【作者】 张健；

【导师】 魏锋；

【作者基本信息】 吉林大学 ， 临床医学硕士（外科学）（专业学位）， 2021， 硕士

【摘要】 目的:胰腺癌是一种恶性程度极高的实体瘤。在美国,胰腺癌位列肿瘤相关死亡人数第4位,而在中国,则位列城市男性恶性肿瘤发病率的第8位。近年来,胰腺癌的发病率还在持续升高,而五年生存率仅9%。由于大多病例诊断为胰腺癌时已处于晚期以及肿瘤的耐药性,其预后极差。近年来,非编码RNA研究的突破很大程度上提高了恶性肿瘤患者的生存率以及生活质量。非编码RNA包括微小RNA(Micro RNA,miRNA)和长链非编码RNA(Long non-coding RNA,Lnc RNA)。他们在多种肿瘤中表达异常,与肿瘤的发生发展有着密切的联系,因此可以作为肿瘤检测的分子标志物以及治疗的作用靶点。其中,MiR-216a-5p在不同肿瘤中有着不同的生物学功能,既有抑癌功能,也有促癌功能。然而,miR-216a-5p在胰腺癌中的功能目前尚未研究清楚。本研究通过多种方法,意在探索miR-216a-5p是否可以成为潜在的胰腺癌治疗靶点,并探究其发挥作用背后的分子机制。方法:通过RNA测序(RNA sequencing),发现miR-216a-5p在胰腺癌组织中的表达水平低于癌旁组织。进一步通过生物信息学方法在Jaspar,Target scan和Miranda三个数据库发现miR-216a-5p与TPT1和LINC01133之间可以靶向结合,并预测他们之间的结合位点。使用双荧光素酶基因报告系统和RNA pull down实验分别检测并验证miR-216a-5p与TPT1和LINC01133相互作用关系。通过实时荧光定量多聚酶链式反应(Quantitative real time polymerase chain reaction,qRT-PCR)检测miR-216a-5p,TPT1,LINC01133在胰腺癌患者组织,血浆和不同细胞系中的表达水平。运用蛋白质印记法和免疫组化(Immunohistochemistry,IHC)的方法分别检测TPT1蛋白以及自噬相关蛋白在转染后胰腺癌细胞中的表达和胰腺癌组织中的表达水平。利用Lipofectamin2000来转染miR-216a-5p mimics/inhibitor,TPT1质粒,LINC01133质粒,siRNA-TPT1,siRNA-LINC01133以及他们相应的阴性对照。通过流式细胞术,Ed U增殖实验,克隆形成实验,划痕实验以及Transwell小室实验来检测miR-216a-5p,TPT1和LINC01133在体外的生物学功能。使用慢病毒感染胰腺癌细胞系SW1990,构建稳转细胞系,经尾静脉注射建立重型联合免疫缺陷鼠(Severe combined immunodeficiency mice,SCID mice)的肺成瘤模型,饲养6周后通过小动物成像系统和苏木精-伊红染色观察肿瘤病理标本。所有的数据结果采用SPSS 20.0和Graphpad Prism软件分析,数据结果用均值±标准差表现,用t检验检测两组数据之间的差异,方差分析检测多组数据之间的差异,临床患者的病理参数的分析则由卡方检验计算,P值小于0.05则表示结果有统计学意义。结果:在本研究中,我们发现miR-216a-5p在胰腺癌组织、血浆和细胞系中都呈低表达,并且与胰腺癌患者TNM分期,胰周淋巴结转移和周围神经浸润相关。功能学实验证明了miR-216a-5p在体内和体外可以抑制胰腺癌细胞的增殖,侵袭和迁移。另外,通过生物信息学分析和荧光素酶基因报告系统,我们发现了TPT1(Tumor controlled transcriptionally protein 1,TCTP/TPT1)是miR-216a-5p的潜在靶点,可以通过介导哺乳动物雷帕霉素靶蛋白(Mammalian target of rapamycin,mTOR)通路相关的自噬来调节胰腺癌的恶性程度。除此之外,生物信息学分析,荧光素酶基因报告系统和RNA pull down实验还证明了miR-216a-5p可以与长链非编码RNA 01133(Long intergenic none-protein coding RNA 1133,LINC01133)相互作用。而LINC01133在体外具有促进胰腺癌细胞增殖,侵袭和迁移的生物学功能。结论:本研究首次发现了miR-216a-5p在胰腺癌中呈低表达状态,并且可以通过TPT1及其下游通路、LINC01133的相互作用,影响胰腺癌的恶性程度。另外,本研究首次发现胰腺癌中的LINC01133/miR-216a-5p/TPT1通路,也为临床上胰腺癌的诊断和治疗提供新的策略。更多还原

【Abstract】 Objective:Pancreatic cancer(PC)is one of the most fatal solid tumors.It is currently the fourth leading cause of cancer-related deaths in the United States and the eighth leading cancer of new cancer cases of urban male in China,respectively.In past decades,there has been a steady increase in PC incidence while the 5 year survival rate has remained the lowest 9%.The dismal prognosis is largely attributed to delayed diagnosis and drug resistance.Recently,the discoveries of non-coding RNAs have significantly promoted the survival rate and life quality of malignant tumor patients.Non-coding RNAs including both micro RNAs(miRNAs)and long non-coding RNAs(lnc RNAs),are aberrantly expressed in different types of cancers.These miRNAs play a key role in the initiation and progression of tumors.Among them,miR-216a-5p was shown to act either as a tumor suppressor or an oncogene.Nevertheless,the role of miR-216a-5p in the initiation and progression of PC is not well characterized.In the currently study,we used many methods to explore whether miR-216a-5p could become the potential target of PC treatment and attempted to uncover the molecular mechanism behind it.Methods:RNA sequencing showed that the expression of miR-216a-5p was significantly down-regulated in adjacent normal normal tissues(ANT)than PC tissues.Then,by employing the bioinformatics analysis we predicted the binding sites of miR-216a-5p,TPT1 and LINC01133 with one another in Jaspar,Target scan and Miranda databases.Using luciferase reporter assay and RNA pull down assay,we demonstrated the interaction between miR-216a-5p with TPT1 or LINC01133.Quantitative real time polymerase chain reaction(qRT-PCR)was used to detect the expression levels of miR-216a-5p,TPT1 and LINC01133 from adjacent normal tissues(ANT),PC tissues,plasma and different cell lines.Western blotting(WB)and Immunohistochemistry(IHC)were performed to detect the expression levels of TPT1 and autophagy-related proteins from different transfected cells or PC tissues.Lipofectamin2000 was adopted to transfect miR-216a-5p mimics,miR-216a-5p inhibitor,TPT1,LINC01133,siRNA-TPT1,siRNA-LINC01133 and their negative controls(NC).Flow cytometry,Edu incorporation assay,colony formation assay,wound healing assay and transwell assay were conducted to validate the bio-functions of miR-216a-5p,TPT1 and LINC01133.Stable cell lines were established using lentivirus and were injected via tail vein into Severe combined immunodeficiency mice’(SCID mice)lungs to establish pulmonary metastasis models.After 6 weeks’,IVIS Lumina XR system and HE staining were used to observe the metastasis specimens.All statistical analyses were conducted using SPSS 20.0 and Graph Pad Prism software.Data are presented as mean±standard deviation(SD).Two-tailed unpaired Student’s t-test was used to assess differences between two groups.The clinicopathological parameters of patients were analyzed byχ~2 test.P values<0.05 were considered indicative of statistical significance.Results:In the current study,we demonstrated that miR-216a-5p is strongly downregulated in PC tissues and cells and is closely associated with TNM stage,peripancreatic lymphatic metastasis,and perineural invasion.Functional assays validated that miR-216a-5p could prohibit cell proliferation,invasion and metastasis both in vitro and vivo.Moreover,via bioinformatics analyses and luciferase reporter assay we found Tumor controlled transcriptionally protein(TCTP/TPT1)is a potential target of miR-216a-5p and is associated with the Mammalian target of rapamycin(mTOR)signaling pathway.Importantly,bioinformatics analyses,luciferase reporter assay and RNA pull down assay proved that miR-216a-5p could interact with Long intergenic none-protein coding RNA 1133(LINC01133),which was confirmed in latter experiments promoting PC cell proliferation,invasion and migration in vitro.Conclusions:Collectively,our study identified that the miR-216a-5p was down-regulated in PC and interacts with TPT1 and LINC01133 to affect the malignance of PC.Moreover,we firstly found LINC01133-miR-216a-5p-TPT1 axis effectively contributed to the tumorigenesis and progression of PC.Thus,our findings may provide new strategies for the diagnosis and treatment of PC.更多还原