Upregulation of microRNA-204 improves insulin resistance of polycystic ovarian syndrome via inhibition of HMGB1 and the inactivation of the TLR4/NF-κB pathway

Abstract

There is growing evidence of the position of microRNAs (miRs) in polycystic ovarian syndrome (PCOS), thus our objective was to discuss the impact of miR-204 on insulin resistance (IR) in PCOS by targeting highmobility group box protein 1(HMGB1)-mediated toll-like receptor 4(TLR4)/nuclear factor-kappa B (NF-κB) pathway.PCOS-IR patients and PCOS non-insulin resistance (PCOS-NIR) patients were included. The levels of serum sex hormones and related insulin were measured, the expression of miR-204, HMGB1, TLR4 and NF-κB p65 was detected, the diagnostic efficacy of miR-204 in PCOS-IR was analyzed, and the correlation between the expression of miR-204 in PCOS-IR and fasting blood glucose (FPG), fasting insulin (FINS), homeostasis model of assessment for insulin resistance index (HOMA-IR) was analyzed. Both in vitro and in vivo experiments were performed to elucidate the capabilities of miR-204 and HMGB1 in proliferation and apoptosis of PCOS-IR granulosa cells.MiR-204 was lowly expressed as well as HMGB1, TLR4 and NF-κB p65 were highly expressed in PCOS-IR patients. Follicule-stimulating hormone was downregulated, while luteinizing hormone, estrogen, progesterone, FPG, FINS and HOMA-IR were elevated in PCOS-IR. Upregulation of miR-204 and downregulation of HMGB1 could repress TLR4/NF-κB pathway activation, degraded insulin release and testosterone (T) leveland ascended ovarian coefficient, boosted cell proliferation and restrained apoptosis of granulosa cells. Overexpression of HMGB1 reverses the effect of upregulation of miR-204 on IR of PCOS.Our study presents that high expression of miR-204 or inhibition of HMGB1 can improve IR of PCOS via the inactivation of TLR4/NF-κB pathway.

Keywords: HMGB1; Polycystic ovarian syndrome; TLR4/NF-κB pathway; microRNA-204.

Figure 1.

In the granulosa cells of PCOS-IR patients, miR-204 is lowly expressed while HMGB1, TLR4 and NF-κB p65 are highly expressed. (a): Expression of miR-204 and HMGB1 mRNA in the granulosa cells of each group by RT-qPCR. (b): Protein bands of HMGB1, TLR4, NF-κB p65. (c): Protein expression of HMGB1, TLR4 and NF-κB p65 in the granulosa cells of each group by western blot analysis. (d): ROC curve to analyze the diagnostic effect of miR-204 on PCOS-IR. * P < 0.05 vs. the control group. # P < 0.05 vs. the PCOS-NIR group. PCOS-IR: n = 68; PCOS-NIR, n = 44; Control, n = 60. Measurement data were depicted as mean ± standard deviation, and data were assessed by one-way analysis of variance followed by LSD-t test.

Figure 2.

FSH is downregulated while LH, E2, P4, FPG, FINS and HOMA-IR are upregulated in PCOSIR. (a) Levels of FSH, LH, E2, and P4 in serum of each group. (b) Levels of FPG, FINS and HOMA-IR in serum of patients in each group. (c) The correlation between expression of miR-204 and FPG, FINS and HOMA-IR in granulosa cells of PCOS-IR patients. * P < 0.05 vs. the control group. # P < 0.05 vs. the PCOS-NIR group. PCOS-IR: n = 68; PCOS-NIR, n = 44; Control, n = 60. Measurement data were depicted as mean ± standard deviation, and data were assessed by one-way analysis of variance followed by LSD-t test. Correlation between miR-204 expression and FPG, FINS and HOMA-IR was conducted by Pearson correlation analysis.

Figure 3.

TLR4/NF-κB pathway activation is repressed, insulin release and T level are reduced, and ovarian coefficient is raised through the upregulation of miR-204 and downregulation of HMGB1. (a): Expression of miR-204 and HMGB1 in each group of rats by RT-qPCR. (b): Protein bands of HMGB1, TLR4 and NF-κB p65. (c): HMGB1, TLR4 and NF-κB p65 protein expression by western blot analysis. (d): Detection of body weight of rats in each group. (e): Insulin release test of rats in each group. (f): T level test results of rats in each group. (g): HOMA-IR detection results in each group. (h): Test results of ovarian coefficient in each group of rats. I: Observation of pathological morphology of ovarian tissues in each group by HE staining . n = 8. * P < 0.05 vs. the control group. # P < 0.05 vs. the mimics-NC group. & P < 0.05 vs. the siRNA-NC group. $ P < 0.05 vs. the miR-204 mimics + pcDNA-NC group. Measurement data were depicted as mean ± standard deviation, and data were assessed by one-way analysis of variance followed by LSD-t test.

Figure 4.

Overexpressed miR-204 and downregulated HMGB1 boosted cell proliferation and suppressed apoptosis of granulosa cells. (a): The culture of primary ovarian granulosa cells in rats under the microscope. (b): Expression of miR-204 and HMGB1 in granulosa cells by RT-qPCR. (c): Protein bands of HMGB1, TLR4 and NF-κB p65. (d): HMGB1, TLR4 and NF-κB p65 protein expression in granulosa cells by western blot analysis. (e): The difference of cell proliferation after transfection of PCOS-IR granulosa cells in each group. (f,g): Western blot analysis to detect PCNA and cyclin D1 protein expression in granulosa cells. (h): Apoptosis of PCOS-IR granulosa cells after transfection in each group by flow cytometry. (i): Comparison of apoptosis rate of PCOS-IR granulosa cells after transfection in each group. J&K: Western blot analysis to detect Bax and Bcl-2 protein expression in granulosa cells. N = 3, * P < 0.05 vs. the mimics NC group. # P < 0.05 vs. the siRNA-NC group. & P < 0.05 vs. miR-204 mimics + pcDNA-NC group. Measurement data were depicted as mean ± standard deviation, and comparisons among multiple groups were assessed by one-way analysis of variance followed by LSD-t test.

Figure 5.

The target relationship among miR-204 and HMGB1. (a): The Targetscan website predicted the target relationship between miR-204 and HMGB1. (b): Dual-luciferase reporter gene assay verified the targeting relationship of miR-204 and HMGB1. Measurement data were depicted as mean ± standard deviation, and comparisons among the two groups were assessed by t test. The experiment was repeated three times.