry assay, and transwell assay, respectively. The relationship between PABPC1 and COL12A1 expression was assessed by Pearson's correlation analysis. The regulatory function of COL12A1 in PABPC1-affected BXPC3 cell behavior was studied after COL12A1 was overexpressed.

Results: PABPC1 and COL12A1 expression was upregulated in patients with PAAD and was linked to poor prognosis. Four hundred and seventy-four DEGs were observed in BXPC3 cells after PABPC1 silencing. GO and KEGG analyses revealed that the top 10 DEGs were enriched in cell adhesion pathways. Additionally, PABPC1 silencing inhibited cell viability, migration, and invasion and accelerated apoptosis in BXPC3 cells. PABPC1 silencing increased AZGP1 and ARHGAP30 expression and decreased CAV1 and COL12A1 expression in BXPC3 cells. PABPC1 positively mediated COL12A1 expression, whereas PABPC1 knockdown induced the inhibition of BXPC3 cell proliferation, migration, and invasion.

Conclusion: The results of this study indicate that PABPC1 may function as a tumor promoter in PAAD, accelerating BXPC3 cell proliferation and metastasis by regulating COL12A1 expression.

Keywords: PABPC1; cell metastasis; collagen type XII α1 chain (COL12A1); pancreatic adenocarcinoma.

Figure 1

Upregulated cytoplasmic poly (A) binding protein‐1 (PABPC1) in pancreatic adenocarcinoma (PAAD) patients is linked to poor prognosis. (A) Based on The Cancer Genome Atlas database, PABPC1 expression level in PAAD patients (N = 179) compared with normal subjects (N = 171) were assessed. (B) The relevance between PABPC1 and overall survival was researched via Kaplan–Meier plotter analysis (n [high] = 89; n [low] = 89). (C) Expression of PABPC1 in PAAD patients (N = 179) at stage I, II, III, IV was analyzed. After transfection with PABPC1 silenced vectors (siPABPC1) and negative controls (siCrtl), (D) the messenger RNA and (E) protein levels of PABPC1 in BXPC3 cells at 0, 24, 48, and 72 h time points were explored via reverse transcription‐quantitative polymerase chain reaction and Western blot assays. These experiments were repeated for three times and analyzed via SPSS 12.0 software using analysis of variance along with Tukey's post hoc test. *p < .05 versus Normal, ***p < .001 versus siCtrl−1, ^### p < .001 versus siCtrl−2, ^{^^^} p < .001 versus siCtrl−3.

Figure 2

Cytoplasmic poly (A) binding protein‐1 (PABPC1) silence affects the gene expression profile of BXPC3 cells. After transfection with PABPC1 silenced vectors (siPABPC1) and negative controls (siCrtl) for 48 h in BXPC3 cells, (A) The significant differences between siPABPC1 group and siCrtl group was analyzed by principal component analysis (PCA) based on fragments per kilo base per million mapped reads value. (B) The differentially expressed genes (DEGs) between siPABPC1 group and siCrtl group were showcased by volcanic distribution map (the downregulated genes: n = 211; the upregulated genes: n = 263). p < .05 and fold change (FC) ≥2 or ≤1/2. (C) The levels of DEGs were presented by hierarchical cluster analysis (the number of differentially expressed genes was 474). (D–G) The enrichment of DEGs (n = 474) was determined by Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis.

Figure 3

Cytoplasmic poly (A) binding protein‐1 (PABPC1) silence inhibits BXPC3 cells growth and metastasis. The vectors of PABPC1 silenced vectors (siPABPC1) and negative controls (siCrtl) were transfected into BXPC3 cells, (A) Cell viability at the different time points (0, 24, 48, and 72 h) was analyzed via Cell Counting Kit‐8 assay. (B) BXPC3 apoptotic cells and (C) Cell cycle was estimated by flow cytometry assay. (D and E) The migration and invasion of BXPC3 cells were studied by transwell assay (magnification = ×200, Scale bar = 50 μM). These experiments were repeated for three times and analyzed via SPSS 12.0 software using analysis of variance along with Tukey's post hoc test. ***p < .001 versus Control, ^### p < .001 versus siCtrl.

Figure 4

Cytoplasmic poly (A) binding protein‐1 (PABPC1) regulates the expression of correlated genes in BXPC3 cells. After transfection with PABPC1 silenced vectors (siPABPC1) (#1, #2 and #3) and negative controls (siCrtl) (#1, #2 and #3) vectors in BXPC3 cells for 48 h, the differential gene expression levels of (A) zinc‐α2‐glycoprotein (AZGP1), (B) Rho GTPase activating protein 30 (ARHGAP30), (C) Caveolin‐1 (CAV1), and (D) collagen type XII α1 chain (COL12A1) were examined with the aid of reverse transcription‐quantitative polymerase chain reaction assay. These experiments were repeated for three times and analyzed via SPSS 12.0 software using analysis of variance along with Tukey's post hoc test. *p < .05, **p < .01 versus siCtrl.

Figure 5

Collagen type XII α1 chain (COL12A1) is upregulated in pancreatic adenocarcinoma (PAAD) patients and is linked to poor prognosis. (A) According to The Cancer Genome Atlas database, COL12A1 expression in PAAD patients (N = 179) compared with normal subjects (N = 171) was analyzed. (B) The relevance between COL12A1 and overall survival was evaluated via Kaplan–Meier plotter analysis (n [high] = 89; n [low] = 89). (C) Expression of COL12A1 in PAAD patients (N = 179) at stage I, II, III, IV was analyzed. (D) The relationship between COL12A1 and cytoplasmic poly (A) binding protein‐1 (PABPC1) in PAAD samples (N = 179) was determined by Pearson's correlation analysis.

Figure 6

Collagen type XII α1 chain (COL12A1) overexpression reverses the prohibitive effect of cytoplasmic poly (A) binding protein‐1 (PABPC1) on BXPC3 cells growth and metastasis. The pcDNA3.1 vector was adopted for establishment of COL12A1 overexpressed vector. After transfection for 48 h, (A) Reverse transcription‐quantitative polymerase chain reaction was performed to assess the efficiency of transfection. (B) Western blot assay was applied to determine the protein level of COL12A1 in transfected BXPC3 cells. After co‐transfection with PABPC1 silenced vectors (siPABPC1) and COL12A1 overexpressed vectors, (C) Cell Counting Kit‐8 assay was utilized to calculate BXPC3 cells viability. (D and E) Transwell assay was carried out to evaluate the abilities of BXPC3 cells migration and invasion (magnification = ×200, Scale bar = 50 μM). These experiments were repeated for three times and analyzed via SPSS 12.0 software using analysis of variance along with Tukey's post hoc test. **p < .01, ***p < .001 versus Control or negative controls (siCrtl) + Vector; ^## p < .01, ^### p < .001 versus Vector or siPABPC1 + Vector, ^{^^^} p < .001 versus siCtrl + COL12A1.