system: [SYSTEM]

user: 3. 结果 1) novel-miR-9在细胞中的表达水平 利用RT-qPCR检测novel-miR-9在正常细胞MCF-10A和人乳腺癌细胞MCF-7、MDA-MB-231中的表达量，发现novel-miR-9在乳腺癌细胞的表达量低于正常细胞(图1)。 图1. novel-miR-9在细胞中的表达水平。(a) novel-miR-9中正常细胞MCF-10A和人乳腺癌细胞MCF-7、MDA-MB-231；(b) (c) 验证novel-miR-9是否导入人乳腺癌MDA-MB-231和MCF-7细胞 2) novel-miR-9对乳腺癌细胞增殖影响 将novel-miR-9类似物导入MCF-7和MDA-MB-231细胞，运用cck-8以及EDU检测乳腺癌细胞增殖能力(图2)。 3) 预测novel-miR-9靶基因 通过TargetScan，miRDB，miRWalk和miRanda预测novel-miR-9的靶基因与GeneCard查询到与乳腺癌相关的致病基因取交集得到同乳腺癌相关的1188个预测靶基因(图3)。 图2. novel-miR-9对乳腺癌细胞增殖影响。(a) cck-8检测novel-miR-9对乳腺癌细胞MCF-7和MDA-MB-231增殖影响；(b) EdU检测novel-miR-9对乳腺癌细胞MDA-MB-231增殖影响；(c) EdU检测novel-miR-9对乳腺癌细胞MCF-7增殖的影响 图3. 预测novel-miR-9同乳腺癌相关的靶基因 4) 找出与增殖相关靶基因 运用DAVID线上网站找出1188个靶基因中与增殖相关的靶基因。发现BRCA1、CDH1、ERBB2、EGFR等26个基因直接调控细胞增殖(表1)并列出26个基因信息(表2)。 Table 1 GO analysis of DEGs in Breast Cance Term Description Count in gene set P-value Benjamini GO:0019899 enzyme binding 209 1.5696323496283148E−15 2.4091839634365897E−12 GO:0001067 regulatory region nucleic acid binding 122 6.3502055620786575E−15 2.4522606167920458E−12 GO:0044877 macromolecular complex binding 162 3.453964263902479E−14 1.0703660180411134E−11 GO:0000989 transcription factor activity， transcription factor binding 92 5.709065010437362E−13 8.84858852856496E−11 GO:0008092 cytoskeletal protein binding 114 2.3582441895158947E−12 3.0460400868292936E−10 GO:0016301 kinase activity 109 1.809990556789057E−8 9.04994877370946E−7 GO:0019838 growth factor binding 26 1.552211434415833E−6 6.014643089469196E−5 GO:0008013 beta-catenin binding 19 8.190108187078926E−6 2.4888509503584455E−4 GO:0035326 enhancer binding 20 2.2386715257633048E−5 5.980934338446486E−4 GO:0005102 receptor binding 134 3.0316297765182315E−4 0.006989990769231791 表1. GO分析乳腺癌的差异基因 GO，基因数据库；DEGs，差异表达基因。 Table 2 Functional roles of 26 gene No. Gene symbol Full name Function 1 GLG1 Golgi Glycoprotein 1 Binds fibroblast growth factor and E-selectin. Membrane sialo glycoprotein of the Golgi apparatus， binding fibroblast growth factor and E selectin， ubiquitous. 2 LTBP3 Latent Transforming Growth Factor Beta Binding Protein 3 Key regulator of transforming growth factor beta that controls TGF-beta activation by maintaining it in a latent state during storage in extracellular space. 3 ERBB2 Erb-B2 Receptor Tyrosine Kinase 2 Protein tyrosine kinase that is part of several cell surface receptor complexes， but that apparently needs a coreceptor for ligand binding. Essential component of a neuregulin-receptor complex， although neuregulins do not interact with it alone. 4 EGFR Epidermal Growth Factor Receptor Receptor tyrosine kinase binding ligands of the EGF family and activating several signaling cascades to convert extracellular cues into appropriate cellular responses. 5 LTBP4 Latent Transforming Growth Factor Beta Binding Protein 4 Key regulator of transforming growth factor beta that controls TGF-beta activation by maintaining it in a latent state during storage in extracellular space. 6 IGF1R Insulin Like Growth Factor 1 Receptor Function: receptor tyrosine kinase which mediates actions of insulin-like growth factor 1. Binds IGF1 with high affinity and IGF2 and insulin (INS) with a lower affinity. 7 ACVR1B Activin A Receptor Type 1B Transmembrane serine/threonine kinase activin type-1 receptor forming an activin receptor complex with activin receptor type-2. Regulating a many physiological and pathological processes. 8 SHC1 SHC Adaptor Protein 1 Signaling adapter that couples activated growth factor receptors to signaling pathways. Participates in a signaling cascade initiated by activated KIT and KITLG/SCF. 9 HTRA3 HtrA Serine Peptidase 3 Serine protease that cleaves beta-casein/CSN2 as well as several extracellular matrix (ECM) proteoglycans such as decorin/DCN， biglycan/BGN and fibronectin/FN1. 10 INSR Insulin Receptor This protein mediates the voltage-dependent sodium ion permeability of excitable membranes. 11 SCN5A Sodium Voltage-Gated Channel Alpha Subunit 5 To be deleted， mutated， or overexpressed in several kinds of cancers. 12 HAP1 Huntingtin Associated Protein 1 Originally identified as neuronal protein that specifically associates with HTT/huntingtin and the binding is enhanced by an expanded polyglutamine repeat within HTT possibly affecting HAP1 interaction properties. 13 RHBDF2 Rhomboid 5 Homolog 2 Regulates ADAM17 protease， a sheddase of the epidermal growth factor (EGF) receptor ligands and TNF， thereby plays a role in sleep， cell survival， proliferation， migration and inflammation. 14 IL2RB Interleukin 2 Receptor Subunit Beta Receptor for interleukin-2. 15 COL4A1 Collagen Type IV Alpha 1 Chain Type IV collagen is the major structural component of glomerular basement membranes (GBM)， forming a ‘chicken-wire’ meshwork together with laminins， proteoglycans and entactin/nidogen. 16 IGFALS Insulin Like Growth Factor Binding Protein Acid Labile Subunit Involved in protein-protein interactions that result in protein complexes， receptor-ligand binding or cell adhesion. 17 FURIN Furin， Paired Basic Amino Acid Cleaving Enzyme Ubiquitous endoprotease within constitutive secretory pathways capable of cleavage at the RX(K/R)R consensus motif. 18 COL5A1 Collagen Type V Alpha 1 Chain Type V collagen is a member of group I collagen. 19 NTRK2 Neurotrophic Receptor Tyrosine Kinase 2 Receptor tyrosine kinase involved in the development and the maturation of the central and the peripheral nervous systems through regulation of neuron survival， proliferation， migration， differentiation， and synapse formation and plasticity. 20 PDGFRB Platelet Derived Growth Factor Receptor Beta Tyrosine-protein kinase that acts as cell-surface receptor for homodimeric PDGFB and PDGFD and for heterodimers formed by PDGFA and PDGFB， and plays an essential role in the regulation of embryonic development， cell proliferation， survival， differentiation， chemotaxis and migration. 21 COL1A1 Collagen Type I Alpha 1 Chain Type I collagen is a member of group I collagen. 22 NGFR Nerve Growth Factor Receptor Low affinity receptor which can bind to NGF， BDNF， NTF3， and NTF4. Forms a heterodimeric receptor with SORCS2 that binds the precursor forms of NGF， BDNF and NTF3 with high affinity， and has much lower affinity for mature NGF and BDNF. 23 IGFBP2 Insulin Like Growth Factor Binding Protein 2 Inhibits IGF-mediated growth and developmental rates. 24 ENG Endoglin Vascular endothelium glycoprotein that plays an important role in the regulation of angiogenesis. 25 IGFBP4 Insulin Like Growth Factor Binding Protein 4 IGF-binding proteins prolong the half-life of the IGFs and have been shown to either inhibit or stimulate the growth promoting effects of the IGFs on cell culture. 26 IGFBP5 Insulin Like Growth Factor Binding Protein 5 IGF-binding proteins prolong the half-life of the IGFs and have been shown to either inhibit or stimulate the growth promoting effects of the IGFs on cell culture. 表2. 26个基因的功能 5) 增殖相关基因相互作用及乳腺癌组织表达 运用STRING线上网站分析相关基因的交互作用；运用UCSC线上网站分析26个基因在乳腺癌组织中的表达量(图4)。 图4. 26个基因相互作用及在乳腺癌组织的表达量。(a)用STRING分析26个基因的相互作用；(b)用UCSC而分析26个基因在乳腺癌组织的表达量 6) 相关基因的GO功能注释及KEGG信号传导通路的富集分析 运用DAVID线上网站分析26个基因的GO功能注释以及KEGG信号传导通路的富集分析(表3)。 Table 3 GO and KEGG pathway enrichment analysis of 26 Gene Term description Count in gene set P-value Benjamini GO:0019838 growth factor binding 26 7.177095130023525E−54 1.4713045016548225E−51 GO:0019199 transmembrane receptor protein kinase activity 9 4.3968967310014277E−13 4.5063952569535104E−11 GO:0005520 insulin-like growth factor binding 7 3.4171081240130494E−12 2.335056592528417E−10 GO:0004714 transmembrane receptor protein tyrosine kinase activity 6 5.5603020389256557E−8 2.2797213016234608E−6 GO:0005102 receptor binding 12 6.568087664376786E−6 1.3463717490713734E−4 GO:0016301 kinase activity 9 6.796957977711867E−5 8.193251938890533E−4 GO:0004872 receptor activity 11 1.554075440077943E−4 0.001516041185204764 GO:0032403 protein complex binding 7 0.001135535736491165 0.008589457035824144 hsa04510 Focal adhesion 8 1.775015314267736E−7 1.1892532947421763E−5 hsa04151 PI3K-Akt signaling pathway 9 3.3386255161809444E−7 1.1184334800629081E−5 hsa04014 Ras signaling pathway 6 1.1890305517812948E−4 0.002652136307670472 hsa04015 Rap1 signaling pathway 5 0.001150670572651469 0.01095940585115629 hsa05200 Pathways in cancer 5 0.011027515410980928 0.011157926385015005 hsa05206 MicroRNAs in cancer 4 0.02664017560230009 0.010776432474605246 表3. GO和KEGG富集化分析26个基因 GO，基因库；KEGG，基因富集化。