【作者】 李娜；

【导师】 邵淑娟；

【作者基本信息】 大连医科大学 ， 人体解剖与组织胚胎学， 2017， 硕士

【摘要】 目的:长链非编码RNA(long noncoding RNA,Lnc RNA)是一类由RNA聚合酶Ⅱ转录形成,转录本长度超过200nt的RNA分子,不编码蛋白,其可在表观遗传学,转录水平和转录后水平等多个层面调控基因表达。其功能的重要性已经受到越来越多人的关注。肺癌的发病率及死亡率都位居恶性肿瘤的前列,这主要是因为肺癌易转移、高侵袭、难治愈。而肺癌的转移主要是由于发生了上皮细胞间质转化(epithelial to mesenchymal transition,EMT),上皮细胞失去自身极性、获得运动能力,发生EMT的细胞离开上皮组织侵入周围组织或向更远端的位置转移。TGF-β可以通过一系列的信号通路活化EMT相关的转录因子,从而诱导EMT。如果可以发现并探索出与EMT相关lnc RNA在此过程中的功能及机制,可为临床的诊断及预后提供有力的科学依据,同时为肺癌的治疗提供可能的生物靶点。目前,国内外研究报道的与癌症相关lnc RNA的文献虽多,但与肺癌EMT相关的较少。我们旨在寻找出与肺癌EMT相关的lnc RNA,并对其功能进行探究。方法:本课题对TGF-β诱导EMT模型中的lnc RNA芯片进行分析,按照一定的筛选原则包括基因长度、变化倍数及与m RNA的位置关系等,筛选出6条lnc RNA序列,使用q PCR的方法进行验证,从中选择一条在EMT模型中下调的lnc RNA;利用抑制剂处理A549细胞,q PCR方法检测MNX1-AS1的表达变化。同时利用数据库LNCipedia检测MNX1-AS1的编码潜能,利用q PCR方法检测MNX1-AS1在21对肺癌临床组织标本及7种肺癌细胞系中的表达情况,并在A549细胞中对MNX1-AS1的细胞定位检测,运用RACE实验方法鉴定MNX1-AS1全长、通过构建sh RNA的方法稳定敲降MNX1-AS1、运用质粒过表达MNX1-AS1,通过划痕、transwell检测其对肺癌细胞侵袭、迁移的影响;用western blot的方法对MNX1-AS1的作用机制进行初步探究。结果:通过对芯片结果的分析,筛选并验证表达下调的目的lnc RNA,该lnc RNA在UCSC数据库名为MNX1-AS1,位于7号染色体上;同时,使用TGF-β通路抑制剂SB431542处理A549细胞,能够抑制TGF-β下调MNX1-AS1的表达。LNCipedia数据库中显示MNX1-AS1无编码潜能,MNX1-AS1在21对癌组织表达均低于癌旁组织,在A549细胞系中表达较高,在H1299细胞中表达较低。RACE方法鉴定MNX1-AS1的全长序列为1035bp,核浆分提实验表明MNX1-AS1在A549细胞核、质中均有表达。功能实验研究表明,稳定敲降MNX1-AS1能够促进肺癌细胞的侵袭和转移能力,过表达MNX1-AS1能够抑制肺癌细胞的侵袭和转移能力。初步的机制研究发现,sh Lnc RNA能够抑制E-cadherin RNA及蛋白水平的表达,同时上调Vimentin、Twist RNA及蛋白水平的表达。结论:本课题在TGF-β诱导EMT模型的芯片结果中,筛选出一条表达下调的lnc RNA MNX1-AS1;使用TGF-β通路抑制剂SB431542会抑制MNX1-AS1的下调;稳定敲降MNX1-AS1能够促进肺癌细胞的侵袭和转移能力过表达MNX1-AS1能够抑制肺癌细胞的侵袭和转移能力。同时敲降MNX1-AS1也能够抑制E-cadherin的表达,上调Vimentin、Twist的表达。综上所述,MNX1-AS1能够影响肺癌EMT的过程,可能为肺癌治疗提供新的生物靶点,同时为肺癌的治疗提供有力的理论依据。更多还原

【Abstract】 Objective: Long non-coding RNA(Lnc RNA)is a class of RNA molecules that are transcribed by RNA polymerase II and its transcribe more than 200 nt,without coding potential,which can regulate the expression of gene on epigenetics,transcription level and Post-transcription level.More and more attention has been paid to its function.The morbidity and mortality of lung cancerare among the forefront of malignant tumors,because of it’s easy to migration,invasion and difficult to cure.Due to the occurrence of EMT,epithelial cells lose their polarity and acquire exercise capacity.Then cells leave the epithelial tissue and transferto surrounding tissue or more distant position.TGF-β can activate EMT-related transcription factors through a series of signaling pathways,so we can induce EMT by TGF-β.If the function and mechanism of EMT-related lnc RNA in this processcan be discovered and explored,it will provide a strong scientific foundation for clinical diagnosis and prognosisand possible biological targets for treatment of lung cancer.At present,the domestic and foreign research has reported that lnc RNA is associate with cancer.Then there werea few research had reported that lnc RNA is associate with EMT.And related researches on lung cancer were less reported.Methods: In this study,we used the EMT model induced by TGF-β1and Lnc RNA microarray analysis.We screened the target lnc RNA related to EMT and verified it by q-PCR.We used the inhibitor of TGF-βto study the upstream of Lnc RNA.We used sh RNA to knockdown lnc RNA orvehicle to overexpress lnc RNA can influence the invasion and migration of lung cancer cells.We used the western blot to explore the mechanism of lnc RNA.Results: Through analysis the results of lnc RNA microarray,we screening and verifying the down regulated lnc RNA.This lnc RNA in the UCSC database were named MNX1-AS1 and located on chromosome 7.At the same time,the A549 lung cancer cells were treated with TGF-β pathway inhibitor SB431542.The results showed that the inhibitior could suppress the function of TGF-β can downregulate the MNX1-AS1 expression.LNCipedia show that MNX1-AS1 has no coding potential.And the expression of MNX1-AS1 in 21 coupleof cancer tissues was lower than adjacent tissues.The expression of MNX1-AS1 was higher in the A549 cell lines,but was lower in H1299 cell lines.We used the RACE method to identify the full-length sequence of lnc RNA(1035bp).The research showed that the MNX1-AS1 was expressed in A549 cell lines nuclear and cytoplasm.Functional experiment shown that knockdown the MNX1-AS1 can promote the invasion and metastasis of lung cancer cells.Overexpression of MNX1-AS1 can inhibit the invasion and metastasis of lung cancer cells.Preliminary mechanism studies have shown that shlnc RNA can inhibit the expression of E-cadherin and up-regulating the expression of vimentin,Twist.Conclusion: Through analysis the results of lnc RNA microarray,one down regulated lnc RNA(MNX1-AS1)was screened.The TGF-βinhibitor(SB431542)could suppress the downregulated expression of MNX1-AS1.Recent experiments have shown thatknockdown the MNX1-AS1 can promote the invasion and metastasis of lung cancer cell.On the contrary,overexpression of MNX1-AS1 can inhibit the ability of the migration and invasion in lung cancer cells.Simultaneously,knockdown MNX1-AS1 can inhibit the expression of E-cadherin and up-regulating the expression of vimentin and Twist.Above all,MNX1-AS1 can affect the EMT process of lung cancer and may provide a new biological target for lung cancer treatment,at the same time provide strong theoretical basis for the treatment of lung cancer.更多还原