新lnc-HZ03和miR-hz03在BPDE导致女性滋养层细胞凋亡而引发复发性流产中的作用及机制研究

【作者】 梁婷婷；

【导师】 张慧东；

【作者基本信息】 四川大学 ， 卫生检验与检疫， 2021， 硕士

【摘要】 目的:苯并(a)芘(BaP)是一种常见的环境污染物,在食品、空气和饮用水中都有检出。体内蓄积过多的BaP可能损害女性生殖健康,诱发复发性流产等疾病。但是BaP诱导复发性流产(recurrent miscarriage,RM)具体的分子机制并不清晰。本文旨在寻找新的lncRNA和miRNA,探讨它们在BaP诱导复发性流产这一过程中发挥的功能和调控作用,为揭示环境毒物诱发复发性流产提供细胞学基础,同时也为复发性流产的预防和治疗提供一定的思路和见解。方法:(1)通过RT-qPCR检测lnc-HZ03在绒毛膜组织中和0.5μM BPDE处理24 h的细胞中的相对表达量。(2)通过CCK8实验和流式细胞术检测lnc-HZ03对细胞增殖和细胞凋亡的影响。(3)通过RT-qPCR和Western blot检测p53和SAT1在组织中的相对表达量,以及检测lnc-HZ03对下游p53/SAT1信号通路的影响。(4)通过ELISA实验检测组织中精胺的含量,以及BPDE或lnc-HZ03对细胞内精胺含量的影响。(5)通过ChIP实验检测lnc-HZ03对p53蛋白结合SAT1启动子区域的影响。(6)通过RT-qPCR检测lnc-HZ03对miR-hz03表达量的影响。(7)通过RT-qPCR和Western blot检测miR-hz03对下游p53/SAT1信号通路的影响。(8)通过RNA稳定性实验检测miR-HZ03对p53 mRNA和lnc-HZ03的影响。(9)通过双荧光素酶报告实验和Biotin-RIP实验检测miR-hz03与lnc-HZ03,以及miR-hz03与p53 mRNA的相互作用。(10)通过使用0、0.05和2 mg/kg的BaP灌胃处理孕鼠13天,从而构建BaP染毒小鼠流产模型,检测BaP对小鼠胚胎吸收率的影响。(11)通过RT-qPCR和Western blot检测小鼠组织中p53和SAT1的相对表达量。结果:(1)RT-qPCR结果显示,lnc-HZ03在复发性流产组织中和BPDE处理的细胞中上调表达(P<0.05)。(2)CCK8实验结果显示,lnc-HZ03抑制细胞增殖(P<0.05)。流式细胞术结果显示,lnc-HZ03促进细胞凋亡(P<0.05)。(3)RT-qPCR和Western blot结果显示,p53和SAT1在复发性流产组织中上调表达(P<0.05);lnc-HZ03也能促进p53和SAT1的表达(P<0.05)。(4)ELISA结果显示,精胺的含量在复发性流产组织中下降(P<0.05);BPDE处理后精胺的含量也下降(P<0.05);lnc-HZ03使得精胺的含量下降(P<0.05)。(5)ChIP实验结果显示,lnc-HZ03促进p53蛋白与SAT1启动子区域的结合(P<0.05)。(6)RT-qPCR 结果显示,lnc-HZ03 促进 miR-hz03 的表达(P<0.05)。(7)RT-qPCR 和 Western blot 结果显示,miR-hz03 促进 p53 和 SAT1 的表达(P<0.05)。(8)RNA稳定性实验结果显示,miR-hz03促进p53 mRNA和lnc-HZ03的稳定性(P<0.05)。(9)双荧光素酶报告实验和Biotin-RIP实验结果显示,miR-hz03能够与lnc-HZ03直接结合,miR-hz03也能与p53直接结合(P<0.05)。(10)BaP染毒小鼠模型显示,BaP增加小鼠胚胎吸收率(P<0.05)。(11)RT-qPCR和Western blot结果显示,BaP处理孕鼠过后,组织中p53和SAT1的相对表达量随着BaP处理浓度增加而增加(P<0.05)。结论:本研究中,我们定义了一个新的lnc-HZ03和miR-hz03。BPDE能够上调lnc-HZ03和miR-HZ03的表达水平。Lnc-HZ03促进miR-hz03的表达,反过来miR-hz03通过“结合位点”直接与lnc-HZ03结合,促进lnc-HZ03的稳定性,增加lnc-HZ03的含量。因此lnc-HZ03与miR-hz03形成正反馈环,激活下游p53/SAT1通路,引发滋养层细胞凋亡和复发性流产,为阐明复发性流产的原因提供了新的思路。更多还原

【Abstract】 Objective:BaP as an environmental pollutant has been widely detected out from food,air and drinking water.Excessive accumulation of BaP could disrupt female reproductive health and induce recurrent miscarriage.However,the regulation mechanisms in the malfunctions of BaP-induced unexplained miscarriage have rarely been explored.The purpose of this paper is to find a novel lncRNA and a novel miRNA,and to explore their regulation mechanisms.These results provided new scientific insights into the occurrence of miscarriage.Methods:(1)The expression of lnc-HZ03 in tissues and 0.5 μM BPDE-treated cells was detected by RT-qPCR.(2)CCK8 assays were used to analyze the effects of lnc-HZ03 on cell proliferation.Flow cytometry was used to analyze the effects of lnc-HZ03 on cell apoptosis.(3)The levels of p53 and SAT1 in tissues and in lnc-HZ03-overexpressed or-silenced cells were detected by RT-qPCR and Western blot analysis.(4)The contents of spermine in tissues,in BPDE-exposed cells and in lnc-HZ03-overexpressed or-silenced cells were detected by ELISA.(5)The effects of lnc-HZ03 on p53 protein binding to SAT1 promoter region were detected by ChIP assays.(6)The level of miR-hz03 in lnc-HZ03-overexpressed or-silenced cells was detected by RT-qPCR.(7)The levels of p53 and SAT1 in miR-hz03-overexpressed or-silenced cells were detected by RT-qPCR and Western blot.(8)The effects of miR-HZ03 on p53 mRNA and lnc-HZ03 were detected by RNA stability tests.(9)The interactions between miR-hz03 and lnc-HZ03,and between miR-hz03 and p53 mRNA were detected by dual-luciferase reporter assay system and Biotin-RIP tests.(10)The pregnant mice were treated with 0,0.05 and 2 mg/kg BaP by gavage for 13 days to establish BaP-exposed mouse model and detect the effects of BaP on the embryo resorptions rates.(11)The expressions of p53 and SAT1 in mouse tissues were detected by RT-qPCR and Western blot.Results:(1)The results of RT-qPCR analysis indicated that lnc-HZ03 was increased in both RM tissues and BPDE-treated trophoblast cells(P<0.05).(2)The results of CCK8 assays showed that lnc-HZ03 inhibited trophoblast cell proliferation(P<0.05)and the results of flow cytometry showed that lnc-HZ03 promoted apoptosis(P<0.05).(3)The results of RT-qPCR and Western blot indicated that the mRNA and protein levels of p53 and SAT1 were increased in RM group,and lnc-HZ03 raised p53 and SAT1 mRNA and protein levels(P<0.05).(4)The results of ELISA showed that the content of spermine decreased in recurrent miscarriage tissues and in BPDE-treated cells(P<0.05).Lnc-HZ03 decreased the content of spermine(P<0.05).(5)The results of ChIP assays showed that lnc-HZ03 promoted p53 protein binding to SAT1 promoter region(P<0.05).(6)The results of RT-qPCR analysis showed that lnc-HZ03 promoted the expression of miR-hz03(P<0.05).(7)The results of RT-qPCR and Western blot showed that miR-hz03 promoted the expression of p53 and SAT1(P<0.05).(8)The results of RNA stability tests showed that miR-hz03 promoted the RNA stability of p53 and lnc-HZ03(P<0.05).(9)The results of double luciferase report assays and Biotin-RIP assays showed that miR-hz03 could directly bind to lnc-HZ03 and p53 mRNA(P<0.05).(10)The BaP-exposed mouse model showed that BaP increased the embryo resorptions rates(P<0.05).(11)The results of RT-qPCR and Western blot showed that the expressions of p53 and SAT1 in the tissues increased with the increase of BaP concentration(P<0.05).Conclusion:In this work,a novel lnc-HZ03 and a novel miR-hz03 were identified to be up-regulated in both BPDE-treated trophoblastic cells and human recurrent miscarriage villous tissues.A positive feedback loop consisting of lnc-HZ03 and miR-hz03,which was activated by BPDE exposure,up-regulated p53/SAT1 pathway to cause trophoblastic cell apoptosis and induce miscarriage.In a mice model,BaP also induces mice miscarriage through murine p53/SAT1 axis.These results provide new scientific insights into the occurrence of miscarriage.更多还原