Isoforms S and L of MRPL33 from alternative splicing have isoform‑specific roles in the chemoresponse to epirubicin in gastric cancer cells via the PI3K/AKT signaling pathway

Abstract

Drug resistance is a major cause of cancer‑associated mortality. Epirubicin‑based chemotherapy initially benefits patients with metastatic or advanced gastric cancer; however, tumor recurrence can occur following several courses of treatment. Mitochondrial ribosomal protein L33 (MRPL33)‑long (L) and MRPL33‑short (S), isoforms of MRPL33 that arise from AS, have been reported to regulate cell growth and apoptosis in cancer; however, few studies have evaluated the roles of MRPL33‑L and MRPL33‑S in gastric cancer. In the present study, MRPL33‑L was demonstrated to be significantly more abundant in gastric tumor tissues than the MRPL33‑S isoform. MRPL33‑S promoted chemosensitivity to epirubicin in gastric cancer as demonstrated by a chemoresponse assay; chemosensitivity was suppressed in response to MRPL33‑L. Gene microarray analysis was performed to investigate the underlying mechanisms. Bioinformatic analysis revealed that overexpression of MRPL33‑L and MRPL33‑S served critical roles in transcription, signal transduction and apoptosis. In particular, the phosphoinositide 3‑kinase (PI3K)/AKT serine/threonine kinase (AKT) signaling pathway was markedly regulated. A total of 36 target genes, including PIK3 regulatory subunit α, AKT2, cAMP response element‑binding protein (CREB) 1, forkhead box 3, glycogen synthase kinase 3β and mammalian target of rapamycin, which are involved in the PI3K/AKT signaling pathway, were selected for further investigation via protein‑protein interaction network and Kyoto Encyclopedia of Genes and Genomes pathway analyses. Furthermore, western blot analysis indicated that MRPL33‑S promoted the chemoresponse to epirubicin by deactivating PI3K/AKT/CREB signaling and inducing apoptosis, while MRPL33‑L had the opposite effects. In conclusion, the results of the present study revealed that isoforms S and L of MRPL33, which arise from alternative splicing, exhibited opposing roles in the chemoresponse to epirubicin in gastric cancer via the PI3K/AKT signaling pathway. These findings may contribute to the development of potential therapeutic strategies for the resensitization of patients with gastric cancer to epirubicin treatment.

Figure 1

Expression of MRPL33-L and MRPL33-S in gastric cancer. (A) Schematic diagram of the splice variants of MRPL33, including or lacking alternative exon 3 (MRPL33-L and MRPL33-S; top). Different amino acid sequences are presented in red font for of MRPL33-L and in blue font for MRPL33-S (bottom). (B) Agarose gel electrophoresis photograph and (C) corresponding scatter diagrams of expression levels of MRPL33-L and MRPL33-S in 10 paired clinical specimens of tumor and matched adjacent normal tissues from patients with gastric cancer. (D) Agarose gel electrophoresis photograph and (E) corresponding histograms of expression levels of MRPL33-L and MRPL33-S in AGS and MGC-803 cells. Three independent biological replicates were performed and data were presented as the mean ± standard deviation. ^***P<0.001 with comparisons shown by brackets. MRPL33, mitochondrial ribosomal protein L33; L, long variant; S, short variant; ACTB, actin β.

Figure 2

Chemoresponse to epirubicin is regulated by MRPL33-L and MRPL33-S in gastric cancer. (A) Histogram of the chemoresponse of AGS cell groups (AGS control, plenti-vector, plenti-MRPL33-L and plenti-MRPL33-S), which were treated with epirubicin (0.003, 0.03, 0.3, 3 and 30 µM) for 72 h. (B) Fluorescent staining of nuclei in AGS cell groups treated with 0.3 µM epirubicin for 72 h. (C) Histogram of the chemoresponse of MGC-803 cell groups (MGC-803 control, plenti-vector, plenti-MRPL33-L and plenti-MRPL33-S), which were treated with epirubicin (0.003, 0.03, 0.3, 3 and 30 µM) for 72 h. (D) Fluorescent staining of nuclei in MGC-803 cell groups treated with 0.3 µM epirubicin for 72 h. Three independent biological replicates were performed and data were presented as the mean ± standard deviation. ^*P<0.05, ^**P<0.01 and ^***P<0.001 with comparisons shown by brackets. MRPL33, mitochondrial ribosomal protein L33; L, long variant; S, short variant.

Figure 3

Effects of MRPL33-L and MRPL33-S overexpression in AGS gastric cancer cells. (A) Volcano plot and (B) heatmap indicating upregu-lated and downregulated genes in plenti-MRPL33-L-transfected cells. (C) Volcano plot and (D) heatmap indicating upregulated and downregulated genes in plenti-MRPL33-S-transfected cells. (E) Venn diagram and (F) heatmap showing the number of overlapping DEGs in plenti-MRPL33-L and plenti-MRPL33-S-transfected cells. (G) Gene Ontology analysis of DEGs in plenti-MRPL33-L and plenti-MRPL33-S-transfected cells. (H) KEGG pathway analysis of DEGs in plenti-MRPL33-L and plenti-MRPL33-S-transfected cells. MRPL33, mitochondrial ribosomal protein L33; L, long variant; S, short variant; DEGs, differentially expressed genes; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 4

Analysis of the PI3K/AKT signaling pathway based on the KEGG database. (A) PPI network analysis of 36 target genes involved in the PI3K/AKT signaling pathway in plenti-MRPL33-L-transfected cells and in (B) plenti-MRPL33-S-transfected cells. (C) Map of the PI3K/AKT signaling pathway with the 36 target genes based on the KEGG database. Red and blue represent the upregulation and downregulation of genes, respectively. The size of circle in (A) and (B) indicates significance based on P-value. The square and rounded square in (C) represent genes in plenti-MRPL33-L- and plenti-MRPL33-S-trans-fected cells, respectively. ^*P<0.05, ^**P<0.01 and ^***P<0.001. PI3K, phosphoinositide 3-kinase; AKT, AKT serine/threonine kinase; KEGG, Kyoto Encyclopedia of Genes and Genomes; PPI, protein-protein interaction; MRPL33, mitochondrial ribosomal protein L33; L, long variant; S, short variant.

Figure 5

Chemoresponse to epirubicin is dependent on the PI3K/AKT/CREB/apoptosis axis, which is regulated by MRPL33-L and MRPL33-S in gastric cancer cells. (A) Western blot analysis and (B) corresponding histogram of ratio of p-AKT/AKT, ratio of p-CREB/CREB, Mcl-1 and Bcl-2 expression levels in the AGS cell groups (control, plenti-vector-plentil-MRPL33-S and plenti-MRPL33-L-transfected), with or without 0.3 µM epirubicin. (C) Western blot analysis and (D) corresponding histogram of ratio of p-AKT/AKT, ratio of p-CREB/CREB, Mcl-1 and Bcl-2 expression levels in the MGC-803 cell groups (control, plenti-vector, plentil-MRPL33-S and plenti-MRPL33-L), with or without 0.3 µM epirubicin. (E) Histograms of chemoresponse in AGS cell groups and (F) MGC-803 cell groups treated with 0.3 µM epirubicin for 72 h. Three independent biological replicates were performed and data were presented as the mean ± standard deviation. ^*P<0.05, ^**P<0.01 and ^***P<0.001 with comparisons shown by brackets. PI3K, phosphoinositide 3-kinase; AKT, AKT serine/threonine kinase; CREB, cAMP response element-binding protein; MRPL33, mitochondrial ribosomal protein L33; L, long variant; S, short variant; p-, phosphorylated; Mcl-1, myeloid cell leukemia 1; Bcl-2, B-cell lymphoma 2.