Participants and clinical measurement
Subgroup 1 including 36 pregnant women with GDM and 40 matched normal glucose tolerance (NGT) pregnant women, subgroup 2 including13 NGT pregnant women, all of them were recruited from May to August 2018, at The Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region, Department of Obstetrics. Inclusion and exclusion criteria are shown in Tab. S1. Written informed consent was obtained from all participants, and the study was approved by The Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region ethics committee.
Pre-pregnancy body mass index (BMI) was calculated according to weight before pregnancy as weight (in kilograms)/height (in square meters). Birthweight, neonatal gender, delivery mode, placental size and weight, neonatal peripheral blood glucose levels were obtained from patient medical records.
Maternal blood samples were taken from a cannulated vein before delivery, gestational diabetes mellitus umbilical vein (Uv) blood and umbilical artery (Ua) blood were taken from the umbilical cord venous and arterial storing in the vacuum blood collection tube during the third stage of labor. All blood samples were centrifuged (3000 rpm at room temperature for 5 minutes), and serum were collected in the 1.5 mL Eppendorf tubes. Placental tissue from the central cotyledon about 1 cm3 was obtained immediately after delivery. Serum aliquots and placental tissues were immediately stored at -80℃ until analysis.
Measurement of serum indicators
The levels of total OCN (tOCN), 1,25-(OH)2-VitD3 (25(OH)D3), parathormone, insulin，glucose, triglycerides (TG), total cholesterol (TC), high density lipoprotein total cholesterol (HDL-C), alkaline phosphatase (ALP), human chorionic gonadotropin (HCG), and estradiol (E2) were analyzed by electrochemiluminescence immunoassay on COBAS 6000 system E601 (Elecsysmodule) immunoassay analyzers (Roche Diagnostics, GmbH, Mannheim, Germany). Leptin (LEP), ADP, and tartrate-resistant acid phosphatase 5b (TRACP-5b) were measured by ELISA kits according to the manufacturers’ instructions (Shanghai Guangrui Biotechnology Co., Ltd).
serum cytokines analysis
8 serum cytokines, IL-2 IL-4 IL-6 IL-8 IL-10 IL-17A IFY γTNF-α, were analyzed on Bio-Plex liquid chip platform. Briefly, the biotinylated detection antibody was added into a 96-well plate, serum sample were mixed with the antigens coupled microspheres and added into each hole, then adding streptavidin phycoerythrin, detecting the fluorescence signal by a Bio-Plex liquid chip system, and judging the positive property/negative property of the sample according to the strength of the fluorescence signal.
RNA extraction and qRT-PCR
RNA extraction and purification were conducted using the E.Z.N.A.™ Total RNA Kit I (Omega). cDNA synthesis was performed using the PrimeScript™ RT Reagent Kit (Takara). SYBR Premix Ex TaqⅡ (Tli RNaseH Plus) was used with the following PCR parameters, 1 cycle of 30 s at 95 ℃ and then 40 cycles of 95 ℃ for 5 s and 1 cycle of 30s at 60 ℃. qRT-PCR was conducted using a LightCycler 96 (Roche). The primer sequences are presented in Tab. S2. The housekeeping gene β-actin was used as control. The relative levels of the mRNA of the genes of interest were normalized to the β-actin mRNA.
Placenta tissues were washed 3 times in PBS for 30 seconds and fixed in 4% formaldehyde for 24 hours. The placental tissues were then embedded in paraffin and sectioned at a thickness of 5 μm. The tissue sections were deparaffinized, subjected to high-temperature antigen exposure, rehydrated in 3% H2O2, and blocked with 10% normal goat serum for 30 minutes. The sections were then incubated with antibodies to OCN (1:1000) (Santa cruz) or GPRC6A (1:1000) (Invitrogen). The secondary antibodies are provided with the DAB Substrate Kit for Peroxidase (Vector Laboratories). Olympus DP11 camera and Olympus Camedia software were used to produce the images.
In vitro experiments
Cell culture: JAR trophoblast cells were giving by professor Longyu, JAR cells were maintained without antibiotics and RPMI-1640 medium (Gibco) containing 10% fetal bovine serum (FBS) (Gibco) at 37 ℃ under 5% CO2.Osteogenic induction medium: α-MEM (Gibco) containing 10% FBS (fetal bovine serum), 100 nM dexamethasone (Sigma), 5 mM β-phosphoglyceride (Sigma) and 5 μg/mL vitamin C (Sigma).
Alizarin red stain: Alizarin red stain positive indicate calcium. JAR cells were maintained in 6 well plate until 90% confluency. Osteogenic induction medium was added confluency to 6 well plate to maintain 7 days, and then JAR cells were fixed by 4% paraforma ldehyde for 10 minutes. Adding alizarin red for 5 minutes. At each of steps, JAR cells were washed for 3 times by PBS.
Scanning electron microscope: JAR cells were maintained in 24 well plate until 80%. Osteogenic induction medium was added to 24 well plate to maintain 7 days. 3% glutaraldehyde was used to fixed JAR cells. The calcium nodules were observed by scanning electron microscope (Czech, TESCAN, VEGA 3 LMU).
Cell proliferation assay: Using the Cell Counting Kit-8 (CCK-8, Solarbio®) according to the manufacturer's instructions. This experiment was performed in triplicate.
shRNA knockdown: Human GPRC6A shRNA lentivirus plasmid pLV[shRNA]-EGFP: T2A: Puro-U6 > hGPRC6A purchases from Victor builder company (Saiye). Lipofectamine 2000 (Invitrogen) was used to package virus in 293T cells; a 5:3:2 ratio of target plasmid: psPAX2: PMD2G was used. Virus supernatant (100 μL) was added to JAR cells in 24-well plates for 24 hours. Green fluorescence could be observed after 72 h.
RNA sequencing and bioinformatics analysis
RNA sequencing: Total RNA was extracted from placental tissues using the E.Z.N.A.TM Total RNA Kit I (Omega). mRNA sequencing was performed on an Illumina HiSeq 4000 RNA-sequencing platform, and the results were uploaded to BaseSpace of the Illumina cloud server. The RNA-Seq Alignment and Cufflinks Assembly procedures were used in the sequencing analysis.
WGCNA: Co-expression networks were constructed using the WGCNA (v1.47) package in R. Without filtering the genes, gene expression values were imported into WGCNA and used to construct co-expression modules using the automatic network construction function block wise modules with default settings except that the power was set to 2. TOM Type was unsigned, and the minimum module size was 250. The genes were clustered into 36 correlated modules. Module eigengenes were used to calculate the correlation coefficients for samples or sample traits.
Gene ontology (GO) analysis: significantly enriched GO terms pathways were defined by the hypergeometric test using a threshold false discovery rate (FDR) ≤ 0.05.
RNA-Seq data to this article can be found online at https://www.ncbi.nlm.nih.gov/sra/, SRR10812151- SRR10812189.
Statistical analyses were undertaken using SPSS 25(IBM, Chicago, IL, USA). The results are reported as the mean ± S.D. unless otherwise noted. Clinical characteristics that followed a normal distribution were compared between the two groups using Student’s t-test. Categorical variables were analyzed using the χ2 test. Spearman’s correlation was used to examine associations between serum OCN and metabolic indices. Statistical analysis and graph plotting were performed using Prism 7 software and Adobe Illustrator. P < 0.05 was considered significant.