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, 40 (3), 1379-1389

miR‑448 Targets Rab2B and Is Pivotal in the Suppression of Pancreatic Cancer

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miR‑448 Targets Rab2B and Is Pivotal in the Suppression of Pancreatic Cancer

Jing Jin et al. Oncol Rep.

Abstract

Improvements in survival rates for pancreatic cancer have been slow and the morality rate continues to increase in patients. MicroRNA (miR)‑448 is reported to be significantly downregulated in several types of cancer. In this study, Rab2B is target of miR‑488 was confirmed by bioinformatics analysis and validated using a luciferase reporter assay. A total of 72 cases of pancreatic cancer in patients diagnosed at The First Affiliated Hospital, School of Medicine, Zhejiang University (Hangzhou, China) were enrolled, and cancer specimens and their adjacent normal tissues were collected for analysis. The expression levels of miR‑448 and Rab2B in these tissues and in pancreatic cancer cell lines were quantified using reverse transcription‑polymerase chain reaction analysis. miR‑448 overexpression was achieved by cell transfection. Protein expression was assessed using western blot analysis. Cell viability, cell cycle and apoptosis were analyzed using CCK‑8 assay and flow cytometry, respectively. The results revealed a negative correlation between miR‑448 and Rab2B in the pancreatic tissues and cell lines. The results of bioinformatics analysis indicated that miR‑448 directly targeted Rab2B. Aberrant miR‑448 levels in PANC‑1 cells downregulated the expression of Rab2B, and significantly decreased cell proliferation and promoted apoptosis of cancer cells. It was also found that miR‑448 mimics resulted in G0/G1 cell cycle arrest and affected the expression of cell cycle regulators, including cyclin D1, p21 and p27. In addition, the miR‑448 mimics led to inactivation of the Akt/Mammalian target of rapamycin signaling pathway. The miR‑448 mimics induced apoptosis and activated the expression of caspase‑3, caspase‑9 and poly(ADP‑ribose) polymerase. The results suggested that miR‑448 was a negative regulator of Rab2B and promoted cell cycle arrest and apoptosis in pancreatic cancer.

Figures

Figure 1.
Figure 1.
Levels of miR-448 and Rab2B in pancreatic tissues and cell lines (HPDE6-C7, AsPC-1, BxPC-3, Capan-1, CFPAC-1, HPAC, Hs 776T and PANC-1). (A) Expression of miR-448 was silenced in pancreatic cancer tissues. (B) Expression of Rab2B was high in pancreatic cancer tissues. (C) Linear correlation of mRNA levels between miR-448 and Rab2B. (D) Survival analysis showed the survival rate of patients with pancreatic cancer with a higher level of Rab2B was lower than that of patients with a lower expression level of Rab2B. (E) Levels of miR-448 in pancreatic cancer cell lines were significantly lower than in normal cells (HPDE6-C7). (F) mRNA levels of Rab2B in pancreatic cancer cell lines were higher compared with that in normal cells (HPDE6-C7). Data are shown as the mean ± standard deviation. *P<0.05, **P<0.01, vs. normal tissues or cells. miR, microRNA.
Figure 2.
Figure 2.
Target of miR-448 identified by bioinformatics analysis and luciferase reporter analysis. (A) A total of 90 reliable target genes of miR-448 were identified through intersection calculation of predicted target genes by miRanda, miRDB, PicTar and TargetScan. (B) GO analysis of gene function found 20 annotations of miR-448-associated biological process, which were mainly enriched in processes of regulation of gene expression, cell proliferation and signal transduction. miR, microRNA; GO, Gene Ontology.
Figure 3.
Figure 3.
(A) Cervical-loop structures of pre-miR-448. (B) Mature hsa-miR-448 (miR-448) was predicted to interact with the Rab2B-3′-UTR at positions 319–349 via a 7-mer seed match interaction. (C) Relative luciferase assay revealed that miR-448 exhibited reduced luciferase reporter activity. *P<0.05, **P<0.01, vs. control. miR, microRNA; UTR, untranslated region; WT, wild-type; Mut, mutant.
Figure 4.
Figure 4.
Overexpression of miR-448 affects cell proliferation, migration and invasion. (A) Ectopic expression of miR-448 was confirmed, which (B) decreased the mRNA level of Rab2B. (C) Protein expression of Rab2B was inhibited by miR-448 mimics. (D) Overexpression of miR-448 decreased the proliferative ability of PANC-1 cells. (E) miR-448 induced G0/G1 cell cycle arrest in PANC-1 cells. (F) miR-448 promoted the apoptotic rate of PANC-1 cells. Data are shown as the mean ± standard deviation of three independent experiments. *P<0.05, **P<0.01, vs. control; ^P<0.05, ^^P<0.01, vs. mock. miR, microRNA; glyceraldehyde-3-phosphate dehydrogenase.
Figure 4.
Figure 4.
Overexpression of miR-448 affects cell proliferation, migration and invasion. (A) Ectopic expression of miR-448 was confirmed, which (B) decreased the mRNA level of Rab2B. (C) Protein expression of Rab2B was inhibited by miR-448 mimics. (D) Overexpression of miR-448 decreased the proliferative ability of PANC-1 cells. (E) miR-448 induced G0/G1 cell cycle arrest in PANC-1 cells. (F) miR-448 promoted the apoptotic rate of PANC-1 cells. Data are shown as the mean ± standard deviation of three independent experiments. *P<0.05, **P<0.01, vs. control; ^P<0.05, ^^P<0.01, vs. mock. miR, microRNA; glyceraldehyde-3-phosphate dehydrogenase.
Figure 5.
Figure 5.
miR-448 mimics affect the expression of cell cycle regulators and inhibit the Akt/mTOR/S6K1 pathway. (A) Ectopic expression of miR-448 inhibited the expression of Cyclin D1 but upregulated the expression of p21 and p27. The phosphorylation of (B) Akt, (C) mTOR and (D) S6K1 was suppressed by miR-448 mimics. Data are shown as the mean ± standard deviation of three independent experiments. **P<0.01, vs. control; ^^P<0.01, vs. mock. miR microRNA; mTOR, Mammalian target of rapamycin; p-, phosphorylated.
Figure 6.
Figure 6.
Overexpression of miR-448 activates caspase-3, caspase-9 and PARP. Aberrant expression of miR-448 increased the activities of (A) caspase-3 and (B) caspase-9. (C) miR-448 mimics upregulated the mRNA expression of PARP. (D) In the miR-448 mimic group, levels of pro-caspase-3 and pro-caspase-9 were decreased, whereas the protein level of PARP was increased. Data are shown as the mean ± standard deviation of three independent experiments. *P<0.05, **P<0.01, vs. control; ^P<0.05, ^^P<0.01, vs. mock. miR, microRNA; PARP, poly (ADP-ribose) polymerase.
Figure 7.
Figure 7.
Summarized model of the findings of the present study. A high expression of Rab2B was associated with poor prognosis of patients with pancreatic cancer. The overexpression of miR-448 modulated the expression of p21, p27, cyclin D1, caspase-3/-9 and PARP, and induced the cell cycle arrest and apoptosis of pancreatic cancer cells. miR, microRNA; UTR, untranslated region; PARP, poly(ADP-ribose) polymerase; mTOR, Mammalian target of rapamycin.

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