H19/let-7c Axis Mediates Adrenal Corticosterone Synthesis Inhibition And Maternal Multigenerational Inheritance Induced By Prenatal Dexamethasone Exposure

Aim Dexamethasone is a synthetic glucocorticoid that can pass through the placental barrier and promotes the development and maturation of fetal tissues and organs.It is widely used in the treatment of pregnant women at risk of preterm birth.However,there is increasing evidences suggest that prenatal dexamethasone exposure(PDE)can also have multiple negative effects on the fetus,including intrauterine growth retardation(IUGR).This study aimed to observe PDE induced adrenal developmental toxicity of IUGR progeny and its maternal multigenerational inheritance at the animal level,and explore the molecular mechanism of intrauterine programming changes in human adrenal cells.At the same time,we also tried to explore the possible mediators that transmited acquired traits through maternal lines on each generation oocytes,so as to provide more evidences for the multigeneration inheritance of acquired traits caused by adverse intrauterine environment.Method At the animal level,pregnant rats were injected intraperitoneally with dexamethasone0.2 mg/kg·d from gestation day 9(GD9)to GD20.Some pregnant rats were sacrificed under anesthesia at GD 20 to obtain fetal rats,while others gave birth naturally to produce F1 generation.Some PDE females mated with wild type males at postnatal week12(PW 12)to produce F2 and F3 generations respectively.Blood and adrenal tissues of F1,F2 and F3 generations of rats were collected after sacrifice under anesthesia in PW 8 for relevant detection.Meanwhile,some female rats of F1 and F2 generations were were subjected to superovulation treatment in PW 12 to obtain oocytes for relevant detection.In human adrenal cortical cell lines,we treated cells with dexamethasone at 500 n M for 24 h and then conducted relevant detection,and further intervened the key molecules(GR/H19/let-7b)in the regulatory pathway to confirm the hypothesis we proposed.Result(1)Fetal weight,adrenal H19/let-7c axis related pathway changes and corticosterone synthesis inhibition: compared with control group,PDE female fetal rats had lower birth weight,higher IUGR rate,and significantly lower m RNA and protein expression of adrenal St AR(P<0.05,P<0.01),while corticosterone level in PDE fetal serum were significantly reduced(P<0.01).In addition,the adrenal GR expression was increased(P< 0.01),and the CTCF expression was decreased(P<0.05)in females PDE rats.Meanwhile,the female PDE fetal rat adrenal H19 showed hypermethylation and low expression accompanied by DNA methylase3a/3b(Dnmt3a/3b)increased,while let-7c expression was significantly increased(P<0.05,P<0.01).(2)Verify the mechanism in vitro: compared with control group,dexamethasone significantly increased the expression of GR in human adrenal cells(P<0.05),and decreased the expression of CTCF(P<0.05).H19 was overexpressed by hypermethylation and accompanied by increased Dnmt3 a expression(P<0.05),and the expression of let-7b was significantly increased(P<0.01)after treated by dexamethasone.The above changes ultimately led to the inhibition of the expression of St AR(P<0.05).After GR interference,the expression of downstream CTCF/H19/Dnmt3a/let-7b/St AR was reversed(P<0.05,P<0.01).When let-7b inhibitor or H19 overexpression vector were co-treated with dexamethasone respectively,the inhibition of St AR by dexamethasone in human adrenal cells returned to the normal level(P=0.09,P<0.01).(3)Adrenal H19/let-7c axis related pathway alterations and corticosterone synthesis inhibition persisted until postnatal: there was no significant difference in body weight between control and PDE group in F1 adult female rats.However,compared with the control group,the female PDE rats adrenal CTCF was decreased(P<0.05,P<0.01),H19 showed hypomethylated and low expression(P<0.05,P<0.01),while adrenal let-7c expression in female PDE rats was significantly increased(P<0.01).These ultimately led to a significant reduction in adrenal St AR expression and serum corticosterone levels in PDE female rats.(P<0.05).(4)Intergenerationgnal inheritance of adrenal H19/let-7c axis related pathway alterations and corticosterone synthesis inhibition: there was no significant difference in body weight between F2 control and PDE adult rats.However,compared with the control group,the expression of CTCF in both male and female rats of PDE was decreased(P<0.05,P<0.01),and H19 showed hypermethylation low expression(P<0.05,P<0.01).Adrenal let-7c expression in male PDE rats was not significantly changed,while that in female PDE rats was significantly increased(P<0.05).The expression of adrenal St AR and serum corticosterone level in both male and female PDE rats were significantly decreased(P<0.05,P<0.01).(5)Transgenerationgnal inheritance of adrenal H19/let-7c axis related pathway alterations and corticosterone synthesis inhibition: there was no significant difference in body weight between F3 control and PDE adult rats.However,compared with the control group,the expression of CTCF in both male and female rats of PDE was decreased(P=0.07,P<0.05),and H19 showed hypermethylation low expression(P=0.07,P<0.05).Adrenal let-7c expression in male PDE rats was not significantly changed,while that in female PDE rats was significantly increased(P<0.01).The expression of adrenal St AR and serum corticosterone level in both male and female PDE rats were significantly decreased(P<0.05,<0.01).(6)H19/let-7c were delivered via oocyte: compared to the control group,the expression of H19 in the F1/F2 PDE oocytes was decreased(P<0.05,P<0.01),and the expression of let-7c was increased(P<0.05,P<0.01).Conclusion PDE inhibited the corticosterone synthesis of adrenal in IUGR offspring and has multigenerational effect.Its intrauterine mechanism may be related to dexamethasone-induced hypomethylation and low expression of adrenal H19,high expression of let-7c,and eventually programming low expression of St AR.The intrauterine epigenetic programming of H19/let-7c regulatory axis mediated the adrenal insufficiency to adulthood in F1 generation,and then to the F2/F3 generation via oocytes,which may be the reason of the inhibition of adrenal St AR expression and the corticosterone synthesis in the PDE progeny to be inherited through multiple generations of maternal lines.And this study provides new experimental basis and directions for the multi-generational inheritance of adrenal developmental toxicity induced by adverse environmental conditions during pregnancy.