1、MicroRNA-95 Promotes Cell Proliferation and Targets Sorting Nexin 1 in Human Colorectal Carcinoma W.T.T2014-3-30第一页，共二十五页。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(dio kn)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
2、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(chngz)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。第二页，共二十五页。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
3、基因芯片高通量筛选的方法，找到了影响(yngxing)结肠癌增殖速度的microRNAmiR-95，并鉴定了miR-95的下游靶基因SNX1。第三页，共二十五页。一、基因芯片(j yn xn pin)高通量筛选发现miR-X与肿瘤细胞增殖有关研究小组首先(shuxin)选取n对肿瘤组织和癌旁组织microRNA芯片（Agilent microRNA microarray）进行分析(fnx)，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
4、、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第四页，共二十五页。microRNA一般(ybn)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(zhngli)细胞增殖用TargetScan预测(yc)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第五页，共二十五页。Introduction In this study,we
5、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.第六页，共二十五页。Materials and me
6、thods 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第七页，共二十五页。Materials and methods Luciferase assay Tumor formati
7、on assay in a nude mouse model Immunohistochemical staining Western blot Statistical analysis第八页，共二十五页。ResultsExpression of miR-95 is frequently increased in human CRC tissuesUnsupervised hierarchical clustering(层次(cngc)聚类）with these 49 significantly dysregulated miRNAs was able to distinguish the C
8、RCs from corresponding NCTs.研究小组首先选取(xunq)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(j)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。第九页，共二十五页。Expression of miR-95 is frequently increased in human CRC tissuesConsistent w
9、ith 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(zzh)中miR-95的表达水平第十页，共二十五页。MiR-95 promotes CRC cell proliferation in vitro and in vivo用慢病毒转导的方法，研究(ynji)小组构建了稳定表达miR-95的HCT-116和LoVo细胞系（两种
10、结肠癌细胞）第十一页，共二十五页。MiR-95 promotes CRC cell proliferation in vitro and in vivoThe cell proliferation assays（细胞(xbo)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(jin c)细胞增殖结晶(jijng)紫染色检测克隆第十二页，共二十五页。RNAi-mediated
11、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(zhngli)细胞的生长速度第十三页，共二十五页。SNX1 is a direct target o
12、f miR-95miR-95 could inhibit the expression of reporter gene in recombinant plasmids ofSNX1,UBE4B,and EMP1 30UTRs,especially SNX1 30UTR.miR-95通过(tnggu)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(fnx)（luciferase reporter assay）发现，SNX1受miR-95抑制最为显著。第十四页，共二十五页。SNX1 is a direct target of
13、miR-95Mutated 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.第十五页，共二十五页。Endogenous SNX1 protein levels were also down-regulated in miR-95-overexpressed cells and could be restored in miR-95depleted
14、 cellsSNX1 is a direct target of miR-95SNX1是miR-95的下游(xiyu)靶基因第十六页，共二十五页。Downregulation of SNX1 inversely correlated with miR-95 expression in CRCA,B Of the 146 cases,114 tumors showed decreased SNX1 expression when compared with paired NCTsThe expression levels of SNX1 in tumor tissues inversely co
15、rrelated 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(rns)第十七页，共二十五页。MiR-95 promotes tumor proliferation via directly targeting SNX1 in CRCwe inhibited the SNX1 expression with siRNA and revealed that SN
16、X1-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.第十八页，共二十五页。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(xbo)中，miR-95通过抑制SNX1来促进细胞增殖。第十九页，共二十五页。Overexpression of miR-95 c
