Long non-coding RNA lnc-CCNL1-3:1 promotes granulosa cell apoptosis and suppresses glucose uptake in women with polycystic ovary syndrome

Figures

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  Figure 1. lnc-CCNL1-3:1 is upregulated in granulosa cells of women with PCOS (A) The expression level of CCNL in 43 women with PCOS and 41 healthy controls. The expression level was detected via quantitative real-time PCR. The first ΔCt normalization was to β-actin, and the second ΔCt normalization was to the control group. (B) Bar graph showing CCNL vector-mediated overexpression efficiency. KGN cells or hLGCs stably expressing empty vector (Enter) or CCNL overexpression vectors (CCNL) were cultured 72 h and then subjected to quantitative real-time PCR and normalized against β-actin. Error bars represent SDs of at least three independent experiments. ∗∗∗∗p < 0.0001 corresponds to two-tailed Student’s tests.

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  Figure 2. lnc-CCNL1-3:1 promoted granulosa cell apoptosis in vitro (A and C) lnc-CCNL1-3:1 promoted cell apoptosis in KGN cells. (B and D) lnc-CCNL1-3:1 promoted cell apoptosis in hLGCs. (A) Enter- or CCNL-overexpression vector (CCNL)-transfected KGN cells were cultured in medium for 72 h and then subjected to annexin V/PI staining followed by fluorescence-activated cell sorting (FACS) analysis to measure the percentages of annexin V-positive/PI-negative (A+/P−) and annexin V-positive/PI-positive (A+/P+) cells or (C) to western blotting analysis to measure relative protein expression (cleaved/total PARP [C/T PARP], cleaved/total caspase-3 [C/T Cap3], Bcl-2, Bax). (B and D) Enter- or CCNL-transfected hLGCs were cultured in medium for 72 h before being subjected to (B) TUNEL (terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling) staining (green) to detect cell apoptosis and DAPI staining (blue) to detect the amount of cells (n = 3) or (D) to western blotting analysis to measure relative protein expression (C/T PARP, C/T Cap3, Bcl-2, Bax). Error bars represent SDs of at least three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p <…

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  Figure 3. lnc-CCNL1-3:1 impaired cell mitochondrial function in vitro (A) lnc-CCNL1-3:1 regulates cell reactive oxygen species (ROS) generation in KGN cells and hLGCs. Enter- or CCNL-transfected cells were cultured in medium for 72 h and then subjected to H2DCFDA staining (green) and Hoechst staining (blue). The level of intracellular ROS production was detected by fluorescence intensity of H2DCFDA and Hoechst 33342 to detect the amount of cells. (B) The concentration of intracellular ATP in Enter- or CCNL-transfected KGN cells. The level of ATP was detected via an Infinite M200 PRO microplate reader. (C) Concentration of intracellular ATP in granulosa cells of eight PCOS patients and eight healthy controls. Error bars represent SDs of at least three independent experiments. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 correspond to two-tailed Student’s tests.

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  Figure 4. lnc-CCNL1-3:1 suppressed cell glucose uptake in vitro (A) The expression level of CCNL was correlated with BMI in patients with PCOS (p = 0.0070). (B) The higher HOMA-IR values had a higher expression level of CCNL (p = 0.0784, tertile I versus II; p = 0.0017, tertile I versus III; p = 0.0267, tertile II versus III). (C and D) Basal and insulin-stimulated glucose uptake levels were reduced in CCNL-transfected KGN cells and hLGCs. The cells were serum starved for 12 h following insulin stimulation (100 nM, 20 min) or PBS as control before the detection. (E) Western blotting analysis to measure relative protein expression (GLUT4, IRS1) from KGN cells and hLGCs after transfection with vectors for 72 h. Error bars represent SDs of at least three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 correspond to two-tailed Student’s tests.

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  Figure 5. FOXO1 was involved in CCNL regulation of granulosa cells (A) The protein level of FOXO1 was upregulated in CCNL-transfected KGN cells and hLGCs. Enter- or CCNL-transfected cells were cultured 72 h then subjected to western blotting. (B) The mRNA level of FOXO1 remained unchanged via quantitative real-time PCR. (C–F) CCNL-induced cell apoptosis and glucose uptake are partially attenuated by knocking down FOXO1 in granulosa cells. (C) Bar graph showing FOXO1-siRNA-mediated knockdown efficiency by quantitative real-time PCR analysis. KGN cells stably transfecting si-Ctrl or si-FOXO1 were cultured 72 h and then subjected to quantitative real-time PCR and normalized against β-actin. (E) Knocking down FOXO1 in granulosa cells can partially rescue the CCNL-induced relative protein expression. Enter- or CCNL-transfected cells were treated with si-Ctrl or si-FOXO1 for 72 h and then subjected to western blotting analysis to measure relative protein expression (C/T PARP, Bcl-2, Bax, IRS1, GLUT4). (D and F) Knocking down FOXO1 in granulosa cells can partially rescue the CCNL effect on cell apoptosis and glucose transport. Enter- or CCNL-transfected cells were treated with si-Ctrl or si-FOXO1…

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  Figure 6. lnc-CCNL1-3:1 interaction with FOXO1 by enhancing its nuclear location (A) Nuclear and cytoplasmic fractions of Enter- or CCNL-transfected cells were subjected to western blotting analysis to measure relative protein expression. The cytoplasmic tubulin and nuclear lamin A/C were used as controls. (B) Nuclear and cytoplasmic fractions of granulosa cells were subjected to quantitative real-time PCR to detect the intracellular localization of CCNL. The cytoplasmic ACTB and nuclear U6 were used as controls. (C) The efficiency of RNA immunoprecipitation (RIP) is shown as quantitative real-time PCR of U1 snRNA. (D) Enrichment of CCNL in granulosa cells was measured by quantitative real-time PCR from RIP with FOXO1 antibody or immunoglobulin G (IgG). IgG immunoprecipitation (IP) was used as a negative control. (E) Cells were incubated with CCNL (pCCNL) or Enter for 72 h. Subcellular localization of FOXO1 was visualized using anti-FOXO1 (green), and the nuclei were counterstained with DAPI (blue). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 correspond to two-tailed Student’s tests.

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  Figure 7. Schematic diagram for lnc-CCNL1-3:1 function in women with PCOS Upregulation of CCNL interacts with the transcription factor FOXO1, impairs the mitochondria function, promotes cell apoptosis, and reduces glucose uptake in women with PCOS.