【作者】 崔巍；

【导师】 吴琼；

【作者基本信息】 哈尔滨工业大学 ， 生物医学工程， 2015， 博士

【摘要】 mi R-1188位于小鼠12号染色体(12q F1)Dlk1-Dio3印记簇内,其宿主基因Meg8是一个母本表达的长非编码RNA。在Dlk1-Dio3印记区内存在着哺乳动物中最大的mi RNAs簇,其区域内的元件Meg3(Gtl2)、anti-sense Rtl1(as-Rtl1)、Meg8(Rian)内含子中以及基因间都有mi RNAs的存在。其中,小鼠内mi RNAs总数量52个,人为54个,它们中的大多数mi RNAs是同源的,且只有mi R-1247为反向转录。已有研究表明,Dlk1-Dio3区域内的元件以及mi RNAs对胚胎发育和癌症发生中都有重要的作用。其中,Meg3的缺失会阻止胚胎发育以及肝癌的发生;而分别位于位于mi R-1188上下游的mi R-127和mi R-370,均影响胚胎发育并诱发肝癌的发生,这暗示着mi R-1188的表达变化可能与簇内其它基因具有相同的生物学功能。故本文以小鼠Dlk1-Dio3区域内的mi R-1188为研究对象,分析了胚胎发育过程中的动态变化,并结合发育相关靶基因对mi R-1188在胚胎发育过程中的功能进行分析;同时以mi R-1188特异性低表达的肝癌细胞Hepa1-6为研究对象,结合肿瘤相关靶基因解析mi R-1188在肝癌细胞中的作用。本研究首先分析了mi R-1188在胚胎发育的各个时期以及不同鼠源癌症细胞系内的表达情况。定量结果表明mi R-1188成熟体广泛表达于胚胎发育的各个时期,在早期胚胎发育的4-细胞内表达较高,至发育中后期E12.5到E18.5天表达逐渐升高;而且在E15.5天的肝脏中特异性高表达,脑、肺脏和心脏次之,肾脏中表达相对较低,原位杂交结果表明mi R-1188在E15.5的表达模式与宿主基因Meg8的表达模式是相一致的;伴随肝脏发育功能的转移,其表达水平逐渐下调,即mi R-1188在胚肝造血期高表达,其中E15.5天达到最高,出生后表达降低;而后对鼠源的各种癌症细胞系内mi R-1188表达分析发现,在肝癌细胞Hepa1-6内的表达量相对最低,同时证实mi R-1188在正常成体肝脏组织和Hepa1-6细胞内差异表达,该结果意味着mi R-1188与癌症发生存在相关性。为一进步了解mi R-1188对胚胎发育的影响,通过尾静脉注射过表达mi R-1188的慢病毒载体至妊娠初期孕鼠,过表达mi R-1188后引起E15.5天胚胎体重减轻,相应胎盘重量增加;定量结果表明mi R-1188成熟体在全胚、胎盘以及羊膜上均有过量富集;其中,在E15.5天肺脏和肝脏内都呈现出高表达;但HE及原位杂交数据表明过表达mi R-1188后并未引起胚胎内各主要脏器细胞的异常;由于mi RNAs是通过调控靶基因而发挥功能,本研究通过生物信息学预测,并结合双荧光素酶报告基因和Western blot,证实胚胎发育相关基因Smad3为mi R-1188的靶基因之一;相关性分析证实,二者在小鼠胚胎发育各个时期的表达存在负相关,即Smad3在小鼠胚胎早期发育中的2-细胞期相对4-细胞高表达,发育中期E15.5天低表达,以及E15.5天内肝脏的特异性低表达等;而且,在过表达mi R-1188的胚胎内检测其Smad3的表达,发现Smad3在胎盘、心脏、肝脏和肾脏中同样存在负相关,该结果表明mi R-1188通过调控发育功能基因Smad3的表达,参与胚胎发育。最后,为确定mi R-1188在癌症中的重要功能,本研究通过改变内源性mi R-1188的表达水平,发现mi R-1188可抑制肝癌细胞Hepa1-6的增殖、侵袭和迁移,引起细胞铺展面积的减小,同时促进细胞凋亡和Caspase-3的激活;而且,mi R-1188直接靶向肿瘤相关基因Bcl-2和Sp1,且在转录后调控二者的表达,同时发现mi R-1188与Bcl-2和Sp1在正常肝脏组织和肝癌细胞Hepa1-6内表达水平存在负相关;过表达mi R-1188后上调相关凋亡基因Bax和p53的表达,以及下调Bcl-XL的表达;进一步分析证明,Hepa1-6细胞内分别共转染mi R-1188模拟物与靶基因Bcl-2和Sp1的干扰片段,确定mi R-1188是通过调控Bcl-2和Sp1的表达参与Hepa1-6细胞的生物学行为;并且体内成瘤实验充分说明了mi R-1188具有肿瘤抑制子的功能,并揭示了mi R-1188参与调控肝癌细胞行为的各种途径。综上所述,mi R-1188是一个广泛表达于胚胎及各种癌症细胞系的mi RNA。mi R-1188引起胚胎发育重量的减少和胎盘重量的增加,以及体内外抑制肝癌细胞的生长等生物学效应,并通过调控不同靶基因的表达参与生物体的各项活动。深入研究mi R-1188的功能对进一步研究小鼠Dlk1-Dio3区域内mi RNAs的调控机制具有重要意义。更多还原

【Abstract】 Mi R-1188 is located in transcripts of Meg8, which has been identified as a maternally expressed gene in the mouse Dlk1-Dio3 imprinted cluster. In mammals, the Dlk1-Dio3 genomic region, located on mouse chromosome 12(12q F1) contains the paternally expressed imprinted genes Dlk1, Rtl1, and Dio3 and the maternally expressed imprinted genes Meg3(Gtl2), anti-sense Rtl1(as-Rtl1), Meg8. This region hosts, in addition to two long intergenic RNAs, the Meg3 and Meg8, one of the largest micro RNA clusters in the genome, with 52 mi RNAs in mouse and 54 mi RNAs in human, in the forward strand and one(mi R-1247) in the reverse strand. Previous research has shown that major regulatory components and mi RNAs in this region are involved in embryo development and pathogenesis of different diseases. And, the deletion of the Meg3 gene could interfere embryo development and contribute to hepatoma cells proliferation; mi R-127 and mi R-370, located in upstream and downstream of mi R-1188, also could involve embryo development and hepatocarcinogenesis. We assumed that there might be the same functions between mi R-1188 and other genes in Dlk1-Dio3 genomic region. In this study, we focus on mi R-1188 and its functions, investigate the dynamic expression patterns of mi R-1188 in embryogenesis. Combined with embryo development related target genes, the research on overexpression of mi R-1188 influences on embryo development. Meanwhile, combined with tumor related target genes, the research on the specific lower expression of mi R-1188 in Hepa1-6 cells is carried out the role of mi R-1188 in liver cancer.Firstly, we analyzed the expression patterns of mature mi R-1188 in different stages of embryo development and different kinds of mouse cancer cells. mi R-1188 expression in embryo was studied by real-time PCR(q RT-PCR) and RNA in situ hybridization(ISH). In early embryonic stage, q RT-PCR showed gradually decreased expression level of mi R-1188(4-cell to morula) and significanly increased expression in 4-cell, increases expression level mi R-1188 in mid-late embryonic stage(E12.5-E18.5), and mi R-1188 is high expression in liver, medium expression in brain, lung and heart, and low expression in kidney. mi R-1188 and host Meg8 share similar expression patterns. With the transfer of function of liver development, the expressions levels of mi R-1188 were gradually reduced, and high during embryo liver hematopoietic period, the highest in E15.5, lower after the birth. Then we used q RT-PCR to describe the expression pattern of mi R-1188 in the various cancer cell lines, the results showed that mi R-1188 in Hepa1-6 cells was the lowest than other cell lines. Meanwhile, comparision of differently expression of mi R-1188 in normal adult liver tissue and Hepa1-6 cells.To get further know about the function of mi R-1188 about embryo development, model of E15.5 with tail vein injection by over-expressed mi R-1188 lentiviral vector to pregant mice, observed that mi R-1188 caused embryo weight loss, and placenta weight increase accordingly. And q RT-PCR results show that mature mi R-1188 in embryo, placenta and amniotic sac were excessive enrichment. Among them, the expressions of mi R-1188 in lung and liver were high. But HE and in situ hybridization showed that mi R-1188 could not cause abnormal changes of major organs. mi RNA are conserved short RNAs that suppress protein expression through base pairing with the 3′-untranslated region(UTR) of target m RNAs. Through bioinformatics prediction, we postulated that Smad3 involved in embry development might represent targets of mi R-1188, combined with luciferase reporter assays and western blot identified that mi R-1188 targets Smad3. Correlation analysis indicated that there was a significant inverse correlation between mi R-1188 and Smad3 m RNA expression in mouse embryo development periods. The results showed that the expression of Smad3 in 2-cells is higher than it in 4-cells in early embryonic stage, the lowest in E15.5 than it in other mid-late embryonic stage, and Smad3 was lower expression in E15.5 liver. Then in the model of E15.5 with over-expressed mi R-1188, there was an inverse correlation between mi R-1188 and Smad3 m RNA in placenta, heart, liver and kidney. All these suggested that mi R-1188 could involve in embryo development though regulating Smad3.At last, in order to identify the function of mi R-1188 about tumorigenesis, the expressions of endogenous mi R-1188 were changed in Hepa1-6 cells. The results revealed that mi R-1188 significantly inhibited Hepa1-6 cells proliferation and colony formation, triggered morphological changes, increased caspase-3 activation and induced hepatoma cells apoptosis. Meanwhile, we postulated that Bcl-2 and Sp1 involved in tumorigenesis are targets of mi R-1188, identified that mi R-1188 could regulate their expressions at the post-transcription level, and correlation analysis indicated that there was a significant inverse correlation between mi R-1188 and Bcl-2 or Sp1 protein levels between normal liver tissues and Hepa1-6 cells, respectively. Besides, to explore the molecules involved in apoptosis, we measured the expressions of Bax, Bcl-XL and p53. Overexpression of mi R-1188 significantly suppressed the expression of Bcl-XL, and the expression of Bax and p53 was up-regulated. To investigate further mi R-1188 mediated apoptosis and invasion by modulating Bcl-2 and Sp1 levels, mi R-1188 analogs and targets si RNAs were co-transfected into Hepa1-6 cells. Finally, cell transplant assay in vivo showed that overexpression of mi R-1188 could impaire the proliferation of Hepa1-6 cells. Therefore, mi R-1188 might serve an important function in apoptosis and invasion pathways.Taken together, mi R-1188 is a widely expressed mi RNA in the embryo development and cancer cell lines. mi R-1188 caused embryo weight reduction and increased placenta weight, as well as inhibited the growth of liver cancer cells in vitro and vivo, and involved in the organism development and carcinogenesis though regulating the exptessions of target genes. The results will contribute to the further investigation of regulatory mechanism and functions of mi RNA in the Dlk1-Dio3 domain.更多还原