Effects Of DEHP Exposure During Rat’s Pregnancy On Hypothalamic LncRNA Expression In Female Offspring

DEHP has clear reproductive toxicity,and the effect on energy metabolism has been gradually confirmed.Effects of pregnancy-exposed DEHP on hypothalamus of offspring female rats are inadequate.This study uses transcriptome sequencing technology to explore the effects of DEHP exposure during pregnancy on the expression of LncRNA in the hypothalamus of rat offspring.By comparing the LncRNA and mRNA differentially expressed in the hypothalamus of the infected group and the control group,the effects of DEHP on the hypothalamus are analyzed and predicted hypothalamus-pituitary-gonad axis and the mechanism of energy metabolism.Our research are done as follows:1.Animal breeding and sample collection: 60-day-old SPF SD rats were selected,and they were divided into an infected group and a control group after conception.From0 to 18 days of pregnancy(G0 to G18),the rats were given by intragastric administration continuously.On the day of birth,the offspring of the female rats were taken from the hypothalamus tissue and stored in the refrigerator at-80℃ until testing..2.The hypothalamic tissues of three rats female offspring in the infected group and control group were collected separately.Samples are mixed and extracted the total RNA.We construct the RNA library and sequenced using the illumina Hiseq sequencing platform.3.The obtained reads is assembled into transcripts to obtain mRNA and LncRNA transcripts.Through data evaluation and analysis,differentially expressed mRNA and LncRNA are screened,and then LncRNA target gene prediction is performed,and the differential LncRNA target gene and mRNA are analyzed for functional enrichment.4.The target LncRNA with significant differential expression will be screened out through correlation analysis(pearson correlation coefficient ≥ 0.9)to obtain the LncRNA-mRNA relationship pair,and the co-expressed mRNA will undergo functional enrichment analysis to predict the molecular function of the target LncRNA and the biological process that it may participate in.5.Real-time fluorescence quantitative PCR detection was used to verify the differential expression of hypothalamic cells in the infected group and the control group.Finally,we confirm that the trend of LncRNA was consistent with the sequencing results.Our Results are as follows:1.Results of RNA-seq sequencing show that 393142752 and 388608118 RNA of hypothalamus reads were obtained in the infected and control group respectively.376311164(95.57%)and 373196206(95.70%)reads are matched with the rats’ gene in the infected and control group respectively.2.Using the DESeq software,we find 108 significantly different LncRNA and 342 significantly different mRNA,with |log2FC|>=1 and Pvalue <|log2FC|>=0.05,Among the LncRNA,60 LncRNA were upregulated,48 downregulated,5954 with no significant change.Screening for 342 significantly differentially expressed mRNA,Among the mRNA,196 were upregulated,146 downregulated,29478 mRNA expression with no significant change.3.GO and KEGG enrichment analysis of differentially expressed mRNA and LncRNA.GO analysis of mRNA shows that it is mainly involved in brain development,hormone synthesis and catabolic processes;KEGG analysis shows: glutamatergic synapses,PPAR signaling pathways,pressure It is significantly enriched by the absorption of water regulated by the element.The GO analysis of LncRNA showed the process of hormone biosynthesis and the regulation of lipid metabolism;KEGG analysis showed that the estrogen signal pathway,Ras signal pathway signal transduction,PI3K-Akt signal pathway,etc.were significantly enriched.4.The target LncRNA and the differentially expressed mRNA were co-expression analysis.With r≥0.90 and p<0.05 as the standard,a total of 15 mRNAs with the same expression pattern as ENSRNOT00000047544 were screened;7 mRNAs with the same expression pattern as MSTRG.6385.1 were screened Pattern mRNA.The co-expressed mRNA was subjected to functional enrichment analysis and found that it was involved in the regulation of the PPAR signaling pathway.5.qPCR results showed that the expression of ENSRNOT00000047544 and MSTRG.6385.1qPCR in hypothalamus of offspring rat was higher than that of control group,which was consistent with RNA-seq sequencing results.Our Conclusion are as follows:1.There were different expression of LncRNA and mRNA in the hypothalamus of female rats 1.pregnancy compared with control group.2.The LncRNA and mRNA function analysis of 2.differential expression showed that it may affect the regulation of gonadal hormone and energy metabolism in hypothalamus.3.Screened out LncRNA ENSRNOT00000047544 and LncRNA MSTRG.6385.1may be involved in energy metabolism regulation.