【作者】 王鹏；

【导师】 徐明清；

【作者基本信息】 四川大学 ， 外科学（普外）， 2022， 博士

【摘要】 目的:肝脏再生是独特的生物学现象,在切除部分肝组织或慢性损伤时肝脏依然能够保持强大的增殖能力,最终恢复到损伤前的状态,维持机体的稳态调节。肝脏这种特殊的再生能力依赖于剩余正常细胞的增殖能力,各种原因导致细胞增殖能力的降低都会影响肝再生结局的转归。肝纤维化背景下的肝再生能力显著减弱,患者肝切除后肝衰风险较大。以往肝再生方面的研究着重针对细胞因子及其信号传导调控途径,环状RNA（circ RNAs）调控肝再生过程的研究相对较少。circ RNAs是一类在细胞中大量存在的单链闭合环状的RNA分子,已有大量研究报道与众多生理活动及多种疾病的发生发展有关,circ RNAs在肝再生方面的研究尚处在起步阶段,有少量研究报道circ RNA参与大鼠肝再生调控,同时有研究发现在肝再生过程中有众多circ RNAs差异性表达,但是circ RNAs参与调控肝再生的分子机制尚不清楚。因此,本课题旨在探讨基因测序发现的在纤维化肝脏再生过程中差异表达的circ RNA circ Spred1（来源于Spred1基因,被命名为circ Spred1）抑制纤维化肝脏再生及其分子机制。材料和方法:首先我们在正常野生型小鼠建立70%肝切除模型（70%PH）,收集术后24h、36h、48h、72h和120h的再生残肝,使用组织病理学技术检测5’-溴-2’-脱氧尿核苷（5-Brd U）的表达,选择肝再生过程DNA合成高峰期即阳性细胞占比最高时相点的肝组织与术前0h肝组织配对进行circ RNA高通量测序,筛选出差异表达排名靠前的候选circ RNAs,通过设计所有候选circ RNAs的小干扰RNA来敲低目标circ RNA后,使用Cell Counting Kit-8细胞计数试剂检测细胞增殖能力的变化趋势,最终筛选出具有功能的circ Spred1。构建大鼠肝纤维化模型,70%PH后记录多个时相点肝重与体重的比值,IHC检测所有时相点增殖指标（Ki67和p H3S10）的表达,对标纤维化与正常组增殖能力的差异,荧光定量聚合酶链反应（q RT-PCR）检测circ Spred1在所有时相点的表达,获得circ Spred1在纤维化与正常无纤维化动物肝再生的表达模型。设计合成携带circ Spred1全部序列的过表达腺相关病毒（AAV）及阴性对照病毒,选择较好靶向肝脏的血清型,经小鼠尾静脉注射,构建体内过表达circ Spred1模型,同时构建四氯化碳（CCl₄）肝纤维化模型,对构建成功的小鼠进行70%PH,收集肝再生重要的时间点（0h、24h、36h、48h、120h）的肝组织,记录各时相点再生肝与手术切除肝重量比值;Masson染色和Sirius Red染色评估肝纤维化模型的质量;q RT-PCR检测所有时相点circ Spred1的表达变化;IHC检测所有时相点增殖指标（Ki67和p H3S10）的阳性细胞占比;血清学分析各时间点小鼠血清中谷丙转氨酶（ALT）及谷草转氨酶（AST）的浓度变化;蛋白印迹（Western Blot）检测各组小鼠不同时相点细胞周期蛋白（cyclins）表达水平。体外细胞实验中,首先设计针对circ Spred1拼接位点的小干扰RNA（si RNA）稳定敲低在小鼠正常肝细胞（AML-12）和肝癌细胞系（Hepa1-6）中circ Spred1的表达水平,同时构建慢病毒过表达circ Spred1稳转细胞株,使用CCK-8检测细胞的增殖能力改变;Ed U增殖实验检测细胞的DNA复制能力的改变;碘化吡啶（PI）染色后借助流式细胞术分析经过干预细胞的周期的阻滞状态变化,最后Western Blot检测circ Spred1调变后细胞周期蛋白（cyclins）表达变化趋势。通过在线工具网站预测circ Spred1的编码能力,确定circ Spred1序列中的内部核糖体进入位点序列（IRES）和开放阅读框（ORF）序列,通过双荧光素酶报告实验验证IRES的生物学活性,使用蛋白质谱分析和Western Blot鉴定circ Spred1所编码的蛋白Spred1-197aa,设计合成过表达Spred1-197aa质粒,转染细胞后,CCK-8细胞实验、Ed U增殖实验及碘化吡啶（PI）染色后借助流式细胞术分析经过干预细胞的周期的阻滞状态变化。利用转录组测序发现si RNA敲低circ Spred1后差异表达的基因。免疫共沉淀（Co-IP）寻找Spred1-197结合的靶蛋白DDX17,检测当使用si RNA敲低细胞中DDX17的后细胞增殖能力的改变;通过尾静脉注射重组DDX17蛋白人为提高纤维化小鼠体内DDX17水平,70%PH后收集术后36h及术前的肝组织,IHC检测Ki67的表达。最后通过q RT-PCR检测si RNA敲低DDX17后细胞周期蛋白的变化。结果:经过免疫组化检测BrdU确认正常小鼠70%PH后36h为增殖高峰期,通过高通量测序发现circ Spred1在肝再生36h表达显著降低,发挥抑制肝再生的作用。采用IHC和q RT-PCR检测临床人纤维化肝组织标本发现,circ Spred1在Ki67高表达肝组织中的表达水平低于Ki67低表达肝组织;纤维化大鼠70%PH后,纤维化大鼠相比正常大鼠,IHC检测所有时相点Ki67为持续低表达,qRT-PCR检测circ Spred1的表达,纤维化大鼠表达在术后6h开始持续升高,72h和96h有显著统计学差异。小鼠体内过表达circ Spred1后,70%PH收集120h的肝组织称重,发现过表达circ Spred1组再生肝与切除肝重比值显著降低,在纤维化组和正常小鼠模型均可观察到这种趋势。血清中ALT、AST在纤维化小鼠中过表达circ Spred1后水平显著升高,而在正常组小鼠过表达circ Spred1后未观察到明显统计学差异;IHC发现过表达circ Spred1后术后多个时相点的肝组织中指标Ki67、p H3S10的阳性细胞染色比例明显减少,Western Blot实验检测多种cyclins表达量也明显减低。体外实验研究发现当敲低circ Spred1后细胞展示出更高的增殖能力,而当过表达circ Spred1后细胞增殖能力显著被抑制,增殖速度缓慢,通过Western Blot实验发现,circ Spred1过表达抑制周期蛋白的表达,敲低后则促进周期蛋白的表达。通过在线工具预测了circ Spred1的编码能力,发现circ Spred1同时具有IRES和ORF,预计能够编码含有197个氨基酸的蛋白Spred1-197aa,通过双荧光素酶报告实验验证IRES能够结合核糖体启动翻译,蛋白质谱成功在细胞蛋白样品中鉴定出Spred1-197aa的氨基酸序列。细胞增殖功能实验验证Spred1-197aa具有体外抑制细胞增殖的功能。转录组测序发现当敲低circ Spred1后多个增殖相关的通路被激活,Co-IP后蛋白质谱鉴定发现DDX17是Spred1-197aa的下游靶蛋白,细胞增殖功能实验验证DDX17具有体外促进细胞增殖的功能,纤维化小鼠尾静脉注射DDX17重组蛋白,70%PH后IHC检测36h肝组织中Ki67的表达显著增加。Western Blot检测发现circ Spred1、Spred1-197aa与DDX17表达呈现明显的负相关性。最后,q RT-PCR检测当用si RNA敲低细胞中DDX17的表达后细胞周期蛋白cyclin A2也随之显著降低。结论:1.我们首次发现了一个全新的具有肝再生调控功能的环状RNA—circ Spred1。circ Spred1能显著抑制正常肝细胞增殖;在纤维化肝脏再生模型中,circ Spred1也显示出显著的肝再生抑制功能。2.circ Spred1通过编码蛋白Spred1-197aa,而发挥肝再生抑制功能;3.Spred1-197aa通过结合并抑制DDX17的功能,进而导致cyclin A2转录水平下降,从而导致细胞增殖能力减弱。本研究揭示了环状RNA在纤维化肝脏再生扮演举足轻重的角色。对circ Spred1的具体功能与机制进行了较为深入的研究,为纤维化肝脏再生的分子生物学基础研究开辟了新的思路。更多还原

【Abstract】 Objective:Liver regeneration is a unique biological phenomenon,the removal of part of liver tissue or chronic injury of the liver can still maintain strong proliferation,eventually restored to the state before the injury,maintaining the steady state regulation of the body.The liver regeneration ability of this special depends on the remaining normal cell proliferation,various reasons resulting in a loss of cell proliferation ability will affect the outcome of liver regeneration.Liver regeneration is significantly reduced under the background of liver fibrosis,with a high risk of post hepatectomy liver failure（PHLF）.Previous studies in the field of liver regeneration focused on cytokines and their signal transduction regulation pathways,but there were relatively few studies on the regulation of liver regeneration by circ RNAs.Circ RNAs are a kind of RNA molecules with single closed loops abundant in cells.Many studies have shown that circ RNAs are related to many physiological activities and the processes of a variety of diseases.There are only scattered reports on circ RNAs in liver regeneration,and a few studies have shown that circ RNAs are involved in the control of rat liver regeneration.At the same time,studies have found that there are many circ RNAs differentially expressed in the process of liver regeneration,but the molecular mechanism of circ RNAs participating in the control of liver regeneration remains unclear.Therefore,the purpose of this study is to explore the differential expression of circ RNA circ Spred1（derived from Spred1 gene and named circ Spred1）in the process of fibrosis liver regeneration and its molecular mechanism as found by gene sequencing.Materials and Methods:First,70%PH model was established in normal wild-type mice.Regenerated liver tissue was collected at 24h,36h,48h,72h,and 120h postoperatively,and the expression of 5-bromodeoxyuridine（Brd U）was detected by immunohistochemistry（IHC）.Liver tissue at the peak of DNA synthesis in the process of liver regeneration,i.e.,the highest proportion of positive cells,was paired with liver tissue 0h before surgery for circ RNA high-throughput sequencing to screen out candidate circ RNAs with high differential expression.After knockdown of target circ RNAs by designing small interfering RNA of all candidate circ RNAs,the effect of CCK-8 on cell proliferation was detected,and the functional circ Spred1 was finally screened out.Rat hepatic fibrosis model was established.The ratio of liver weight to body weight at multiple time points was recorded after 70%PH,and the expression of proliferation indicators（Ki67 and p H3S10）at all time points was detected by IHC,and the difference of proliferation ability between fibrosis and normal group was marked.The expression of circ Spred1 was detected by q RT-PCR,and the expression model of circ Spred1 in liver regeneration of fibrotic and normal non-fibrotic animals was obtained.The overexpressed adeno-associated virus（AAV）and negative control virus carrying all circ Spred1 sequences were designed and synthesized,and the serum type that was better targeted to the liver was selected and injected into the tail vein of mice to build the circ Spred1 overexpressed model in vivo,and carbon tetrachloride（CCl₄）liver fibrosis model.The successfully constructed mice were treated with70%PH,and the liver tissues at important time points of liver regeneration（0h,24h,36h,48h,120h）were collected,and the weight ratio of the regenerated liver to the surgically resected liver at each time point was recorded.Masson staining and Sirius Red staining were used to evaluate the quality of liver fibrosis models.The expression of circ Spred1 at all time points was detected by q RT-PCR.IHC detected the percentage of positive cells of all the time point proliferation indicators（Ki67 and p H3S10）.Serum ALT and AST concentrations were analyzed by serology at each time point.Western Blot was used to test the expression of cyclins at different time points in each group.In vitro cell experiments,the expression level of circ Spred1 in mouse normal liver cells（AML-12）and hepatoma cell lines（Hepa1-6）was stably knocked down by small interfering RNA（si RNA）targeting the circ Spred1 splicing site.Meanwhile,stable cell lines overexpressing circ Spred1 were constructed.CCK-8 was used to detect changes about cell proliferation.Ed U proliferation assay was used to detect the changes of DNA replication ability of cells.After pyridinium iodide（PI）staining,flow cytometry was used to analyze the change of cycle arrest status of the cells after intervention.Finally,Western Blot was applied to test the change trend of cyclins expression after circ Spred1 modulation.The online tool website was used to predict the coding ability of circ Spred1,determine the internal ribosome entry site（IRES）and open reading frame（ORF）sequences of circ Spred1,and verify the biological activity of the IRES by dual lucifase reporting assay.Protein spectrum analysis and Western Blot were used to identify the protein Spred1-197aa encoded by circ Spred1.The overexpressed Spred1-197aa plasmid was designed and synthesized.After transfection,cell proliferation was tested by CCK-8 cell assay,Ed U proliferation assay and flow cytometry.Transcriptome sequencing was used to identify differentially expressed genes after si RNA knockdown of circ Spred1.Immunoprecipitation（Co-IP）was used to search for the target protein DDX17 bound to Spred1-197,and to detect the changes in cell proliferation ability of DDX17 after si RNA knockdown.The level of DDX17in fibrotic mice was artificially increased by tail vein injection of recombinant DDX17protein.Liver tissue was collected 36h after operation and before operation after70%PH,and Ki67 expression was detected by immunohistochemical.Finally,the changes of cyclin after DDX17 knockdown by si RNA were detected by q RT-PCR.Results:Brd U immunohistochemistry test confirmed that the proliferation peak was 36h after 70%PH in normal mice.Through high-throughput sequencing,circ Spred1expression was significantly decreased at 36h of liver regeneration,playing a role in inhibiting liver regeneration.IHC and q RT-PCR were used to detect clinical human fibrotic liver tissue samples.It was found that the expression level of circ Spred1 in liver tissue with high Ki67 expression was lower than that in liver tissue with low Ki67 expression.After 70%PH in fibrotic rats,Ki67 was continuously low expression at all time points detected by IHC compared with normal rats,and circ Spred1expression was detected by q RT-PCR.The expression of fibrotic rats began to increase continuously at 6h after surgery,and there was significant statistical difference at 72h and 96h.After circ Spred1 was overexpressed in mice,120h of liver tissue was collected and weighed with 70%PH,and it was found that the weight ratio of regenerated liver to resected liver in the overexpressed circ Spred1 group was significantly reduced,which could be observed in both the fibrosis group and the normal mouse model.The levels of ALT and AST in serum were significantly increased after overexpression of circ Spred1 in fibrotic mice,while no significant statistical difference was observed after overexpression of circ Spred1 in normal mice.IHC found that after overexpression of circ Spred1,the proportion of Ki67 and p H3S10 positive cells in liver tissues at multiple postoperative time points was significantly reduced,and the expression level of cyclin detected by Western Blot was also significantly reduced.In vitro experimental studies found that when circ Spred1 was knocked down,the cells showed higher proliferation ability,while when circ Spred1 was overexpressed,the cell proliferation capacity was significantly restrained,and the proliferation rate was slow.Western Blot analysis of cyclin showed that circ Spred1 overexpression inhibited the expression of cyclin.Knockdown promotes the expression of cyclin.Online tools were used to predict the coding capacity of circ Spred1.It was found that circ Spred1 had both IRES and ORF,which were expected to encode Spred1-197aa containing 197 amino acids.Dual luciferase reporting assay confirmed that IRES could bind ribosome to initiate translation.The amino acid sequence of Spred1-197aa was successfully identified in cell protein samples.Cell proliferation assay confirmed that Spred1-197aa could inhibit cell proliferation in vitro.Transcriptional sequencing showed that multiple proliferation-related pathways were activated when circ Spred1 was knocked down,and DDX17 was identified as the downstream target protein of Spred1-197aa by Co-IP protein profile.Cell proliferation assay verified that DDX17 could promote cell proliferation in vitro.After tail vein injection of DDX17 recombinant protein in hepatofibrosis mice,Ki67expression in liver tissue was obvious elevated by IHC 36h after 70%PH detection.Western Blot analysis showed that circ Spred1,Spred1-197aa showed significant negative correlation with DDX17 expression.Finally,q RT-PCR showed that cyclin A2 was significantly reduced when DDX17 expression was knocked down by si RNA.Conclusion:1.We firstly found a novel circ Spred1 with the function of regulating liver regeneration.Circ Spred1 could significantly inhibit the proliferation of normal hepatocytes.In the fibrosis liver regeneration model,circ Spred1 also showed significant inhibitory function of liver regeneration.2.Circ Spred1 encodes protein Spred1-197aa to inhibit liver regeneration.3.Spred1-197aa binds to and inhibits the function of DDX17,causing a decrease in cyclin A2 transcription level,resulting in decreased cell proliferation capacity.This study revealed that circ RNAs play an important regulatory role in the impaired regenerative ability of fibrotic liver.The specific function and mechanism of circ Spred1 have been studied,which opens a new idea for the molecular biology of liver regeneration in fibrosis.更多还原