Fig 2: Low villous SDHB expression reduces succinate accumulation and correlates with RSA.a Western blot analysis of glycolysis and citrate cycle enzymes in villi from individuals with RSA or normal controls (n = 6 persons/group). Right panel: quantitative western blot results of six samples per group. b mRNA expression of glycolytic and citrate cycle genes in villi from individuals with RSA and normal controls (n = 6 persons/group). Exact P values in b are listed below: HK1 0.800, GPI 0.326, TPI 0.369, PGK1 0.202, ENO1 0.145, PKM2 0.685, 6PGD 0.715, TKT 0.914, PDHA1 0.691, CS 0.841, IDH1 0.546, IDH2 0.801, IDH3A 0.969, OGDH 0.163, SUCLA2 0.816, SDHA 0.00134, SDHB 0.00243, SDHC 0.000233, FH 0.222, and MDH2 0.938. c Relative SDH activities in SDHB-overexpressing JEG3 and HTR8 cells and control cells (n = 5 independent experiments). The SDHB overexpression efficiency is shown in the right panel. d Succinate levels in SDHB-overexpressing JEG3 and HTR8 cells and control cells (n = 5 independent experiments). e Relative SDH activities in SDHB-knockdown JEG3 and HTR8 cells and control cells (n = 5 independent experiments). The SDHB-knockdown efficiency is shown in the right panel. f Succinate levels in SDHB-knockdown JEG3 and HTR8 cells and control cells (n = 5 independent experiments). g The relative SDH activities in villi and decidua tissues from both RSA patients and normal controls (n = 10 persons/group). a–g Data represent the mean ± standard error; ns not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ns not significant. Two-tailed unpaired Student’s t tests in a–g. Source data are provided as a Source Data file.

Fig 3: OIP5-AS1 regulates miR-124-5p expression by both directly targeting and Ago2-dependent manner, and miR-124-5p target inhibition of IDH2 expression in cervical cancer cell. (A) Predicted binding sites of miR-124-5p on OIP5-AS1 transcript (black) and IDH2 transcript (black). (B) RT-qPCR analysis of the indicated miR-124-5p levels in Hela, Caski, and Siha cells under normoxic or hypoxic conditions for 24 h. Data were shown as mean ± SD of three individual experiments. *** was P < 0.001 and P value was calculated by Student’s t test. (C,D) RT-qPCR analysis of the indicated miR-124-5p or OIP5-AS1 levels in Hela, Caski, and Siha cells over-expressing miR-124-5p or expressing knockdown miR-124-5p under normoxic or hypoxic conditions for 24 h. Data were shown as mean ± SD of three individual experiments. * was P < 0.05, ** was P < 0.01 and, *** was P < 0.001, and P value was calculated by Student’s t test. (E) miR-124-5p-NC, mimic and inhibitor was co-transferred with OIP5-AS1 wild type (WT) or mutant (MUT) transcript, and then detected luciferase activity. Data were shown as mean ± SD of three individual experiments. ns was P > 0.05 and *** was P < 0.001, and P value was calculated by post hoc comparisons. (F) RT-qPCR analysis of the indicated miR-124-5p and OIP5-AS1 levels associated with AGO2 after RIP assay in Hela cells. Data were shown as mean ± SD of three individual experiments. *** was P < 0.001, and P value was calculated by post hoc comparisons. (G,H) Dual-luciferase reporter assay shows that miR-124-5p targets inhibition of IDH2 expression (G), and Western blot analysis shows that OIP5-AS1 promotes IDH2 expression via inhibiting miR-124-5p expression (H). Data were shown as mean ± SD of three individual experiments. ns was P > 0.05 and *** was P < 0.001, and P value was calculated by post hoc comparisons in panel (G) and by Student’s t test in panel (H). (I) Schematic illustration of the proposed model depicting OIP5-AS1 promotes IDH2 expression via inhibiting miR-124-5p expression.

Fig 4: ME1 interference adaptively upregulates IDH2-mediated NADPH production as a compensatory mechanism.A. Effect of ME1 knockdown on the relative NADPH/NADP+ ratio in MCF-7 cells cultured for prolonged durations (n = 4).B. Apoptosis rate of ME1 knockdown cells cultured in suspension at the indicated times (n = 3).C. Immunoblot analysis of ME1 and IDH2 expression in MCF-7 and A549 cells under the indicated treatments. ß-Tubulin was used as the loading control.D. Analysis of citrate, fumarate and malate mass isotopologues in MCF-7 cells cultured in medium containing 2 mM [U-13C] glutamine for 24 h (n = 5).E. Percentage of NADP2H labeling from [2,3,3-2H] aspartate (2 mM, 24 h) in MCF-7 cells with control transfection or ME1 knockdown (n = 3).F. Citrate m+4 and m+5 isotopologues in MCF-7 cells treated as indicated and cultured in medium containing 2 mM [U-13C] glutamine for 24 h (n = 3 for the siME1 DMSO group and n = 5 for others). The concentration of AGI-6780 used in this study is 2.5 µM.G. Effect of combinatorial targeting ME1 and IDH2 (2.5 µM AGI-6780 for 24 h) on relative NADPH/NADP+ ratio in MCF-7 cells (n = 3).H. Mitochondrial reductive carboxylation and subsequent isocitrate/citrate transport is adaptively upregulated by ME1 knockdown and functions as a compensatory route to replenish the cytosolic NADPH pool.Data are represented as mean ± SD. The experiments described in A and F were conducted once, and all other experiments were repeated at least twice. Statistical significance was determined by Student's t-test. *p < 0.05, **p < 0.01, ***p < 0.001. See also Supplementary Fig. 4.

Fig 5: OIP5-AS1 regulates the Warburg effect through IDH2 is HIF-1a dependent. (A,B) Levels of IDH2, HIF-1a, GLUT1, and LDHA protein expression in Hela cells using western blot. (C–F) Levels of lactate (C) in the culture medium and intracellular glucose (D) of Hela cells after culturing for 24 h under hypoxia, and RT-qPCR indicated GLUT1/LDHA mRNA levels, western blot analysis showed IDH2, HIF-1a, GLUT1, and LDHA protein expression. Data were shown as mean ± SD of three individual experiments. P value was calculated by post hoc comparisons. ***P < 0.001.