Fig 1.
Expression of SSTR2 after octreotide treatment in INS-1 cells.
(A) INS-1 cells were incubated with 1 μmol/L octreotide. After the indicated time, SSTR2 and β-actin levels were analyzed by western blotting. The images shown here are cropped and the full-length original blots are shown in S6 Fig. (B) INS-1 cells were plated on coverslips in 24-well plates. On the next day, the cells were incubated with 1 μmol/L octreotide for the indicated time. Cells were fixed and processed for immunofluorescence staining of SSTR2 protein. Scale bar: 20 μm.
Fig 2.
Microarray analysis of miRNA expression.
miRNA profiling was performed to determine the (A) number and (B) profile of differentially expressed miRNAs. Red and green indicate significantly upregulated or downregulated miRNAs, respectively. INS-1 cells (C) and GH3 cells (D) were incubated with 1 μmol/L octreotide. After the indicated incubation time, total RNA was isolated, after which miR-16-5p levels were measured by qRT-PCR and normalized for input RNA using control miRNA (U87) with the 2−ΔΔCt method. Data represent the mean of three independent experiments ± SD; *p < 0.05, **p < 0.01.
Fig 3.
Expression of SSTR2 after treatment with miR-16-5p mimic or inhibitor in INS-1 cells.
INS-1 cells were transfected with 50 or 100 pmol of mimic miR-16-5p or control miRNA. (A) miR-16-5p levels were examined by RT-qPCR at 48 h after transfection. miRNA expression of target genes was normalized for input RNA using endogenous control miRNA (U87) and calculated by the 2−ΔΔCt method. (B) SSTR2, phospho-ERK (Thr202/Tyr204), ERK, and β-actin levels were analyzed by western blotting. The images shown here are cropped and the full-length original blots are shown in S7 Fig. (C) INS-1 cells were plated on coverslips in 24-well plates and transfected with 100 pmol of mimic miR-16-5p or control miRNA. After 48 h, the cells were fixed and processed for immunofluorescent staining of SSTR2 protein. Scale bar: 20 μm (D) Fluorescence intensity of SSTR2 was measured using IMT i-Solution Industrial Image Analysis Software. (E) INS-1 cells were treated with 1 or 10 nmol/L of miR-16-5p inhibitor or negative control. miR-16-5p levels at 24 h after treatment were determined by qRT-PCR. (F) Western blotting of SSTR2, phospho-ERK (Thr202/Tyr204), ERK, and β-actin levels. The images shown here are cropped and the full-length original blots are shown in S8 Fig.
Fig 4.
Upregulation of SSTR2 expression in INS-1 cells after octreotide treatment with mimic miR-16-5p.
(A) INS-1 cells were transfected with 100 pmol of mimic miR-16-5p or control miRNA for 24 h and then treated with 1 μmol/L octreotide for the indicated durations. The cells were then fixed and processed for immunofluorescence staining of SSTR2 protein. Scale bar: 20 μm. (B) Fluorescence intensity of SSTR2 was measured with IMT i-Solution Industrial Image Analysis Software.
Fig 5.
Sensitivity of INS-1 cells to octreotide after treatment with mimic miR-16-5p.
(A) INS-1 cells were transfected with 2.5 pmol of mimic miR-16-5p or control miRNA for 24 h and treated with the indicated concentration of octreotide for 24 h. Cell proliferation was measured in a CCK-8 assay. (B) Spheroids from miR-16-5p mimic- or control-transfected INS-1 cells were treated with octreotide for 48 h. The proliferation of six spheroids were measured by CellTiter-Glo® 3D Cell Viability Assay. Values represent the mean of three independent experiments ± SD; *p < 0.05, **p < 0.01.