發(fā)布時間：2018-05-05 22:10

  本文選題：鼻咽癌 + lncRNA��； 參考：《廣州醫(yī)科大學》2017年碩士論文

【摘要】：【背景】長鏈非編碼RNA(long non-coding RNA,lnc RNA)是一類長度超過200nt的RNA,它們本身并不編碼蛋白,而是以RNA的形式在多層面上(表觀遺傳調控,轉錄調控以及轉錄后調控等)調節(jié)基因的表達【1】。近年來的研究表明,Lnc RNA廣泛參與各種生物學的過程,Lnc RNA的異常表達與包括腫瘤在內的多種疾病密切相關。已有證據(jù)證明lnc RNA參與肝癌、胰腺癌、膠質瘤等多種惡性腫瘤的發(fā)生發(fā)展【2-5】,但lnc RNA在鼻咽癌中的功能機制研究鮮有報道。鼻咽癌的發(fā)病率有明顯的地域差異性,在我國南方、北非及部分地中海地區(qū)是高發(fā)區(qū)域,特別在我國南方地區(qū)鼻咽癌明顯高發(fā)。鼻咽癌預后與疾病分期密切相關,由于鼻咽腫瘤生長部位隱蔽,早期沒有任何癥狀和體征,確診時已有70%的患者是Ⅲ或ⅣA期的局部晚期鼻咽癌【6】。近年來,盡管鼻咽癌的診治水平有了較大的提高,但其具體的發(fā)生機制仍不十分明確,臨床治療仍缺乏有效手段,5年生存率僅70%左右【7】,局部治療失敗和遠處轉是治療失敗的重要原因,因此,提高局部控制率和預防遠處轉移是局部晚期鼻咽癌患者治療上面臨的巨大挑戰(zhàn)。近年來,lnc RNA越來越多的成為人們研究的熱點,但lnc RNA與鼻咽癌的作用關系尚有待研究,尤其lnc RNA與局部晚期鼻咽癌的關系更值得我們去探討。LncRNA基因芯片是目前廣泛應用與研究1nc RNA表達譜的一種理想有效的方法。通過Lnc RNA基因芯片,能夠快速高通量的獲得與特定生物學過程或疾病相關的Lnc RNA的表達變化,從而為后續(xù)的Lnc RNA功能研究或生物標志物篩選提供極大便利�！灸康摹勘菊n題旨在研究與局部晚期鼻咽癌相關的lnc RNA及其相關生物學功能,并初步探討其發(fā)生機制,為后續(xù)鼻咽癌的研究提供理論和科學依據(jù)�！痉椒ā�1、采用Human Lnc RNA Array V4.0檢測局部晚期鼻咽癌組織及其對照的癌旁組織,得到兩種組織之間lnc RNA和m RNA差異表達譜,綜合NCBI Refseq,UCSC,RNAdb及Lnc RNAs等數(shù)據(jù)庫資源,對芯片結果進行聚類分析、GO分析及Pathway分析等初步生物信息學分析。2、體外培養(yǎng)鼻咽癌細胞CNE1和HONE1以及鼻咽正常上皮細胞NP69,運用Real-time PCR技術驗證目標lnc RNA在CNE1和HONE1與NP69的表達水平。3、采用si RNA干擾技術干擾目標lnc RNA表達后,運用Real-time PCR方法檢測si RNA的轉染效率,干擾目標lnc RNA后檢測目標lnc RNA對鼻咽癌細胞生物學特性的影響。4、應用CCK8法、Boyden侵襲實驗、Transwell遷移實驗和細胞劃痕實驗以及細胞凋亡實驗,分別檢測干擾目標lnc RNA后,對鼻咽癌細胞株CNE1和HONE1增殖、侵襲、遷移以及凋亡能力的影響�！窘Y果】第一部分:1、通過基因芯片技術成功篩選差異表達的lnc RNA和m RNA,設定篩選標準:P值≤0.05,Foldchange≥1.5,篩選得到324個差異表達lnc RNAs,上調226個,下調98個;同時有188個差異表達m RNAs,上調98個,下調90個。2、Pathway結果顯示:lnc RNA潛在的靶基因m RNA可能參與的信號通路共有22條,其中表達上調的信號通路6條,下調的13條。GO分析表明,上調表達的m RNA生物過程、細胞組成、分子功能富集程度最高的條目分別是cell chemotaxis,collagen trimer和chemokine activity;下調表達的分別是protein localization to cytoskeleton,cell cortex和protein homodimerization activity。第二部分:1、實時熒光定量PCR實驗結果顯示,AW450413、ENST00000501541、ENST00000457325、uc003owl.1相對表達量增加,ENST00000448834、ENST00000554458、ENST00000433377相對表達量降低,與基因芯片結果一致。更重要的是,ENST00000501541在CNE1和HONE1上表達量明顯高于NP69表達量,具有明顯統(tǒng)計學差異(p0.05)。2、CCK8增殖實驗結果顯示:與對照組(si RNA-NC)相比,實驗組(si RNA-1)、實驗組(si RNA-2)鼻咽癌細胞CNE1和HONE1增殖能力顯著下降(P0.05)。3、Transwell遷移實驗顯示:與對照組(si RNA-NC)相比,實驗組(si RNA-1)每高倍鏡下穿過的細胞數(shù)較對照組明顯減少(P0.05)。4、Boyden侵襲實驗結果顯示:與對照組(si RNA-NC)相比,實驗組(si RNA-1)每高倍鏡下穿過Matrigel膜的細胞數(shù)較對照組明顯減少(P0.05)。5、劃痕實驗結果顯示:與空白組及陰性對照組相比,在48小時和72小時檢測,實驗組(si RNA-1)細胞劃痕愈合能力較對照組顯著下降(P0.05)。6、凋亡實驗結果顯示:實驗組(si RNA-1)相對于陰性對照組(sh RNA-NC),鼻咽癌細胞CNE1和HONE1凋亡細胞百分比顯著增加(p0.05)�！窘Y論】1、我們利用lnc RNA基因芯片研究發(fā)現(xiàn)了324個差異表達lnc RNAs,并在鼻咽癌細胞株上驗證了基因芯片結果的可靠性。2、實驗證明ENST00000501541在鼻咽癌組織及鼻咽癌細胞株中均上調表達,干擾細胞ENST00000501541表達水平能夠抑制鼻咽癌細胞株增殖、遷移和侵襲能力以及促進細胞凋亡,結果提示ENST00000501541可能參與鼻咽癌的發(fā)生發(fā)展過程。綜上所述,我們的實驗初步研究了ENST00000501541在體外對鼻咽癌細胞株的影響,為后續(xù)鼻咽癌的功能機制研究打下了基礎,也為局部晚期鼻咽癌的研究提供了新的視角。
[Abstract]:[background] long chain non coded RNA (long non-coding RNA, LNC RNA) is a class of RNA with a length exceeding 200nt. They do not encode proteins themselves, but regulate the expression of genes in the form of RNA (epigenetic regulation, transcriptional regulation and post transcriptional regulation). [1]. Recent studies have shown that Lnc RNA is widely involved in a variety of life. The abnormal expression of Lnc RNA is closely related to a variety of diseases including tumors. There is evidence that LNC RNA is involved in the development of a variety of malignant tumors, such as liver cancer, pancreatic cancer, and glioma [2-5], but the functional mechanism of LNC RNA in nasopharyngeal carcinoma is rarely reported. The incidence of nasopharyngeal carcinoma has a distinct regional difference. In the south of China, North Africa and some Mediterranean regions are high incidence areas, especially in the southern part of China. Nasopharyngeal carcinoma has a high incidence of nasopharyngeal carcinoma. The prognosis of nasopharyngeal carcinoma is closely related to the staging of the disease. There are no symptoms and signs in the early stage of nasopharyngeal tumor, and 70% of the patients are locally advanced nasopharyngeal carcinoma (6) at the stage of stage III or IV A. In recent years, although the level of diagnosis and treatment of nasopharyngeal carcinoma has been greatly improved, its specific mechanism is still not very clear, the clinical treatment is still lack of effective means, the 5 year survival rate is only about 70% [7], local treatment failure and distant transfer are important reasons for the failure of treatment. Therefore, the improvement of local control rate and the prevention of distant metastasis are locally late. LNC RNA has become a hot topic in the treatment of nasopharyngeal carcinoma in recent years. In recent years, more and more people have been the hot spot of research, but the relationship between LNC RNA and nasopharyngeal carcinoma remains to be studied. Especially, the relationship between LNC RNA and local advanced nasopharyngeal carcinoma is worth exploring the.LncRNA gene core is widely used and studied the RNA expression spectrum of 1nc at present. An ideal and effective method. Through the Lnc RNA gene chip, the expression of Lnc RNA related to specific biological processes or diseases can be obtained quickly and high fluxes, thus providing a great convenience for subsequent Lnc RNA function research or biomarker screening. [Objective] this lesson aims to study l related to locally advanced nasopharyngeal carcinoma. NC RNA and its related biological functions, and preliminarily explore its mechanism to provide theoretical and scientific basis for the study of subsequent nasopharyngeal carcinoma. [method] 1, Human Lnc RNA Array V4.0 was used to detect the adjacent tissues of locally advanced nasopharyngeal carcinoma and its control, and the differential expression profiles of LNC RNA and m RNA were obtained between the two tissues. CSC, RNAdb and Lnc RNAs and other database resources, carry out cluster analysis, GO analysis and Pathway analysis for the preliminary bioinformatics analysis,.2, and in vitro culture of nasopharyngeal carcinoma cells CNE1 and HONE1, and normal nasopharyngeal epithelial cells NP69. After the A interference technique interference target LNC RNA expression, the Real-time PCR method was used to detect the transfection efficiency of Si RNA, and the effect of LNC RNA on target LNC RNA on the biological characteristics of nasopharyngeal carcinoma cells was detected after the target LNC RNA, and the interference test, the migration experiment, the cell scratch test and the cell apoptosis experiment were used to detect the interference, respectively. Target LNC RNA, the effect on proliferation, invasion, migration and apoptosis of nasopharyngeal carcinoma cell lines CNE1 and HONE1. [results] 1: 1, the differential expression of LNC RNA and m RNA were successfully screened by gene chip technology, and the screening criteria were set: P value less than 0.05, Foldchange > 1.5, 324 differential expression LNC RNAs, up 226, down 9 8, 188 differentially expressed m RNAs, up 98, and 90.2, and Pathway results showed that LNC RNA potential target gene m RNA may participate in 22 signal pathways, of which 6 of up regulated signaling pathway, and 13.GO analysis of down regulation indicated that the up regulation of M RNA biological process, cell composition, and the highest enrichment degree of molecular function The items are cell chemotaxis, collagen trimer and chemokine activity, respectively. The downregulated expressions are protein localization to cytoskeleton, cell cortex and second parts respectively. In addition, the relative expression of ENST00000448834, ENST00000554458 and ENST00000433377 decreased, which was consistent with the results of gene chip. More importantly, the expression of ENST00000501541 on CNE1 and HONE1 was significantly higher than that of NP69, with a significant statistical difference (P0.05).2, and the results of CCK8 colonization showed that compared with the control group (Si RNA-NC), the experimental group was compared with the control group (Si RNA-NC). RNA-1), the proliferation ability of CNE1 and HONE1 cells in the experimental group (Si RNA-2) decreased significantly (P0.05).3. The Transwell migration experiment showed that compared with the control group (Si RNA-NC), the number of cells passed under each high magnification microscope in the experimental group was significantly lower than that in the control group. The experimental results showed that compared with the control group, the experiment showed that the experiment group was compared with the control group. In group (Si RNA-1), the number of cells passing through Matrigel membrane under each high magnification was significantly lower than that of the control group (P0.05).5. The results of scratch test showed that compared with the blank group and negative control group, the scar healing ability of the experimental group (Si RNA-1) was significantly lower than that of the control group (P0.05).6 compared with the blank group and the negative control group (P0.05). The experimental group (Si RN) showed that the experimental group (Si RN) A-1) compared to the negative control group (SH RNA-NC), the percentage of CNE1 and HONE1 apoptotic cells in nasopharyngeal carcinoma cells increased significantly (P0.05). [Conclusion] 1, we found 324 differentially expressed LNC RNAs using LNC RNA gene chip study, and verified the reliability.2 of the gene chip results on the nasopharyngeal carcinoma cell line, and the experiment proved ENST00000501541 in nose. Both pharyngeal and nasopharyngeal carcinoma cell lines are all up expression. The interference of ENST00000501541 expression level can inhibit the proliferation, migration and invasion of nasopharyngeal carcinoma cell lines and promote cell apoptosis. The results suggest that ENST00000501541 may be involved in the development and development of nasopharyngeal carcinoma. In summary, our experiment preliminarily studied ENST00000501 The effect of 541 on nasopharyngeal carcinoma cell line in vitro lays a foundation for the study of the functional mechanism of the subsequent nasopharyngeal carcinoma and provides a new perspective for the study of local advanced nasopharyngeal carcinoma.

【學位授予單位】：廣州醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R739.63

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本文編號：1849502