【作者】 刘杰；

【导师】 姚吉龙；

【作者基本信息】 山东大学 ， 妇产科学（专业学位）， 2021， 博士

【摘要】 研究背景小于孕龄儿（small for gestational age,SGA）定义为出生胎儿体重小于同孕龄第10百分位数,全球发生率为2.3%至10%,我国发生率6.5%。SGA胎儿各器官发育不完善,可导致新生儿呼吸窘迫综合征、颅内出血等疾病发生率明显升高,增加新生儿患病率和死亡率。SGA在胎儿近期及远期发育均可造成不良影响,给社会家庭造成沉重负担。SGA的病因复杂多样,母体因素、胎儿因素、胎盘脐带因素等可解释约60%的SGA病因,而40%SGA病因不明。目前SGA病因学研究多以欧美人群为主,国内缺少大样本量的SGA病因学研究,且具体影响因素仍缺乏高质量循证证据支持,而对于不明原因SGA的病因研究仍处于探索阶段。病因管理不足给SGA防控带来重大挑战。目前研究显示,人类基因组中仅2%的序列能够编码蛋白质,对占大多数的非编码RNA的研究仍不确切。其中microRNA（miRNA）的研究相对成熟。长链非编码RNA（long non-coding RNA,lncRNA）是一类长度大于200nt,由基因组转录,但无法编码成蛋白质的RNA,其参与体内多种生物学过程的调控。环状RNA（circularRNA,circRNA）是lncRNA的一类特殊类别,经特殊剪切形成的闭环结构,参与转录及转录后的调控。胎盘中滋养细胞良好的迁移侵袭及增殖功能是维持正常胎盘功能的关键因素,当滋养细胞分化发育不良,对子宫螺旋动脉侵蚀不足,致其生长受限,胎盘进入不深,导致胎盘血流灌注不足,引发SGA。非编码RNA在胎盘中表达,并对滋养层细胞的增殖、凋亡、迁移和侵袭等功能进行调控,包括circRNA在内的非编码RNA功能变化可能是引发SGA的重要因素。本文通过meta分析及回顾性研究进行分析,探讨SGA发生的影响因素。将找到的影响因素作为参考,确定不明原因SGA研究标本的纳入及排除标准,对不明原因SGA标本进行测序、验证、细胞动物实验等探索circRNA引发疾病的作用机制。第一部分 小于孕龄儿发生影响因素的meta分析及回顾性研究目的对小于孕龄儿发生的影响因素进行meta分析及回顾性分析,重视小于孕龄儿病因管理,制定适宜的干预措施,预防小于孕龄儿的发生。方法1.通过检索截止至2018.12.28发表的关于SGA发生影响因素的中英文研究,筛选文献,提取资料,采用stata12.0软件对各影响因素进行meta分析。2.回顾性分析2013年1月1日至2017年12月31日深圳市妇幼保健院81939例产妇单胎分娩新生儿病例资料,对研究对象的一般临床特征进行统计描述,使用均数±标准差描述符合正态分布的连续型变量;对胎儿体重及身长等不服从正态分布的连续型变量采用中位数,25th和75th百分位数进行描述;分类变量采用频数（n）和构成比（%）描述。对连续型变量胎儿体重及身长采用秩和检验、对分类变量χ2检验分析组间差异。采用单因素和多因素logistic回归分析小于孕龄儿与子痫前期早发晚发的关系。结果1.meta分析共纳入高质量文献31篇,其中病例对照研究11篇,队列研究20篇。meta分析结果显示低龄、偏瘦、孕期体重增长不足、矮小、文化程度低、家庭收入低、早产史、流产史、死胎史、吸烟、被动吸烟、饮酒、妊娠期高血压疾病、子痫前期、产前出血、脐带异常是SGA发生的危险因素,其OR值及 95%CI 分别为 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.回顾性研究中,SGA共3996例,发生率为4.88%。SGA组子痫前期有294例（7.40%）,非SGA组子痫前期有2839例（3.60%）,有统计学差异（P<0.05）。3.早发子痫前期组与非子痫前期组比较,在年龄、受教育程度、BMI、产次、孕次、户籍状态、胎儿是否有先心病、是否为SGA、分娩方式和分娩孕周等全部纳入因素中均有显著差异（P<0.05）。晚发子痫前期组与非子痫前期组比较,除在胎儿是否有先心病因素中无显著差异外,其余纳入因素均有显著差异（P<0.05）。早发子痫前期组晚发子痫前期组比较,除在年龄、BMI及产次等因素无显著差异外,其余纳入因素均有显著差异（P<0.05）。早发子痫前期发生SGA比例显著高于晚发子痫前期。4.对各因素与SGA行多因素Logistic回归分析。发现流动人员、胎儿患有先心病、孕期有子痫前期及助产分娩情况下发生SGA的风险增高,危险系数分别为1.248,2.189,1.59,1.921（P<0.05）,而二次生育、多次生育、较晚分娩孕周的情况下发生SGA风险降低。结论1.SGA发生的影响因素包括母体因素、胎儿因素及胎盘脐带因素。母体因素中,低龄、高龄、矮小、偏瘦、孕期体重增长不足、早产史、流产史、死胎史、妊娠期高血压、子痫前期、产前出血、文化程度低、家庭收入低、初产、脐带异常是SGA发生的危险因素。2.相较于晚发型子痫前期,早发型子痫前期引发SGA发生的风险更高。第二部分不明原因小于孕龄儿胎盘组织中circRNAs的表达及hsa_circ₀035897 对 HTR-8/SVneo 和 JEG-3 细胞生物学行为的影响目的筛选在不明原因小于孕龄儿（SGA）胎盘组织中差异表达的环状RNA,并进行环状特性的鉴定和环化位点测序。明确hsa circ₀035897（简写为circ-0035897）对人绒毛膜滋养层细胞HTR-8/SVneo和人绒毛膜癌细胞JEG-3增殖、迁移、侵袭和凋亡等生物学行为的影响。方法1.对3例不明原因SGA胎盘组织和3例正常对照胎盘组织进行环状RNA高通量测序,筛选差异表达的circRNAs。2.荧光实时定量PCR技术检测32例不明原因SGA胎盘组织和32例对照胎盘组织中候选差异表达circRNAs的表达量。3.提取组织中总RNA和基因组DNA,RNase R处理后利用实时定量PCR检测circ-0035897同源线性RNA和circ-0035897相对表达丰度变化情况,对circ-0035897的环化特性进行验证,并测序确定环化位点。4.提取孕妇血清中外泌体,qRT-PCR检测不明原因SGA和正常对照组胎盘来源外泌体中circ-0035897的表达情况。5.构建circ-0035897过表达和敲低载体,转染HTR-8/SVneo和JEG-3细胞,建立circ-0035897过表达和敲低的细胞模型。6.MTS法检测circ-0035897过表达和敲低后对HTR-8/SVneo和JEG-3细胞增殖水平的影响。7.transwell法检测circ-0035897过表达和敲低后对HTR-8/SVneo和JEG-3细胞迁移和侵袭水平的影响,8.流式细胞术检测circ-0035897过表达和敲低后对HTR-8/SVneo和JEG-3细胞凋亡水平的影响。9.qRT-PCR 和 western blot 检测 circ-0035897 过表达和敲低后对 HTR-8/SVneo和JEG-3细胞中迁移、侵袭和凋亡相关基因表达水平的影响。结果1.通过高通量环状RNA测序以及生物信息学分析,筛选出差异表达circRNAs 19 个（log Fold Change≥ 1.5,P<0.05）,其中 9 个上调,10 个下调。2.qRT-PCR验证结果发现,与正常胎盘组织相比,circ-0035897在不明原因SGA胎盘组织中表达明显下降（P<0.01）。3.以未经RNase消化的总RNA为模板,应用背靠背引物和对向引物都能扩增出环状和线性circ-0035897;以RNase R消化后的总RNA为模板,应用背靠背引物能扩增出环状circ-0035897,应用对向引物不能扩增出线性circ-0035897;以基因组DNA为模板,应用背靠背引物不能扩增出环状circ-0035897,而应用对向引物能扩增出同源线性circ-0035897。确定了circ-0035897为闭合环状的特性。4.测序结果显示了 circ-0035897的环化位点。5.qRT-PCR结果显示,与正常对照组相比,不明原因SGA血清中胎盘来源外泌体circ-0035897的表达水平显著降低（P<0.01）。6.circ-0035897 过表达（circ-0035897 组）和敲低（circ-0035897 siRNA 组）载体转染HTR-8/SVneo和JEG-3细胞后,qRT-PCR结果发现,与空载体组（NC组）相比,circ-0035897过表达组circ-0035897表达水平显著升高（P<0.01）,与敲低空载体（siRNANC）相比,circ-0035897 siRNA 组 circ-0035897 表达水平显著降低（P<0.01）,说明circ-0035897过表达和敲低的HTR-8/SVneo和JEG-3细胞模型构建成功,可用于后续实验。7.MTS实验结果显示,与对照组（NC或siRNA NC）相比,circ-0035897过表达组,HTR-8/SVneo和JEG-3细胞增殖水平显著升高（P<0.01）,而circ-0035897 siRNA组细胞增殖水平显著降低（P<0.01）。说明circ-0035897能显著促进HTR-8/SVneo和JEG-3细胞增殖。8.与对照组（NC 或 siRNANC）相比,circ-0035897 过表达组,HTR-8/SVneo和JEG-3细胞迁移和侵袭水平显著升高（P<0.01）,而 circ-0035897 siRNA组细胞迁移和侵袭水平显著降低[P<0.01)。说明circ-0035897能显著促进HTR-8/SVneo和JEG-3细胞的迁移和侵袭能力。9.与对照组（NC 或 siRNA NC）相比,circ-0035897 过表达组,HTR-8/SVneo和JEG-3细胞早期凋亡、晚期凋亡水平和总凋亡水平显著降低（P<0.01）,而circ-0035897 siRNA组细胞早期凋亡、晚期凋亡水平和总凋亡水平显著升高（P<0.01）。说明 circ-0035897 能显著抑制 HTR-8/SVneo 和 JEG-3 细胞的凋亡水平。10.与对照组（NC 或 siRNA NC）相比,circ-0035897 过表达组,HTR-8/SVneo和JEG-3中凋亡相关蛋白caspase3和caspase9表达水平显著降低（P<0.01）,BCL2/BAX的比值显升高,迁移和侵袭相关蛋白MMP2和MMP9表达水平显著升高;而circ-0035897 siRNA组凋亡相关蛋白caspase3和caspase9表达水平显著升高（P<0.01）,BCL2/BAX的比值显著降低,迁移和侵袭相关蛋白MMP2和MMP9表达水平显著降低。说明circ-0035897通过影响HTR-8/SVneo和JEG-3细胞凋亡、迁移和侵袭相关蛋白的表达从而影响其生物学行为。结论1.circ-0035897在不明原因SGA胎盘组织中表达显著下调,说明其表达降低与不明原因SGA发病有相关性,并通过实验验证了circ-0035897的环状特性。2.circ-0035897显著促进HTR-8/SVneo和JEG-3细胞的增殖、迁移和侵袭水平,抑制细胞的凋亡水平,并通过影响HTR-8/SVneo和JEG-3细胞BCL2/BAX比值、Caspase3、Caspase9、MMP2和MMP9表达影响其生物学行为。第三部分 hsa_c i rc₀035897 调控 HTR-8/SVneo 和 JEG-3 细胞生物学行为的机制目的明确circ-0035897作为miR-450b-5p的分子海绵调控PTPRB的表达参与HTR-8/SVneo和JEG-3细胞生物学行为调控。方法1.circ-0035897 靶向 miR-450b-5p 的预测和验证（1）生物信息学预测circ-0035897靶向的miRNA;（2）过表达或敲低circ-0035897后检测HTR-8/SVneo和JEG-3细胞中候选miRNA的表达量;（3）荧光原位杂交技术确定circ-0035897和miR-450b-5p在HTR-8/SVneo细胞中的定位;（4）RNA免疫沉淀技术检测miR-450b-5p和circ-0035897的结合情况;（5）双荧光素酶报告基因实验明确circ-0035897和miR-450b-5p靶向关系。2.miR-450b-5p靶基因PTPRB的预测和验证（1）生物信息学预测miR-450b-5p的靶基因;（2）过表达或敲低miR-450b-5p检测HTR-8/SVneo和JEG-3细胞中候选mRNA的表达量;（3）敲低 miR-450b-5p,western blot 检测 HTR-8/SVneo 和 JEG-3 细胞中靶基因PTPRB蛋白表达水平变化;（4）双荧光素酶报告基因实验检测miR-450b-5p与靶基因PTPRB的结合情况。3.circ-0035897对PTPRB表达水平的影响（1）过表达或敲低circ-0035897,应用qRT-PCR检测HTR-8/SVneo和JEG-3细胞中PTPRB的表达量;（2）过表达或敲低 circ-0035897,应用 western blot 检测 HTR-8/SVneo 和 JEG-3细胞中PTPRB的表达量;4.将细胞分为 3 组行 rescue 实验:NC+miRNANC,circ-0035897+miRNANC,circ-0035897+miR-450b-5p 组,qRT-PCR 检测 HTR-8/SVneo 和 JEG-3 细胞中circ-0035897、miR-450b-5p 和 PTPRB 的表达水平的变化,western blot检测PTPRB蛋白的表达水平。5.将细胞分为 3 组行 rescue 实验:NC+miRNA NC,circ-0035897+miRNANC,circ-0035897+miR-450b-5p组,通过MTS法、transwell迁移侵袭实验和细胞凋亡流式分析,检测HTR-8/SVneo和JEG-3细胞增殖、迁移、侵袭和凋亡生物学行为的变化;6.将细胞分为 3 组行 rescue 实验:NC+miRNA NC,circ-0035897+miRNANC,circ-0035897+miR-450b-5p 组,并采用 qRT-PCR 和 western blot,检测HTR-8/SVneo和JEG-3细胞中凋亡、迁移和侵袭相关基因的表达水平变化。7.构建PTPRB敲低病毒载体,尾静脉注射孕鼠后,评估胎鼠体重。结果1.circ-0035897和miR-450b-5p的靶向关系预测和验证（1）采用 miRanda,targetscan 和 RNAhybrid 软件预测可以与 circ-0035897结合的 8 个候选 miRNAs:miR-19b-1,miR-19b-2,miR-19a,miR-450b-5p,miR-9,miR-340,miR-765,miR-302f。（2）在HTR-8/SVneo和JEG-3细胞中,与对照组相比,转染circ-0035897过表达载体组细胞中miR-450b-5p的表达量显著降低（P<0.01）,转染circ-0035897 siRNA 组细胞中 miR-450b-5p 表达显著增高（P<0.01）。（3）FISH 结果显示,circ-0035897 和 miR-450b-5p 主要位于 HTR-8/SVneo细胞的胞质,说明circ-0035897可能通过ceRNA机制发挥其调控作用。（4）RNA免疫沉淀实验结果显示,与miRNANC组相比,转染miR-450b-5p的HTR-8/SVneo细胞组中AGO2结合的circ-0035897水平显著上升（P<0.01）。（5）双荧光素酶报告实验结果显示,与miRNANC组相比,共转染circ-0035897-WT和miR-450b-5p mimic组细胞中荧光素酶活性显著降低（P<0.01）,而共转染 circ-0035897-WT 和 miR-450b-5p inhibitor 后,与inhibitor NC相比,荧光素酶活性显著升高（P<0.01）。将circ-0035897与miR-450b-5p的结合位点突变后,共转染circ-0035897-mut和miR-450b-5p mimic 或 miR-450b-5p inhibitor 后,与 NC 组相比,荧光素酶活性没有显著变化。说明circ-0035897和miR-450b-5p之间存在结合关系。2.miR-450b-5p和PTPRB的靶向关系预测和验证（1）采用 miRanda,targetscan 和 RNAhybrid 软件预测可以与 miR-450b-5p结合的 5 个候选靶基因:DCAF5、PTPRB、CLOCK、PLCG2、FAM107B。（2）在HTR-8/SVneo和JEG-3细胞中,与阴性对照组相比,转染miR-450b-5p mimic细胞中PTPRB mRNA的表达量显著降低（P<0.01）,转染miR-450b-5p inhibitor组细胞中PTPRB mRNA和蛋白表达量显著升高（P<0.01）。（3）共转染 PTPRB-3’ UTR 和 miR-450b-5p mimic 后,与 NC 组相比,荧光素酶活性显著降低（P<0.05）,而共转染PTPRB-3’ UTR和miR-450b-5p inhibitor后,与inhibitor NC组相比,荧光素酶活性显著升高（P<0.01）。将PTPRB-3’ UTR与miR-450b-5p的两个结合位点分别突变后,与NC组相比,共转染PTPRB-3’ UTR-mut和miR-450b-5p mimic组荧光素酶活性显著降低（P<0.05）,与inhibitor NC组相比,共转染PTPRB-3’UTR-mut和miR-450b-5p mimic组荧光素酶活性显著升高（P<0.01）;而同时将两个结合位点突变后,与NC组相比,共转染PTPRB-3’ UTR-mut和miR-450b-5p mimic或miR-450b-5p inhibitor组荧光素酶活性无显著变化。说明miR-450b-5p与PTPRB-3’ UTR之间存在结合关系。3.circ-0035897 调控 PTPRB 的表达（1）在HTR-8/SVneo和JEG-3细胞中,与阴性对照组相比,circ-0035897过表达组细胞中PTPRB mRNA的表达量显著升高（P<0.01）,circ-0035897敲低组细胞中PTPRB mRNA表达量显著降低（P<0.01）。（2）在HTR-8/SVneo和JEG-3细胞中,与阴性对照组相比,circ-0035897过表达组细胞中PTPRB蛋白的表达量显著升高（P<0.01）,circ-0035897敲低组细胞中PTPRB蛋白表达量显著降低（P<0.01）。4.将细胞分为 3 组:NC+miRNA NC,circ-0035897+miRNA NC,circ-0035897+miR-450b-5p 组。qRT-PCR 结果显示,与 circ-0035897+miRNA NC 组相比,circ-0035897+miR-450b-5p 组中 circ-0035897 和 PTPRB 的表达水平显著降低（P<0.01）,miR-450b-5p表达水平显著升高（P<0.01）。5.MTS 结果显示,在 HTR-8/SVneo 和 JEG3 细胞中,与 circ-0035897+miRNA NC组相比,circ-0035897+miR-450b-5p组细胞增殖水平显著降低（P<0.01）。6.细胞迁移和侵袭实验结果显示,在HTR-8/SVneo和JEG3细胞中,与circ-0035897+miRNA NC 组相比,circ-0035897+miR-450b-5p 组细胞的迁移和侵袭水平显著降低（P<0.01）。7.流式细胞术结果显示,在HTR-8/SVneo和JEG3细胞中,与circ-0035897+miRNA NC 组相比,circ-0035897+miR-450b-5p 组细胞的早期凋亡、晚期凋亡和总凋亡水平显著升高（P<0.01）。8.qRT-PCR 和 western blot 结果显示,在 HTR-8/SVneo 和 JEG3 细胞中,与circ-0035897+miRNA NC 组相比,circ-0035897+miR-450b-5p 组细胞中caspase3和caspase9表达水平显著升高,BCL2/BAX 比值显著降低,MMP2和MMP9表达水平显著降低（P<0.01）。9.动物实验结果显示,PTPRB敲低组小鼠的子鼠出生体重显著降低（P<0.01）。结论circ-0035897以竞争性内源RNA（ceRNA）的形式与miR-450b-5p结合,调控下游靶基因PTPRB的表达,增强人胎盘滋养层细胞的迁移侵袭能力,抑制细胞凋亡。更多还原

【Abstract】 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 hsa_circ₀035897 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 has_circ₀035897（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 hsa_circ₀035897 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.更多还原