| BACKGROUND AND OBJECTIVEGestational Diabetes Mellitus(GDM)is an important subtype of diabetes,which means women who did not have diabetes before pregnancy have different degrees of glucose metabolism during pregnancy and have symptoms of high blood sugar.GDM is one of the most common complications of pregnancy.Due to differences in population characteristics of different regions,previous countries or regions lacked consensus and uniformity in the screening and diagnosis of GDM,and the diagnostic criteria for GDM also varis over the years.GDM in different countries or regions worldwide is about 3%～30%.The exact pathogenesis of gestational diabetes has not been clarified.Studies have found that factors such as insulin sensitivity,obesity,and genetic factors in pregnant women are related to the incidence of gestational diabetes.Multiple studies have shown that overweight or obesity,advanced age,family history of type 2 diabetes,race,history of gestational diabetes,and genetic factors are all risk factors for gestational diabetes.Women with gestational diabetes need to control blood sugar,otherwise it will cause great harm and impact to the health and development of pregnant women and their fetuses.Women with gestational diabetes have an increased risk of metabolic syndrome,cardiovascular disease,preterm birth,miscarriage,cesarean section,postpartum hemorrhage,infection and an increased incidence of hypertension(including preeclampsia and eclampsia)throughout pregnancy.They also have a high recurrence rate of GDM when they pregnancy again,and may develop type 2 diabetes in the long time.Women with gestational diabetes are more likely to have respiratory distress syndrome and fetal growth restriction,and even congenital malformations and stillbirths.For neonates with gestational diabetes,hyperglycemia caused by hyperglycemia during pregnancy can cause shoulder dystocia,fractures,neonatal ischemic hypoxic encephalopathyand even death.The offspring of women with gestational diabetes have a significantly increased risk of obesity,overweight,and type 2 diabetes.Complications caused by gestational diabetes have a huge health impact on pregnant women,fetuses and newborns.Studies have shown that these complications may be related to abnormal placenta during pregnancy.The placenta,which originates from extra-embryonic trophoblast cells,is a highly specialized organ.It is an important organ for the mother and fetus to exchange material.The placenta can anchor the fetus to the uterus during uterine and embryonic development,and can establish a proper exchange cycle between the mother and the fetus,including oxygen/carbon dioxide exchange,nutrients exchange and removal of waste.The fetus receives nutrients from the mother through the placenta to maintain its normal development in the womb.Moreover,the placenta can serve as a protective barrier against xenobiotics by synthesizing and releasing a variety of steroids,hormones,enzymes and cytokines.Placental dysregulation or dysfunction can cause a variety of pregnancy complications,such as preeclampsia,premature birth,restricted fetal growth,repeated abortions and stillbirth.Placenta originates from the proliferation,differentiation,and invasion of trophoblasts in the periphery of the embryo sac cavity.The underlying of pathological of placenta in diabetic pregnant women is not clear,but some evidence indicates that the trophoblast proliferation,apoptosis and cell cycle control have changed.Inhibiting the apoptosis of trophoblast cells in women with gestational diabetes is expected to ensure that the fetus gets sufficient nutrition and avoid adverse pregnancy outcomes such as abortion and fetal death.miRNA is an endogenous non-coding single-stranded small RNA with a variety of biological functions.It can regulate the expression of target genes by binding to mRNA of the target gene,thereby participating in the regulation of various cellular processes in the organism,including cell differentiation,proliferation,autophagy and apoptosis.Studies have found that miRNA is closely related to pancreatic development,insulin production and secretion,and insulin resistance,and may play an important role in the pathogenesis of diabetes.Gestational diabetes shares similar incidence and genetic factors with type 2 diabetes,so miRNAs may also play a role in the pathogenesis of gestational diabetes.A study collected peripheral blood of gestational diabetes patients and healthy pregnant women to exploring miRNA expression levels in gestational diabetes patients by miRNA chip.The results showed that a series of miRNAs significantly differential expressed between the two groups.Among them,miR-193 b is one of the most down-regulated miRNAs in patients with gestational diabetes.The evolutionarily conserved miR-193b belongs to the miR-193 family and is located on chromosome 16(chr16:14303967-14304049).It was found that miR-193b can promote autophagy and non-apoptotic cell death of esophageal cancer cells.Down-regulation of miR-193b-3p can also promote autophagy and cell survival of NSC-34 cells.However,the role of miR-193b in autophagy and apoptosis in trophoblasts needs further explanation.In this study,miR-193b was selected as the research object,to explore the effects of miR-193b and its target genes on autophagy,proliferation and apoptosis of high glucose-treated trophoblast HTR8 cells were studied.RESEARCH METHODS:1.Peripheral blood and part of placenta tissues from 20 gestational diabetes patients and 20 normal pregnant women were collected clinically.2.The expressions of Mir-193 b and IGFBP5 in peripheral blood and placenta were detected by immunofluorescence and RT-PCR.3.Human chorionic trophoblast cells(HTR8 cells)were cultured with high concentration of glucose(25 mmol/L glucose)to simulate the diabetic environment in vitro.4.RT-PCR was used to detect the expressions of Mir-193b and IGFBP5 in HTR8 cells cultured with high glucose.5.Dual luciferase assay was used to detect the direct downstream target genes of Mir-193b.6.The expression of autophagy and apoptosis-related proteins in HTR8 cells was detected by Western-bloD.7.Proliferation of HTR8 cells in each group was detected by CCK-8 assay and Edu staining,and apoptosis of HTR8 cells was detected by flow cytometry.RESULTS:1.Down-regulation of Mir-193b leads to significantly enhanced autophagy and apoptosis in HTR8 cells treated with high glucose。1.1 Results showed that compared with the healthy pregnant women,miR-193b was significantly down-regulated in GDM patients.1.2 We then cultured human trophoblast cells HTR8 with high concentration(25 mmol/L glucose)glucose to simulate the diabetic environment in vitro.It was found that the expression of miR-193b in HTR8 cells treated with high glucose was also significantly down-regulated,and cell autophagy was significantly enhanced.1.3 By transfecting miR-193b mimics and inhibitors into high-glucose-treated HTR8 cells,we found that down-regulation of miR-193b exacerbates autophagy levels in high-glucose-treated HTR8 cells.1.4 In order to study the effect of cell autophagy on the proliferation and apoptosis of HTR8 cells,we added 3-MA,an autophagy inhibitor,to HTR8 and then detected the cell proliferation and apoptosis.The results showed that HTR8 cell proliferation was inhibited and apoptosis increased in HTR8 cells treated with high glucose.Attenuation of cell autophagy could promote HTR8 cell proliferation and inhibit apoptosis.At the same time,enhancing miR-193b expression or adding 3-MA can reverse the effect of miR-193b down-regulation on HTR8 cell apoptosis.2.miR-193b through targeted IGFBP5 sugar induced inhibition of sertoli cell autophagy and apoptosis.2.1 To further study the mechanism of miR-193b’s effect on the autophagy,proliferation and apoptosis of high glucose treated HTR8 cells,we first used miRBase database to predict the downstream target gene of miR-193b,and insulin-like growth factor-binding protein 5(IGFBP5)was identified as one of the downstream target genes of miR-193b.To verify this finding,we first detected the expression of IGFBP5 in GDM patients,and found that the expression level of IGFBP5 in GDM patients was significantly increased.Then our further in vitro studies showed that IGFBP5 is a direct downstream target gene of miR-193b,and miR-193b can negatively regulate IGFBP5.2.2 To study the role of miR-193b-targeted IGFBP5 in high glucose-induced autophagy of HTR8 cells,we designed and synthesized IGFBP5 siRNA(si-IGFBP5),and transfected si-IGFBP5 alone or with miR-193b inhibitor to HTR8 cells treated with high glucose to detect the effects of IGFBP5 expression on autophagy,proliferation and apoptosis of HTR8 cells.The results showed that silencing IGFBP5 can inhibit autophagy induced by high glucose in HTR8 cells,while inhibiting miR-193b expression in HTR8 cells can reduce this effect.Down-regulation of miR-193b in high-glucose-treated HTR8 cells can lead to the increasing expression of IGFBP5,thereby inhibiting cell proliferation and promoting apoptosis.2.3 In order to further confirm the pro-apoptotic effect of downregulation miR193b-IGFBP5 pathway was induced by the pro-autophagic effect of IGFBP5 in HG-treated HTR-8 cells,we silenced IGFBP5 and induced autophagy using RAPA on the basis of si-IGFBP5 in HG-treated HTR-8 cells,and conducted CCK-8,Edu staining,flow cytometry and western blot to detect cell proliferation and apoptosis.Results showed that activating autophagy eliminates the alleviation effect of si-IGFBP5 on HG-induced autophagy and apoptosis in HTR8 cells.CONCLUSIONS:1.Compared with healthy pregnant women,the expression level of Mir-193b in placental tissues and peripheral blood of GDM patients was significantly down-regulated.Meanwhile,the expression level of Mir-193b in HTR8 cells was also significantly down-regulated after high glucose treatment.Down-regulation of Mir-193b leads to significantly enhanced autophagy and apoptosis in HTR8 cells treated with high glucose,which can be reversed by enhanced expression of Mir-193b or additional use of autophagy inhibitor 3-MA.The results indicated that the down-regulation of Mir-193b expression in HTR8 cells treated with high glucose promoted autophagy,thereby inhibiting cell proliferation and promoting cell apoptosis.2.MiRBase database predicted that IGFBP5 was one of the downstream target genes of Mir-193b.The expression levels of IGFBP5 in placental tissues and peripheral blood of GDM patients were significantly increased.In vitro studies such as RT-PCR,WB and double lucifase reporter assay demonstrated that IGFBP5 is a direct downstream target gene of Mir-193b,and Mir-193b can negatively regulate IGFBP5.3.Silencing IGFBP5 by Si-IGFBP5 can inhibit the autophagy and apoptosis of HTR8 cells induced by high glucose,while inhibiting Mir-193b can reverse the alleviating effect of Si-IGFBP5 on the autophagy and apoptosis of HTR8 cells induced by high glucose.Meanwhile,activation of autophagy reversed the inhibitory effect of Si-IGFBP5 on autophagy and apoptosis of HTR8 cells induced by high glucose.Our study revealed that down-regulation of miR-193b in high-glucose-treated trophoblasts leads to the up-regulation of IGFBP5,which can mediate autophagy in HTR8 cells and eventually trigger a large number of apoptosis events.The large number of apoptosis in trophoblast cells may prevent the exchange of nutrients or gases between the mother and fetus,which may be one of the main inducements of complications and adverse pregnancy outcomes during pregnancy in patients with gestational diabetes.Therefore,miR-193b may be a brand-new,useful and potential clinical treatment target to avoid complications and adverse pregnancy outcomes in patients with gestational diabetes. |
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