Contributed equally
Radioresistance has become a challenge in the treatment of pancreatic cancer, which limits the efficacy and outcomes of radiotherapy in clinical treatment. Autophagy, recognized as an adaptive response to cell stress, has recently been involved in the radioresistance of cancer cells. MicroRNAs (miRNAs) are also involved in the radioresistance of pancreatic cancer cells. In the present study, we established a radioresistant pancreatic cancer cell line and found that miRNA-216a was significantly downregulated whereas the autophagy activity was increased as compared with the control. Forced expression of miR-216a was found to inhibit the expression of beclin-1, a critical autophagic gene, as well as autophagy. Using bioinformatics analysis and the dual-luciferase reporter gene assay, we found that miR-216a directly interacted with 3′-untranslated region (UTR) of beclin-1. Furthermore, the forced expression of miR-216a inhibited cell growth and colony formation ability and promoted the cell apoptosis of radioresistant pancreatic cancer cells in response to irradiation. By contrast, over-expression of beclin-1 abrogated the effects of miR-216a. Furthermore, miR-216a sensitized xenograft tumor to irradiation treatment and inhibited irradiation-induced autophagy by regulating beclin-1. Collectively, the results demonstrated that miR-216a enhanced the radiosensitivity of pancreatic cancer cells by inhibiting beclin-1-mediated autophagy, suggesting a promising molecular target for improving the radiotherapy of pancreatic cancer.
Pancreatic cancer, one of the most lethal types of cancer, lacks effective therapeutic methods and exhibits a low 5-year survival rate of <5% (
Autophagy, a catabolic process for the degradation of cytoplasmic proteins and organelles as an adaptive response to cell stress such as nutrient starvation or metabolic stress, is considered a potential mechanism for the radioresistance of cancer cells (
MicroRNAs (miRNAs), the post transcriptional regulators of gene expression, have been identified as an important regulator in a variety of cell processes (
Previous findings have indicated that miR-216a was markedly decreased in pancreatic cancer (
The human pancreatic cancer cell line, PANC-1, was obtained from the Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen-Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Invitrogen-Life Technologies) containing penicillin/streptomycin. The cells were grown in a humidified 5% CO2 at 37°C in an incubator. The irradiation of PANC-1 cells was performed according to a previously reported method (
Female 6-week-old BALB/c nude mice (25–30 g) were obtained from the Medical Experimental Animal Center (Guangdong, China) and housed under pathogen-free conditions with free access to water and food. The animal experimental procedures were approved and reviewed by the Institutional Animal Care and Use Committee of Guangzhou Medical University.
Following irradiation or miR-216a treatment, cell growth and viability was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sangon, Shanghai, China) assay. Briefly, the cells were seeded in 96-well plates at a density of 5×103 cells/200
Apoptotic cells were stained using the TUNEL (terminal deoxynucleotidyl transferase dUTP nick end-labeling) apoptosis kit (Genmed Scientifics, Arlington, MA, USA) according to the supplier's instructions. Briefly, the cells were fixed with 4% paraformaldehyde followed by incubation with TUNEL reaction mixtures for 1 h at 37°C. The stained cells were visualized and counted using a fluorescence microscope (Olympus, Tokyo, Japan). Five fields (magnification, ×400) were randomly selected for the measurement of apoptotic cells in a blinded manner.
The beclin-1 3′-UTR and mutated 3′-UTR constructs were amplified and subcloned into pGL3 Luciferase Promoter Vector (Promega, Madison, WI, USA) with
Total RNA was isolated by using TRIzol (Invitrogen-Life Technologies) and small RNAs were extracted by using mirVana kits (Ambion Inc, Austin, TX, USA) according to the manufacturer's instructions. Corresponding cDNA was generated by using M-MLV reverse transcriptase (Clontech, Palo Alto, CA, USA) and the TaqMan miRNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. To analyze the gene expression levels, the RT-qPCR mixture system containing cDNA templates, primers and SYBR-Green qPCR Master Mix were subjected to RT-qPCR quantification. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; for beclin-1) and U6 SnRNA (for miR-216a) were used as an internal reference and relative gene expression was quantified by 2−ΔΔCt method.
Total cell lysates were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Amersham, Little Chalfont, UK). Non-fat dry milk (2.5%) was used for blocking the membrane and primary antibodies including anti-LC3 antibody (ab63817) and anti-beclin-1 (ab62557) (both from Abcam, Cambridge, UK), anti-cleaved caspase-3 (PC679-50UG) (from Millipore, Boston, MA, USA) and anti-GAPDH antibody (bs-2188R) (Bioss, Beijing, China) were used for detection of the target protein. The target protein was visualized by using an enhanced chemiluminescence (ECL) detection system (Amersham).
Cells (2×106) diluted in 200
Data were presented as the mean ± standard deviation (SD) of three or more independent experiments. Levels of significance between or among groups were analyzed by the two-tailed Student's t-test or the one-way ANOVA, respectively. Results were considered statistically significant at P<0.05.
To investigate the potential role of miR-216a in radioresistance of pancreatic cancer cells, we first examined the miR-216a levels in radioresistant cells using RT-qPCR. The results showed that miR-216a expression levels were significantly inhibited in radioresistant (RR)-PANC-1 cells as compared with the control PANC-1 cells (
To investigate whether the inhibited miR-216a expression has a certain correlation with the increased autophagy activity, we treated the RR-PANC-1 cells with miR-216a mimics and determined the alterations of autophagy activity. The results exhibited that forced expression of miR-216a significantly suppressed beclin-1 and LC3-II protein expression levels (
To investigate the relationship between miR-216a and autophagy, we screened whether miR-216a has a direct interaction with autophagy-related genes using bioinformatics analysis. As expected, we found that beclin-1 was a putative target gene of miR-216a (
To assess whether miR-216a-mediated autophagy inhibition via beclin-1 plays an important role in the growth of RR cells in response to irradiation, we forced the expression of miR-216a in RR cells and analyzed the cell growth and colony formation after irradiation. RR-PANC-1 cells were pretreated with miR-216a mimics or control scramble miRNA for 1 h and then subjected to irradiation (2 Gy/min). Using the MTT assay, we found that the forced expression of miR-216a markedly sensitized RR cells to cell death in response to irradiation, as compared with the control group (
To study the role of miR-216a on RR cells, we detected the effect of miR-216a on cell apoptosis in RR cells in response to irradiation. The results of the TUNEL assay indicated that a forced expression of miR-216a significantly increased the cell apoptosis of RR-PANC-1 cells in comparison with the control (
To verify whether miR-216a sensitized RR-PANC-1 cells to irradiation by regulating beclin-1, we co-transfected miR-216a with beclin-1 overexpression vectors harboring no specific miR-216a binding specific sequences in 3′-UTR in RR-PANC-1 cells. The results showed that over-expression of beclin-1 (
To assess whether miR-216a can increase the efficiency of irradiation in killing implanted tumors in nude mice, nude mice were subcutaneously injected with PANC-1 cells pre-transfected with lentiviral vector expressing miR-216a or scramble miRNA control. When the tumors reached ~500 mm3 on day 16, the tumors received a single dose of 10-Gy irradiation. The results showed that force expression of miR-216a significantly increased the radiosensitivity of PANC-1-derived tumors on irradiation treatment (
The role of miR-216a in cancer has increasingly drawn attention. The transcription of miR-216a has been found to be stimulated by the androgen pathway which targets the tumor suppressor in lung cancer-1 gene in the early stage of carcinogenesis (
As expected, we found that miR-216a was involved in the regulation of radioresistance through beclin-1-mediated autophagy. Autophagy, an adaptive response against cellular streak, has been currently suggested to be involved in the radioresistance of cancer cells (
The role of autophagy in the regulation of radioresistance of pancreatic cancer has been investigated. Profilin1 has been indicated to be capable of sensitizing pancreatic cancer cells to irradiation by inhibiting autophagy (
miR-30a sensitizes cancer cells to cis-platinum via suppression of beclin 1-mediated autophagy (
The present study was supported by the Guangdong Province Natural Science Foundation (S2013010016662), the Health Bureau of Guangdong Province (A2014224 and B2014196), the Science and Technology Planning Project of Guangdong Province (2013B021800284) and the National Natural Science Foundation of China (81201932 and 81372493).
microRNAs
untranslated region
microtubule-associated protein light chain 3
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide
terminal deoxynucleotidyl transferase dUTP nick end-labeling
Effect of irradiation on miR-216a expression and autophagy activity in PANC-1 cells. (A) qRT-PCR was performed to analyze the expression levels of miR-216a in RR-PANC-1 cells. (B) Western blot analysis was performed to detect the autophagy activity indicated by beclin-1 and LC3 I/II protein expression levels. Relative protein expression levels of beclin-1 (C) and LC3-II (D) were quantified using Image-Pro Plus 6.0 software and normalized to GAPDH. PANC-1 denotes PANC-1 cells without irradiation, RR-PANC-1 denotes radioresistant PANC-1 cells.*P<0.05 and **P<0.01. PANC-1, human pancreatic cancer cell line; RT-qPCR, reverse transcriptase-quantitative polymerase chain reaction; LC3, microtubule-associated protein light chain 3.
Effect of miR-216a on autophagy in RR-PANC-1 cells. (A) Beclin-1 and LC3 I/II protein expression was detected by western blot analysis using the indicated antibodies. Relative protein expression levels of (B) beclin-1 and (C) LC3-II were quantified using Image-Pro Plus 6.0 software and normalized to GAPDH *P<0.05. PANC-1, human pancreatic cancer cell line; LC3, microtubule-associated protein light chain 3.
miR-216a directly targets 3′-UTR of beclin-1. (A) The predicted binding sequences for beclin-1 3′-UTR with miR-216a. (B) Luciferase activity assays were used to detect the interaction between miR-216a and beclin-1 3′-UTR. The wild-type or mutant beclin-1 3′-UTR was co-transfected with miR-216a mimics or scrambled miRNA and incubated for 48 h in PANC-1 cells followed using a detection by dual-luciferase reporter assay kit. Wt, pGL3 vectors containing beclin-1 3′-UTR; Mut, pG3 vectors containing mutated Beclin-1 3′-UTR as mentioned above.*P<0.05. PANC-1, human pancreatic cancer cell line; UTR, 3′-untranslated region.
Effect of miR-216a on cell growth and colony formation in RR-PANC-1 cells. (A) Cell growth was detected by MTT assay. Control PANC-1 cells, or RR-PANC-1 cells pretreated with control scramble miRNA and miR-216a mimics were irradiated (2 Gy/min). After 24 and 48 h treatment, cell growth was determined. (B) Cells subjected to irradiation were continued to be cultured for 15 days. Crystal violet staining was performed and the cell colonies were counted. *P<0.05. PANC-1, human pancreatic cancer cell line.
Effect of miR-216a on cells apoptosis in RR-PANC-1 cells. (A) Apoptotic cells were detected by TUNEL assay. Control PANC-1 cells or RR-PANC-1 cells pretreated with control scramble miRNA and miR-216a mimics were irradiated (2 Gy/min) and cultured for 48 h. Apoptotic cells were counted in a field of 500×500
Effect of beclin-1 overexpression on miR-216a-induced irradiation sensitivity in RR-PANC-1 cells. (A) Western blot analysis to detect the expression levels of beclin-1 in different treated groups. RR-PANC-1 cells treated with miR-216a with or without beclin-1 overexpression vectors were subjected to irradiation (2 Gy/min) and cultured for 48 h. Cells treated with scramble oligonucleotides were regarded as the control. (B) Relative protein expression was quantified using Image-Pro Plus 6.0 software and normalized to GAPDH. Cell growth (C) and apoptosis (D) was detected by MTT assay and TUNEL assay, respectively. *P<0.05 and **P<0.01. PANC-1, human pancreatic cancer cell line.
Effect of miR-216a on xenograft tumor growth