本文選題：子宮頸癌　切入點：ΔNp63α　出處：《安徽醫(yī)科大學》2017年碩士論文　論文類型：學位論文

【摘要】：研究背景作為p53家族的成員,p63根據(jù)其N端轉錄激活區(qū)不同分為TA型和ΔN型,根據(jù)轉錄本3’端的選擇性剪切產(chǎn)生不同的C端,至少包含6種亞型TAp63a,TAp63β,TAp63γ,ΔNp63a,ΔNp63β,ΔNp63γ[2-4]。p63是誘導基底細胞分化的重要調控基因,并且特別是在上皮分層和終末分化過程中。本課題組前期研究結果發(fā)現(xiàn),ΔΝp63α是宮頸上皮組織中主要的表達亞型,在低分化的宮頸鱗癌中表達量低。LncRNA是一類大于200個核苷酸的非編碼RNA[24]。因與腫瘤的緊密關聯(lián)而備受矚目。LncRNAs可以在各種水平上調節(jié)基因表達,包括染色質修飾,轉錄和轉錄后調控。LncRNA的異常表達會引起癌癥的發(fā)生和進展,包括乳腺癌,肺腺癌和宮頸癌。本研究的目的是探討子宮頸癌中ΔNp63α的LncRNA表達譜。通過分析這些篩選出mRNA和LncRNA,來構建ΔNp63α調控上皮增殖和分化的調控網(wǎng)絡。方法通過慢病毒載體的構建和包裝體系,構建了ΔNp63α穩(wěn)定表達的SiHa細胞系及其對照細胞,分別命名為SiHa/ΔNp63α和SiHa/con。通過對SiHa/ΔNp63α細胞系及其對照細胞進行芯片測序,檢測出有明顯表達差異的mRNA和LncRNA。通過qRT-PCR在SiHa/ΔNp63α細胞系和瞬時沉默ΔNp63α的ME-180的細胞中多次驗證候選序列,篩選出與ΔNp63α有明顯相關性的mRNA和LncRNA,并對其進行分析。結果1.通過ΔNp63α的LncRNA表達譜分析和qRT-PCR驗證,我們篩選出9個與ΔNp63α具有相關性的LncRNA,包括ENST00000447565(Lnc-LIF-AS),TCONS00012062,ENST00000570197,ENST00000563036,TCONS00011964,ENST00000439517,TCONS00004869,TCONS00014003,ENST00000469965。2.對這些LncRNA的UCSC分析之后,我們發(fā)現(xiàn)了由Lnc-LIF-AS和ΔNp63α共同調節(jié)的靶基因白血病抑制因子(Leukemia inhibitory factor,LIF)。ΔNp63α可在轉錄水平直接抑制LIF的表達,并通過抑制Lnc-LIF-AS的表達間接抑制LIF的表達。3.Lnc-LIF-AS通過與LIF mRNA的3'端的重疊區(qū),吸附可能降解LIF的非編碼RNA,促進LIF基因的表達。結論1.ΔNp63α通過下調Lnc-LIF-AS來間接抑制LIF的表達;2.ΔNp63α通過直接結合作用抑制LIF的轉錄,3.Lnc-LIF-AS與LIF mRNA的3'端UTR區(qū)重疊,吸附可能降解LIF的非編碼RNA,促進LIF基因的表達。
[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 緯, 螖 Np63a, 螖 Np63 尾, 螖 Np63 緯 [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 偽 in cervical carcinoma. By analyzing these mRNA and LncRNAs, we constructed the regulatory network of 螖 Np63 偽 regulating epithelial proliferation and differentiation. Construction and packaging system, The stable expression of 螖 Np63 偽 in SiHa cell line and its control cell line were constructed, named SiHa/ 螖 Np63 偽 and SiHarcon. the SiHa/ 螖 Np63 偽 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 偽 cell line and ME-180 cell line with transient silence 螖 Np63 偽. MRNA and LncRNAs with significant correlation with 螖 Np63 偽 were screened and analyzed. Results 1.Through the LncRNA expression profiling and qRT-PCR verification of 螖 Np63 偽, we screened out 9 LncRNAs associated with 螖 Np63 偽, 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 偽, 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 偽 indirectly inhibits LIF expression by down-regulating Lnc-LIF-AS. 螖 Np63 偽 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.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R737.33

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