王婷婷--miR-95通过靶标SNX1促进结肠癌细胞增殖修改.ppt

MicroRNA-95 Promotes Cell Proliferation and Targets Sorting Nexin 1 in Human Colorectal Carcinoma W.T.T 2014-3-30 microRNA是一类约22个核苷酸长度的非编码RNA小分子，一般通过与mRNA的3UTR区域相互作用从而抑制相关基因的表达。据研究表明，microRNAs在转录后水平上至少调控了三分之一的人类基因。目前最新的microRNA数据库（miRBase）版本号是17.0（April 2011，www.mirbase.org），涵盖了153个物种、共19724条成熟microRNA序列。虽然microRNA的研究进展神速，但是大多数microRNAs在生理或病理过程中发挥作用的机制仍未被揭示出来。结肠癌（colorectal carcinoma，CRC）是世界第三大癌症，虽然在过去几十年中结肠癌的治疗技术取得了长足的进步，但是结肠癌的分子病理机制仍未被了解清楚。过去的研究发现了一些蛋白编码基因（例如：P53，APC，K-ras，DCC等）可能与结肠癌的形成有关，但是在结肠癌形成过程中发挥作用的被发现的microRNA却少之又少。复旦大学附属肿瘤医院和上海市肿瘤研究所癌基因和相关基因国家重点实验室利用基因芯片高通量筛选的方法，找到了影响结肠癌增殖速度的microRNAmiR-95，并鉴定了miR-95的下游靶基因SNX1。一、基因芯片高通量筛选发现miR-X与肿瘤细胞增殖有关 研究小组首先选取n对肿瘤组织和癌旁组织 microRNA芯片（Agilent microRNA microarray）进行分析，共发现有表达差异的microRNA。从中选取了几个microRNA用miRNA拟似物转染分析实验（miRNA mimics transfection assay）分析它们对肿瘤细胞生长的影响，通过一系列体内、体外实验证实miR-X确实能促进（抑制）肿瘤细胞的增殖。获得对肿瘤影响最强的miR-X microRNA一般通过抑制下游基因来发挥作用，由于前面的研究结果表明miR-X促进（抑制）肿瘤细胞的增殖，那么它所抑制的下游基因应该是肿瘤抑制（激活）基因。二、miR-X通过作用于靶基因Y促进(抑制）肿瘤细胞增殖 用TargetScan预测一些miR-X的靶基因 采用荧光素酶报告基因分析筛选靶基因 分析靶基因功能及证实是否是最佳靶标基因 进一步阐明miR-X在肿瘤中的功能和分子机制 Introduction In this study,we investigated the miRNA expression profiles in RC and identified a new proliferation-promoting miRNA,miR-95,which is frequ-ently up-regulated in CRC.Moreover,we identified sorting nexin 1(SNX1),a putative tumor suppressor in CRC(14),as the direct functional target of miR-95.Materials and methods Human tissues and cell lines Microarray analysis Vector constructs Lentivirus production and transduction RNA extraction and quantitative real-time PCR Oligonucleotide transfection Cell proliferation assay and colony formation assay Materials and methods Luciferase assay Tumor formation assay in a nude mouse model Immunohistochemical staining Western blot Statistical analysis Results Expression of miR-95 is frequently increased in human CRC tissues Unsupervised hierarchical clustering(层次聚类）with these 49 significantly dysregulated miRNAs was able to distinguish the CRCs from corresponding NCTs.研究小组首先选取了10对结肠癌组织和癌旁组织，用microRNA芯片（Agilent microRNA microarray）进行分析 随后从中选取了几个microRNA用miRNA拟似物转染分析实验（miRNA mimics transfection assay）分析它们对结肠癌细胞生长的影响，结果发现miR-95有最强的促进结肠癌细胞增殖的能力。Expression of miR-95 is frequently increased in human CRC tissues Consistent with the microarray data,miR-95 expression was up-regulated in nearly half of the tumors examined(42/87)when compared with the adjacent NCTs.qRT-PCR检测分析87对CRC和旁癌组织中miR-95的表达水平 MiR-95 promotes CRC cell proliferation in vitro and in vivo 用慢病毒转导的方法，研究小组构建了稳定表达miR-95的HCT-116和LoVo细胞系（两种结肠癌细胞）MiR-95 promotes CRC cell proliferation in vitro and in vivo The cell proliferation assays（细胞增殖实验）and colony formation assays（克隆形成实验）revealed that overexpression of miR-95 can significantly promote CRC cell proliferation.CCK-8检测细胞增殖 结晶紫染色检测克隆 RNAi-mediated silencing of miR-95 decreased cell growthratio.Overexpression of miR-95 could significantly promote the tumorigenicity of CRC cells in nude mice.MiR-95 promotes CRC cell proliferation in vitro and in vivo 稳定转染miR-95的LoVo细胞和稳定转染慢病毒空载体的LoVo细胞注射到裸鼠中观察肿瘤细胞的生长速度 SNX1 is a direct target of miR-95 miR-95 could inhibit the expression of reporter gene in recombinant plasmids of SNX1,UBE4B,and EMP1 30UTRs,especially SNX1 30UTR.miR-95通过作用于靶基因SNX1促进结肠癌细胞增殖 用TargetScan预测了一些miR-95的靶基因，采用荧光素酶报告基因分析（luciferase reporter assay）发现，SNX1受miR-95抑制最为显著。SNX1 is a direct target of miR-95 Mutated the predicted binding site of miR-95 on the SNX1 3UTR.The mutant SNX1 3UTR was completely refractory to miR-95-ediated luciferase reporter repression.Endogenous SNX1 protein levels were also down-regulated in miR-95-overexpressed cells and could be restored in miR-95depleted cells SNX1 is a direct target of miR-95 SNX1是miR-95的下游靶基因 Downregulation of SNX1 inversely correlated with miR-95 expression in CRC A,B Of the 146 cases,114 tumors showed decreased SNX1 expression when compared with paired NCTs The expression levels of SNX1 in tumor tissues inversely correlated with the miR-95 levels suggesting that the decreased SNX1 expression might result from miR-95 overexpression in human CRC.C and Table 1 免疫组化染色 MiR-95 promotes tumor proliferation via directly targeting SNX1 in CRC we inhibited the SNX1 expression with siRNA and revealed that SNX1-depleted cells showed increased proliferation,which phenocopied the proliferation-promoting effect of miR-95.Cotransfection experiments using siSNX1 and antimiR-95 also showed that miR-95 silencing could not repress proliferation in SNX1-depleted HCT-8 cells.SNX1 overexpression could significantly abrogate miR-95induced cell growth.Downregulation of SNX1 inversely correlated with miR-95 expression in CRC 这些现象表明，在结肠癌细胞中，miR-95通过抑制SNX1来促进细胞增殖。Overexpression of miR-95 could increase EGFR phosphorylation and inhibition of miR-95 led to decreased EGFR phosphorylation which phenocopied the modulating function of SNX1 on EGFR signaling.Downregulation of SNX1 inversely correlated with miR-95 expression in CRC Discussion 表皮生长因子受体EGFR是一个分子量为170kD的跨膜酪氨酸激酶受体，是人类表皮生长因子受体HER（ErbB）家族中的一员