A total of 121 pairs of ECa tumor and normal para-cancerous tissues were obtained from patients undergoing surgery at the Fujian Provincial Hospital. Enrolled patients had not undergone preoperative systemic or local anti-tumor therapy. Following collection, pathological examination was used to confirm ECa diagnosis, and samples were snap frozen prior to storage at -80 °C. All patients supplied informed consent to participate, and the Ethics Committee of the Fujian Provincial Hospital approved all human studies, the Ethics number was NO. FJPH-2013.06.05. All experiments were performed in accordance with the relevant guidelines and regulations.
Human endothelial HET-1A cells and ECa cells including EC9706 and TE-1 were grown in DMEM + 10% FBS at 37°C. ECa ECA109 and TE-8 cells were grown in RPMI-1640 + 10% FBS at 37°C. HEK293 cells were grown in MEM + 10% FBS at 37°C. For overexpression studies, TE-1 cells were transfected with miR-195-5p mimics or miR-195-5p negative controls (NC; 40 nM) for 24 h. For suppression studies, TE-8 cells were transfected with miR-574-3p inhibitors or miR-195-5p inhibitor NCs.
Potential miR-195-5p targets were identified using the TargetScan database, which highlighted IGF-1R as a potential target. Based on this prediction, pmiR-RB-report plasmids (RiboBio Inc) were cloned to contain either a mutant or wild-type version of the 3’-UTR of IGF-1R. Plasmids were then co-transfected with appropriate miRNA mimics, inhibitors, or controls for 48 h. Commercial kits (Promega) were used to analyze luciferase activity. Firefly activity was normalized to Renilla.
Cell lines or tissues were lysed in RIPA (Beyotime) buffer and protein content of the lysates was assessed via BCA assays (Thermo Fisher). Proteins (~20 ng) were resolved by SDS-PAGE electrophoresis and transferred. PVDF membranes were blocked in 5% milk and probed overnight with antibodies against IGF-1R (1:1000, 9750S, CST), p-AKT (Ser473, 1:1000, 9271S; CST), and total-AKT (1:1000, 9272S, CST), GLUT1 (1:1000, sc-377228; Santa Cruz) at 4˚C. Blots were then labeled for 1 h with anti-mouse or anti-rabbit HRP-secondary antibodies. Proteins were visualized using the ECL system on a ChemiDoc Imaging Platform (Bio-Rad).
RNA was isolated from cell lines or paraffin-embedded tissue using a miRNeasy FFPE kits (Qiagen) or miRNeasy Mini kits (Qiagen). cDNA was generated using a miScript II RT kit (Qiagen) from 1 µg of input RNA or miRNA. RT-PCR parameters were as follows: 37°C for 60 min, 95°C for 5 min. The miScript SYBR Green PCR kit was used for qRT-PCR. Reactions were performed as follows: 95°C for 15 min; 40 cycles of 94°C for 15 s, 55°C for 30 s, and 70°C for 30 s on a 7500 Fast Real-Time PCR platform. Values were normalized to GAPDH and U6 and quantified. All reactions were performed in triplicate.
MiR-195-5p Forward 5’-ACACTCCAGCTGGGTGTCAGTTTGTCAAAT-3’;
MiR-195-5p Reverse 5’-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGT-3’,
U6 Forward 5’-TGCGGGTGCTCGCTTCGCAGC-3’;
U6 Reverse 5’-CCAGTGCAGGGTCCGAGGT-3’,
GAPDH Forward 5’-TTGGTATCGTGGAAGGACTCA-3’;
GAPDH Reverse 5’-TGTCATCATATTTGGCAGGTT-3’.
Cell Proliferation Assays
CCK-8 assays were used to assess ECa and non-ECa cell proliferation. Cells were co-transfected as described in 96-well plates (1,000 cells/well) for 24 h, and 10 μl of CCK-8 was added to the wells in normal culture conditions for 3 h. Absorbances at 450 nm were read on a Bio-Rad microplate reader.
Colony Formation Assays
Treated cells (1 × 103 per well) were seeded into 10 cm culture dishes for 2 weeks. Cell colonies were stained in crystal violet (1%) and colony numbers were counted.
For migration/invasion assays, 12-well plates containing Transwell inserts (Corning, MA, USA) with 8 μm pore sizes were used. For migration assays, ~100,000 cells were resuspended in serum-free media and added to the upper regions of the Transwell chambers for 8 h at 37 °C. Cells in the lower chambers were then removed and methanol fixed prior to staining using 0.04% crystal violet. Cells were then quantified. For invasion assays, the protocols were identical to those above, but the transwell inserts had first been coated using Matrigel (BD Biosciences, MA, USA), and plates were instead incubated for 24 h.
Glucose uptake, lactate, and ATP assays
Colorimetric assays were performed to assay glucose uptake. Briefly, cells (1×104 per 96 wells) were glucose starved in Krebs-solution plus 2% BSA for 40 min. Cells were then treated with 2-DG (10 mM) for 20 min and color changes were assessed on a plate reader. For the assessment of lactate and ATP production, cells (5×105) were homogenized in commercial assay buffers at 4°C. Soluble fractions were assayed using Lactate Assay Kit II reagent or ATP colometric assays.
Data are shown and the mean ± SD analyzed via SPSS 17.0. Inter-group differences were assessed via a student’s t-test. Multiple groups were compared using a one-way ANOVA analysis of variance. Kaplan Meier (KM) curves were plotted for survival analysis, and groups were compared via log-rank assessments (n=3 for all). P-values < 0.05 were deemed significant differences