Effect Of Circadian Clock Gene Clock Induced By Hypoxia On Biological Behavior Of Trophoblast And Its Correlation With Pathogenesis Of Preeclampsia

Objective: preeclampsia is a multi-system progressive disease peculiar to pregnancy,which seriously affects the health of mother and infant.And it is one of the main reasons for the increase of maternal and perinatal mortality.Poor trophoblast invasion and impaired spiral arteriolar remodeling lead to placental ischemia and hypoxia are currently recognized causes of preeclampsia,but the specific pathogenesis is still unknown.Circadian clock genes are ubiquitous in the biological world,regulating our biological rhythms.It has been found that circadian clock genes may be related to hormone secretion and placental development during pregnancy.As the core gene of biological clock,clock is not only expressed in placenta with circadian rhythm,but also related to the invasiveness of many tumor cells.The invasion ability of trophoblast is similar to that of tumor cells,and the difference lies in its controllability.Some studies have proved that preeclampsia is related to trophoblast invasion and biological rhythm.We have reason to think that biological clock gene may play an important role in the pathogenesis of preeclampsia.In addition,it has been found that hypoxia response pathway in vivo is monitored by circadian clock genes.HIF-1 ? is similar to Clock protein in structure,and both of them belong to the basic-he-lix-loop-helix(b HLH)family.It is the core regulatory node connecting hypoxia regulation pathway and clock regulation.Therefore,we think that the circadian clock gene Clock induced by hypoxia may be involved in the pathogenesis of preeclampsia by regulating the biological behavior of placental trophoblasts.In this study,we investigated the relationship among Clock,hypoxia and preeclampsia by human placenta,Co Cl2 induced trophoblast hypoxia model and reduced uterine perfusion pressure(RUPP)rat models.Methods: 1.From August 2017 to December 2018,70 pregnant women delivered in Shengjing Hospital of China Medical University were selected as the experimental objects(20 cases in the early-onset preeclampsia group,10 cases in the early-onset control group,20 cases in the late-onset preeclampsia group,20 cases in the late-onset control group).The placenta tissues of them were collected and processed.RT-q PCR,western blot and immunohistochemistry were used to detect the expression of Clock m RNA and protein in placenta of four groups.2.HTR-8/SVneo,a sub cultured stably trophoblast cell line,was selected for the experiment.The optimal concentration of Co Cl2 was determined by detecting the expression of HIF-1 ?.The circadian clock genes in trophoblasts were synchronized by serum shock.The expression and the circadian rhythm within 96 hours after hypoxia of Clock m RNA and protein were detected.The migration,invasion and proliferation ability of trophoblasts in hypoxia group and control group were detected by wound healing assay,transwell assay and MTT assay.The si-Clock interference sequence was introduced into the hypoxic model of trophoblasts.The changes of cell migration,invasion and proliferation ability were detected to verify the reversal effect of si-Clock on hypoxia.3.The RUPP rat model was established by constricting the abdominal aorta and one side of the uterine artery.In the control group,the arteries were isolated but not constricted.The placentas of the RUPP rat model group and the control group were collected.The expression of Clock m RNA and protein was detected by RT-q PCR,western blot and immunohistochemistry.The changes of blood pressure and urine protein in RUPP rat models were detected after injecting lentivirus into the placenta,and the reversal effect of Clock on RUPP rats was verified.4.The synchronized hypoxic trophoblasts and control trophoblasts were collected.The degree of sumoylation of Clock protein was detected by immunoprecipitation(IP).In the same way,the changes of sumoylation of Clock protein in placentas of RUPP rat model group and the control group were detected.Results: 1.RT-q PCR showed that the expression of Clock m RNA in placenta of early and late onset preeclampsia was significantly higher than that of the corresponding control group.While western blot and immunohistochemistry showed that the expression of Clock protein was significantly lower than that of the control groups.2.HTR-8/SVneo was cultured in medium containing Co Cl2 with final concentrations of 0,50,100,200,300 and 400 ? M,respectively.It was found that the expression of HIF-1 ? protein in 200 ? M group was the most significant.It is suggested that Co Cl2 can induce hypoxia in cells,and the optimal concentration is 200 ? M.After hypoxia,the expression of Clock m RNA increased,while the expression of protein decreased.The expression of Clock m RNA was rhythmic,and the rhythm became shorter after hypoxia.But the expression rhythm of protein was not obvious within 96 hours.The biological behavior of HTR-8/SVneo cells was compared with that of the control group.The results showed that the ability of migration,invasion and proliferation ability of trophoblast cells decreased after hypoxia.The results showed that the ability of migration,invasion and proliferation of si-Clock group were improved.3.The expression of HIF-1 ? in placenta of RUPP model rats was significantly higher than that of the control group.The expression of Clock m RNA in placenta of RUPP model rats was significantly lower than that of the control group,and the protein was significantly higher than that of the control group.The changes of blood pressure,24-hour urinary protein and placental absorption rate in preeclampsia rats were significantly improved after the expression of Clock was decreased by injecting lentivirus into placenta.4.The sumoylation of Clock protein in hypoxic trophoblasts was significantly higher than that in control group.The sumoylation of Clock protein in placenta of RUPP model rats was significantly lower than that of control rats.Conclusion: 1.The expression of Clock m RNA and protein in placenta of early and late onset preeclampsia is increased.Clock may be related to the pathogenesis of preeclampsia.2.It may be that hypoxia induces the expression and rhythm change of Clock and affects the biological behavior of trophoblasts.3.Clock may be involved in the process of trophoblast invasion and placenta development in rats,and affect the changes of blood pressure and urinary protein.4.Sumoylation may affect the stability of Clock protein,resulting in the inconsistent expression of Clock m RNA and protein.