【作者】 朱浩；

【导师】 周光新；

【作者基本信息】 南京大学 ， 临床医学（骨科）， 2017， 硕士

【摘要】 世界上第一个被发现的miRNA是在1993年在线虫中发现。时隔7年,2000年在人体中也发现了 miRNA的存在,到目前为止已经有2000多种miRNA被报道能够调控基因的表达。miRNA是一种长度为19-24个nt的单链RNA,通过结合到靶基因mRNA的3’-UTR区域在转录后水平调控基因的表达,已经被预测与分化、发生、发展、代谢及凋亡等等大量生物进程相关。miRNAs与肿瘤相关的研究也是日益增多。本实验选取了骨巨细胞瘤中的RANK以及肺癌中的Ral作为靶点,分别研究miR-129和miR-215在各自肿瘤中的作用,了解这些miRNAs在其肿瘤的生物学进程中的所扮演的角色,对于寻找肿瘤早期诊断及治疗的新靶点有着积极的作用。在第一部分中,我们以骨巨细胞瘤作为对象。骨巨细胞瘤一种常见的原发性中间性骨肿瘤。我国是骨巨细胞瘤的高发国家,原发性骨肿瘤中有20%都是骨巨细胞瘤。骨巨细胞瘤目前的治疗以外科手术治疗为主,但是针对难以手术切除以及术后复发的骨巨细胞瘤,仍缺少一种安全、副作用下、适用性广的治疗方法。我们希望通过miRNA来解决这个问题。我们使用miRNA芯片技术检测2个瘤旁组织和4个骨巨细胞瘤组织的miRNA表达谱,找出了其中有显著性表达差异的miRNA,并使用生物信息学软件预测了在RANK的3’-UTR端存在结合位点的miRNA,从中筛选出了 miR-129作为研究对象。转染miR-129 mimic后在蛋白水平验证miR-129对RANK的调控。但是目前未取得进展,拟调整实验来进一步验证。在第二部分中,我们以肺癌为研究对象。肺癌是恶性肿瘤中发病率和死亡率最高的。目前肺癌的治疗手段主要有手术治疗、化疗、放疗及靶向药物等,但是疗效一般,肺癌的死亡率依然居高不下。我们希望通过miRNA找到新的治疗靶点。在这个实验中,我们通过查阅相关资料,发现在多种肺癌组织中,Ral家族蛋白都呈现高表达的情况,选定为研究靶点。向目标细胞系中转染Ral的过表达质粒,再通过功能实验验证Ral家族在肺癌中的功能。使用targetscan网站预测相关的miRNAs。通过将预测可能的miRNAsmimic转染入肺癌细胞,在蛋白水平验证各种miRNA对Ral家族的影响,从中筛选出对Ral家族调控作用最为明显的miR-215,将其作为主要的研究对象。接着通过Luciferase实验在细胞水平验证了 miR-215能够和Ral家族的mRNA的3’-UTR直接结合从而影响Ral家族的表达。之后通过CCK8、EdU、FCM、Transwell等的回复实验,验证了 miR-215 通过调控 Ral 家族的表达从而调节肺癌的增殖、侵袭等生物进程,为肺癌的治疗提供了新的思路及实验依据。更多还原

【Abstract】 microRNAs(miRNAs)are small(19-24 nucleotides)non-coding RNA molecules that were first discovered at nematos in 1993.miRNAs act as endogenous suppressors of gene expression by binding to 3’-UTR of the target gene’s mRNAs.A large number of mi RNAs including miR-31,miR-86,miR-183 and miR-221/222 were reported playing an important role in many biological process of tumors,such as poliferation,differentiation,apoptosis and invasion.More and more researchs are carried on the connection between miRNAs and tumor.In this study,wo focused on giant cell tumor of bone(GCTB)and lung cancer,We studied how miR-129 influences it’s target RANK and how miR-125 influences the target Ral to inhibit the tumor.We hope that we could find new targets of tumor therapy.In the first part,wo focoused on GCTB.GCTB is a primary skeletal neoplasm.China is a high-risk group of GCTB.Usually,the remedial method of GCTB is surgery.However,in some typical cases,it’s difficult to have a operation and the existing therapy ia not satisfied.We need new therapeutic methods.We showed differential expression of miRNA profiles between GCTB tissues and normal control by miRNA gene chip technology and predicted that miR-129 can bind to RANK.We then tranfected miR-129 into A549 by mimic and checked the protein level of RANK.But unfortunately,these preliminary work failed and we put forward a corresponding adjusting scheme.In the secondary part,we focoused on lung cancer.Lung cancer is the most prevalent tumor,with the highest morbidity and mortality over the wolrd.But unfortunately,the therapeutic methods of lung cancer did not get satisfied results.All of this remind us that more attention should be paid on the lung cancer and the new potential therapeutic targets are required.By consulting a source,we found that in most of lung cancer,the Ral was overexpression and Ral was selected as our target of the study.We up-regulated the level of RalA and RalB in A549,and check the fuction of RalA and RalB in A549 by CCK8,EdU and Transwell.We then used TargetScan to find some miRNAs that can bind to Ral.We then tranfected these miRNAs in A549 by mimic and checked the protein level of RalA and RalB in these transfected A549.We found that these A549 were weak at poliferation and invasion.We screened these miRNAs,and focused on miR-215.We use CCK8,Transwell and EdU recovery tests to prove that miR-215 can restrain lung caner by targeting the RalA and RalB.We provide theoretical basis for new target of lung cancer therapy.更多还原