Downregulation of miR-21 inhibits the malignant phenotype of pancreatic cancer cells by targeting VHL

Abstract

Background: MicroRNA (miR)-21 is overexpressed in numerous types of malignancy and participates in the development of cancer. However, the basic mechanism of the influence of miR-21 on the malignant phenotype of pancreatic cancer remains unclear.

Purpose: The present study aimed to investigate the role of miR-21 in pancreatic cancer development and explore its molecular mechanism.

Patients and methods: The tissue samples were collected at the Second Hospital of Tianjin Medical University (Tianjin, China) between January 2013 and December 2015. The expression of VHL in tissue samples was evaluated by IHC staining. The expression of miR-21 was measured by quantitative real-time polymerase chain reaction (qRT-PCR). MiR-21 target gene was detected by real-time PCR, Western blot and the luciferase reporter assay. Cell viability, cell proliferation, cell migration and invasion were evaluated by the MTT assays, the colony formation assays and the transwell assays. The nude mouse tumor xenograft model was performed to detect the effect of miR-21 on tumor growth in vivo.

Results: Von Hippel-Lindau tumor suppressor (VHL) was downregulated in pancreatic cancer tissues compared with pancreatic non-tumor tissues. VHL was identified as a novel direct target of miR-21, by which it is negatively regulated. In PANC-1 cells, inhibition of miR-21 and upregulation of VHL significantly suppressed cell proliferation, migration and invasion. Knockdown of miR-21 inhibited the hypoxia-inducible factor (HIF)-1α/vascular endothelial growth factor (VEGF) pathway, while inhibiting the expression of matrix metallopeptidase (MMP)-2 and MMP-9. Silencing of miR-21 inhibited tumor growth in vivo.

Conclusion: Knockdown miR-21 increased the expression of VHL, and thus modulated the HIF-1α/VEGF pathway and the expression of MMP-2 and MMP-9, which led to the inhibition of the proliferation, migration and invasion of pancreatic cancer cells. All of these results suggest that the miR-21/VHL interaction may be a novel potential target for pancreatic cancer prevention and therapy.

Keywords: VHL; invasion; miR-21; migration; pancreatic cancer; proliferation.

Figure 1

Expression levels of VHL in human pancreatic cancer tissues and pancreatic non-tumor tissues. (A) Representative images of VHL protein expression determined by immunohistochemical staining of pancreatic non-tumor tissues (left) and human pancreatic cancer tissues (right). (B) Semi-quantitative analysis of VHL protein expression using the mean density in human pancreatic cancer tissues and pancreatic non-tumor tissues. Values are expressed as the average values from several fields of view from several slices of the same sample and from several samples per group. Values are expressed as the mean ± standard deviation. Notes: **P<0.01. The image color is RGB. Abbreviations: VHL, Von Hippel-Lindau tumor suppressor.

Figure 2

VHL is a direct target of miR-21. (A) The 3′-UTR fragments containing the predicted miR-21 targeting sites of VHL (wild type or mutant type) were cloned into the reporter plasmid downstream of the firefly luciferase gene. (B) PANC-1 and MiaPaca-2 cells were co-transfected with human VHL 3′-UTR (wild type or mutant type) firefly luciferase reporter plasmid and lenti-NC or lenti-AS-21 as indicated. After 48 h, firefly luciferase activity was measured. (C) The expression levels of VHL protein in PANC-1 and MiaPaca-2 cells treated with lenti-NC or lenti-AS-21 were detected by Western blot analysis. (D) Quantification of VHL protein expression detected by Western blot and normalized to GAPDH. Values are expressed as the mean ± standard deviation from three independent experiments. Notes: *P<0.05; **P<0.01; ⁺No significance. Abbreviations: VHL, Von Hippel-Lindau tumor suppressor; UTR, untranslated region; lenti-NC, negative control lentivirus; lenti-AS-21, microRNA-21 inhibitor vector; PANC-1, human pancreatic cancer cell; Mia Paca-2, human pancreatic cancer cell.

Figure 3

VHL inhibits the proliferation, migration and invasion of pancreatic cancer cells. (A) PANC-1 cells were transfected with either control plasmid or pcDNA3-VHL The mRNA expression levels of VHL at 24 h post-transfection were detected by reverse transcription-quantitative polymerase chain reaction analysis with normalization to β-actin. (B) The protein expression levels of VHL at 48 h post-transfection were detected by Western blot analysis. (C) Cell viability was measured using an MTT assay at 24, 72 and 120 h post-transfection. (D) Colony formation assay was performed at 24 h post-transfection. (E) Transwell migration and (F) invasion assays were carried out at 24 h post-transfection. Values are expressed as the mean ± standard deviation from three independent experiments. Notes: *P<0.05; **P<0.01; ***P<0.001. The image color is RGB. Abbreviations: VHL, Von Hippel-Lindau tumor suppressor; pcDNA3-VHL, overexpression plasmid for Von Hippel-Lindau tumor suppressor; PANC-1, human pancreatic cancer cell.

Figure 4

Silencing of miR-21 suppresses the proliferation, migration and invasion of pancreatic cancer cells. (A) PANC-1 and MiaPaca-2 cells were infected with lenti-AS-21 or lenti-NC. Reverse transcription-quantitative polymerase chain reaction analysis was used to confirm the knockdown of miR-21 in PANC-1 and MiaPaca-2 cells after 24 h infection. (B) An MTT assay was used to detect the viability of PANC-1 cells infected with lenti-AS-21 or lenti-NC for 24, 72 or 120 h. (C) A colony formation assay was used to detect the colony formation rate of PANC-1 cells infected with lenti-AS-21 or lenti-NC. The colony formation rate was calculated using the following equation: Colony formation rate = (number of colonies/number of plated cells) x100%. (D) Transwell migration and (E) invasion assays were performed with PANC-1 cells infected with lenti-AS-21 or lenti-NC. Values are expressed as the mean ± standard deviation from three independent experiments. Notes: *P<0.05; **P<0.01; ***P<0.001. The image color is RGB. Abbreviations: miR, microRNA; lenti-NC, negative control lentivirus; lenti-AS-21, microRNA-21 inhibitor vector; PANC-1, human pancreatic cancer cell; Mia Paca-2, human pancreatic cancer cell.

Figure 5

Silencing of miR-21 inhibits the HIF-1α/VEGF pathway, and the expression of MMP-2 and MMP-9. The expression levels of HIF-1α and VEGF (A), MMP-2 and MMP-9 (B) in PANC-1 cells treated with lenti-NC or lenti-AS-21 were determined by Western blot analysis with normalization to GAPDH. Abbreviations: Lenti-NC, negative control lentivirus; lenti-AS-21, microRNA-21 inhibitor vector. HIF-1α, hypoxia-inducible factor-1α; VEGF, vascular endothelial growth factor; MMP, matrix metallopeptidase; PANC-1, human pancreatic cancer cell; GAPDH, internal control.

Figure 6

Knockdown miR-21 inhibits tumor growth in vivo. (A) The nude mouse tumor xenograft model was utilized to study the effect of miR-21 on tumor growth in vivo. The tumor growth curve is presented on the left. The tumor volume was calculated as follows: Tumor volume = (length x width²)/2. *P<0.05, n=6 (Student’s t-test). The mice were euthanized and the tumors were isolated 21 days after implantation (right panel). (B) The VHL expression levels in the tumors of the mice were detected by immunohistochemistry, and representative photomicrographs of the tumor sections are presented. (C) The VHL expression levels in the tumors of the mice were detected by Western blot with normalization to GAPDH. Values are expressed as the mean ± standard deviation from six nude mouse tumor samples. Notes: GAPDH: internal control; nude mice: female BALB/c nude mice (age, 6 weeks). The image color is RGB. Abbreviations: VHL, Von Hippel-Lindau tumor suppressor; miR, microRNA Lenti-NC, negative control lentivirus; lenti-AS-21, microRNA-21 inhibitor vector.