高通量測序研究卵巢子宮內(nèi)膜異位癥中異常lncRNA表達
發(fā)布時間：2018-12-30 17:11
【摘要】：子宮內(nèi)膜異位癥(endometriosis, EMs),是一種常見的婦科疾病,育齡期婦女發(fā)病率高達10-15%,病灶可侵犯全身任何組織,卵巢子宮內(nèi)膜異位癥是其常見的類型之一。盡管其具體發(fā)病機制不明,但一般認為內(nèi)異癥是一種多基因導致的疾病。研究表明長鏈非編碼RNA (long non-coding RNA, lncRNA)的異常表達與多種疾病相關,目前與子宮內(nèi)膜異位癥相關的lncRNA研究還較少。高通量測序技術是研究lncRNA的一種重要方法,國內(nèi)外目前尚無高通量測序對于子宮內(nèi)膜異位癥的研究,本實驗首次采用高通量測序技術對異位內(nèi)膜(ectopic endometrium, EC)、在位內(nèi)膜(eutopic endometrium,EU )及正常內(nèi)膜(normal endometrium, N)組織進行檢測,擬為內(nèi)異癥相關的lncRNA的后續(xù)研究提供候選基因庫,希望有助于進一步揭示lncRNA在內(nèi)異癥發(fā)生發(fā)展過程中的具體作用機制,及為后期尋找新的靶向治療藥物提供實驗基礎。第一部分:IncRNA在卵巢子宮內(nèi)膜異位癥中的表達譜分析目的:高通量測序技術篩選卵巢子宮內(nèi)膜異位癥中異位內(nèi)膜、在位內(nèi)膜和正常內(nèi)膜組織間差異表達的lncRNAs,并進行初步的功能研究,探尋內(nèi)異癥可能得發(fā)病機制。方法:采集8對人異位內(nèi)膜、在位內(nèi)膜組織及5例正常內(nèi)膜組織進行高通量測序檢測,并用生物信息技術對測序得到的海量信息進行分析,篩選三者間差異表達的lncRNAs,對差異lncRNAs進行功能注釋。結果:比較異位內(nèi)膜、在位內(nèi)膜及正常內(nèi)膜組之間差異表達lncRNAs,發(fā)現(xiàn)異位內(nèi)膜與在位內(nèi)膜之間有683個已知lncRNAs和443個新發(fā)現(xiàn)的lncRNAs異常表達;在位內(nèi)膜與正常內(nèi)膜之間有5個已知lncRNAs和56個新發(fā)現(xiàn)的lncRNAs異常表達;異位內(nèi)膜與正常內(nèi)膜之間有603個已知lncRNAs和408個新發(fā)現(xiàn)的lncRNAs異常表達。對其進行生物信息學分析發(fā)現(xiàn),異常表達的lncRNAs參與多種內(nèi)異癥的分子功能、細胞組分和生物過程中。結論:本研究首次采用了高通量測序技術對正常內(nèi)膜、在位內(nèi)膜及異位內(nèi)膜組織進行檢測,發(fā)現(xiàn)三者間有大量異常表達的lncRNAs,這些lncRNAs可能在卵巢子宮內(nèi)膜異位癥的發(fā)生發(fā)展中發(fā)揮著重要的作用。第二部分:IncRNAGAPLINC、CTD-2207P18.1 和 RP11-1100L3.8 在卵巢子宮內(nèi)膜異位癥中的表達分析目的:驗證 lncRNAGAPLINC、CTD-2207P18.1 和 RP11-1100L3.8 在異位內(nèi)膜、在位內(nèi)膜組織及正常內(nèi)膜組織中的差異表達,并探尋其可能作用途徑。方法:提取20對配對的人異位內(nèi)膜、在位內(nèi)膜組織及5例正常內(nèi)膜組織的總RNA,利用實時定量聚合酶鏈鎖反應(quantitative real time PCR,qRT-PCR)方法對3個差異表達的lncRNAs進行檢測,2-△△Ct的方法計算不同組織的實時定量數(shù)據(jù),組間差異采用t檢驗,P0.05認為差異有統(tǒng)計學意義。并對這3個異常表達的lncRNAs進行生物信息學分析。結果:lncRNAGAPLINC和CTD-2207P18.1異位內(nèi)膜中表達水平明顯高于在位內(nèi)膜,;ncRNARP11-1100L3.8在位內(nèi)膜中表達水平高于正常內(nèi)膜,三者參與到內(nèi)異癥的多個GO條目的富集及KEGG通路。結論:qRT-PCR結果分析與高通量測序一致。異位內(nèi)膜、在位內(nèi)膜中;ncRNA GAPLINC、CTD-2207P18.1、RP11-1100L3.8存在差異表達,這三者可能參與卵巢子宮內(nèi)膜異位癥的的發(fā)生發(fā)展。
[Abstract]:Endometriosis (EMs) is a common gynecological disease. The incidence of women in the childbearing potential is up to 10-15%. Although the specific pathogenesis is unknown, it is generally considered to be a multi-gene-induced disease. The research shows that the abnormal expression of long-chain non-coding RNA (lncRNA) is associated with a variety of diseases, and the research on the ncRNA associated with endometriosis is still less. high-throughput sequencing technology is an important method to study lncRNA, and there is no high-throughput sequencing at home and abroad for endometriosis. N) The tissue was tested to provide a candidate gene bank for the follow-up study of the lncRNA associated with the endosiosis, and it was hoped to help to further reveal the specific mechanism of the lncRNA in the development of the isodisease, and to provide an experimental basis for finding new targeted therapeutic drugs in the later stage. The first part: The purpose of the expression of IncRNA in the diagnosis of ovarian endometriosis: the high-throughput sequencing technique is used to screen the expression of the ncRNAs in the ectopic endometrium, the in-place and the normal endometrium of the endometriosis, and to carry out a preliminary functional study. It is possible to explore the possible mechanism of internal heterosis. Methods: 8 pairs of ectopic endometrium, in-place and 5 normal endometrium were collected for high-throughput sequencing, and the mass information obtained by sequencing was analyzed by using the biological information technology. Results: The abnormal expression of lncRNAs was found between the ectopic endometrium, the in-place and the normal endometrium group, and there were 683 known lncRNAs and 443 newly discovered lncRNAs, and there were 5 known lncRNAs and 56 newly discovered lncRNAs in the endometrium and the normal endometrium. There were 603 known lncRNAs and 408 newly discovered lncRNAs abnormal expression between the ectopic endometrium and the normal endometrium. Bioinformatics analysis shows that the abnormal expression of lncRNAs is involved in the molecular function, cellular components and biological processes of a variety of endothers. Conclusion: The first time in this study, high-throughput sequencing technology was used to detect the normal endometrium, in-place and ectopic endometrium, and it was found that there was a large number of abnormal expression of lncRNAs, which could play an important role in the development of endometriosis. The second part: The purpose of the expression and analysis of IncRNA GAPLINC, CTD-2207P18.1 and RP11-1100L3. 8 in the diagnosis of ovarian endometriosis: to verify the differential expression of lncRNAGAPLINC, CTD-2207P18.1 and RP11-1100L3. 8 in the ectopic endometrium, in-place and in the normal endometrium, and to explore the possible ways of action. Methods: The total RNA of 20 pairs of human heterotopic endometrium, in-place and 5 normal endometrium was extracted, and 3 differentially expressed lncRNAs were detected by the method of real-time quantitative polymerase chain reaction (qRT-PCR). The real-time quantitative data of different tissues was calculated by the method of 2-Thiophans. The difference between the groups was t-test, and the difference was considered to be of statistical significance. The bioinformatics analysis of the three abnormal expression of lncRNAs was carried out. Results: The expression level of ncRNAGAPLINC and CTD-2207P18. 1 was significantly higher than that in the in-place, and the expression of ncRNARP11-1100L3. 8 was higher than that in the normal endometrium. Conclusion: The results of qRT-PCR are consistent with high-throughput sequencing. The differential expression of ncRNA GAPLINC, CTD-2207P18.1, RP11-1100L3. 8 may be involved in the development of endometriosis.
【學位授予單位】：中國人民解放軍醫(yī)學院
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R711.71
本文編號：2395876
[Abstract]:Endometriosis (EMs) is a common gynecological disease. The incidence of women in the childbearing potential is up to 10-15%. Although the specific pathogenesis is unknown, it is generally considered to be a multi-gene-induced disease. The research shows that the abnormal expression of long-chain non-coding RNA (lncRNA) is associated with a variety of diseases, and the research on the ncRNA associated with endometriosis is still less. high-throughput sequencing technology is an important method to study lncRNA, and there is no high-throughput sequencing at home and abroad for endometriosis. N) The tissue was tested to provide a candidate gene bank for the follow-up study of the lncRNA associated with the endosiosis, and it was hoped to help to further reveal the specific mechanism of the lncRNA in the development of the isodisease, and to provide an experimental basis for finding new targeted therapeutic drugs in the later stage. The first part: The purpose of the expression of IncRNA in the diagnosis of ovarian endometriosis: the high-throughput sequencing technique is used to screen the expression of the ncRNAs in the ectopic endometrium, the in-place and the normal endometrium of the endometriosis, and to carry out a preliminary functional study. It is possible to explore the possible mechanism of internal heterosis. Methods: 8 pairs of ectopic endometrium, in-place and 5 normal endometrium were collected for high-throughput sequencing, and the mass information obtained by sequencing was analyzed by using the biological information technology. Results: The abnormal expression of lncRNAs was found between the ectopic endometrium, the in-place and the normal endometrium group, and there were 683 known lncRNAs and 443 newly discovered lncRNAs, and there were 5 known lncRNAs and 56 newly discovered lncRNAs in the endometrium and the normal endometrium. There were 603 known lncRNAs and 408 newly discovered lncRNAs abnormal expression between the ectopic endometrium and the normal endometrium. Bioinformatics analysis shows that the abnormal expression of lncRNAs is involved in the molecular function, cellular components and biological processes of a variety of endothers. Conclusion: The first time in this study, high-throughput sequencing technology was used to detect the normal endometrium, in-place and ectopic endometrium, and it was found that there was a large number of abnormal expression of lncRNAs, which could play an important role in the development of endometriosis. The second part: The purpose of the expression and analysis of IncRNA GAPLINC, CTD-2207P18.1 and RP11-1100L3. 8 in the diagnosis of ovarian endometriosis: to verify the differential expression of lncRNAGAPLINC, CTD-2207P18.1 and RP11-1100L3. 8 in the ectopic endometrium, in-place and in the normal endometrium, and to explore the possible ways of action. Methods: The total RNA of 20 pairs of human heterotopic endometrium, in-place and 5 normal endometrium was extracted, and 3 differentially expressed lncRNAs were detected by the method of real-time quantitative polymerase chain reaction (qRT-PCR). The real-time quantitative data of different tissues was calculated by the method of 2-Thiophans. The difference between the groups was t-test, and the difference was considered to be of statistical significance. The bioinformatics analysis of the three abnormal expression of lncRNAs was carried out. Results: The expression level of ncRNAGAPLINC and CTD-2207P18. 1 was significantly higher than that in the in-place, and the expression of ncRNARP11-1100L3. 8 was higher than that in the normal endometrium. Conclusion: The results of qRT-PCR are consistent with high-throughput sequencing. The differential expression of ncRNA GAPLINC, CTD-2207P18.1, RP11-1100L3. 8 may be involved in the development of endometriosis.
【學位授予單位】：中國人民解放軍醫(yī)學院
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R711.71
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