Downregulation of miR-374b-5p promotes chemotherapeutic resistance in pancreatic cancer by upregulating multiple anti-apoptotic proteins

Abstract

Resistance to first-line chemotherapeutic drugs such as gemcitabine contributes to the poor prognosis of patients with pancreatic cancer. MicroRNAs (miRNA) regulate chemoresistance in pancreatic cancer. By analyzing the miRNA sequencing dataset of pancreatic cancer from The Cancer Genome Atlas, it was demonstrated that miR-374b-5p expression was dramatically reduced in pancreatic cancer tissues compared with adjacent normal tissues, as well as decreased in chemoresistant compared with chemosensitive pancreatic carcinoma tissues. The decreased expression of miR-374-5p was associated with poor overall and progression-free survival in patients with pancreatic cancer. Furthermore, increased expression of miR-374b-5p abrogated, while the silencing miR-374b-5p increased the chemoresistance of pancreatic cancer cells to gemcitabine in vitro. Importantly, the upregulation of miR-374b-5p ameliorated the chemoresistance of pancreatic cancer cells to gemcitabine in vivo. It was also demonstrated that miR-374b-5p targeted several anti-apoptotic proteins, including B-cell lymphoma 2, Baculoviral IAP Repeat Containing 3 and X-linked inhibitor of apoptosis in pancreatic cancer cells, which further attenuated chemoresistance in pancreatic cancer. Therefore, the results of the current study indicate that miR-374b-5p serves as a potential diagnostic marker. It also suggests that miR-374b-5p sensitizes cells to chemotherapy and may be used in combination with chemotherapeutic agents such as gemcitabine to treat patients with pancreatic cancer.

Figure 1

miR-374b-5p expression is decreased in pancreatic cancer tissues. (A) Analyzing the TCGA pancreatic cancer miRNA sequencing dataset indicated that miR-374b-5p expression was significantly decreased in pancreatic cancer tissues compared with ANTs. (B) Analysis of the GSE24270 pancreatic cancer miRNA sequencing dataset indicated that miR-374b-5p expression was decreased in pancreatic cancer tissues compared with ANTs. (C) Analysis of the GSE29352 pancreatic cancer miRNA sequencing dataset indicated that miR-374b-5p expression was decreased in pancreatic cancer tissues compared with ANTs. (D) miR-374b-5p expression was decreased in pancreatic cancer tissues compared with the 8 paired matched normal pancreatic tissues taken from patients in the current study. ^*P<0.05 vs. corresponding ANT (E) Analysis of patients included in the current study indicated that miR-374b-5p expression was decreased in pancreatic cancer tissues compared with ANTs. Kaplan-Meier analysis of (F) overall and (G) progression-free survival curves of patients with pancreatic cancer with low miR-374b-5p expression compared with those with high miR-374b-5p expression included in the current study. Kaplan-Meier analysis of (H) overall and (I) progression-free survival curves of patients with low miR-374b-5p expression compared with those with high miR-374b-5p expression in the TCGA pancreatic cancer dataset. The optimal cutoff point was selected using the log-rank test using X-tile software. The results are presented as the mean ± standard deviation of three independent experiments. ANT, adjacent normal tissue; miR, microRNA; TCGA, The Cancer Genome Atlas; L, low; H, high.

Figure 2

The decreased expression of miR-374b-5p is associated with chemoresistance in pancreatic cancer. (A) In the TCGA dataset, miR-374b-5p expression was reduced in chemoresistant pancreatic cancer tissues compared with chemosensitive pancreatic cancer tissues. (B) In tissues taken from patients included in the present study, miR-374b-5p expression was reduced in chemoresistant pancreatic cancer tissues compared with chemosensitive pancreatic cancer tissues. (C) Reverse transcription-quantitative polymerase chain reaction analysis of miR-374b-5p expression in pancreatic cancer cells. Transcript levels were normalized to U6 expression. (D) The apoptotic ratio of different pancreatic cancer cell lines following treatment with GEM. (E) The correlation between miR-374b-5p expression and the apoptotic ratio in pancreatic cancer cells following treatment with GEM. The results are presented as the mean ± standard deviation of three independent experiments. ^*P<0.05. TCGA, The Cancer Genome Atlas; miR-374-5p, microRNA-374-5p; GEM, gemcitabine.

Figure 3

Downregulation of miR-374b-5p induces chemoresistance in BxPC-3 and SW1990 cells. (A) Reverse transcription-quantitative polymerase chain reaction analysis of miR-374b-5p expression. Transcript levels were normalized to U6 expression. (B) Annexin V-FITC/PI staining of cells treated with gemcitabine (10 µM) for 36 h. (C) JC-1 staining of the cells treated with GEM. (D) Western blotting analysis measuring CytoC expression. α-tubulin served as the loading control. (E) Analysis of caspase-3 and -9 activity. All results are presented as the mean ± standard deviation of three independent experiments. ^*P<0.05 vs. vector. miR-374b-5p, microRNA-374b-5p; GEM, gemcitabine; CytoC, cytochrome c; FITC, fluorescin isothiocyanate; PI, propidium iodide.

Figure 4

Upregulation of miR-374b-5p decreases the chemoresistance of AsPC-1 and PANC-1 cells. (A) Reverse transcription-quantitative polymerase chain reaction analysis of miR-374b-5p expression in the indicated pancreatic cancer cells. Transcript levels were normalized to U6 expression (B) Annexin V-FITC/PI staining of the cells treated with gemcitabine (10 µM) for 36 h. (C) JC-1 staining of cells treated with GEM. (D) Western blotting measuring CytoC expression. α-tubulin served as the loading control. (E) Analysis of caspase-3 and -9 activity. Results are presented as the mean ± standard deviation of three independent experiments. ^*P<0.05 vs. respective control. miR-374b-5p, microRNA-374b-5p; FITC, fluorescin isothiocyanate; PI, propidium iodide; CytoC, cytochrome c; GEM, gemcitabine.

Figure 5

Upregulation of miR-374b-5p restores the sensitivity of pancreatic cancer cells to gemcitabine in vivo. (A) Representative images of the tumors in the xenograft model of nude mice are presented. (B) Following 10 days of inoculating the indicated AsPC-1 cells, mice were intraperitoneally injected with 50 µg/g GEM twice a week for 4 weeks. Tumor volumes were measured from the fifth day at intervals of 5 days. (C) Tumor weights of each group. (D) The overall survival of mice in the indicated group. (E) Caspase-3 and -9 activity in the indicated tumors. Data are presented are the mean ± standard deviation. ^*P<0.05. miR-374b-5p, microRNA-374b-5p; GEM, gemcitabine.

Figure 6

miR-374b-5p targets BCL2, BIRC3 and XIAP in pancreatic cancer cells. (A) Predicted miR-374b-5p target sequence in the 3′-UTRs of BCL2, BIRC3 and XIAP. (B) Western blotting measuring BCL2, BIRC3 and XIAP expression. α-tubulin served as the loading control. (C and D) Luciferase assay of the cells transfected with the indicated reporter following transfection with (C) miR-374b-5p mimic or (D) anti-miR-374b-5p. ^*P<0.05 vs. control or vector, respectively. (E and F) RNA immunoprecipitation assay indicating the association between miR-374b-5p and BCL2, BIRC3 and XIAP transcripts in the indicated cells. ^*P<0.05 vs. vector (G) Reverse transcription-quantitative polymerase chain reaction analysis of BCL2, BIRC3 and XIAP expression in the indicated pancreatic cancer cells. Transcript levels were normalized to U6 expression. ^*P<0.05 vs. scramble. (H) The protective effect of anti-miR-374b-5p on the apoptotic rate of pancreatic cancer cells was attenuated by the individual silencing of BCL2, BIRC3 and XIAP. (I) The stimulatory effect of anti-miR-374b-5p on the mitochondrial potential of pancreatic cancer cells was attenuated by the individual silencing of BCL2, BIRC3 and XIAP. (J and K) The stimulatory effects of anti-miR-374b-5p on caspase-3 and -9 activity of pancreatic cancer cells was attenuated by the silencing of BCL2, BIRC3 and XIAP. Data are presented as the mean ± standard deviation of three independent experiments. ^*P<0.05. miR-374b-5p, microRNA-374b-5p; BCL2, B-cell lymphoma 2; BRIC3, Baculoviral IAP Repeat Containing 3; XIAP, X-linked inhibitor of apoptosis; UTR, untranslated region; mut, mutant; wt, wild-type; si, small interfering; GEM, gemcitabine.