miR-3065-5p和miR-1307-5p在肝癌细胞中的功能研究

发布时间：2018-04-03 06:14

本文选题：肝癌　切入点：微小核糖核酸　出处：《内蒙古大学》2017年硕士论文

【摘要】：肝细胞癌(Hepatocellular carcinoma,HCC)是一种常见的恶性肿瘤,多发于成人,肝癌的发生、发展和转移是一个复杂的过程,受多个因子和基因的调节[1]。微小核糖核酸RNA(MicroRNA,miRNA)是一类长度在19-24个核苷酸范围内的内源性非编码RNA,miRNA能够通过与靶基因mRNA3' UTR互补结合来抑制翻译或者促进mRNA降解,从而调控相关基因的表达。大量研究报道称miRNA在许多肿瘤细胞中都存在差异性表达,并且在肿瘤的发生发展中都可能发挥着重要的作用。我们实验室前期的研究发现一定浓度的脐带间充质干细胞(Umbilical cord-derived mesenchymal stem cells,UC-MSCs)条件培养基能够抑制肝癌细胞HepG2的增殖,并且通过高通量测序技术,分析了 UC-MSCs条件培养基共培养后HepG2 miRNAs的差异表达谱,找到了一些可能相关的miRNAs。其中miR-3065-5p和miR-1307-5p在UC-MSCs条件培养基处理HepG2后表达水平显著上调。首先,我们通过qRT-PCR检测了三种肝癌细胞系(HepG2、MHCC97-L、MHCC87-H)和正常肝细胞系L02中miR-3065-5p的表达是否存在差异;结果显示,相较正常肝细胞L02,miR-3065-5p在肝癌细胞系中的表达明显下调,接下来我们通过体外合成miR-3065-5p质粒转染三种肝癌细胞,结果发现过表达miR-3065-5p能够显著抑制三种肝癌细胞的增殖、侵袭、克隆形成能力。此外,我们通过生物信息学预测到SATB1可能是miR-3065-5p的相关靶基因,通过双荧光素酶报告基因实验进一步检测了预测的可靠性,实验结果显示SATB1与miR-3065-5p存在互补结合位点。qRT-PCR与Western blot结果显示过表达的miR-3065-5p能够显著抑制SATB1表达水平。最后通过体外合成siRNA干扰SATB1在肝癌细胞中的表达,结果显示,干扰SATB1后也能显著抑制肝癌细胞的增殖,其效果与miR-3065-5p类似。在miR-1307-5p的研究中,我们发现过表达的miR-1307-5p也能显著抑制肝癌细胞的增殖、侵袭等生物学特性,而在靶基因的筛选验证中,我们通过生物信息学软件预测了 PIM3可能是miR-1307-5p的靶基因,但是双荧光素酶报告基因实验效果并不明显,说明PIM3并不是miR-1307-5p的相关靶基因,其靶基因有待进一步筛选验证。
[Abstract]:Hepatocellular carcinoma (HCC) is a common malignant tumor, mostly in adults. The occurrence, development and metastasis of HCC is a complicated process, which is regulated by many factors and genes.MicroRNAs miRNAs are a class of endogenous non-coding RNAs in the range of 19-24 nucleotides, which can inhibit translation or promote mRNA degradation by complementing with target gene mRNA3'#en0#, thus regulating the expression of related genes.A large number of studies have reported that miRNA is differentially expressed in many tumor cells and may play an important role in tumorigenesis and development.Our previous study in our laboratory found that Umbilical cord-derived mesenchymal stem stem cells (UC-MSCs) conditioned medium could inhibit the proliferation of HepG2 cells, and high throughput sequencing technique was used.The differential expression profiles of HepG2 miRNAs after co-culture on UC-MSCs conditioned medium were analyzed, and some possible related miRNAs were found.The expression levels of miR-3065-5p and miR-1307-5p were upregulated significantly after HepG2 treatment in UC-MSCs conditioned medium.First, we detected the difference of the expression of miR-3065-5p in three kinds of hepatoma cell lines (HepG2MHCC97-LHCC87-H) and normal liver cell line L02 by qRT-PCR, the results showed that the expression of MHCC97-MHCC87-H2MHCC97-L02miR-3065-5p was significantly down-regulated than that of normal hepatoma cell line L02miR-3065-5p.Then we synthesized miR-3065-5p plasmid in vitro and transfected into three kinds of hepatoma cells. The results showed that overexpression of miR-3065-5p could significantly inhibit the proliferation invasion and clone formation of three kinds of hepatoma cells.In addition, we predicted that SATB1 might be the target gene of miR-3065-5p by bioinformatics, and further tested the reliability of the prediction by double luciferase reporter gene experiment.The results showed that there were complementary binding sites between SATB1 and miR-3065-5p. QRT-PCR and Western blot showed that overexpression of miR-3065-5p could significantly inhibit the expression of SATB1.Finally, siRNA was synthesized in vitro to interfere the expression of SATB1 in hepatoma cells. The results showed that interfering with SATB1 could significantly inhibit the proliferation of HCC cells, and its effect was similar to that of miR-3065-5p.In the study of miR-1307-5p, we found that overexpression of miR-1307-5p could significantly inhibit the proliferation and invasion of hepatoma cells. In the screening and verification of target genes, we predicted that PIM3 might be the target gene of miR-1307-5p by bioinformatics software.But the effect of double luciferase reporter gene is not obvious, which indicates that PIM3 is not the target gene of miR-1307-5p, and its target gene needs further screening and verification.
【学位授予单位】：内蒙古大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

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