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MicroRNA-17-5p acts as a biomarker and regulates mitochondrial dynamics in trophoblasts and endothelial cells by targeting the mitofusins Mfn1/Mfn2 in gestational diabetes mellitus

Introduction Gestational diabetes mellitus (GDM) is a metabolic disease that endangers pregnant women and their offspring. Insights into biomarkers and GDM pathogenesis are crucial. Ectopic expression of microRNA-17-5p was found in GDM, but its the diagnostic value and role of miR-17-5p remain unclear. Material and methods Detection of miRNA microarray and quantitative PCR (qPCR) found that miR-17-5p was significantly increased and positively associated with biochemical indicators of GDM in 30 GDM plasma samples and 28 matched control plasma samples. Results The area under the ROC curve was 0.827 (P < 0.01), which showed good diagnostic potential. Mitochondrial staining showed that compared with controls, trophoblasts exhibited more mitochondrial fusion and endothelial cells exhibited more mitochondrial fission in GDM than these in controls. Western blot and qPCR assays further revealed that expression of the mitofusin Mfn1/Mfn2 was lower in primary endothelial cells from GDM patients, whereas their expression was significantly higher in primary trophoblasts from GDM patients compared with those from controls. Conversely, miR-17-5p expression was higher in primary endothelial cells from GDM patients, whereas their expression was significantly lower in primary trophoblasts from GDM patients compared with those from controls. Bioinformatics and luciferase reporter assays confirmed that both Mfn1 and Mfn2 are targets of miR-17-5p. Last, decreased Mfn1/2 was observed not only to increase the apoptotic rate of primary endothelial cells from GDM, but also to reverse anti-apoptotic effects of miR-17-5p inhibitor. Conclusions MiR-17-5p regulates Mfn1/Mfn2-mediated mitochondrial dynamics involved in GDM. MiR-17-5p may serve as a promising biomarker and therapeutic target for GDM. Powered by TCPDF (www.tcpdf.org) rep rin t MicroRNA-17-5p acts as a biomarker and regulates mitochondrial 1 dynamics in trophoblasts and endothelial cells by targeting the 2 mitofusins Mfn1/Mfn2 in gestational diabetes mellitus 3 4 Jie Li 1, Zhi-Hui Liu 1, Tong Wu 2, Si Li 3, Ya-nan Sun 4* 5 6 1 Department of Obstetrics and Gynecology, Tangshan Gongren Hospital; 2 Department of 7 Internal Medicine, Tangshan Gongren Hospital; 3 Department of Cardiology, Tangshan 8 Gongren Hospital; 4 Department of Endocrinology,Tangshan Gongren Hospital, Wenhua road 9 27, Tangshan, 063000, Hebei, China. 10 11 *Address for correspondence: Ya-nan Sun, MD, Department of Endocrinology, Tangshan 12 Gongren Hospital, 27 Wenhua Road, Tangshan. E-mail: 826270793@qq.com. 13 14 Abstract 15 Background: Gestational diabetes mellitus (GDM) is a metabolic disease that endangers 16 pregnant women and their offspring. Insights into biomarkers and GDM pathogenesis are 17 crucial. Ectopic expression of microRNA-17-5p was found in GDM, but its diagnostic value and 18 role of miR-17-5p remain unclear. 1915 Background: Gestational diabetes mellitus (GDM) is a metabolic disease that endangers 16 pregnant women and their offspring. Insights into biomarkers and GDM pathogenesis are 17 crucial. Ectopic expression of microRNA-17-5p was found in GDM, but its diagnostic value and 18 role of miR-17-5p remain unclear. 19 Method and result: Detection of miRNA microarray and qRT-PCR found that miR-17-5p was 20 significantly increased and positively associated with biochemical indicators of GDM in 30 21 GDM plasma samples and 28 matched control plasma samples. The area under the ROC 22 curve was 0.827 (P < 0.01), which showed good diagnostic potential. Mitochondrial staining 23 showed that compared with controls, trophoblasts exhibited more mitochondrial fusion and 24 endothelial cells exhibited more mitochondrial fission in GDM than these in controls. Western 25 blot and qRT-PCR assays further revealed that expression of the mitofusin Mfn1/Mfn2 was 26 lower in primary endothelial cells from GDM patients, whereas their expression was 27 significantly higher in primary trophoblasts from GDM patients compared with those from 28 controls. Conversely, miR-17-5p expression was higher in primary endothelial cells from GDM 29 patients, whereas their expression was significantly lower in primary trophoblasts from GDM 30 patients compared with those from controls. Bioinformatics and luciferase reporter assays 31 confirmed that both Mfn1 and Mfn2 are targets of miR-17-5p. Last, decreased Mfn1/2 was 32 observed not only to increase the apoptotic rate of primary endothelial cells from GDM, but 33 also to reverse anti-apoptotic effects of miR-17-5p inhibitor. 34 Conclusion: MiR-17-5p regulates Mfn1/Mfn2-mediated mitochondrial dynamics involved in 35 GDM. MiR-17-5p may serve as a promising biomarker and therapeutic target for GDM. 36 37


P r e p r i n t
Gestational diabetes mellitus (GDM), which is characterized by glucose intolerance with 45 onset or first recognition during pregnancy, has become a common health problem worldwide 46 in the context of increased obesity [1]. Its incidence is high in Asians, and its prevalence in 47 China is 14.8% [2]. Unless properly diagnosed and treated, GDM can result in short-term and 48 long-term adverse outcomes for both the mother and fetus, including cesarean section, 49 shoulder dystocia, macrosomia, and neonatal hypoglycemia [3]. Although detection of GDM as 50 early as possible is crucial to avoid poor pregnancy outcomes, the current diagnostic test for 51 GDM is an oral glucose tolerance test (OGTT), which is performed at 24-28 weeks of

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Interestingly, literatures reported that mitochondrial fission associated with hyperglycemia 66 induces endothelial cell apoptosis and blood vessel damage [11,12]. Therefore, these findings 67 urged us to explore whether these miRNAs could be the diagnostic, prognostic, and 68 therapeutic targets in GDM.

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Accordingly, in the present study, we studied the expression patterns and diagnostic value 75 of miR-17-5p in a large cohort of GDM patients from the gynaecology and obstetrics 76 departments of Tangshan Gongren Hospital and explored the pathophysiological role of 77 miR-17-5p in trophoblasts and endothelial cells in GDM.

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Clinical specimens 82 Thirty GDM-complicated pregnancies and 28 normal pregnant women as control were 83 admitted to the gynaecology and obstetrics departments of Tangshan Gongren Hospital 84 between May 2019 and August 2020 were included in this study. Healthy pregnant women as 85 controls not only have normal OGTT, but also meet matched age, body mass index (BMI) and 86 gestational age. The medical history and examination of all enrolled participants should be between the 10th and 90th percentile. None of the women showed signs of hypertension or 90 any other disease. Basic clinical data of the subjects are presented in Table 1. Venous blood 91 samples (5 mL) were centrifuged at ~1,000 x g for 30 min at room temperature, and plasma 92 was stored at -80˚C until subsequent analysis. GDM patients were diagnosed by OGTT at 28-93 32 weeks of gestation as previously described [15]. All patients and controls provided written 94 informed consent. According to the agency guidelines, all these patients had got informed 95 consent before sample collection, and to approve the study by the Research Ethics Committee 96 of XXXXX Hospital (the reference number is GRYY-LL-2019-21).

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Diagnostic criteria of GDM [15] 99 The diagnosis of GDM is confirmed using the diagnostic criteria of the IADPSG as

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Up-regulation of miR-17-5p expression in plasma from women with GDM 205 Using a high-throughput human miRNA microarray, we found the miRNA expression 206 profiles between 3 GDM plasma specimens and 3 age-and pregnancy-matched normal 207 plasma specimens, which served as corresponding controls. The results of hierarchical 208 clustering and qRT-PCR validation showed top 10 up-regulated miRNAs among the 6 samples 209 ( Fig. 1a and 1b).

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Subsequently, we expanded the sample size (30 GDM specimens: age range from 25 to 211 34, 29.4±3.2 years, and responding 28 control: age range from 24 to 33, 28.9±4.1 years) to 212 confirm increased plasma miR-17-5p expression in GDM. As presented in Fig. 1c, miR-17-5p 213 expression was significantly higher in plasma from GDM patients than that in controls (P = 214 0.009). Importantly, the diagnostic value of miR-17-5p in plasma from GDM patients was also was 0.827 and that the sensitivity and specificity were 0.816 and 0.865, respectively (Fig. 1d).

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In addition, we also determined whether a high level of miR-17-5p was associated with 218 biochemical parameters of GDM patients. As shown in Table 2 patients than in controls ( Fig. 2b and 2c). Furthermore, the mitochondrial fusion-related genes 233 targeted by miR-17-5p were predicted online using TargetScan (Fig. 2d), which implied that  Fig. 3a-3b).