Anticancer effects of miR-124 delivered by BM-MSC derived exosomes on cell proliferation, epithelial mesenchymal transition, and chemotherapy sensitivity of pancreatic cancer cells

Abstract

Objective: This study aims to explore the roles of miR-124 in pancreatic tumor and potential vehicles.

Results: The miR-124 expression levels decreased in pancreatic adenocarcinoma tissues and cancer cell lines AsPC-1, PANC1, BxPC-3 and SW1990. Furthermore, the elevated expression of miR-124 in AsPC-1 and PANC1 via miR-124 mimic transfection-induced apoptosis, metastasis and epithelial mesenchymal transition was suppressed, and the EZH2 overexpression partly reversed the protective effects of miR-124 against pancreatic tumors. In addition, the expression of miR-124 was detected in exosomes extracted from miR-124-transfected BM-MSCs, and these exosomes delivered miR-124 into pancreatic cancer cells, and presented the anti-tumor effects in vitro and in vivo.

Conclusion: MiR-124-carried BM-MSC-derived exosomes have potential applications for the treatment of pancreatic tumors.

Methods: The expression of miR-124 and EZH2 was determined in both pancreatic cancer tissues and cell lines. miR-124 or EZH2 was overexpressed in AsPC-1 and PANC1 cells. Then, the effects on cell viability. apoptosis, invasion, migration and epithelial mesenchymal transition were evaluated. Afterwards, the roles of miR-124 on the expression and function of EZH2 in pancreatic tumors were determined by dual luciferase reporter assay. Subsequently, miR-124 was transfected to bone marrow mesenchymal stromal cells (BM-MSCs), and the BM-MSCs derived exosomes were isolated and co-cultured with AsPC-1 and PANC1 cells, or injected into pancreatic cancer tumor-bearing mice.

Keywords: BM-MSCs; cancer; chemotherapy sensitivity; epithelial mesenchymal transition; miR-124.

Figure 1

MiR-124 expression was downregulated and the expression of EZH2 was stimulated in pancreatic cancer tissues and cell lines. Relative expression levels of miRNA-124 or EZH2 in pancreatic cancer tissues (A) or pancreatic cancer cell lines (B) AsPC-1, PANC1, BxPC-3 and sw1990, and pancreatic duct epithelial cell line HPDE6 were determined by qRT-PCR or western blot. The results were presented as the mean ± standard deviation (SD) of three independent experiments. ***P<0.001, compared with normal tissues or HPDE6 cell lines.

Figure 2

The miR-124-mimic transfection significantly decreased the cell viability in AsPC-1 and PANC1 cell lines, and induced apoptosis, while these effects were removed by the miR-124 inhibitor or EZH2 overexpression. Transfection efficacy was determined by RT-qPCR (A). Cell viability was determined by MTT (B), the apoptosis ratio was determined using Annexin V-propidium iodide (PI) by flow cytometry (C), and the expression of cleaved caspase-3, BAX and Bcl2 were determined by western blot (D). The results were presented as the mean ± standard deviation (SD) of three independent experiments.

Figure 3

The miR-124-mimic transfection inhibited the invasion, migration of AsPC-1 and PANC1 cells, which were partly reversed by the miR-124 inhibitor or EZH2 overexpression. The capability of cell invasion was determined by transwell assay (A). The wound-healing assay was used for measurement for cell migration (B).

Figure 4

The miR-124-mimic transfection inhibited the EMT of AsPC-1 and PANC1 cells, which were partly reversed by the miR-124 inhibitor or EZH2 overexpression. The expression of N1CD, Hes1, MMP-9, E-cadherin and vimentin were determined by western blot. These results were presented as the mean ± standard deviation (SD) of three independent experiments. **P<0.01, compared with the control.

Figure 5

EZH2 is a target of miR-124. The binding site between miR-124 and EZH2 was predicted (A), dual-luciferase reporter systems were built (B) and the report data illustrated that EZH2 is the target gene of miR-124-3p. The expression of EZH2 in miR-124 inhibitor-treated cells were determined by western blot (C, D). These results were presented as the mean ± standard deviation (SD) of three independent experiments. **P<0.01.

Figure 6

Exosomes were extracted from BM-MSCs and delivered miR-124 into pancreatic cancers. The BM-MSCs were identified using flow cytometry (A) since CD90 and CD105 were positive, and CD34 and CD45 were negative. The extracted exosomes were examined by transmission electron microscopy (B) The expression level of miR-124 in BM-MSCs were determined by RT-PCR (C) The expression level of miR-124 in exosomes extracted from the BM-MSCs were determined by RT-PCR, and the protein level of CD9, CD63 and CD81 were determined by western blot (D) The expression levels of miR-124 in miR-124-exo co-cultured AsPC-1 or PANC1 cells were determined by RT-PCR (E) and cells were stained using Dil C16 dye (F). These results present the mean ± standard deviation (SD) of three independent experiments. ***P<0.001.

Figure 7

Exosomes-delivered miR-124 inhibited the proliferation, invasion, migration and induced the apoptosis in pancreatic cancers. The effects of BM-MSC-derived exosomes on AsPC-1 or PANC1 proliferation, apoptosis was determined using MTT (A) Annexin V-propidium iodide (PI) by flow cytometry (B) respectively and western blot was used to measure the apoptosis related proteins (C) Transwell invasion assay was conducted (D) and the wound-healing assay was used for migration assay (E). These results present the mean ± standard deviation (SD) of three independent experiments. ***P<0.001.

Figure 8

Exosomes-delivered miR-124 inhibited the EMT in pancreatic cancers. The protein levels of EZH2, N1CD, Hes1, MMP-9, vimentin and E-cadherin were determined by western blot (D). These results present the mean ± standard deviation (SD) of three independent experiments. ***P<0.001.

Figure 9

Exosomes-delivered miR-124 enhanced the chemotherapy on pancreatic cancers. The effects of BM-MSC-derived exosomes on AsPC-1 or PANC1 cell viability (A) were measured by MTT and apoptosis were determined using AnnexinV-propidium iodide (PI) by flow cytometry (B), while the apoptosis related protein (C) levels of caspase-3, Bax, Bcl-2 and EZH2 and EMT related proteins (D) N1CD, Hes1, MMP-9, vimentin and E-cadherin were determined by western blot. These results present the mean ± standard deviation (SD) of three independent experiments. ***P<0.001.

Figure 10

Exosomes-delivered miR-124 enhanced the chemotherapy on pancreatic tumors in vivo. MiR-124-carried exosomes enhanced the anti-tumor effects of 5-FU on pancreatic cancer (A) The expression of miR-124-3p was remarkably higher in miR-124-EXO group (B) And the apoptosis related protein (C) levels of caspase-3, Bax, Bcl-2 and EZH2 and EMT related proteins (D) N1CD, Hes1, MMP-9, vimentin and E-cadherin on pancreatic tumors were determined by western blot. These results present the mean ± standard deviation (SD) of three independent experiments. ***P<0.001.