| There are growing evidences show that,fetal development and physiological status are closely related to maternal nutrition and placenta nutrient transfer.Markly differencesare noted between fetal weight,placental structure and nutrient transport capacity in different position within the same uterine.Studies have confirmed that miR-17-5p retarded cell and tissue growth,and there are also evidences suggesting that circRNAs bind the microRNAs act as their sponge.Hitherto,despite these representative clinical and animal experimental datums,the precise molecular mechanisms were responsible for placental-fetal growth and physiological function as a result of altering the maternal nutrients transport from placenta to fetus remains poorly elucidated.Furthermore,the functions of non-coding RNAs in the progression of adiposity and the relationship between non-coding RNAs and imprinted genes remain an enigma.The objective of our study was to evaluate the effect of the maternal energy levels during the whole gestation on placental and fetal growing development during the whole gestation and potential mechanism.Additionally,we anticipated that the present study could disclose a original regulatory pathway which is composed of circ-Amotll,miR-17-5p,and imprinted genes.As well as this could act as a model for studying on the prevalence of maternal high energy and offspring obesity predisposition.The main contents and results are presented as follows.Experiment 1.The effect of dietary energy levels on placental nutrient transport and fetal development.At day 0 of pregnancy,42 gilts(Large White breeding,body weight at 135.74±0.88 kg,age at 255± 11 days)were allocated to one of two experimental diets(HE group:3.40 Mcal/kg,n=21;LE group:3000 Mcal/kg,n=21)using a completely randomized design.The artificial insemination was carried out at the three estrus of gilts and recorded as first day.On 35,55,90 days of pregnancy and delivery,carotid blood from the mothers and cord blood(except day 35)from fetuses were collected for serum parameters andmaternal placentas and fetal livers were obtained for analysis of morphological,biochemical and molecular analysis.The concentration of hormone in serum were carried out by using the ELISA kit.Plasma levels of glucose,triglycerides and FFAs followed the manufacturers’ recommendations.Non coding RNAs and mRNA expression in placenta and fetal livers were detected by RT-PCR.The results are presented as follows:1.Compared with the LE group,body weight was increased markedly in HE group(P<0.05)on days 55,90 and 110 of gestation,and there was no significant difference between the two group on day 35;placental weight and fetal body weight in HE group were significantly higher than the LE group at Day 90 of gestation and Day 0 of delivery(P<0.05),and there was no significant difference at day 35,55.2.HE resulted in higher maternal and fetal serum glucose,triglyceride,FFA and leptin concentration on day 90 and Day 0 of delivery(P<0.05),there was no significant difference between the two group on day 35,55.Maternal and fetal serum total cholesterol and glucocorticoid were not affected by HE diet during the whole gestation(P>0.05).3.Compared with the LE group,the expression of circ-Amotll were increased markedly(P<0.05),and the expression of miR-17-5p were decreased significantly(P<0.05)in HE group on day 90 and Day 0 of delivery.4.HE resulted in higher expression of IFF-2,Stat3,Slc2a3,SLC27A1 and Dnmt3a in placenta and liver of fetus(P<0.05),and the expression of Slc2al,amino acid transporter,FABP3,Dnmtl,Dnmt3b were no significant difference between the two group during the whole gestation(P>0.05).The results of this study implicated that,HE diet during pregnancy promoted the placental and fetal nutrient transfer,fetal development.The epigenetic regulation of circ-Amotll and miR-17-5p and Stat3 signaling pathway may play important roles during these processes.Experiment 2.Role of circ-Amotll in the energy levels dependent regulation of proteomic alternations of placenta with different position in uterineThe results of experiment 1 showed that HE diet during pregnancy promoted the placental and fetal nutrient transfer,fetal development and the expression of circ-Amotll and miR-17-5p had significant difference between the two group.There were lots of studies with pigs models have shown that the negative effects of abnormal fetal growth may be contribute to a great within-litter variation of piglet birth weight(CVBW0).But the effect of energy level on fetal development of different plant site of placenta is still unclear.So we designed the experiment 2.The experimental treatment was same as experiment 1.On day 55 and 90 of gestation,umbilical vein blood was collected from each fetal which located toward the uterine cervix and utero-tubal junction.Serum samples were collected and stored at-20℃ for future analysis.The fetuses weight located in each uterine horn from the cervix to utero-tubal junction were recorded on day 35,55 and 90 of gestation.The placenta were carefully isolated and weighed from the uterus of individual fetal on day 35,55 and 90 of gestation.On day 90 of gestation,these placenta tissue samples which surrounding toward the cervix and utero-tubal junction were frozen rapidly in liquid nitrogen after rinsing with cold sterile saline for iTRAQ and MRM assay,and to measure the expression of circ-Amotll and miR-17-5p.The results are presented as follows:1.Maternal dietary treatment had no effect on coefficient of variation of weight(CVweight)on day 55 of gestation(P>0.05).Compared with the LE group,the fetal weight on day 90 and the piglet weight at parturition were higher in HE group(p<0.05).It is noteworthy that increased dietary energy level during gestation influenced on CVweight in the fetal and piglet.2.The glucose and FFA concentrations in LE group were significant higher in the fetal located toward the utero-tubal junction than the cervix on day 90 of gestation(P<0.05).3.Using iTRAQ analysis,a total of 3011 and 3018 proteins were identified within the false discovery rate of 1%in different uterine location of LE and HE groups,respectively.For LE group,a total of 78 proteins showed>1.2-fold changes between the cervix and the utero-tubal junction among the identified proteins(P<0.05),including 36 up-regulated proteins and 42 down-regulated proteins.The protein involved nutritional transport and metabolism were predominant and accounting for about 62.3%of the differentially expressed proteins.Among these protein,lipid transport and metabolism were predominant and accounted for 18.9%.However,for HE group,a total of 50 proteins showed>1.2-fold changes between the cervix and the utero-tubal junction among the identified proteins(P<0.05),with 27 being up-regulated and 23 down-regulated.Although these differentially expressed proteins were also connected with nutrient transport and metabolism,only about 35.6%differentially expressed proteins participation,which are main involved carbohydrate,coenzyme,lipid and inorganic ion transport and metabolism.The differentially expressed proteins related to lipid transport and metabolism onlyaccounted for 6.5%.4.The expression of circ-Amotll were significant up-regulated and miR-17-5p weresignificant down-regulated in the fetal located toward the utero-tubal junction than the cervix on day 90 of gestation in the two group(P<0.05).Our study provide the first evidence for an alteration of placental between utero-tubal junction and cervix proteome in response to increase dietary energy intake in gilts.Placental lipid and energy metabolism and nutrient transport may be crucial importance in difference weight fetuses.In addition,this study demonstrates that a high energy supply during gestation enhanced fetus development,improved the placental nutrients supply and decreased CVBw,ultimately,increased the fetus weight and uniformity of piglet birth weight.These findings provide new information that can help to understanding the mechanisms that maternal nutrition regulation of fetal growth and improve our understanding of placental efficiency.No-coding RNA effected the fetal development and placental nutrient transport.Experiment 3.The regulation of circ-Amotll on cells or tissues growth and its molecular mechanismsThe results of experimentl and 2 showed that circ-Amotll and miR-17-5p effected the placental nutrient transport and promotes fetal growth,but the exact mechanism is unclear.So we designed the experiment 3.Cell culture test:NIH-3T3 cells were transfected with vector,circ-Amotll,Stat3,si-Stat3 and miR-17-5p to test their effect on cell growth and its exact mechanism.Animal experiment:A one-factor experiment was designed to investigate the effects of circ-Amotll on animal growth.Thirty C57BL/6 mice(4 weeks old,similar body weights)were allocated to one of two experimental group(control:vector plasmid,n=15;circ-Amotll overexpress group:n=15,150 μg/kg by injecting abdominal every two days)using a completely randomized design.4 weeks after treatment harvested the liver,heart,kidney and pancreas were obtained for analysis of morphological,biochemical and molecular analysis.The results are presented as follows:1.HE resulted in higher expression of circ-Amotll,and promoted the cell proliferation,survival,adhesion and migration.2.circ-Amotll overexpression increased the the cell proliferation,survival,adhesion and migration,and promoted cell growth.3.The results of western blotting,luciferase assay,and immunostaining showed that circ-Amotll increased the Stat3 nuclear translocation,which led to promotion of cell viability.Stat3 nuclear translocation can bind the Dnmt3a promoter,and promoted the transcription and translation.Dnmt3a induced the methylation of miR-17 promoter,decreased the expression of miR-17-5p and increased the expression of IGF-2.4.The results of animal experiment showed that circ-Amotll overexpression increased the mice body,organ weight.Compared with the control group,circ-Amotll resulted in higher expression of Stat3,Dnmt3a and IGF-2,and the expression of miR-17-5p and H19 were decreased significantly.Our results indicated that the expression of circ-Amotll enhanced Stat3 nuclear translocation,which promoted Dnmt3a transcription.Increased Dnmt3a expression repressed miR-17 transcription.MiR-17-5p could repress Stat3 levels resulting in down regulating circ-Amotll function have added an additional layer to understand the mutual regulation of these two classes of non-coding RNAs.A feed-back loop in the mutual regulation of circ-Amotll and miR-17-5p expression,suggesting a mutual regulation of circ-Amotll and miR-17-5p functions,which can regulate the growth of cells and animals.In summary,the main conclusions of this study are as follows:(1)Energy levels regulates the placental nutrient transport and fetal intrauterine growth at late stages of gestation by affecting the expression of circ-Amotll and miR-17-5p.(2)Energy levels regulates CVBW by affecting the number of differentially expressed proteins associated with nutrient transport and metabolismin placenta of cervical and tubal;The expression of non-coding RNAs can affect the fetal development and corresponding placental nutrient transport with different position in uterine.(3)Circ-Amotll promotes the cells and tissues growth by inhibiting the expression of miR-17-5p. |
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