Low glucose enhanced metformin’s inhibitory effect on pancreatic cancer cells by suppressing glycolysis and inducing energy stress via up-regulation of miR-210-5p

Figures & data

Figure 1. Effects of metformin on proliferation, viability, and apoptosis of PANC-1 cells cultured in high and low glucose conditions.

Note: PANC-1 cells were cultured in DMEM with 25 mM or 5 mM glucose for 24 h and then treated with metformin (0/5/10/20 mM) for 24 h or 48 h. Cell proliferation was detected with EdU assay, and the cell proliferation rate (%) was calculated by providing the number of proliferative cells by the number of total cells (a). Cell apoptosis was assayed using Annexin V, FITC Apoptosis kit, and the ratio of apoptotic cells and total cells was presented (b). Cell viability was measured using the CCK8 kit, and the OD value represented cell viability. Two-way ANOVA follows by Tukey post-hoc was used to analyze the differences among groups. Met: metformin, Glu: glucose. *P < 0.05, **P < 0.01, *** P < 0.001 when compared with the control group without metformin. #P < 0.05, ##P < 0.01, ###P < 0.001 when compared between the high and low glucose groups.

Figure 2. Relative expression of miR-210-5p in PANC-1 cells treated with metformin in high and low glucose conditions.

Note: PANC-1 cells were cultured in DMEM with 25 mM or 5 mM glucose for 24 h and then treated with metformin (0/1/10 mM) for 48 h. RNA was extracted, and real-time PCR was used to detect the relative expression of miR-210-5p. a) Comparison of the relative expression of miR-210-5p in cells treated with different concentrations of metformin. Cells treated without metformin were defined as the control group. Thus, cells under the 5 mM and 25 mM glucose conditions had their own control group when calculated the relative expression of miR-210-5p using the comparative Ct method. b) Differences in the relative expression of miR-210-5p in cells cultured in different concentrations of glucose. Cells treated without metformin under 25 mM glucose conditions were defined as the control group to calculate the relative expression of miR-210-5p using the comparative Ct method. *P < 0.05, **P < 0.01, *** P < 0.001.

Figure 3. Effects of miR-210-5p mimic and inhibitor on the viability of PANC-1 cells cultured in high and low glucose conditions.

Note: PANC-1 cells were transfected with miR-210-5p mimic or inhibitory or their respective negative control (NC) for 24 h, then treated with or without metformin for 24 h, 48 h, 72 h respectively. Cell viability was detected and analyzed using the CCK8 kit (a/b/c/d). Cell apoptosis was assayed using Annexin V, FITC Apoptosis kit, and the ratio of apoptotic cells and total cells was presented (e/f). *P < 0.05, **P < 0.01, ***P < 0.001 when compared with the NC group. #P < 0.05, ##P < 0.01, ###P < 0.001 when compared between the high and low glucose groups.

Figure 4. Effects of miR-210-5p mimic combined with or without metformin on the expression of PFKFB2 and p-PFKFB2 in PANC-1 cells.

Note: PANC-1 cells were transfected with miR-210-5p mimic or inhibitor or negative control (NC) for 24 h and then treated with or without metformin for 48 h. Then the protein was extracted, and western blot was used to test the expression of PFKFB2 and p-PFKFB2. *P < 0.05, **P < 0.01, ***P < 0.001 when compared with the NC group. #P < 0.05, ##P < 0.01, ###P < 0.001 when compared between cells transfected with miR-210-5p mimic or inhibitor combined treatment with metformin and those without metformin.

Figure 5. Effects of miR-210-5p mimic combined with or without metformin on glycolysis related enzyme and products in PANC-1 cells.

Note: PANC-1 cells were transfected with miR-210-5p mimic or inhibitor or negative control (NC) for 24 h and then treated with or without metformin for 48 h. Then the media was collected for analysis of the activity of PFK1 (a) and LDH (b), consumption of glucose (c), and pyruvate (d), the content of lactate (e), and ATP (f). *P < 0.05, **P < 0.01, ***P < 0.001 when compared with the NC group. #P < 0.05, ##P < 0.01, ###P < 0.001 when compared between the high and low glucose groups. *P < 0.05, **P < 0.01, ***P < 0.001 when compared between cells treated with or without metformin.

Figure 6. Possible mechanism of the difference in the effect of metformin on PANC-1 cells cultured in high and low glucose.

Note: Met: metformin; G-6-P: glucose 6-phosphate; F-6-P: fructose 6-phosphate; F-2,6-BP: fructose 2,6-bisphosphate; F-1,6-BP: fructose 1,6-bisphosphate; PFKFB2: 6-phosphofructo-2-kinase; PFK1: phosphofructokinases; LDH: lactate dehydrogenase; TCA Cycle: tricarboxylic acid cycle.