分子流行病学实验课2018
分子流行病学实验课 2018
数据计算公式相对定量 ：2-A ct△△ct=实验组（目的ct-内参ct）-对照组（目的ct-内参ct）假设检测A基因，CT分别为>普通组/Testl ：28> 实验组1/Test2 : 29>实验组2/Test3 ：30假设Test1为对照组（普通组）那么，相对含量为：普通组/Testl ：1实验组1/Test2 :2^-(29-28)=1/2=0.5实验组2/Test3 : 2^-(30-28)=1/4=0.25
数据计算公式 相对定量：2 -△△ ct △△ct=实验组（目的ct-内参ct）-对照组（目的ct-内参ct） 假设检测A基因，CT分别为 普通组/Test1：28 实验组1/Test2：29 实验组2/Test3：30 假设Test1为对照组（普通组）那么，相对含量为： 普通组/Test1：1 实验组1/Test2：2^-(29-28)=1/2=0.5 实验组2/Test3：2^-(30-28)=1/4=0.25
数据分析A基因内参GAPDHCon29.3499698616.0867519429.0263500216.20421791处理组129.1854953815.95644283293035430915.87710857处理组229.5239067115.897140529.3277893116.07045364处理组330.9853000618.9686927818.9292812331.61320114
数据分析
QsearchCancerResearchIF:9.122AdvancedSearchHomeAboutArticlesForAuthorsAlertsMolecular and Cellular PathobiologyLongNoncodingRNAGCASPC,aTargetofmiR-17-3p,NegativelyRegulatesPyruvateCarboxylase-DependentCellProliferationinGallbladderCancerMing-zhe Ma, Yan Zhang,Ming-zhe Weng, Shou-hua Wang, Ye Hu, Zhao-yuan Hou, Yi-yu Qin, Wei Gong, Yong-Jie Zhang,Xiang Kong, Jian-dong Wang, and Zhi-wei QuanDOl: 10.1158/0008-5472.CAN-15-3047 Published September 2016PDFArticleInfo&MetricsSeptember2016Volume76,Issue17ResearchTableofContentsAbstractTable of Contents (PDF)IndexbyauthorLong noncoding RNAs (lncRNA) are being implicated in the development of.many cancers. Here,we report the discoveryofa critical rolefor the IncRNA GCASPC indetermining theprogressionofgallbladder cancer.Differentially expressed IncRNAs andmRNAs between gallbladder cancerspecimensandpairedadjacentnontumortissuesfromfivepatientswereidentifiedandvalidatedbyan expression microarray analysis. Quantitative real-time PCR was used to measure GCASPClevelsintissuesfrom42gallbladdercancerpatients,andlevelsofGCASPCwereconfirmedfurtherina separate cohort of 89gallbladder cancerpatients.GCASPC was overexpressed or silenced inSearch this issueQseveral gallbladder cancer cellines where molecular and biological analyses were performed.GCASPC levelsweresignificantlyloweringallbladdercancerthanadjacentnontumortissuesandSign up for alertswereassociatedwithtumorsize,AmericanJointCommitteeonCancertumorstage,andpatientoutcomes.GCASPCoverexpressionsuppressedcellproliferationinvitroandinvivo,whereasGCASPC silencing had opposite effects. By RNA pull-down and mass spectrometry, we identified
IF:9.122
dFigurel.GCASPC downregulation inGBC tissues100Figure 1ABCGCASPCexpressionp<0.001,n=42100**10.00-中80-二1.0060-R40-AUC=0.6970.10(0.584 to0.810)20-p=0.0020-0.01-206004080100TumorNon-tumor100%-Specificity%EDLoWGCASPCLowGCASPC100HighGCASPCHighGCASPC100808060604040P20202313p<0.001p<0.0010-0.102040Tumor60800204060-3,70.0+4.3Non-Tumor(log2 fold)Overallsurvival(monthsDisease-freesurvival(months)
Figure1. GCASPC downregulation in GBC tissues
elFigure2.GCASPCinhibitsGBC cell proliferation and tumorgrowth100Figure 2BE-,GnC-SD scramble1.5MockSGC-996GBC-SDSHRNA!aGC9SGCASPO3eavectorof04o80.5SGC-996nLEGBCSOCoMBeRsPevectSGC-996vectot..SGC-996GCASPC6PCNA业(29 kDa)ph1.000.980.241.001.143.473.0720.82oIB-Tublin(50 kDa)3010152025SGC-906SBOR.00DayspostinoculationDGCASPChRNA1shRNA2SGC-996SGC-996Z80-AvectorASP4040ki67joa203OO0"G0/G1AG0/G1sG2/MG2/Mki67FGBC-SDGBC-SD scrambleGBC-SD三scrambleGBC-SDShRNA(ol. paonscrambleInNGBC-SDShRNA-2GBC-SDshRNA-IGBC-SDShRNA-20-GBC-SD300101520255:shRNA-10.8-Dayspostinoculationa280.4XIPALOX0.2GBC-SDshRNA-20.5-90GBC-SDGBC-SDGBC-SDShRNA-ShRNA-290scrambleGBC-SDGRCIADGRCAD
Figure 2. GCASPC inhibits GBC cell proliferation and tumor growth
Figure 3.GCASPC binds to pyruvate carboxylase proteinure3BIS10SGC-996170PCSGC-996130NOZPCNOZ95D5'Antisense7253'GCASPCPBSKA1(1-472nt)55PBSK△2(1-233nt)43kDaEF1.0(oaoo)0.5-CU2nRNA0.0COXIVGCASPC0.5T0
Figure 3. GCASPC binds to pyruvate carboxylase protein
Figure 4. GCASPC decreases the protein level and activity of pyruvatecarboxylasebyinhibitingitsproteinstability.Figure4A20*t(uedBnl)KuaeeodBentPC10(130 kDa)1.871.001.651.000.38β-Tublin(50 kDa)GBC-SDSGC-996GBC-SDGBC-SDoceSARNA-1ShRNA-2scramblevectorBCDCHXMGI32DMSOcontrolPC(130kDa)1.000.310.41PCNAPCPC(29 kDa)0.451.920.161.002.251.001.901.002.531.000.340.30B-Tublirβ-TublinB-Tublin(50 kDa)ENA3.1-PCS1.5-scramblePeDCPCDNA3.1-PC+GCASPCGCASPC0-0PCDNA3.1-PC1.0PC1.000.422.091.090.5PCNA0.441.961.001.160.0-β-Tublin0n3
Figure 4. GCASPC decreases the protein level and activity of pyruvate carboxylase by inhibiting its protein stability
Figure 5. GCASPC is a target of miR-17-3p100Figure5BA1.5scrambleGCASPCshRNA-1ss1.25'-GAGTGCCAAAAACTGCAGA-3'GCASPC-wt-GCASPCshRNA-2scrambleCmiR-17-3p1.0m1.00.8miR-17-3p3-UUCACGGAAG--UGACGUCA-5"T0.6-萃8华de0.5GCASPC-mut5'-GATGTTAAAAAGTGTAGTA-3'0.00.01.5-GBC-SDOCUG-1GBC-SDOCUG-1**CeDS01.0-1.5scramble+miR-17-3p mimicsnw ayoog0.5-1.0PC30.50.01.000.52wtytmutASPC.3p-17-3PGCAS-aC0.0Cβ-Tublin0.51.01.52.02.53.03.50.0alpha-amanitin (hours)FE=-0.498,p=0.00110.1.5scramblessshRNA-GCASPC+miR-17-3pinhibitor8shRNA-GCASPC+miR-17-3pinhibitor1.06-40.5-2o.0.0161214234105048miR-17-3pexpression (μCt)
Figure 5. GCASPC is a target of miR-17-3p
上海交通大学医学院CasShanghai Jiao Tong University School of Medicinc细胞的传代与培养
细胞的传代与培养