Professor Qi-Qiang He, Shenzhen Research Institute, Wuhan University, Keyuan South Road, Nanshan, Shenzhen, Guangdong 518000, P.R. China
*Contributed equally
Maternal obesity is associated with disturbance of lipid metabolism and obesity in offspring; however, the pathogenesis is still unclear. The present study elucidated the role of potential lipid metabolism-associated long non-coding RNA (lncRNA) and identified the pathways involved in mice born to obese dams. In the present study, maternal obesity was induced by feeding a high-fat diet for 10 weeks in female C57/BL6 mice, whereas control mice were fed a standard diet. All female mice mated with healthy male mice and were allowed to deliver spontaneously. The results demonstrated that female offspring from obese dams presented a tendency to become overweight in the first 8 weeks after birth; however, maternal obesity did not significantly alter the body weight of male offspring. RNA-sequencing analysis was performed on female offspring liver at 3 weeks old. Significantly dysregulated lncRNAs and downstream targets in female offspring liver were identified using bioinformatics analysis. lncRNA, microRNA (miRNA or miR) and mRNA expression levels in liver and AML12 cells were assessed using reverse transcription-quantitative PCR. A total of 8 upregulated and 17 downregulated lncRNAs were demonstrated in offspring from obese dams and lncRNA Lockd was indicated to be a key dysregulated lncRNA. Competing endogenous RNA (ceRNA) models suggested that the lncRNA Lockd/miR-582-5p/Elovl5 pathway was key for lipid metabolism in the liver of offspring from obese dams. Finally, small interfering RNA and miRNA inhibitor transfection was used to evaluate the ceRNA models in AML12 cells. Taken together, the results of the present study indicated that lncRNA Lockd-miR-582-5p-Elovl5 network may be disrupted in lipid metabolism and lead to obesity in the offspring of obese dams. This research will provide new insights into the molecular mechanism of obesity and lipid metabolism disorder.
The prevalence of female obesity around the world has roughly doubled in the past four decades (from 6.4 to 14.9%) (
Coding genes form <2% of the total human genome. However, ~70% of the human genome is transcribed into RNA, which generates thousands of non-coding (nc)RNAs (
A previous study has focused on the metabolic characteristics of offspring obesity or specific genes (
It is unclear whether lncRNA regulation is involved in metabolic abnormalities of offspring caused by overfeeding in early life. The present study assessed the potential lipid metabolism-associated lncRNA and pathways in mice born from obese dams using RNA-sequencing and bioinformatic analyses. The findings of the present study may contribute to understanding of the effects of maternal obesity on offspring liver lipid metabolism and suggest novel therapeutic possibilities for obesity-associated disease.
A total of 14 C57/BL6 female mice (age, 4 weeks; weight, 12±1 g; Hubei Experimental Animal Research Center) were housed individually in woodchip-bedded plastic cages at a constant temperature (25±2˚C) and humidity (60±5%) with a 12/12-h light/dark cycle and free access to water. Maternal obesity (n=10) was induced by feeding a high-fat diet (45% of energy from fat; Research Diets, Inc.) for 10 weeks while the control mice (n=4) were fed using a standard diet (15% of energy from fat; Research Diets, Inc.), 7 female mice became obese in maternal obesity group. All female mice mated with 6 healthy male mice (age, 14 weeks; weight, 36±2 g; Hubei Experimental Animal Research Center) in a 2:1 ratio, randomly as previously described, the housing conditions of male mice are the same as the female mice (
The library construction and sequencing were performed by Annoroad Gene Technology Co. Ltd. A total of three liver samples each were retrieved from female offspring of the CON and OB groups. Total RNA was extracted from the tissues using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). A total of 3 µg total RNA of each sample was used to construct the lncRNA library. A Kaiao K5500 spectrophotometer (Beijing Kaiao Technology Development Co., Ltd.) was used to assess purity of samples and the Agilent 2100 RNA Nano 6000 Assay kit (Agilent Technologies, Inc.) was used to assess integrity and concentration of RNA samples. The ribosomal RNA was removed using Ribo-Zero™ Gold kit (Guangzhou RiboBio Co., Ltd.). Different index tags were selected using NEB Next Ultra Directional RNA Library Prep kit for Illumina (New England BioLabs, Inc.) according to the manufacturer's protocol. The constructed library was used for lncRNA and mRNA sequencing on the Illumina sequencing platform.
The raw reads obtained using Illumina sequencing were processed by Annoroad Gene Technology Co. Ltd. to remove low-quality sequences, adapter contamination and rRNA to obtain clean reads. All subsequent analyses were based on clean reads. lncRNA and mRNA sequencing analysis process was as follows: Quality control of sequencing data, data comparison analysis, expression quantification, novel lncRNA recognition, differential expression analysis, feature analysis and target prediction of novel lncRNA, and functional enrichment (
Dysregulated gene analysis was performed using the DEseq package in R 4.0.3 (
The liver tissue of 3-week-old female offspring was collected and lncRNA, miRNA and mRNA expression levels were assessed using RT-qPCR. Total RNA was extracted from liver tissue using TRIzol® (Invitrogen; Thermo Fisher Scientific, Inc.). RNA was reverse-transcribed into complementary (c)DNA using the PrimeScript™ 1st Strand cDNA Synthesis kit (Takara Bio, Inc.) and miRcute Plus miRNA First-Strand cDNA kit with poly(A) tailing reaction (Tiangen Biotech Co., Ltd.) according to the manufacturer's protocols. The expression levels of lncRNA and mRNA were quantified using TB Green® Premix Ex Taq™ II kit (Takara Bio, Inc.) according to the manufacturer's protocol. Expression levels of lncRNA and mRNA relative to β-actin and expression of miRNA relative to U6 were quantified using the 2-ΔΔCq method (
The mouse hepatocyte AML12 cell line (Shenzhen Haodi Huatuo Biotechnology Co., Ltd.) was plated in 6-well plates (1.2x106/well) for RT-qPCR using Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 5 insulin, 5 transferrin, 5 selenium and 40 mg/l dexamethasone, 100,000 U/l penicillin and 100 mg/l streptomycin. Cultures were maintained at 37˚C in a humidified atmosphere containing 5% CO2 for experiments. RT-qPCR was performed according to the aforementioned method.
siRNA of lncRNA Lockd and miR-582-5p inhibitor were used to inhibit the expression of lncRNA Lockd and miR-582-5p in AML12 cells, respectively. siRNA of lncRNA Lockd and miR-582-5p inhibitor were obtained from Guangzhou RiboBio Co., Ltd. The siRNA was 19 nt + dTdT 3' overhanging structure. The transfection of these plasmids was performed using ribo FECT™ CP Transfection kit (Guangzhou RiboBio Co., Ltd.) in AML12 cells according to the manufacturer's protocol. RT-qPCR was performed to determine the expression of lncRNA Lockd, miR-582-5p, Elovl5 according to the aforementioned method. The siRNA primer sequences of lncRNA Lockd and miR-582-5p inhibitor are presented in
All data were presented as mean ± SD and analyzed using SPSS 25.0 (IBM Corp.) or R 4.0.3 (
From the 8th to 10th week of feeding, the weight of maternal mice in the OB group was significantly increased compared with CON (
A total of 4,393 lncRNAs was identified from the total RNA libraries. The classification of lncRNA in different samples was based on the genomic origin (
Compared with CON offspring, 81 differentially expressed lncRNAs, including 37 up- and 44 downregulated genes, were demonstrated in OB offspring (
A total of 81 differentially expressed lncRNAs were identified, of which 56 were unknown and 25 were known. The known lncRNAs included 17 down- and 8 upregulated genes (
In the liver of 3-week-old female offspring, it was demonstrated that compared with the offspring from CON group, the expression of LncRNA Lockd (P=0.013) and Elovl5 (P=0.024) was significantly lower in OB offspring and expression of miR-582-5p (P=0.022) was significantly higher (
In the present study, an animal model of maternal obesity was constructed to assess expression of lncRNA in offspring liver using whole transcriptome sequencing technology and bioinformatics analysis. Illumina sequencing platform-based next-generation lncRNA sequencing technology accurately and quickly determines the number and structure of transcripts (such as mRNA, known and novel lncRNA) using high-performance computing clusters and powerful bioinformatics analysis techniques (
To the best of our knowledge, previous studies on the mechanism of maternal obesity on metabolic disease of offspring are limited (
At present, the literature reports that lncRNA liver-specific triglyceride regulator, maternally expressed gene 3 and metastasis-associated lung adenocarcinoma transcript 1 regulate liver lipid metabolism signaling pathways (
Competing endogenous RNAs (ceRNAs) are transcripts that regulate each other at a post-transcription level by competing for shared miRNAs. ceRNA networks link the function of mRNAs with that of ncRNAs (such as, lncRNA, circular RNA and miRNA) (
Elovl5 is a key PPARα regulatory enzyme involved in synthesis of mono- and polyunsaturated fatty acids (PUFAs) and it is downregulated in the liver of diet-induced obese mice (
There are potential limitations in the present study. First, only three offspring from CON and OB dams were used for RNA-sequencing. As such, validation in larger cohorts needs to be performed in the future. Moreover, additional experiments such as dual-luciferase reporter and fluorescence in situ hybridization should be performed to elucidate the regulatory mechanism of the lncRNA-miRNA-mRNA network.
To summarize, the present study indicated that maternal obesity is an important risk factor for obesity and adult liver lipid metabolism disorder in offspring. Excessive nutrient intake in the early life disrupts liver metabolism in offspring and increases risk of liver lipid metabolism disorder in adulthood. In this process, the expression of miR-582-5p and Elovl5 is regulated by lncRNA Lockd, which affects key components in the lipid metabolism pathway; this may provide novel approaches for effective prevention and treatment of obesity and associated metabolic disease.
Not applicable.
The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request. The RNA-sequencing data are available from the Figshare repository (
YS, HLG and QQH conceptualized the study and designed the research. HL, ZLZ and KJ made substantial contributions to the acquisition of data. YS and MZZ performed the experiments. YS, ZLZ and HL analyzed and interpreted the data. ZLZ and QQH drafted and edited the manuscript. KJ edited the manuscript. QQH supervised the project. YS and HLG confirm the authenticity of all the raw data. All authors have read and approved the final manuscript.
All experimental procedures were approved by Ethics Committee of Wuhan University School of Medicine (approval no. 2018YF0165).
Not applicable.
The authors declare that they have no competing interests.
Weight gain in dams and offspring. (A) Body weight gain in CON and OB dams after 10 weeks. (B) Birth weight of offspring from CON and OB dams. (C) Survival rate of pups at 7 days. Body weight gain in (D) female and (E) male offspring from birth to 8 weeks. Data are presented as the mean ± SD. *P<0.05 vs. CON. CON, control offspring; OB, obese offspring.
Expression profiles of lncRNA in the liver of postnatal mice. (A) Classification of all lncRNAs based on genomic origin. (B) Circos plot of distribution of lncRNA transcripts in chromosomes, the outer circle represents the chromosome and the inner circle indicates lncRNA transcripts. It comprises six concentric rings, and each corresponds to a different sample. From outer to inner, they are OB1, OB2, OB3, CON1, CON2 and CON3 samples, respectively. (C) Violin plot of distribution of identified lncRNA following normalization. lncRNA, long non-coding RNA; CON, control; OB, obese; lincRNA, long intergenic non-coding RNA.
Identification of differentially expressed lncRNAs. (A) Heatmap of significant differently expressed lncRNAs (>4-fold difference in expression and P<0.05). (B) Volcano plot. (C) Scatter plot of variation in lncRNA expression levels between samples. Red and green represent up- and downregulated lncRNAs, respectively. (D) Classification of differently expressed lncRNAs based on genomic origin. (E) Distribution of up- and downregulated lncRNAs by Chr. lncRNA, long non-coding RNA; CON, control; OB, obese; lincRNA, long intergenic non-coding RNA; Chr, chromosome; FPKM, Fragments Per Kilobase per Million mapped fragments.
lncRNA-miRNA-mRNA network. (A) Downstream targets of lncRNA Lockd were predicted using the online tool Starbase version 2.0. (B) Intersection of 1,208 predicted target and differentially expressed genes were analyzed and 28 differential target genes were indicated. (C) LncRNA Lockd as CeRNA in lncRNA-miRNA-mRNA network. (D) Gene Ontology and Kyoto Encyclopedia of Genes and Genomes gene enrichment analysis of target genes. (E) ceRNA model and binding sites in lncRNA Lockd, miRNA-582-5p and Elovl5. lncRNA, long non-coding RNA; lincRNA, long intergenic non-coding RNA; ceRNA, competing endogenous RNA; miRNA, microRNA.
mRNA expression levels of lncRNA Lockd, mi-582-5p and Elovl5. mRNA expression levels of lncRNA Lockd, mi-582-5p and Elovl5 in (A) 3- and (B) 8-week-old female offspring mice from CON and OB dams. *P<0.05 vs. CON. The mRNA expression levels of lncRNA Lockd, miR-582-5p and Elovl5 following treatment using (C) siRNA of lncRNA Lockd and (D) miR-582-5p inhibitor in AML12 cells. *P<0.05 vs. Control. lncRNA, long non-coding RNA; CON, control; OB, obese; miR, microRNA; NC, negative control; si, small interfering.
Up- and downregulated genes in differentially expressed known lncRNAs.
Expression, n | Long non-coding RNA |
---|---|
Downregulated, 17 | AC122326.1, Gm44787, AC091458.3, Gm14097, Gm6135, Tbx3os1, Gm15611, Lockd, Gm45792, Gm44963, 1810008I18Rik, Gm32540, Gm45836, Gm4316, Gm42031, AC159886.3, AC159895.1 |
Upregulated, 8 | AC079680.3, Hnf4aos, Gm10804, Gm15860, Gm3054, 9530026P05Rik, 2010007H06Rik, Xist |
Lnc, long non-coding.