Fig 2: Regulation of glucocorticoid receptor (GR) and miR-183-5p expression in HEK293 cells. (A,B) Cells were treated with DEX as indicated. MiR-183-5p and Nr3c1 expression level was analyzed and normalized to U6 and ß-actin level. Data are presented as the fold change relative to the control group. Statistical analyses were performed using the one-way ANOVA. Error bars represent mean ± SD (n = 3). * p < 0.05, *** p < 0.001. (C) Western blots showing the nuclear translocation of GR protein following DEX treatment. GAPDH and Lamin B1 were used as the cytoplasmic and nuclear loading controls, respectively. Quantification of the relative blot intensity of the GR protein was performed using ImageJ software. Statistical analyses were performed using the one-way ANOVA. Error bars represent mean ± SD (n = 3). ** p < 0.01, *** p < 0.001. (D) Cells seeded on sterile coverslips were treated with DEX for 6 h, then fixed with methanol, incubated with fluorescently tagged mouse monoclonal GR antibody, and stained with Alexa Flour 488 (green)-labeled anti-mouse antisera and DAPI. GR signals were then visualized by confocal microscopy. Quantification of the relative fluorescence intensity of GR was performed using ImageJ software. Data are presented as the fold change relative to the control group. Statistical analyses were performed using the one-way ANOVA. Error bars represent mean ± SD (n = 3–6). ** p < 0.01. (E) Cells were treated with 1 µM of DEX and 3 µM of RU486 as indicated for 24 h. (F) Cells were treated with DEX and RU486 as indicated. MiR-183-5p expression level was analyzed and normalized to U6 level. Data are presented as the fold change relative to the control group. Statistical analyses were performed using the one-way ANOVA. Error bars represent mean ± SD (n = 3). ** p < 0.01. (G,H) Cells were treated with 1 nM miR-183-5p mimic for 48 h. MiR-183-5p and Nr3c1 expression level was analyzed by qRT-PCR and normalized to U6 and ß-actin level. Data are presented as the fold change relative to the control group. Statistical analyses were performed using the Student’s t-test. Error bars represent mean ± SD (n = 3). *** p < 0.001. (I) GR protein expression level. (J,K) Cells were treated with 10 nM miR-183-5p inhibitor for 48 h. MiR-183-5p and Nr3c1 expression level was analyzed by qRT-PCR and normalized to U6 and ß-actin level. Data are presented as the fold change relative to the control group. Statistical analyses were performed using the one-way ANOVA. Error bars represent mean ± SD (n = 3). *** p < 0.001. (I,L) GR protein expression level. (M) Cells were treated with the indicated combinations of DEX, miR-183-5p mimic, and miR-183-5p inhibitor, and GR protein expressions were measured by western blot analyses. (N) miR-183-5p expression level was analyzed and normalized to U6 level. Data are presented as the fold change relative to the control group. Statistical analyses were performed using the one-way ANOVA. Error bars represent mean ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig 3: Effects of METH injections on the locomotor activity in rats. (A) Flowchart showing an overview of the experimental procedure. Rats were injected with single or repeated METH injections. (B,G) The total travelled distance was recorded 1 h after single or repeated METH injections. Statistical analyses were performed using the Student’s t-test. Error bars represent mean ± SEM (n = 8). *** p < 0.001. (C,H) Plasma corticosterone levels in rats following single or repeated injections of saline or METH. Statistical analyses were performed using the Student’s t-test. Error bars represent mean ± SEM (n = 8). *** p < 0.001. (D,I) Western blot analysis showing p-GR (S211) expression in rat striatum. The bands were normalized to ß-actin. Quantification of the relative blot intensity of proteins was performed using ImageJ software. Data are presented as the fold change relative to the saline group. Statistical analyses were performed using the Student’s t-test. Error bars represent mean ± SEM (n = 8). * p < 0.05, ** p < 0.01. (E,J) qRT-PCR analysis of miR-183-5p expression. The value was normalized to the U6 level. Data are presented as the fold change relative to the saline group. Statistical analyses were performed using the Student’s t-test. Error bars represent mean ± SEM (n = 8). ** p < 0.01. (F,K) qRT-PCR analysis of Nr3c1. The level was normalized to the ß-actin level. Data are presented as the fold change relative to the saline group. Statistical analyses were performed using the Student’s t-test. Error bars represent mean ± SEM (n = 8). *** p < 0.001.

Fig 4: Identification of miRNA transcripts in METH self-administered rats. (A) Flowchart showing an overview of the experimental procedure. (B) The overall workflow for identification of miRNA and its target gene. Nr3c1 expression level in the striatum was analyzed by qRT-PCR and normalized to ß-actin level. Data are presented as the fold change relative to the saline group. Statistical analyses were performed using the Student’s t-test. Error bars represent mean ± SEM (n = 4). *** p < 0.001.

Fig 5: GR correlates with PD-L1 expression, low MHC-I expression, and poor survival in human PDAC.a Representative immunohistochemical (IHC) staining of GR in the normal pancreatic duct and PDAC. Scale bars, 100 µm (left), 50 µm (middle), and 50 µm (right). b Quantification of GR-positive and GR-negative cases of PDAC and adjacent normal pancreatic tissue. n = 101 patients. Statistical significance was determined by a two-tailed Fisher’s exact test. c GR (encoded by NR3C1) mRNA levels in paired normal pancreatic tissue and PDAC based on the GSE15471 dataset. n = 36 patients. Statistical significance was determined by a two-tailed paired t-test. d, e Representative IHC staining (d) and statistical analysis (e) of the correlation of GR protein levels with PD-L1, MHC-I, and CD8 proteins levels in patients with PDAC. n = 101 patients. f, g Correlation of NR3C1 (encoding GR) mRNA levels with CD274 (encoding PD-L1; f) and HLA-A (g) mRNA levels in PDAC based on TCGA data. n = 178 patients. h Kaplan–Meier curves of overall survival of pancreatic cancer patients stratified by GR protein levels based on IHC. n = 101 patients. i Kaplan–Meier curves of overall survival of pancreatic patients stratified by GR (encoded by NR3C1) mRNA levels. Data were obtained from the ICGC (International Cancer Genome Consortium). n = 95 patients. Statistical significance was determined by the log-rank test in h and i. j Plasma cortisol levels in healthy volunteers (n = 122) and PDAC patients (n = 82). Statistical significance was determined by a two-tailed unpaired t-test. Error bars are s.e.m. k–m Correlation of plasma cortisol levels with PD-L1 (k), MHC-I (l), or GR (m) protein levels in pancreatic tumors. n = 32 patients. Statistical significance was determined by a two-tailed Pearson correlation test in e–g and k–m. Source data are provided as a Source Data file.