MicroRNA-21 induces 5-fluorouracil resistance in human pancreatic cancer cells by regulating PTEN and PDCD4

Abstract

Pancreatic cancer patients are often resistant to chemotherapy treatment, which results in poor prognosis. The objective of this study was to delineate the mechanism by which miR-21 induces drug resistance to 5-fluorouracil (5-FU) in human pancreatic cancer cells (PATU8988 and PANC-1). We report that PATU8988 cells resistant to 5-FU express high levels of miR-21 in comparison to sensitive primary PATU8988 cells. Suppression of miR-21 expression in 5-Fu-resistant PATU8988 cells can alleviate its 5-FU resistance. Meanwhile, lentiviral vector-mediated overexpression of miR-21 not only conferred resistance to 5-FU but also promoted proliferation, migration, and invasion of PATU8988 and PANC-1 cells. The proresistance effects of miR-21 were attributed to the attenuated expression of tumor suppressor genes, including PTEN and PDCD4. Overexpression of PTEN and PDCD4 antagonized miR-21-induced resistance to 5-FU and migration activity. Our work demonstrates that miR-21 can confer drug resistance to 5-FU in pancreatic cancer cells by regulating the expression of tumor suppressor genes, as the target genes of miR-21, PTEN and PDCD4 can rescue 5-FU sensitivity and the phenotypic characteristics disrupted by miR-21.

Keywords: 5-Fluorouracil; PDCD4; PTEN; miR-21; pancreatic cancer.

Figure 1

IC50s and expression of miR‐21 in PATU8988/5‐FU and PATU8988 cells. (A) Representative curves of growth inhibitory in 5‐FU‐resistant PATU8988/5‐FU and PATU8988 cells. IC50, half maximal inhibitory concentration. (B) The expression of miR‐21‐5p and miR‐21‐3p in 5‐FU‐resistant PATU8988/5‐FU cells and its parental PATU8988 cells. The error bars represent the standard deviation obtained from three independent experiments. ***P < 0.001 **P<0.01.

Figure 2

Cytotoxicity activity and proliferation rates of pancreatic cancer cells with miR‐21 overexpression. (A) The expression of miR‐21 in PATU8988/5‐FU cells transfected with negative control (nc) or miR‐21 inhibitor (anti‐miR‐21). (B) Representative curves of growth inhibitory in PATU8988/5‐FU cells transfected with negative control(nc) or miR‐21 inhibitor. (C) RT‐PCR showed the expression of miR‐21 in PATU8988 cells infected with lenti_miR‐21 and control group lenti_GFP. (D) Representative curves of growth‐inhibitory effects of 5‐FU in PATU8988 cells after treatments. (E) The expression of miR‐21 in PANC‐1 cells. (F) Representative curves of growth inhibitory effects of 5‐FU in PANC‐1 cells. (G) The cell growth curve of PATU8988 cells. (H) The cell growth curve of PANC‐1 cells. *P < 0.05, ** P < 0.01 and ***P < 0.001.

Figure 3

Overexpression of miR‐21 promotes PATU8988 cells migration and invasion. (A) The pictures of wound healing of PATU8988 cells infected with lenti_miR‐21 or lenti_GFP at times 0, 12, 24 and 36 h from the scratch. (B) The relative ratio of wound closure per field at each time point is shown. (C) Cells migrated to the bottom of membranes were stained and photographed. (D) Matrigel cell invasion assay. In (C and D), microscopic magnification (100×). The relative ratio of invasive cells per field is shown. ***P < 0.001.

Figure 4

Overexpression of miR‐21 promotes PANC‐1 cells migration and invasion. (A) The pictures of wound healing of PANC‐1 cells infected with lenti_miR‐21 or lenti_GFP at times 0, 12, 24 and 36 h from the scratch. (B) The relative ratio of wound closure per field at each time point is shown. (C) Cells migrated to the bottom of membranes were stained and photographed. (D) Matrigel cell invasion assay. In (C and D), microscopic magnification (100×). The relative ratio of invasive cells per field is shown. ***P < 0.001.

Figure 5

MiR‐21 promotes 5‐fluorouracil (5‐FU) resistant in pancreatic cancer by targeting PTEN directly. (A) Western blot showed PTEN protein levels in PANC‐1 and PATU8988 transfected with pcDNA3.1_miR‐21 or pcDNA3.1. (B) Western blot showed PTEN protein levels in PATU8988/5‐FU and PATU8988 cells. (C) Rescue assays by transfection with pcDNA3.1 (nc), pcDNA3.1_miR‐21 (miR‐21), pcDNA3.1_PTEN (PTEN) or pcDNA3.1_miR‐21 plus pcDNA3.1_PTEN (rescue) in PATU8988 and western blot. (D) Rescue assays and western blot in PANC‐1. (E) Representative curves of growth‐inhibitory effects of 72 h 5‐FU exposure in PATU8988 in rescue assay. (F) Representative curves of growth‐inhibitory effects of 72 h 5‐FU exposure in PANC‐1 in rescue assay. (G) Transwell assay after transfection in PATU8988 cells for 24 h. (H) Transwell assay after transfection in PANC‐1 cells for 24 h. In (G and F), microscopic magnification (100×). The relative ratio of invasive cells per field is shown. *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 6

MiR‐21 promotes 5‐fluorouracil (5‐FU) resistant in pancreatic cancer by targeting PDCD4 directly. (A) Western blot showed PDCD4 protein levels in PATU8988 and PANC‐1 transfected with pcDNA3.1_miR‐21 or pcDNA3.1. (B) Western blot showed PTEN protein levels in PATU8988/5‐FU and PATU8988 cells. (C) Rescue assays by transfection with pcDNA3.1 (nc), pcDNA3.1_miR‐21 (miR‐21), pcDNA3.1_PDCD4 (PDCD4) or pcDNA3.1_miR‐21 plus pcDNA3.1_PDCD4 (rescue) in PATU8988 and western blot. (D) Rescue assays and western blot in PANC‐1. (E) Representative curves of growth‐inhibitory effects of 72 h 5‐FU exposure in PATU8988 in rescue assay. (F) Representative curves of growth‐inhibitory effects of 72 h 5‐FU exposure in PANC‐1 in rescue assay. (G) Transwell assay after transfection in PATU8988 cells for 24 h. (H) Transwell assay after transfection in PANC‐1 cells for 24 h. In (G and F), microscopic magnification (100×). The relative ratio of invasive cells per field is shown. *P < 0.05, **P < 0.01 and ***P < 0.001.