宫颈癌中ΔNp63α的LncRNA表达谱及其抑制LIF表达的分子机制

发布时间：2018-03-05 08:34

本文选题：子宫颈癌　切入点：ΔNp63α　出处：《安徽医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景作为p53家族的成员,p63根据其N端转录激活区不同分为TA型和ΔN型,根据转录本3’端的选择性剪切产生不同的C端,至少包含6种亚型TAp63a,TAp63β,TAp63γ,ΔNp63a,ΔNp63β,ΔNp63γ[2-4]。p63是诱导基底细胞分化的重要调控基因,并且特别是在上皮分层和终末分化过程中。本课题组前期研究结果发现,ΔΝp63α是宫颈上皮组织中主要的表达亚型,在低分化的宫颈鳞癌中表达量低。LncRNA是一类大于200个核苷酸的非编码RNA[24]。因与肿瘤的紧密关联而备受瞩目。LncRNAs可以在各种水平上调节基因表达,包括染色质修饰,转录和转录后调控。LncRNA的异常表达会引起癌症的发生和进展,包括乳腺癌,肺腺癌和宫颈癌。本研究的目的是探讨子宫颈癌中ΔNp63α的LncRNA表达谱。通过分析这些筛选出mRNA和LncRNA,来构建ΔNp63α调控上皮增殖和分化的调控网络。方法通过慢病毒载体的构建和包装体系,构建了ΔNp63α稳定表达的SiHa细胞系及其对照细胞,分别命名为SiHa/ΔNp63α和SiHa/con。通过对SiHa/ΔNp63α细胞系及其对照细胞进行芯片测序,检测出有明显表达差异的mRNA和LncRNA。通过qRT-PCR在SiHa/ΔNp63α细胞系和瞬时沉默ΔNp63α的ME-180的细胞中多次验证候选序列,筛选出与ΔNp63α有明显相关性的mRNA和LncRNA,并对其进行分析。结果1.通过ΔNp63α的LncRNA表达谱分析和qRT-PCR验证,我们筛选出9个与ΔNp63α具有相关性的LncRNA,包括ENST00000447565(Lnc-LIF-AS),TCONS00012062,ENST00000570197,ENST00000563036,TCONS00011964,ENST00000439517,TCONS00004869,TCONS00014003,ENST00000469965。2.对这些LncRNA的UCSC分析之后,我们发现了由Lnc-LIF-AS和ΔNp63α共同调节的靶基因白血病抑制因子(Leukemia inhibitory factor,LIF)。ΔNp63α可在转录水平直接抑制LIF的表达,并通过抑制Lnc-LIF-AS的表达间接抑制LIF的表达。3.Lnc-LIF-AS通过与LIF mRNA的3'端的重叠区,吸附可能降解LIF的非编码RNA,促进LIF基因的表达。结论1.ΔNp63α通过下调Lnc-LIF-AS来间接抑制LIF的表达;2.ΔNp63α通过直接结合作用抑制LIF的转录,3.Lnc-LIF-AS与LIF mRNA的3'端UTR区重叠,吸附可能降解LIF的非编码RNA,促进LIF基因的表达。
[Abstract]:Background: p63, a member of p53 family, is divided into TA type and 螖 N type according to its N-terminal transcriptional activation region. There are at least 6 subtypes of TAp63 尾 TAp63 纬, 螖 Np63a, 螖 Np63 尾, 螖 Np63 纬 [2-4] .p63 are important regulatory genes to induce basal cell differentiation, especially in the process of epithelial stratification and terminal differentiation. In poorly differentiated squamous cell carcinoma of the cervix, low expression of .LncRNAs is a class of noncoding RNA [24] that is more than 200 nucleotides. LncRNAs have attracted attention because of their close association with tumors. LncRNAs can regulate gene expression at various levels, including chromatin modification. Abnormal expression of transcriptional and posttranscriptional regulation of. LncRNA can lead to the development and progression of cancer, including breast cancer, The aim of this study was to investigate the LncRNA expression profiles of 螖 Np63 伪 in cervical carcinoma. By analyzing these mRNA and LncRNAs, we constructed the regulatory network of 螖 Np63 伪 regulating epithelial proliferation and differentiation. Construction and packaging system, The stable expression of 螖 Np63 伪 in SiHa cell line and its control cell line were constructed, named SiHa/ 螖 Np63 伪 and SiHarcon. the SiHa/ 螖 Np63 伪 cell line and its control cell line were sequenced by microarray sequencing. MRNA and LncRNA, which had obvious difference in expression, were detected. The candidate sequences were verified by qRT-PCR in SiHa/ 螖 Np63 伪 cell line and ME-180 cell line with transient silence 螖 Np63 伪. MRNA and LncRNAs with significant correlation with 螖 Np63 伪 were screened and analyzed. Results 1.Through the LncRNA expression profiling and qRT-PCR verification of 螖 Np63 伪, we screened out 9 LncRNAs associated with 螖 Np63 伪, including ENST00000447565Lnc-LIF-ASTCONS00012062NST0000000563036-TCONS00011964nST00000439517TCONS00004869TCONS0001400000000000469965.After UCSC analysis of these LncRNA, We found that the target gene leukemia inhibitor, Leukemia inhibitory factor-lif, regulated by Lnc-LIF-AS and 螖 Np63 伪, could directly inhibit the expression of LIF at the transcriptional level, and indirectly inhibit the expression of LIF by inhibiting the expression of Lnc-LIF-AS. 3. Lnc-LIF-AS was found to be an overlapping region with the 3 'end of LIF mRNA. Conclusion: 1. 螖 Np63 伪 indirectly inhibits LIF expression by down-regulating Lnc-LIF-AS. 螖 Np63 伪 inhibits LIF transcription by direct binding. 3. Lnc-LIF-AS and LIF mRNA 3 'UTR region overlap. Adsorption may degrade noncoding RNAs of LIF and promote the expression of LIF gene.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.33

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