Gestational diabetes mellitus (GDM) is a pathological condition, characterized by glucose intolerance or hyperglycemia resulted from insufficient insulin production or signaling in pregnant women [
MicroRNAs (miRNAs) are a class of RNA molecules which play important roles in many biological processes [
In this study, we utilized a high-fat diet-induced GDM model [
Animal experiments were approved by the Animal Care and Use Committee of Jinan University (approval no. 2017031705005). Animal experiments were performed in the laboratory animal research center of Jinan University. The methods were carried out in accordance with the approved guidelines. Sixty female C57BL/6 J mice at the age of 8 weeks were obtained from Guangdong Medical Laboratory Animal Center (approval no. SCXK (Yue) 2013–0002). All the animals were maintained in a temperature-controlled room (22°C–25°C; 35–55% humidity) with a twelve-hour light/dark cycle. Mice were allowed free access to food and water. Mice were randomly divided into three groups (
Body weight was recorded on a top-loading balance (Fisher Scientific) before dietary intervention after 6 weeks of HFD and at GD7 and GD16. Lipid levels were measured at gestation day 20. Mice were anesthetized with pentobarbital sodium (60 mg/kg ip), and blood was collected by removing the left eyeball of the mice. Then, the blood samples were rapidly centrifuged at 1000 g at 4°C for 10 min. Plasma levels of triglycerides (TG), total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C), and high-density lipoprotein-cholesterol (HDL-C) were measured using a multifunctional biochemistry analyzer (Olympus AU2700, Tokyo, Japan).
Glucose tolerance was measured at GD16. Following a 6 h fast, mice were given 2 g/kg glucose solution via oral gavage [
At GD20, mice livers from all groups were removed and fixed immediately in 10% neutral buffered formalin, dehydrated in gradual ethanol (50%–100%), cleared in xylene, and embedded in paraffin. Sections (4–5
Liver tissues were homogenized in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor cocktail and phenylmethylsulfonyl fluoride (PMSF). Liver extracts were centrifuged at 13,000 rpm for 10 min, and the supernatant was collected for use in western blot. Protein concentration in supernatants was quantified using the BCA reagent. Aliquots of proteins were analyzed by 12% SDS-PAGE and transferred to the nitrocellulose membrane. The membrane was then immersed in blocking buffer (PBS, 0.1% Tween 20) containing 5% nonfat milk for 1 h and then incubated with primary antibodies HMGCR (ab174830) and CPT1A (CST-12252) (1 : 1000 dilution in blocking buffer) overnight at 4°C. The membranes were further incubated with horseradish peroxidase-conjugated secondary antibodies (1 : 2000; Santa Cruz, USA). Chemiluminescence was detected by the Pierce® ECL western blotting substrate (Thermo Fisher Scientific, USA). The intensity of the bands was quantified using the western blotting detection system Quantity One 4.31 (Bio-Rad, USA).
Twelve of the pregnant mice in each group were used to perform miRNA sequencing. Exosomes from plasma were isolated using RiboTM Exosome Isolation Reagent (RiboBio, Guangzhou, China) according to the manufacturer’s instructions. For exosomal RNA extraction, total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA). The concentration and purification of RNA were determined by a spectrophotometer (NanoDrop Technologies, Wilmington, DE). The RNA integrity was evaluated by the Agilent 2200 TapeStation (Agilent, Santa Clara, CA). Fixed quantities of RNA of four samples from one group were combined into a single sample. Thus, each group has three biological repeats.
Library preparation and sequencing was conducted at RiboBio Co., Ltd. Total RNAs from exosomes were subjected to RNA 3′ adapter ligation and RNA 5′ adapter ligation. Then, the first strand cDNA was synthesized, and PCR amplification was performed. Small RNAs ranging between 18 and 40 nucleotides (nt) were used for library preparation. Finally, sequencing was performed using the Illumina HiSeq 2500 next-generation sequencing platform.
We performed several filtering steps after obtaining the raw reads. Reads that met the following filtering criteria were removed: (1) no 3′ adapter, (2) 5′adapter, (3) excessively long poly A/T sequence, (4) short-sequence reads (length < 18 nt), or (5) low quality. A low-quality read was defined as a read in which >20% of the read bases had a quality value (the error rate of each base sequencing) of ≤20. We also removed reads containing >10% of N bases among the total. Then, further analysis can be conducted. Further analysis identified several categories of small RNAs (miRNA, rRNA, tRNA, snRNA, snoRNA, and piRNA). The annotation of measured small RNAs (rRNA, tRNA, snRNA, and snoRNA) was mapped to Rfam 12.1 (
Target genes of the differentially expressed miRNAs with
To further confirm the findings from the RNA-seq analysis, we used the real-time quantitative RT-PCR method to examine the common differentially expressed miRNAs (miR-93-3p, miR-188-5p, miR-466k, miR-1188-5p, miR-7001-3p, and miR-7115-5p) identified in the HFD/control and HFD + LBP/HFD comparisons. Total RNA was extracted from liver tissues of mice in each group using the TRIzol reagent (Invitrogen, CA, USA) according to manufacturer’s protocol. The miRNAs’ primers were designed by RiboBio Co., Ltd (Guangzhou, China). qRT-PCR was performed with SYBR Premix ExTaqTM II (Takara, Dalian, China) using CFX96 PCR System (Bio-Rad). Relative expressions were normalized to the expression of U6 and calculated using the 2-ΔΔCT method.
All data are expressed as mean ± SEM. One-way analysis of variance (ANOVA) was used to detect statistical significance followed by Tukey post hoc multiple comparisons using software SPSS 22.0. Values of
As shown in Table
Effect of LBP on body weights of mice in the three groups.
Time | Control group | HFD group | HFD + LBP group |
---|---|---|---|
Before HFD | 17.9 ± 0.2 | 17.8 ± 0.3 | 17.6 ± 0.2 |
End of HFD | 20.8 ± 0.2 | 22.4 ± 0.6 | 22.7 ± 0.4 |
GD7 | 23.1 ± 0.3 | 25.1 ± 0.5 | 24.9 ± 0.3 |
GD16 | 29.3 ± 0.5 | 30.9 ± 0.9 | 31.0 ± 0.4 |
At the end of HFD and GD16, glucose tolerance was examined by oral glucose tolerance test. HFD feeding tended to increase glucose AUC at the end of HFD, but this effect failed to reach statistical significance (Figures
Effect of LBP on glucose intolerance in experiment mice. Glucose tolerance in dams at the end of HFD (a, b) and GD16 (c, d) of mice fed control, HFD, or HFD + LBP diets.
Table
Effect of LBP on lipid profiles of mice in the three groups.
Control group | HFD group | HFD + LBP group | |
---|---|---|---|
Plasma TC (mmol/L) | 0.94 ± 0.06 | 2.03 ± 0.08 | 1.49 ± 0.08 |
Plasma TG (mmol/L) | 0.53 ± 0.03 | 0.99 ± 0.06 | 0.70 ± 0.04 |
Plasma LDL (mmol/L) | 0.14 ± 0.01 | 0.31 ± 0.02 | 0.20 ± 0.02 |
Plasma HDL (mmol/L) | 0.45 ± 0.04 | 0.28 ± 0.02 | 0.48 ± 0.05 |
As shown in Figure
Photomicrograph of a section of the liver of a control diet-fed mouse (a), a HFD-fed mouse (b), and a HFD + LBP diet-fed mouse (c). HFD: high-fat diet; LBP: Lycium barbarum polysaccharide.
Exosomes have been demonstrated to contain several categories of small RNAs, including miRNA, tRNA, rRNA, snRNA, snoRNA, piRNA, Y_RNA, and unannotated RNAs. The percentages of miRNA in the total small RNA isolated from the control group, HFD group, and HFD + LBP group corresponded to 16.76, 17.83, and 15.77%, respectively (Figure
The percentage of small RNA categories in all reads mapped to noncoding RNA databases of mice in the control, HFD, and HFD + LBP groups. HFD: high-fat diet; LBP: Lycium barbarum polysaccharide.
Summary of sequence statistics of the samples.
Group | Total reads | Clean reads | Mapped clean reads | Mapping ratio (%) |
---|---|---|---|---|
Control | 15,260,745 | 13,801,051 | 10,901,115 | 79.0 |
HFD | 13,924,992 | 12,783,536 | 10,191,842 | 79.7 |
HFD + LBP | 14,557,650 | 13,240,453 | 10,955,990 | 82.7 |
HFD: high-fat diet; LBP: Lycium barbarum polysaccharide.
Using
Differentially expressed miRNAs in the three groups.
miRNA_ID | Log2 (fold change) | |||
---|---|---|---|---|
HFD/control | HFD + LBP/HFD | HFD/control | HFD + LBP/HFD | |
mmu-miR-466k | 10.414 | −10.414 | 0.011 | 0.010 |
mmu-miR-93-3p | 10.274 | −10.274 | 0.013 | 0.013 |
mmu-miR-7115-5p | 10.009 | −10.009 | 0.019 | 0.018 |
mmu-miR-1188-5p | 9.570 | −9.570 | 0.032 | 0.030 |
mmu-miR-7001-3p | 9.421 | −9.421 | 0.046 | 0.039 |
mmu-miR-188-5p | 9.403 | −9.403 | 0.049 | 0.042 |
mmu-miR-666-5p | 10.124 | — | 0.001 | — |
mmu-miR-30c-2-3p | −10.867 | 8.184 | 0.002 | 0.161 |
mmu-miR-7048-3p | −9.827 | 8.638 | 0.004 | 0.104 |
mmu-miR-6981-5p | 9.998 | −1.511 | 0.019 | 0.508 |
mmu-miR-369-5p | −9.812 | 8.858 | 0.024 | 0.099 |
mmu-miR-6540-5p | −10.048 | — | 0.025 | — |
mmu-miR-374b-5p | 3.731 | — | 0.026 | — |
mmu-miR-709 | −3.26 | 7.534 | 0.031 | 0.338 |
mmu-miR-8097 | 9.616 | −2.344 | 0.031 | 0.365 |
mmu-miR-5129-5p | 9.437 | −1.336 | 0.037 | 0.470 |
mmu-miR-547-3p | −9.506 | 6.849 | 0.046 | 0.465 |
mmu-miR-7676-3p | −9.187 | — | 0.049 | — |
mmu-miR-7073-5p | — | −10.009 | — | 0.002 |
mmu-miR-29b-3p | −1.625 | 7.143 | 1 | 0.004 |
mmu-miR-190b-5p | 3.26 | −6.839 | 0.239 | 0.019 |
mmu-miR-378a-5p | — | −9.311 | — | 0.024 |
mmu-miR-6952-5p | 1.158 | −9.586 | 0.615 | 0.028 |
mmu-miR-382-3p | −− | 9.597 | — | 0.035 |
mmu-miR-10b-3p | −4.143 | 4.737 | 0.308 | 0.036 |
mmu-miR-6967-3p | −9.202 | 9.462 | 0.072 | 0.040 |
mmu-miR-6994-3p | — | 9.431 | — | 0.048 |
mmu-miR-7036b-5p | — | 9.588 | — | 0.049 |
HFD: high-fat diet; LBP: Lycium barbarum polysaccharide.
Candidate target genes for 6 common differentially expressed miRNAs in the two comparisons were predicted bioinformatically. GO analysis classified genes by biological process, molecular function, and cellular component. In biological process, genes were mainly enriched in protein modification by small protein conjugation or removal, cellular protein catabolic process, and ubiquitin-dependent protein catabolic process. In the cellular component, genes were mainly enriched in the synapse part, postsynapse, and synapse. In molecular function, genes were mainly enriched in ubiquitin-like protein transferase activity, ubiquitin-protein transferase activity, and GABA receptor activity (Figure
GO enrichment analysis of 6 common differentially expressed miRNAs identified in the two comparisons of HFD/control and HFD + LBP/HFD. HFD: high-fat diet; LBP: Lycium barbarum polysaccharide.
KEGG pathway analysis suggested that the genes were evidently enriched in the phospholipase D signaling pathway, MAPK signaling pathway, FoxO signaling pathway, dorsoventral axis formation, insulin resistance, choline metabolism in cancer, renal cell carcinoma, insulin signaling pathway, and cAMP signaling pathway (Figure
KEGG pathway enrichment analysis of 6 common differentially expressed miRNAs identified in the two comparisons of HFD/control and HFD + LBP/HFD. HFD: high-fat diet; LBP: Lycium barbarum polysaccharide.
qRT-PCR was used to validate the expression levels measured by RNA-seq for common differentially expressed miRNAs (miR-93-3p, miR-188-5p, miR-466k, miR-1188-5p, miR-7001-3p, and miR-7115-5p) in livers. As demonstrated in Figure
Effect of LBP on common differentially expressed miRNAs by qRT-PCR in livers. Relative mRNA expression of miR-93-3p (a), miR-188-5p (b), miR-466k (c), miR-1188-5p (d), miR-7001-3p (e), and miR-7115-5p (f) in the control, HFD, and HFD + LBP group mice.
We used western blot to determine the effect of LBP on proteins’ expression level of the candidate target genes involved in insulin resistance in mice livers. In HFD and HFD + LBP groups, the protein expression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), which is the target gene of miR-93-3p, was similar to that of the control group (Figure
Effect of LBP on proteins associated with insulin resistance in livers. Representative immunoblot (a) and quantification (b) of HMGCR and CPT1A in the control, HFD, and HFD + LBP group mice.
The pathogenesis of GDM is complex. Previous studies have demonstrated that genetic predisposition and pregnancy hormones contribute to the development of GDM [
Although increasing studies have been conducted on diabetes, very few research studies were specifically tailored towards GDM. LBP has been reported to possess a beneficial effect on diabetes [
To further explore the mechanism of the beneficial role of LBP on GDM, we examined the changes of exosomal microRNA expression profiling in all three groups of mice. To our knowledge, this is the first study investigating miRNAs in GDM mice. Previous studies investigating miRNAs in GDM all focused on humans. For example, Zhao et al. reported that miR-29a, miR-222, and miR-132 were decreased in serum of women with GDM [
We did the GO and KEGG pathway analysis of the candidate target genes for the 6 miRNAs, and KEGG pathway analysis showed that the genes were evidently enriched in the phospholipase D signaling pathway, MAPK signaling pathway, FoxO signaling pathway, dorsoventral axis formation, insulin resistance, etc. Insulin resistance plays an important role in the development of diabetes. HMGCR and CPT1A, the target gene of miR-93-3p and miR-188-5p, respectively, are closely related not only to insulin resistance but also to abnormal lipid metabolism. Our results showed that GDM mice had hyperlipidemia. Therefore, we choose the insulin resistance-related proteins HMGCR and CPT1A to be verified by western blot. We found that the protein expression of HMGCR, which is the target gene of miR-93-3p, was similar in control and GDM mice, while the protein expression of the miR-188-5p target gene CPT1A was significantly reduced in GDM mice, and it can be notably reversed by LBP treatment. CPT1A can catalyze the entrance of fatty acids into the mitochondria, and it is the rate-limiting enzyme of hepatic fatty acid
LBP significantly relieved glucose intolerance, abnormal plasma lipid levels, and pathomorphological changes of liver histopathology in HFD-induced GDM mice. Moreover, we found that this effect of LBP was mediated by downregulation of the increase of 6 miRNAs (miR-93-3p, miR-188-5p, miR-466k, miR-1188-5p, miR-7001-3p, and miR-7115-5p) and reversing the increase of the protein expression of CPT1A, which is the target gene of miR-188-5p. The results provide novel insights into the biological activities of LBP in the treatment of GDM.
The data used to support the findings of this study are available from the corresponding author on reasonable request.
The authors declare that they have no conflicts of interest.
Ya Xiao, Weihao Chen, and Ruixue Chen contributed equally to this work. YX conceived and designed the experiments. WC and RC acquired the data. AL, DC, and QL analyzed and interpreted the data. YX, WC, and RC drafted the manuscript. XC and TL revised the manuscript for important intellectual content. WT supervised the study. All authors were involved in the formulation of the research questions. All authors read and approved the final manuscript.
This work was supported by the National Natural Science Foundation of China (no. 81603520), the Natural Science Foundation of Guangdong Province (nos. 2016A030310084 and 2017A030313658), the Science and Technical Plan of Guangzhou, Guangdong, China (no. 201804010213), the Administration of Traditional Medicine of Guangdong Province (no. 20181068), and the Medical Scientific Research Foundation of Guangdong Province (no. A2017552).