| Background:Preeclampsia(PE)is one of the most serious complications of pregnant women.It is one of the importaant causes of perinatal mortality,which seriously threatens the health of mothers and infants.The incidence rate of PE is about 2-8%,and China has a higher incidence of 9.4%.Clinically,maternal syndrome is associated with hypertension after 20 weeks of pregnancy(such as proteinuria,liver and kidney dysfunction)and fetal syndrome(fetal growth restriction or oligohydramnios).According to the onset of gestational age,they were divided into early-onset preeclampsia(<34 weeks)and late-onset PE(?34w).Early onset preeclampsia often progresses rapidly,more likely to produce serious maternal and infant complications[4],and even placental abruption,fetal death and other adverse outcomes.As for preeclampsia,the general clinical monitoring means are only sedation,spasmolysis,blood pressure reduction,correction of hypoproteinemia and other symptomatic treatment.The definite treatment method is still termination of pregnancy.At the same time,it brings a series of short-term and long-term complications such as premature delivery.Therefore,it is very important to predict the incidence of preeclampsia as soon as possible,to prevent it as soon as possible,and to intervene and treat it effectively.PE has a complex pathogenesis,which has not been fully clarified.At present,the generally accepted theory is that "the ability of trophoblast infiltration is impaired,the remodeling of uterine spiral artery is impaired,resulting in shallow placental implantation".According to the theory,in the early stage of placental formation in preeclampsia patients,abnormal proliferation and differentiation of trophoblasts,increased apoptosis and decreased activity of trophoblasts led to decreased invasion of trophoblast and limited invasion of uterine muscle wall,which led to poor remodeling of uterine spiral arterioles and shallow implantation of placenta,resulting in low blood flow of placenta,which led to the onset of preeclampsia.The oxidative stress induced by hypoxia and ischemia is one of the mechanisms.KISS1 gene,located in 1q32-q41 region of long arm of chromosome 1,was first discovered by Lee et al.[6]in 1996 when using subtractive hybridization and differential display technology to study the different metastatic ability of human melanoma cell lines.KiSS1 gene is a tumor metastasis suppressor gene isolated from non metastatic malignant melanoma[7,8].KiSS1 can inhibit cell infiltration and migration in vitro and in vivo[9,10].In existing studies,KiSS1,as a tumor suppressor gene,It significantly affects the formation and development of tumors,such as liver cancer(HCC)[11],breast cancer(BC)[12],and colorectal cancer(CRC)[13].Trophoblast cells are a major component of placental tissues and significantly affect the whole pregnancy.Trophoblasts form villous trophoblast and extravillous trophoblast(evts)with the development of pregnancy.Villous trophoblast cells can form chorionic villi,whose function is to transport nutrients to the fetus;while extravillous trophoblasts invade decidual tissue,invade vascular endothelium and reconstruct spiral artery,resulting in the loss of contractility of spiral artery,so as to make enough maternal blood flow into the villi,so as to maintain the normal physiological function of placenta.The characteristics of trophoblast lie between normal cells and tumor cells.The invasion and migration of normal trophoblast cells are very important for the development of placenta.The changes of these functions of trophoblast in early stage will lead to the changes of blood flow in uterus and placenta,and affect the structure and function of placenta.Studies have shown that KiSS1 is highly expressed in human placental trophoblast cells,which may affect the proliferation and infiltration of trophoblast cells[14].Moreover,KiSSl expression increased in preeclampsia placenta,which inhibited trophoblast infiltration during placental development[15].This leads to a series of pregnancy complications caused by placental shallow implantation.Therefore,we speculate that in the pathogenesis of preeclampsia,the abnormal epigenetic regulation of KiSS1 gene leads to the change of gene expression,and then affects the biological behavior of trophoblast,which may be involved in the pathogenesis of preeclampsia.The purpose of this study is to explore the molecular mechanism of KiSS1 gene expression in preeclampsia.Part ? the expression of KiSS1 gene in placenta of patients with preeclampsiaObjective:To investigate the expression status of KISS1 and related proteins in the placenta of PE patients and normal women in the third trimester of pregnancy,and to investigate the expression characteristics of KISS1 gene in the placenta of PE patients.Methods:1.Participants:The study was conducted in in the Affiliated Hospital of Qingdao University,Qingdao,Shandong,China,from January 2017 to January 2018.28 patients with severe preeclampsia and delivered by cesarean section were selected as preeclampsia group(PE group,n=28),Thirty women with normal pregnancy and elective cesarean section during the same period were assigned to the normal control group(NC group,n=30).2.experimental methods:The expression level of KISS1 mRNA in placental tissues of severe PE patients and normal women in the third trimester of pregnancy was determined by QRT-PCR,and the expression of KISS1 protein was measured by WestemBlot(WB)technique.Results:1.Comparison between PE group and NC group,In terms of age,body mass index and parity between severe preeclampsia group and normal pregnancy group(P>0.05).There were significant differences in SBP,gestational weeks,DBP,24-hour urine protein volume and birth weight of newborns(P<0.05).2.Qrt-pcr showed that KISS1mRNA expression in placental tissues was significantly higher in PE group than in NC group(P<0.05).3.Compared with the NC group,the PE group was significantly higher in the expression of KISS1 in placental tissues by WB.Conclusion:1.KISS 1 gene and related protein were expressed in normal pregnant women and severe preeclampsia patients.2.The expression levels of KISS 1 gene and related proteins in severe preeclampsia were higher than those in normal pregnant women.Part ? Knockout of KISS1 gene has protective effect on human placental trophoblast injury under oxidative stress by regulating PI3K/Akt1 mTOR signaling pathwayObjective:To establish a trophoblast oxidative stress model by pretreating HTR8/Svneo cells with 250?mol hydrogen peroxide for 4 hours to simulate the oxidative stress environment of preeclampsia placenta,and to study the changes of autophagy,apoptosis and biological behavior of trophoblast.After transfection with si-KISS 1 and 3-MA,the above biological processes were reevaluated to determine the effect of KISS1 knockout on hydrogen peroxide injured HTR8/Svneo cells.Detecting the phosphorylation of PI3K/Akt/mTOR signaling pathway is to explore its mechanism,so as to provide new theoretical for early prediction and effective treatment of preeclampsia.Methods:HTR8/Svneo trophoblast cells were pretreated with 250?mol hydrogen peroxide for 4 hours.KISS1 by qRT-PCR technique,gene,as well as Beclinl,LCII these 2 types of autophagy to determine the expression of related gene(ATG),through the WB method was developed for the determination of related proteins expression,using method(CCK8)used in the determination of the cell vitality,Transwell invasion experiment and flow cytometry(FCM),and the detection of hydrogen peroxide intervention group and normal group HTR8 cell autophagy changes and biological behavior(ability)the cell vitality,and apoptosis.After transfection with siRNA and 3-mA(selective PI3K inhibitor),the above biological processes were reevaluated to determine the effect of KiSS1 silencing on hydrogen peroxide injured htr8 cells.The protein levels of p-pi3k,p-Akt and p-mTOR related genes in PI3K/Akt/mTOR pathway were detected by Western blot,and the possible mechanism was speculated.Results:1.qRT-PCR showed that htr8/svneo cells had excessive autophagy under oxidative stress.Compared with the NC group,H2O2 treatment enhanced the mRNA expression levels of Beclin-1 and LC3B,two types of autophagy markers(P<0.01).Similarly,Western.blot analysis also revealed that beclin-1 and lc3b protein levels in htr8 cells were increased after hydrogen peroxide treatment(P<0.01).The difference was statistically significant.2.CCK8 cell viability test,Transwell experiment and FCM showed that the HTR8 cell activity of H2O2 group was significantly lower than that of NC group(P<0.01),the invasiveness was significantly decreased(P<0.01),and apoptosis was induced(P<0.01).Western blot analysis showed that MMP-9 protein level decreased(P<0.1),while Bax and caspase-3 cleavage increased(P<0.001),which promoted the occurrence of apoptosis.The differences were statistically significant.3.Qrt-pcr and WB assay showed that KISS1 mRNA level of the hydrogen peroxide intervention group was higher than that of the NC group(P<0.01).KiSS1 protein level also increased.These results indicate that the expression of KISS1 in HTR8 cells is increased under oxidative stress.The difference was statistically significant.4.The mRNA and protein levels of KISS1 in HTR8 cells were decreased,compared with Si-NC transfection group(P<0.01).The difference was statistically significant.5.Beclin-1 and LC3B levels were lower in siKISSl+H2O2+HTR8 group than in H2O2+HTR8 group(P<0.001),and the levels of beclin-1 and lc3b in 3mA+H2O2+htr8 group were lower than those in H2O2+htr8 group(P<0.001).The difference was statistically significant.6.The results of CCK8 cell viability test,Transwell test and flow cytometry showed that compared with H2O2+htr8 group,sikiss1+H2O2+htr8 group had higher cell viability(P<0.001),stronger invasion ability(P<0.001),higher invasion related protein(MMP9)and expression level of apoptotic related protein(cleaved caspases 3,P<0.001),Bax decreased(P<0.001).Similar results were found in 3mA+H2O2+htr8 group.7.Western blot analysis showed that the expression levels of P/t-pi3k,P/t-akt and P/t-mtor in PI3K/Akt/mTOR signaling pathway in h2o2-htr8 group were lower than those in htr8 group,indicating that PI3K/Akt/mTOR signaling pathway was inhibited under oxidative stress,and the results were statistically significant(P<0.001).8.Western blot showed that the protein expression levels of P/t-pi3k,P/t-akt and P/t-mtor in 3mA+h2o2-htr8 group were significantly higher than those in H2O2-htr8 group(P<0.001).9.Western blot showed that the protein expression levels of P/t-pi3k,P/t-akt and P/t-mtor in Si+KiSS1+H2O2 group were significantly higher than those in h2o2-htr8 group(P<0.001).Conclusion:1.After the intervention of hydrogen peroxide,the expression of KISS1 in trophoblasts(H2O2+htr8)was increased,and the expression of autophagy related genes and proteins was higher than that of normal trophoblasts.2.H2O2+htr8 decreased the activity and invasiveness of trophoblast(H2O2+htr8)under oxidative stress,and promoted cell apoptosis.3.Silencing KiSS1 and 3-mA could reverse the damage of htr8 trophoblast induced by hydrogen peroxide.It can inhibit excessive autophagy,It promotes the invasion and proliferation of human chorionic trophoblast cells and inhibits apoptosis under the state of oxidative stress.The mechanism of action may be the regulation of PI3K/AKT/mTOR,a classic autophagy signaling pathway.Trophoblast is the main part of placental formation.Therefore,we speculate that KiSS1 may participate in the pathogenesis of preeclampsia by regulating PI3K/Akt/mTOR signaling pathway. |
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