GSE38642, GSE20966 and GSE25724 were mRNA expression profiles from GEO DataSets including human pancreatic islet samples, human β-cells samples and human islet samples with or without T2D. The differentially expressed genes (DEGs) were screened by limma 3.26.8 with P-value < 0.05. GSE26168 was the miRNA expression profile from GEO DateSets including blood samples with or without T2D. Then, the two online tools (WebGestalt and Metascape) were applied to analyze the biological processes of DEGs. TargetScan and miRwalk were performed to predicted the miRNAs targeting our interested gene. Finally, the key genes and miRNAs were overlapped using Venny 2.1.0.
Blood sample collection
Peripheral blood was sampled from 40 patients with T2D and 40 non-T2D patients in Wuhan Wuchang Hospital Affiliated to Wuhan University of Science and Technology. Patients who had been diagnosed with T2D accompanied by high blood pressure, obesity and dyslipidemia were included. This study gained the approval from the Ethics Committee of Wuhan Wuchang Hospital Affiliated to Wuhan University of Science and Technology and performed in keeping with the Declaration of Helsinki. Written informed consent from all subjects was obtained before sampling blood. Clinical characteristics of 40 patients with T2D were listed in Table 1.
Pancreatic β-cell line, the rat INS-1E cell line (AddexBio, US), was cultured in RPMI 1640 medium supplemented with 11 mM glucose, 5% FBS, 10 mM HEPES, 50 μM β-mercaptoethanol and 2 mM glutamine at 37°C with 5% CO2. For glucose induction, INS-1E cells cultured with 11 mM glucose served as control group and INS-1E cells cultured with 25 mM glucose was built up to be the model group.
miR-765 mimic, miR-765 inhibitor, and negative controls (NC) were provided by RiboBio (China). PDX1 overexpression vector (PDX1-OE), knockdown of PDX1 (si-PDX1), and their NC were purchased from GenePharma (China). Before cell transfection, INS-1E cells would be seeded in different plates depending on the subsequent corresponding assay. Cell density was 2.5×106·mL-1 for a 6-well plate, 1×106·mL-1 for a 12-well plate, 5×104 ·mL-1 for a 96-well plate, respectively. Cell transfection was performed utilizing Lipofectamine 3000 reagent (Invitrogen, US) and cell transfection concentration was 100 ng for a 96-well plate, 1000 ng for a 12-well plate, 2500 ng for a 6-well plate as stated in the protocol. After 48 h transfection, INS-1E cells were collected for further detections on condition that the transfection efficiency detected via qRT-PCR was satisfied for the present experiment.
Quantitative real-time PCR (qRT-PCR)
Total RNA from tissues or cells was extracted using RNeasy Micro Kit (QIAGEN, Germany) following manufacturer’s protocol. Titan One Tube RT-PCR Kit (Roche, Switzerland) was applied to get the cDNA for mRNA. One Step miRNA cDNA Synthesis Kit (HaiGene, China) was used to obtain the cDNA for miRNA. Relative expression of PDX1 or miR-765 was measured by SYBR-Green real-time PCR kit (Takara, Japan) in Bio-Rad CFX96 instrument (Bio-Rad, US). Internal reference for PDX1 was GAPDH, and for miR-765 was U6. The relative mRNA expression level was quantified through 2-ΔΔCt method. The sequences of primers were list in Table 2.
INS-1E cells with a 48-h transfection were treated by RIPA buffer with 1 mM PMSF (Sigma-Aldrich, US) at 4°C for 15 to 20 min. After being gathered, the proteins (10 μL for each hole) were separated by a 10% SDS-polyacrylamide gel at 100 V for 1 h. By the time the separation finished, proteins were transferred onto polyvinylidene diﬂuoride membranes (PVDF; Millipore, US) at 4°C. After the transfer and PBS washing, 5% skim milk was added to the PVDF membrane for 1-h blocking at 25°C. PBS was used to wash the PVDF membrane again when the blocking ended. Then, PVDF membrane was incubated in anti-PDX1 (1:1000, arigobio, China) and the reference β-actin (1:500, SANTA CRUZ BIOTECHNOLOGY, US) at 4°C overnight. The next morning, the secondary antibodies Goat-Anti-Rabbit IgG (1:5000, arigobio, China) for anti-PDX1 and Goat-Anti-Mouse IgG (1:5000, arigobio, China) for β-actin were added to the proteins for 1-h incubation at 25°C. Finally, the protein bands were added ECL luminescence reagent (Sangon Biotech, China) and then exposed by Image-Pro Plus 6.0 software (Media Cybernetics, US).
Glucose-stimulated insulin secretion determination (GSIS)
GSIS was performed to confirm the function of INS-1E cells. Briefly, the transfected INS-1E cells were seeded in a 96-well plate for another 24-h culture and then incubated under the condition of 3.3 mM glucose (basal glucose) or 16.7 mM glucose (stimulatory glucose) for 1 h. After this step, cell supernatant was collected for the insulin level determination using Insulin-1 ELISA Kit (Sigma-Aldrich, US) following the manufacturer’s instructions. In brief, 100 μL of sample was added into appropriate well for a 2.5-hour incubation. Then, 100 μL of 1 × prepared Biotinylated Detection Antibody was added to each well to incubate for 1 h. 100 μL of prepared HRP-Streptavidin solution was added to each well to incubate for 45 minutes. 100 μL of ELISA Colorimetric TMB Reagent was added to each well to incubate for 30 minutes avoiding light. 50 μL of Stop Solution was added to each well. After each incubation at room temperature, the solution was discarded and the sample was washed 4 times with 1 × Wash Solution. Finally, the data was read at 450 nm in a microplate reader (BIOBASE, China) immediately.
Cell viability detection
Cell viability was detected by CCK-8 assay. INS-1E cells seeded in a 96-well plate were treated with 10 μL CCK-8 solution per well using CCK-8 kit (TransGen Biotech, China) 1 h in advance of each detection at the point of 0, 24, 48, 72 h. The absorbance value was read in a microplate reader (BIOBASE, China) at 450 nm.
Cell apoptosis detection
Cell apoptosis was detected by flow cytometry assay. After 48-h transfection, INS-1E cells seeded in a 12-well plate were treated with a transient trypsin digestion and the digestion was terminated by complete medium. Then the cells were washed by PBS twice and then stained using Annexin V-FITC/DAPI Apoptosis Detection Kit (Elabscience, China) for 15 min protected from light at 25°C. Finally, the CytoFLEX (BECKMAN COULTER, US) was used to analyze the cell apoptosis of INS-1E cells.
Luciferase reporter assay
Rat gene PDX1 3’UTR wild type (PDX1-WT) and mutant type (PDX1-MUT) were obtained by subcloning into pmiRGLO vector. PDX1-MUT contained the sequence “GGAAGG” in place of “CCUUCC” which was the predicted binding site of miR-765. The predicted binding site of miR-765 remained unchanged in PDX1-WT. INS-1E cells were seeded in a 96-well plate and co-transfected with miR-765 mimic or mimic-NC and PDX1-WT or PDX1-MUT using Lipofectamine 3000 reagent (Invitrogen, US). After 48 h, luciferase activities were quantiﬁed by Luciferase reporter assay (Promega, US).
All data from at least three repeated experiments were depicted as mean ± SD in this study. We applied GraphPad Prism 6 software (US) to deal with statistical analysis as well as process correspondent graphs. The correlation between PDX1 or miR-765 expression and blood glucose was obtained by Spearman correlation analysis. Student’s t-test or one-way ANOVA was used to analyze differences. P < 0.05 was accounted as statistically significant difference between value.