Volume 145, Issue 5, Pages 1133-1143.e12 (November 2013) MicroRNA 23b Regulates Autophagy Associated With Radioresistance of Pancreatic Cancer Cells  Peng Wang, Juan Zhang, Li Zhang, Zhengfei Zhu, Jie Fan, Lianyu Chen, Liping Zhuang, Jianmin Luo, Hao Chen, Luming Liu, Zhen Chen, Zhiqiang Meng  Gastroenterology  Volume 145, Issue 5, Pages 1133-1143.e12 (November 2013) DOI: 10.1053/j.gastro.2013.07.048 Copyright © 2013 AGA Institute Terms and Conditions

Figure 1 miRNA expression profile of RR pancreatic cancer cells. (A) The surviving fraction of established RR pancreatic cancer cells and their parental cell lines after irradiation with 0, 2, 4, 6, 8, or 10 Gy. Various numbers of cancer cells (for BxPC3, the numbers were 200, 400, 1000, 4000, 10,000, and 40,000; for PANC1, the numbers were 200, 400, 600, 2000, 6000, and 20,000) were plated into 6-well plates. Fourteen days after culturing, cells were fixed in paraformaldehyde and stained with crystal violet. The number of colonies with >50 cells was counted under a dissecting microscope, and the percentage of cell survival was calculated. The results are presented as the means ± SD of values obtained in 3 independent experiments. Statistical significance (P < .05) is indicated vs RR1 (*) and RR2 (†). (B) Representative crystal violet staining of the colonies formed by RR BxPC3 cells and their parental cells 14 days after irradiation with 0, 2, 4, 6, 8, or 10 Gy. The cells were seeded at a density of 2 × 104 cells per well in a 6-well plate. (C) Heat map of 15 qPCR-verified miRNAs (4 up-regulated and 11 down-regulated) based on the relative miRNA expression of RR cells compared with parental cells. (D) miR-23b expression in pancreatic cancer cell lines using semiquantitative real-time PCR. U6 served as an internal control. An ANOVA test was used to determine the statistical significance of differences between the groups. The results are presented as the means ± SD of values obtained in 3 independent experiments. *P < .05. Gastroenterology 2013 145, 1133-1143.e12DOI: (10.1053/j.gastro.2013.07.048) Copyright © 2013 AGA Institute Terms and Conditions

Figure 2 RR pancreatic cancer cells exhibit increased autophagy. (A) Autophagy was evaluated in pancreatic cancer cell lines that exhibited varying degrees of radiosensitivity using transmission electron microscopy. Scale bars, 500 nm. The data were quantified by counting the number of autophagosomes per cross-sectioned cell. The results are presented as the means ± SD of values obtained in 3 independent experiments. *P < .05. (B) Pancreatic cancer cells that stably expressed the GFP-LC3 fusion protein were established. Cells were then observed under a fluorescent microscope (200× magnification). The percentage of cells with GFP-LC3 puncta was used to quantify the percentage of autophagic cells. (C) Autophagosome formation in whole cell lysates was determined by Western blot analysis using LC3 and p62 antibodies. The top band (16 kilodaltons) represents LC3-I, and the bottom band (14 kilodaltons) represents LC3-II. (D) RR cells and their parental cell lines were treated with 10 μmol/L CQ for 24 hours before being subjected to Western blot analysis for LC3 expression. (E) The indicated cell lines were treated with 10 μmol/L CQ 24 hours before irradiation. Survival fractions were calculated as described in Figure 1A. The results are presented as the means ± SD of values obtained in 3 independent experiments. *P < .05. Gastroenterology 2013 145, 1133-1143.e12DOI: (10.1053/j.gastro.2013.07.048) Copyright © 2013 AGA Institute Terms and Conditions

Figure 3 miR-23b inhibits autophagy in pancreatic cancer cells. (A) BxPC3/RR2 cells were transfected with the indicated miRNA mimics or inhibitors. Forty-eight hours later, LC3-I/II proteins were detected by Western blot. (B) RR pancreatic cells were transfected with miR-23b mimics or NC as indicated. Forty-eight hours after transfection, LC3-II and p62 expression were examined by Western blot. (C) RR pancreatic cells were transfected with miR-23b mimics or NC as indicated. Forty-eight hours later, autophagy was evaluated using transmission electron microscopy. *P < .05. (D) RR pancreatic cells were transfected with miR-23b mimics or NC as indicated. Forty-eight hours later, cells were observed under a fluorescent microscope. *P < .05. (E) RR pancreatic cells were transfected with miR-23b mimics or NC as indicated. Forty-eight hours later, cells were treated with 10 μmol/L CQ for 24 hours before being harvested for Western blot analysis for LC3 expression. (F) Cells were transfected with duplexes as indicated. Forty-eight hours after transfection, cells were treated with a single fraction of 4 Gy IR. Twenty-four hours after IR, total protein as well as cytoplasmic protein fractions were isolated, and the indicated proteins were detected by Western blot. Gastroenterology 2013 145, 1133-1143.e12DOI: (10.1053/j.gastro.2013.07.048) Copyright © 2013 AGA Institute Terms and Conditions

Figure 4 miR-23b sensitizes pancreatic cancer cells to irradiation treatment. (A) BxPC3 or PANC-1 cells were transfected with miR-23b mimics, inhibitors, or NC as indicated. Forty-eight hours after transfection, cells were treated with 0, 2, 4, 6, 8, or 10 Gy of IR. Survival fractions were calculated as described in Figure 1A. The results are presented as the means ± SD of values obtained in 3 independent experiments. The statistical significance of differences between the groups was calculated using Student t tests. *P < .05. (B) Radiosensitivity parameters of respective cell lines. D0, dose to reduce survival to 37%; SF2, surviving fraction at 2 Gy; SER10, sensitizer enhancement ratio at 10% survival. Gastroenterology 2013 145, 1133-1143.e12DOI: (10.1053/j.gastro.2013.07.048) Copyright © 2013 AGA Institute Terms and Conditions

Figure 5 ATG12 is a direct target of miR-23b. (A) The predicted binding sequences for miR-23b within the human ATG12 3′UTR. Seed sequences are highlighted and underlined. (B) Validation of ATG12 expression among pancreatic cancer cell lines using semiquantitative real-time PCR and Western blot analysis. (C) The correlation between miR-23b and ATG12 mRNA expression in clinical specimens obtained from patients with pancreatic cancer (n = 10) was evaluated using Spearman correlation analysis. (D) Luciferase activity assays using a luciferase reporter with wild-type or mutant human ATG12 3′UTRs were performed after cotransfection of miR-23b mimics or NC into HEK293 cells. The luciferase activity recorded for the NC transfection in each experiment was used to normalize the data; luciferase activity of the NC transfection was set as 1. n.s., not significant. *P < .05. (E) Pancreatic cancer cells were transfected with miR-23b mimics, a miR-23b inhibitor, or NC, and ATG12 expression was determined using real-time PCR and Western blot analysis. ANOVA and Student t tests were used to determine the statistical significance of the differences between groups. *P < .05. (F) BxPC3 cells were transfected with siATG12, NC, empty vector, miR-23b, or ATG12 (open reading frame lacking 3′UTR), as indicated, followed by treatment with IR. Twenty-four hours after IR, LC3-II, ATG12, and p62 expression were evaluated by Western blot. Gastroenterology 2013 145, 1133-1143.e12DOI: (10.1053/j.gastro.2013.07.048) Copyright © 2013 AGA Institute Terms and Conditions

Figure 6 miR-23b sensitizes pancreatic cancer cells to radiation treatment by blocking radiation-induced autophagy. (A) Pancreatic cancer cells were transfected with siATG12, miR-23b, NC, or ATG12 (ORF without 3′UTR), as indicated, followed by treatment of the cells as described in Figure 1A. Survival fractions were calculated for each group. The results are presented as the means ± SD of values obtained in 3 independent experiments. ANOVA or Student t tests were used to determine the statistical significance of the differences between groups. Statistical significance (P < .05) is indicated vs NC (*) and miR-23b+ATG12 (†). (B) Nude mice were subcutaneously injected (into the right axilla) with 2 × 106 cells infected with a control lentiviral vector, an miR-23b overexpression vector, or an ATG12 vector expressing the entire coding sequence of ATG12 but lacking the 3′UTR as indicated. When the average tumor volume reached approximately 500 mm3, the tumors were irradiated with a single 10-Gy dose of IR (arrow indicates the time of irradiation). Data are presented as tumor growth curves. Each group was composed of 6 mice. (C) Nude mice were subcutaneously injected with 2 × 106 control lentivector-infected cells (ShLuc) or shATG12 vector-infected cells as indicated. Mice were then treated as in panel B. Each group was composed of 6 mice. (D) Autophagy was evaluated using transmission electron microscopy in cells derived from 3 representative xenograft samples from each group. ANOVA was used to determine the statistical significance of the differences between groups. *P < .05. (E) Total protein and the cytoplasmic protein fractions were isolated from cells derived from 3 representative xenograft samples from each group, and the indicated proteins were detected by Western blot analysis. (F) Pancreatic cancer cells were transfected with miR-23b, anti–miR-23b inhibitor, or negative control as indicated. Cells were then maintained in the presence or absence of CQ (10 μmol/L) before treatment with IR. Survival fractions were calculated for each group. ANOVA was used to determine the statistical significance of the differences between the groups. Gastroenterology 2013 145, 1133-1143.e12DOI: (10.1053/j.gastro.2013.07.048) Copyright © 2013 AGA Institute Terms and Conditions

Figure 7 Inverse correlation between miR-23b expression and autophagy activity in human pancreatic cancer tissues. (A) In situ hybridization (ISH) was used to detect mature miR-23b in human pancreatic cancer specimens (n = 65) using LNA-miRNA probes. Immunohistochemistry (IHC) was performed on serial sections using antibodies against ATG12 and LC3. Original magnification: 200× (insets: 400×). (B) The correlation between miR-23b and ATG12 and LC3 expression in clinical specimens. *miR-23b expression was detected with ISH. †ATG12 and LC3 expression were detected with IHC. P values were obtained using Pearson χ2 test. Gastroenterology 2013 145, 1133-1143.e12DOI: (10.1053/j.gastro.2013.07.048) Copyright © 2013 AGA Institute Terms and Conditions

Supplementary Figure 1 Schemes for the establishment of RR pancreatic cancer sublines. BxPC3 and PANC-1 cells were seeded at a density of 1 × 106/T25 flask in complete medium. When cell confluence reached 70%, cells were treated with 2 Gy 60Co radiation at 2 Gy/min. When they reached 80% confluence, the cells were trypsinized and subcultured into new flasks. When they reached 70% confluence, the cells were serially irradiated with increasing doses (2, 4, 6, 8, or 10 Gy) of IR. We then selected 2 clones from each group and designated them BxPC3/RR1, BxPC3/RR2, PANC-1/RR1, and PANC-1/RR2. Sublines were then treated with another 5 cycles of 10-Gy irradiation. The newly generated cell lines were then evaluated for radiosensitivity using clonogenic survival assays. Gastroenterology 2013 145, 1133-1143.e12DOI: (10.1053/j.gastro.2013.07.048) Copyright © 2013 AGA Institute Terms and Conditions

Supplementary Figure 2 Survival fraction, cell proliferation, and colony formation in the established RR cell sublines. (A) The surviving fraction of established RR pancreatic cancer cells and their parental cell lines after irradiation with 0, 2, 4, 6, 8, or 10 Gy. The cells were seeded at a density of 2 × 104 cells per well in a 6-well plate. Fourteen days after culturing, cells were fixed in paraformaldehyde and stained with crystal violet. (B) Colony formation among RR cell sublines and their parental cell lines. A total of 800 transfected cells were seeded in a fresh 6-well plate. The number of colonies with >50 cells was counted under a dissecting microscope. The results are presented as the means ± SD of values obtained in 3 independent experiments. n.s., not significant. (C) The effect of miR-23b on pancreatic cancer cell growth, as measured using the cholecystokinin-8 assay. The results are presented as the means ± SD of values obtained in 3 independent experiments. Gastroenterology 2013 145, 1133-1143.e12DOI: (10.1053/j.gastro.2013.07.048) Copyright © 2013 AGA Institute Terms and Conditions

Supplementary Figure 3 The effects of CQ or ATG12 siRNA on pancreatic cancer cell colony formation. (A) The effect of CQ on colony formation. A total of 800 cells were seeded in a fresh 6-well plate. Cells were then treated with 10 μmol/L CQ for 7 days. The number of colonies with >50 cells was counted under a dissecting microscope. The results are presented as the means ± SD of values obtained in 3 independent experiments. n.s., not significant. (B) The effect of ATG12 siRNA on colony formation. Pancreatic cancer cells were transiently transfected with ATG12 siRNA (siATG12) or an NC using Lipofectamine. The number of colonies with >50 cells was counted under a dissecting microscope. The results are presented as the means ± SD of values obtained in 3 independent experiments. n.s., not significant. Gastroenterology 2013 145, 1133-1143.e12DOI: (10.1053/j.gastro.2013.07.048) Copyright © 2013 AGA Institute Terms and Conditions

Supplementary Figure 4 The effect of miR-23b on pancreatic cancer cell colony formation and proliferation. (A) BxPC3 and PANC-1 cells were transiently transfected with miR-23b mimics or an anti–miR-23b inhibitor using Lipofectamine. miR-23b expression was confirmed 48 hours after transfection using quantitative PCR. U6 served as an internal control. (B) The effect of miR-23b on colony formation in BxPC3 and PANC-1 cells. The results are presented as the means ± SD of values obtained in 3 independent experiments. (C) The effect of miR-23b on pancreatic cancer cell growth, as measured using the cholecystokinin-8 assay. The results are presented as the means ± SD of the values obtained in 3 independent experiments. n.s., not significant. Gastroenterology 2013 145, 1133-1143.e12DOI: (10.1053/j.gastro.2013.07.048) Copyright © 2013 AGA Institute Terms and Conditions

Supplementary Figure 5 Screening for miR-23b regulated targets in pancreatic cancer cells. (A) RR BxPC3 cells were transfected with miR-23b mimics or a control as indicated. After 48 hours, the mRNA expression of the indicated autophagy-related genes was measured using real-time PCR. Student t tests were used to determine the statistical significance of the differences between the groups. n.s., not significant. *P < .05; **P < .01. (B) Validation of ATG3 expression in pancreatic cancer cell lines using semiquantitative real-time PCR. The base pairing complement was derived using miRanda (microrna.org and miRBase). (C) RR PANC-1 cells were transfected with miR-23b mimics or NC. After 48 hours, the mRNA expression of BECN1 was quantified using real-time PCR. The base pairing complement was derived using miRanda. Gastroenterology 2013 145, 1133-1143.e12DOI: (10.1053/j.gastro.2013.07.048) Copyright © 2013 AGA Institute Terms and Conditions

Supplementary Figure 6 ATG12 is a direct target of miR-23b. BxPC3 cells were cotransfected with miR-23b or NC and a luciferase reporter construct containing the wild-type or mutant ATG12 3′UTR. For each experiment, the data were normalized to the luciferase activity detected in cells transfected with NC, which was set to 1. n.s., not significant. *P < .05. Gastroenterology 2013 145, 1133-1143.e12DOI: (10.1053/j.gastro.2013.07.048) Copyright © 2013 AGA Institute Terms and Conditions

Supplementary Figure 7 Knockdown efficiencies of siRNA and shRNA against ATG12 in pancreatic cancer cells. (A) Pancreatic cancer cells were transiently transfected with ATG12 siRNA (siATG12) or an NC using Lipofectamine. ATG12 expression levels were measured 48 hours after transfection by Western blot. (B) BxPC3 cells stably expressing shATG12 were generated using a lentiviral infection system. ATG12 protein expression levels were measured by Western blot. (C) ATG12 protein levels after knockdown in xenografts expressing shATG12, detected by Western blot. GAPDH served as an internal control. Gastroenterology 2013 145, 1133-1143.e12DOI: (10.1053/j.gastro.2013.07.048) Copyright © 2013 AGA Institute Terms and Conditions

Supplementary Figure 8 miR-23b sensitizes pancreatic cancer cells to radiation treatment in vivo. (A) (related to Figure 6B) Nude mice were subcutaneously injected into the right axilla with 2 × 106 cells infected with control lentiviral vector, miR-23b vector, or an ATG12-expressing vector encoding the entire coding sequence of ATG12 but lacking the 3′UTR as indicated. When the average tumor volume reached approximately 500 mm3, the tumors were irradiated with a single 10-Gy dose of IR. Tumor growth was then monitored using calipers, and the mice were killed when the tumors reached 1.5 cm in diameter. The tumors were removed, weighed, and compared between the groups using ANOVA. (B) (related to Figure 6C) The indicated tumor cells were injected as described previously. Forty-one days later, the primary tumor weights were compared between the groups using ANOVA. n.s., not significant. Gastroenterology 2013 145, 1133-1143.e12DOI: (10.1053/j.gastro.2013.07.048) Copyright © 2013 AGA Institute Terms and Conditions

Supplementary Figure 9 The sensitization effects of CQ on cells with differing miR-23b expression levels. Pancreatic cancer cells were transfected with miR-23b, anti–miR-23b, or negative control as indicated. Cells were then maintained in the presence or absence of CQ (10 μmol/L) before treatment with 0, 2, 4, 6, 8, or 10 Gy of IR. Survival fractions were calculated for each group. The results are presented as the means ± SD of values obtained in 3 independent experiments. Gastroenterology 2013 145, 1133-1143.e12DOI: (10.1053/j.gastro.2013.07.048) Copyright © 2013 AGA Institute Terms and Conditions