| BackgroundSmall for gestational age（SGA）,defined as a birth weight less than the 10th percentile for the same gestational age,occurs in 2.3%to 10%of cases worldwide and 6.5%of cases in our country.The underdevelopment of all organs of SGA infant can lead to a significantly higher incidence of neonatal respiratory distress syndrome and intracranial hemorrhage,and increase neonatal morbidity and mortality.SGA can cause adverse effects in both the near-term and long-term development of the fetus,resulting in a heavy burden on social and family.The etiology of SGA is complex and diverse,and maternal factors,fetal factors,placental umbilical factors,etc.,may explain about 60%of SGA etiologies,while 40%of SGA etiologies are unknown.At present,most studies on the etiology of SGA are based on European and American populations.The large sample size of studies on the etiology of SGA in China are lacking,and the specific influencing factors still lack high-quality evidence support,while studies on the etiology of idiopathic SGA are still in the stage of exploration.Inadequate etiological management poses major challenges for SGA prevention and control.Existing studies suggest that only about 2%of the sequence in the human genome is capable of encoding proteins,and the mechanism of action of the large number of noncoding RNAs remains poorly understood.Among the current studies on noncoding RNAs,the study of microRNAs（miRNAs）is relatively mature,but the study of long non coding RNAs（lncRNAs）is still insufficient.Long noncoding RNAs,a class of RNAs greater than 200 nt in length that are transcribed from the genome but not translated into proteins,function to regulate the expression of genes encoding proteins.circular RNAs（circRNAs）are also a member in lncRNA family,which have multiple biological functions and played an important role in the occurrence and development of diseases.Effective migration and invasion of trophoblasts in the placenta and proliferative function are key factors in maintaining normal placental function,and when trophoblast differentiation is dysplastic,there is insufficient erosion into the uterine spiral arteries,resulting in poor uteroplacental vascular recasting and shallow placental implantation,followed by placental hypoperfusion,which triggers SGA.A large number of noncoding RNAs are expressed in the human placenta and are involved in regulating trophoblast cell proliferation,apoptosis,migration,and invasion,suggesting that noncoding RNAs,including circRNAs,may be important factors in triggering SGA.In this paper,we conducted a meta-analysis and retrospective analysis to explore the factors involved in the occurrence of SGA.We used the identified influencing factors as references to determine the inclusion and exclusion criteria of idiopathic SGA,then sequenced idiopathic SGA samples,verified them and conducted cellular animal experiments to explore the mechanism of circRNA triggering disease.Part one Influencing factors of small for gestational age:a meta-analysis and retrospective studyObjectiveConducting meta-analysis and retrospective study on the influencing factors of small for gestational age,focusing on etiological management of small for gestational age infants and developed appropriate interventions to prevent the occurrence of small for gestational age infants.Methods1.Searching the Chinese and English studies published until 2018.12.28 about the influencing factors of SGA,screening the literature,extracting data,Stata 12.0 software was used to perform the meta-analysis of each influencing factor.2.The data of 81939 neonates with a singleton delivery from January 1,2013 to December 31,2017 from（Examines Anonymous）maternity and child health care hospital were retrospectively analysised to provide a statistical description of the general clinical characteristics of the study subjects,using mean ± standard deviation to describe continuous type variables that fit a normal distribution,and median,25th and 75th percentiles to describe continuous type variables that do not follow a normal distribution with respect to gestational weight and length quantiles were described;categorical variables were described by frequency（n）and compositional ratio（%）.For continuous variables fetal weight and length,the rank sum test was used,and for categorical variables,the x 2 test was used to analyze the difference between groups.Univariate and multivariate logistic regression were used to analyze the association between small for gestational age infants and early and late onset of preeclampsia.Results1.The meta-analysis included 31 articles,including 11 case-control studies and 20 cohort studies.The results of the meta-analysis showed that low age,leanness,lack of gestational weight growth,short stature,low educational level,low family income,history of preterm delivery,abortion,stillbirth,smoking,passive smoking,alcohol consumption,hypertensive disorders of pregnancy,preeclampsia,antepartum hemorrhage,and abnormal umbilical cord,the OR and 95%CI were 1.23（1.11-1.36）、1.59（1.40-1.81）、1.43（1.35-1.50）、1.51（1.36-1.69）、1.64（1.39-1.93）、1.28（1.09-1.49）、1.29（1.01-1.65）、1.21（1.07-1.36）、3.17（2.14-4.70）、1.86（1.56-2.22）、1.75（1.15-2.64）、1.32（1.00-1.74）、2.37（1.76-3.19）、3.08（1.61-5.88）、3.48（1.66-7.33）、3.76（2.14-6.61）.2.A total of 3996 cases were SGA,with an incidence of 4.88%.There were 294 cases（7.40%）of preeclampsia in the SGA group and 2839 cases（3.60%）of preeclampsia in the non SGA group,which was statistically different（P<0.05）.3.Significant differences（P<0.05）were observed between the early-onset preeclampsia and non preeclampsia groups for all included factors:age,education,BMI,parity,gravidity,household registration status,whether the infant had CHD,whether the infant was SGA,mode of delivery,and gestational age at delivery.There were significant differences between the late-onset preeclampsia group and the non preeclampsia group in any of the included factors except for whether the infant had preeclamptic factors（P<0.05）.All the included factors were significantly different（P<0.05）except for age,BMI and parity,which were not significantly different between the early-onset preeclampsia group and late-onset preeclampsia group.SGA occurred significantly more frequently in early-onset preeclampsia than in late-onset preeclampsia.4.Multivariate logistic regression analysis was performed between each factor and SGA.The risk of developing SGA was found to be higher in the case of migrant,infant with CHD,pregnancy with preeclampsia and midwifery delivery,with hazard coefficients of 1.248,2.189,1.59,1.921（P<0.05）,respectively,whereas the risk of developing SGA was lower in the case of second birth,multiple birth and later gestational age at delivery.Conclusions1.Maternal and fetal factors and placental cord factors that may contribute to the development of SGA include low age,advanced age,short stature,leanness,lack of gestational weight gain,history of preterm delivery,miscarriage,history of stillbirth,gestational hypertension,preeclampsia,antepartum haemorrhage,low education,low family income,primiparity,fetal factors and placenta umbilical among the cord factors,umbilical abnormalities are risk factors for the development of SGA.2.Early-onset preeclampsia triggers a higher risk of SGA development than late-onset preeclampsia.Part Two Expression of circRNAs in placenta from idiopathic small for gestational age infant and effects of hsacirc0035897 on biological behavior of HTR-8/SV neo and JEG-3 cellsObjectiveTo screen and identify differentially expressed circular RNA in placental tissues of idiopathic small for gestational age infants（SGA）.To determine the effects of hascirc0035897（abbreviated as circ-0035897）on the proliferation,migration,invasion and apoptosis of human chorionic trophoblast cell line HTR-8/svneo and human choriocarcinoma cell line JEG-3.Methods1.High throughput sequencing of circular RNA was performed in 3 idiopathic SGA placental tissues and 3 normal control placental tissues to establish the expression profile of circular RNA in idiopathic SGA placental tissues.2.Real-time quantitative PCR was used to detect the expression of candidate differentially expressed circRNAs in 32 idiopathic SGA placental tissues and 32 control placental tissues.3.Total RNA and genomic DNA were extracted from placental tissues,and circ-0035897 and homologous linear RNA relative abundance expression changes was detected by real-time quantitative PCR after RNase R treatment,and sequencing was performed to verify the cyclization sites.4.The expression of circ-0035897 in placental exosomes from maternal serum of SGA and normal control group by qRT-PCR.5.To establish circ-0035897 overexpression and knockdown cell model,we construct circ-0035897 overexpression and knockdown vector and transfect them into HTR-8/svneo and JEG-3 cells.6.The effects of circ-0035897 overexpression and knockdown on the proliferation of HTR-8/svneo and JEG-3 cells were detected by MTS.7.Transwell assay was used to detect the effects of overexpression and knockdown of circ-0035897 on the migration and invasion of HTR-8/svneo and JEG-3 cells,8.The effects of circ-0035897 overexpression and knockdown on the apoptosis of HTR-8/svneo and JEG-3 cells were detected by flow cytometry.9.qRT-PCR and western blot were used to detect the effects of circ-0035897 overexpression and knockdown on the expression levels of migration,invasion and apoptosis related genes in HTR-8/svneo and JEG-3 cells.Results1.Through high-throughput circular RNA sequencing and bioinformatics analysis,19 differentially expressed circRNAs were screened（log fold change≥1.5,P<0.05）,of which 9 were up-regulated and 10 were down-regulated.2.qRT-PCR showed that the expression of circ-0035897 in idiopathic SGA placenta was significantly lower than that in normal placenta（P<0.01）.3.Circular and linear circ-0035897 could be amplified by using back-to-back primers and opposite primers with total RNA not digested by RNase R as template;Using the total RNA digested by RNase R as the template,circular circ-0035897 can be amplified by back-to-back primers,but linear circ-0035897 can not be amplified by opposite primers;Using genomic DNA as template,circular circ-0035897 could not be amplified by back-to-back primers,but homologous linear circ-0035897 could be amplified by opposite primers.It shows that circ-0035897 is a closed ring.4.The sequencing results showed the cyclization site of circ-0035897.5.qRT-PCR showed that the expression level of circ-0035897 in serum exosomes from placental in idiopathic SGA group was significantly lower than that in normal control group（P<0.01）.6.After HTR-8/svneo and JEG-3 cells were transfected with circ-0035897 overexpression（circ-0035897）and knockdown（circ-0035897 siRNA）vectors,qRT-PCR results showed that the expression level of circ-0035897 in circ-0035897 overexpression group was significantly increased（P<0.01）,while the expression level of circ-0035897 in circ-0035897 siRNA group was significantly decreased（P<0.01）.It shows that circ-0035897 overexpressed and knockeddown HTR-8/svneo and JEG-3 cell models are successfully constructed and can be used in subsequent experiments.7.MTS results showed that compared with the negative control group（NC or siRNA NC）,the proliferation levels of HTR-8/svneo and JEG-3 cells in circ-0035897 overexpression group were significantly higher（P<0.01）,while the proliferation levels of cells in circ-0035897 siRNA group were significantly lower（P<0.01）.It shows that circ-0035897 can significantly promote the proliferation of HTR-8/svneo and JEG-3 cells.8.Compared with the negative control group（NC or siRNA NC）,the migration and invasion levels of HTR-8/svneo and JEG-3 cells in circ-0035897 overexpression group were significantly higher（P<0.01）,while the migration and invasion levels of cells in circ-0035897 siRNA group were significantly lower（P<0.01）.It shows that circ-0035897 can significantly promote the migration and invasion of HTR-8/svneo and JEG-3 cells.9.Compared with the negative control group（NC or siRNA NC）,the levels of early apoptosis,late apoptosis and total apoptosis of HTR-8/svneo and JEG-3 cells in circ-0035897 overexpression group were significantly lower（P<0.01）,while the levels of early apoptosis,late apoptosis and total apoptosis in circ-0035897 siRNA group were significantly higher（P<0.01）.It shows that circ-0035897 can significantly inhibit the apoptosis of HTR-8/svneo and JEG-3 cells.10.Compared with the negative control group（NC or siRNA NC）,the expression levels of apoptosis related proteins Caspase3 and caspase9 in HTR-8/svneo and JEG-3 were significantly decreased（P<0.01）,the ratio of BCL2/Bax was significantly increased,and the expression levels of migration and invasion related proteins MMP2 and MMP9 were significantly increased in circ-0035897 overexpression group;In circ-0035897 siRNA group,the expression levels of apoptosis related proteins caspase 3 and caspase 9 increased significantly（P<0.01）,the ratio of BCL2/Bax was significantly decreased,and the expression levels of migration and invasion related proteins MMP2 and MMP9 were significantly decreased.It shows that circ-0035897 affects the biological behavior of HTR-8/svneo and JEG-3 cells by affecting the expression of apoptosis,migration and invasion related proteins.ConclusionThis experiment confirmed that the expression of circ-0035897 was significantly down-regulated in idiopathic SGA placenta,indicating that its reduced expression was related to the pathogenesis of SGA.Through experiments,the circular characteristics of circ-0035897 were verified,which laid a foundation for functional research and mechanism research.circ-0035897 significantly promoted the proliferation,migration and invasion of HTR-8/svneo and JEG3 cells,inhibited the level of apoptosis,and affected their biological behavior by affecting the expression of apoptosis,migration and invasion related proteins of HTR-8/svneo and JEG3 cells.Part Three Mechanism of hsacirc0035897 regulating biological behavior of HTR-8/svneo and JEG-3 cellsObjectiveTo clarify the relationship between circ-0035897,miR-450b-5p and PTPRB and to identify that circ-0035897 participates in the regulation of biological behavior of HTR-8/svneo and JEG-3 cells through ceRNA mechanism.Methods1.Prediction and verification of circ-0035897 targeted miR-450b-5p（1）Bioinformatics predicted the miRNA targeted of circ-0035897;（2）After overexpression or knockdown of circ-0035897,the expression of candidate miRNAs in HTR8/SVneo and JEG-3 cells was detected;（3）The localization of circ-0035897 and miR-450b-5p was determined by fluorescence in situ hybridization;（4）The binding of miR-450b-5p and circ-0035897 was detected by RNA immunoprecipitation;（5）The binding of circ-0035897 and miR-450b-5p was detected by dual luciferase reporter gene assay.2.Prediction and verification of miR-450b-5p target gene PTPRB（1）Overexpression or knockdown of miR-450b-5p detected the expression of candidate mRNA in HTR8/SVneo and JEG-3 cells;（2）miR-450b-5p was knocked down,and the expression of target gene ptprb protein in HTR8/SVneo and JEG-3 cells was detected by Western blot;（3）The binding of miR-450b-5p to the target gene ptprb was detected by double luciferase reporter gene experiment.3.Effect of circ-0035897 on PTPRB expression level（1）Overexpression or knockdown of circ-0035897.The expression of ptprb in htr-8/svneo and JEG-3 cells was detected by qRT-CR;（2）Western blot was used to detect the expression of PTPRB in HTR8/SVneo and JEG-3 cells;4.HTR-8/SVneo or JEG-3 cells were divided into three groups:NC+miRNA NC,circ-0035897+miRNA NC,circ-0035897+miR-450b-5p group.The expression level of circ-0035897,miR-450b-5p and PTPRB were detected by qRT-PCR and western blot.5.HTR-8/SVneo or JEG-3 cells were divided into three groups:NC+miRNA NC,circ-0035897+miRNA NC,circ-0035897+miR-450b-5p group.The effects of proliferation,migration,invasion and apoptosis of circ-0035897 on HTR-8/svneo and JEG-3 cells were detected by MTS method,transwell migration and invasion assay and flow cytometry.6.HTR-8/SVneo or JEG-3 cells were divided into three groups:NC+miRNA NC,circ-0035897+miRNA NC,circ-0035897+miR-450b-5p group.The expression levels of apoptosis,migration and invasion related genes in HTR-8/svneo and JEG-3 cells were detected by qRT-PCR and western blot.7.We construct PTPRB knockdown virus vector and evaluate the body weight of pregnant mice after tail vein injection.Results1.Prediction and verification of targeting relationship between circ-0035897 and miR-450b-5p（1）miRanda,Targetscan and Rnahybrid software were used to predict eight candidate miRNAs that can be combined with circ-0035897:miR-19b-1,miR-19b-2,miR-19a,miR-450b-5p,miR-9,miR-340,miR-765 and miR-302f.（2）Compared with the negative control group,the expression of miR-450b-5p in HTR-8/svneo and JEG-3 cells transfected with circ-0035897 overexpression vector decreased significantly（P<0.01）,and the expression of miR-450b-5p in cells transfected with circ-0035897 siRNA increased significantly（P<0.01）;（3）Fluorescence in situ hybridization showed that circ-0035897 and miR-450b-5p were mainly located in the cytoplasm of HTR-8/SVneo cells,indicating that circ-0035897 may play its regulatory role through ceRNA mechanism.（4）RNA immunoprecipitation assay showed that compared with miRNA NC group,the level of AGO2 bound circ-0035897 in HTR-8/SVneo cell group transfected with miR-450b-5p was significantly increased（P<0.01）.（5）The results of double luciferase report experiment showed that compared with miRNA NC group,the luciferase activity of co-transfected circ-0035897-WT and miR-450b-5p mimic group was significantly decreased（P<0.01）,while the luciferase activity of co-transfected circ-0035897-WT and miR-450b-5p inhibitor was significantly increased compared with inhibitor NC（P<0.01）.After mutating the binding site of circ-0035897-mut and miR-450b-5p,there was no significant change in luciferase activity compared with NC group.It indicates that there is a binding relationship between circ-0035897 and miR-450b-5p.2.Prediction and verification of targeting relationship between miR-450b-5p and PTPRB.（1）miRanda,targetscan and rnahybrid software were used to predict five candidate target genes that can bind to miR-450b-5p:DCAF5,PTPRB,CLOCK,PLCG2 and FAM107B.（2）In HTR-8/svneo and JEG-3 cells,compared with the negative control group,the expression of PTPRB mRNA in miR-450b-5p cells decreased significantly（P<0.01）,and the expression of ptprb mRNA and protein in miR-450b-5p inhibitor group increased significantly（P<0.01）.（3）After co-transfection of PTPRB-3’UTR and miR-450b-5p mimic,the luciferase activity decreased significantly compared with NC group（P<0.05）,while after co-transfection of circr-0035897 and miR-450b-5p inhibitor,the luciferase activity was significantly increased compared with inhibitor NC group（P<0.01）.After mutating the two binding sites of PTPRB-3’UTR and miR-450b-5p respectively,compared with NC group,the luciferase activity of co-transfected PTPRB-3’UTR mut and miR-450b-5p mic group decreased significantly（P<0.05）,and the luciferase activity of co-transfected PTPRB-3’UTR mut and miR-450b-5p mimic group increased significantly（P<0.01）;When the two binding sites were mutated at the same time,compared with NC group,there was no significant change in luciferase activity in co-transfected PTPRB-3’UTR mut,miR-450b-5p mimic or miR-450b-5p inhibitor group.It indicates that there is a binding relationship between miR-450b-5p and PTPRB-3’UTR.3.The cells were divided into three groups:NC+miRNA NC,circ-0035897+miRNA NC,circ-0035897+miR-450b-5p group.qRT-PCR results showed that compared with circ-003 5 897+miRNA NC group,the expression levels of circ-0035897 and ptprb in circ-0035897+miR-450b-5p group decreased significantly（P<0.01）,and the expression level of miR-450b-5p increased significantly（P<0.01）.4.MTS results showed that the proliferation level of HTR-8/SVneo and JEG-3 cells in circ-0035897+miR-450b-5p group was significantly lower than that in circ-0035897+miRNANC group（P<0.01）.5.The results of cell migration and invasion showed that in HTR-8/SVneo and JEG-3 cells,the migration and invasion levels of cells in circ-0035897+miR-450b-5p group were significantly lower than those in circ-0035897+miRNA NC group（P<0.01）.6.Flow cytometry showed that in HTR-8/SVneo and JEG-3 cells,the levels of early apoptosis,late apoptosis and total apoptosis in circ-0035897+miR-450b-5p group were significantly higher than those in circ-0035897+miRNA NC group（P<0.01）.7.qRT-PCR and western blot showed that in HTR-8/SVneo and JEG-3 cells,compared with circ-0035897+miRNA NC group,the expression levels of caspase3 and caspase9 in circ-0035897+miR-450b-5p group were significantly increased,the ratio of BCL2/Bax was significantly decreased,and the expression levels of MMP2 and MMP9 were significantly decreased（P<0.01）.8.The results of animal experiments showed that the birth weight of mice in PTPRB knockdown group decreased significantly（P<0.01）.Conclusioncirc-0035897 regulates the expression level of PTPRB in HTR-8/SVneo and JEG-3 cells via sponging miR-450b-5p. |
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