課件文獻(xiàn)一點(diǎn)通單變量

1、文 獻(xiàn) 一 點(diǎn) 通醫(yī)學(xué)文獻(xiàn)結(jié)構(gòu)化泛讀方法案例匯總聚焦單變量論證Longnon-codingRNA (LncRNA)HOXA11-ASpromotes breast cancer invaand metastasisby regulatingthelial-mesenymal transition.Li W, et al. 2017;23:3393-3403. IF=1.59. PMID:28701685Ku70 inhibits gemcitabine-induced DNA damage and pancreatic cancer cell apoptosis.Ma J, et al. 20

2、17;484(4):746-752. IF=2.47. PMID: 28153717YKL-40regulatedthelial-mesenchymal enhancement intransition and migration/innon-small cellg cancer.Jefri M, et al. 2015;15:590. IF=3.27. PMID: 26275425解 螺 旋|表型背景知識(shí)上皮間質(zhì)轉(zhuǎn)化（thelial- mesenchymal transition, EMT）是腫瘤侵襲和早期轉(zhuǎn)移的重要過(guò)程拼課vx：88166945Kalluri R, Weinberg RA.

3、 The basics of J Clin Invest. 2009 Jun;119(6):1420-8.thelial-mesenchymal transition.解 螺 旋|泛讀四步驟題目要素假設(shè)邏輯數(shù)據(jù)泛讀套路總結(jié)疾病表型分子機(jī)制表達(dá)差異正反回復(fù)拼課vx：88166945分組恒量套路變量套路數(shù)據(jù)維度細(xì)胞動(dòng)物解 螺 旋|題目要素案例一分子疾病表型Longnon-codingRNA(LncRNA)HOXA11-ASpromotes breast cancer invaand metastasisby regulatingthelial-mesenchymal transition.表型Li

4、 W, et al. 2017;23:3393-3403.IF=1.59. PMID:28701685表型科學(xué)假設(shè)：在【疾病】中，【分子】經(jīng)由【機(jī)制】介導(dǎo)了【表型】。在【】中，【 HOXA11 】介導(dǎo)【侵襲、轉(zhuǎn)移、EMT】（無(wú)機(jī)制）。解 螺 旋|假設(shè)邏輯案例一需證明已知分子表型疾病侵襲轉(zhuǎn)移 EMTHOXA11解 螺 旋|細(xì)胞動(dòng)物細(xì)胞實(shí)驗(yàn)動(dòng)物模型正反回復(fù)加減HOXA11回復(fù)HOXA11表達(dá)差異組織標(biāo)本細(xì)胞培養(yǎng)數(shù)據(jù)泛讀ABqRT PCR10案例一組織表達(dá)1086420-2-48細(xì)胞表達(dá)6qRT PCR420MCF-10AMDA MCF MDA SKBR3 MDA-MB-231-MB-468-MB-

5、435DC1.21.21.01.0操作驗(yàn)證操作驗(yàn)證0.80.80.60.60.40.4qRT PCR0.2表達(dá)差異0.20.00.0si-NC si-HOXA11 si-HOXA11 si-HOXA11si-NC si-HOXA11 si-HOXA11 si-HOXA11-AS1#-AS2#-AS3#-AS1#-AS2#-AS3#Fig.1 The expresty of lncRNA HOXA11-AS in breast cancer ties and cells.解 螺 旋|Relative HOXA11-ASRelative HOXA11-AS expres（Fold Change）e

6、xpresRelative HOXA11-ASRelative HOXA11-AS expres（Fold Change）expresLow expresHigh expres數(shù)據(jù)泛讀對(duì)照敲減B1.01.2A案例一MDA-MB-231MCF-70.80.9si-NCsi-NCsi-HOXA11-AS#2MTT試驗(yàn)MTT試驗(yàn)si-HOXA11-AS#20.60.60.40.30.20.00.002448Hours729602448Hours7296MDA-MB-231MCF-7CD350300250200150100500克隆形成克隆形成細(xì)胞表型增殖表型MDA-MB-231MCF-7Fig.2

7、The influence of HOXA11-AS on the proliferation capacity of breast cancer cells.解 螺 旋|Cell viability（OD 490 nm）si-HOXA11-ASsi-NCCell viability（OD 490 nm）Number ofclonessi-NCsi-HOXA11-AS數(shù)據(jù)泛讀MDA-MB-231MCF-7對(duì)照敲減案例一104103102101100100 101 102 103 104104103102101100100 101 102 103 104104103102101100100 10

8、1 102 103 104AVAsi-NCsi-HOXA11-ASB201816流式凋亡104103102101100100 101 102 103 104MDA-MB-231MCF-7AVMDA-MB-231C6005004003002001000MCF-76004002000Dsi-NCsi-HOXA11-AS1009080706050403020100流式細(xì)胞周期20 40 60 80 100DNA content(PI)2040 60 80 100DNA content(PI)6004002000600400200細(xì)胞表型020 40 60 80 10020 40 60 80 100D

9、NA content(PI)MDA-MB-231MCF-7DNA content(PI)Fig.3 The influence of HOXA11-AS on the biological function of breast cancer cells.解 螺 旋|si-HOXA11-ASCell numbersi-NC Cell numbersi-HOXA11-AS PIsi-NC PIPIPICell numberCell numberG1-G0 distribution(%)Apoptotic rate(%)數(shù)據(jù)泛讀案例一MDA-MB-231CAsi-NCsi-HOXA11-ASsi-N

10、Csi-HOXA11-AS劃痕試驗(yàn)Trans wellMigration0hInva24hMCF-7DBsi-NCsi-HOXA11-ASsi-NCsi-HOXA11-AS劃痕試驗(yàn)Trans well0hMigration24hInva遷移侵襲細(xì)胞表型Fig.4 The influence of HOXA11-AS on cell migration and invacapacity.解 螺 旋|數(shù)據(jù)泛讀案例一腫瘤組織對(duì)照敲減ABsi-NCsi-HOXA11-AS造模驗(yàn)證D1.11.00.90.80.70.60.50.40.30.20.10.0C100轉(zhuǎn)移表型qRT PCR806040動(dòng)物實(shí)驗(yàn)

11、200si-NCsi-HOXA11-ASsi-NCsi-HOXA11-ASFig.5 Study on the influence of HOXA11-AS on the migration capacity of breast cancer cells via experiment in vitro.解 螺 旋|Nicumber of metas-tumorH&ERelative HOXA11-AS expres數(shù)據(jù)泛讀對(duì)照敲減Csi-NCsi-HOXA11-ASAsi-NCsi-HOXA11-AS案例一免疫組化VimentinWBN-cadherinE-cadherinGAPDHBE-ca

12、dherinDAPIMergeE-cadherinDAPIMerge免疫熒光細(xì)胞表型Fig.6 The influence of HOXA11-AS on EMT.解 螺 旋|si-HOXA11-ASsi-NC套路總結(jié)案例一恒量變量疾型模法標(biāo)分子in vitro MCF-7MDA-MB-231Breast CancerProliferationqRT-PCRVimentinHOXA-11in vivonude miceMTTclone表達(dá)差異正反回復(fù)細(xì)胞動(dòng)物ApoptosisN-Cadherinin vitro MCF-7MDA-MB-231InvaFCME-CaherinTiesiRNATr

13、answellWoundin vivonude miceMetastasisCellEMTWB , IHC, IF解 螺 旋|題目要素案例二分子表型Ku70 inhibits gemcitabine-induced DNA damage andpancreatic cancer cell apoptosis.Ma J, et al. 2017;484(4):746-752 IF=2.47. PMID: 28153717疾病表型科學(xué)假設(shè)：在【疾病】中，【分子】經(jīng)由【機(jī)制】介導(dǎo)了【表型】。在【胰】中，【 Ku70 】介導(dǎo)【DNA損傷、凋亡】（無(wú)機(jī)制）。解 螺 旋|假設(shè)邏輯案例二需證明已知表型分子疾

14、病胰DNA損傷細(xì)胞凋亡Ku70解 螺 旋|細(xì)胞動(dòng)物細(xì)胞實(shí)驗(yàn)動(dòng)物模型正反回復(fù)加減Ku70回復(fù)Ku70表達(dá)差異組織標(biāo)本細(xì)胞培養(yǎng)數(shù)據(jù)泛讀案例二AMIA-PaCa-2MIA-PaCa-212010080MIA-PaCa-2C40120100806040200Bsh-NCsh-Ku70(a) sh-Ku70(b) sh-Ku70(c)70KD-Ku70300.82 0.75 0.02 0.01 0.00增殖表型克隆形成86KD-Ku80增殖表型MTT操作驗(yàn)證PCR WB60402000.33 0.29 0.29 0.29 0.312055KD-Tublinsh-NCsh-Ku70(a) sh-Ku70(

15、b) sh-Ku70(c)10增殖表型MTT增殖表型MTT操作驗(yàn)證WB0Ctrl1050100500CtrlGemcitabine(100nM)7-daysGemcitabine(nM), 72-hrsPrimary pancreatic cancer cellsFDE120100806040200GPANC-1Primary pancreatic cancer cellssi-NCsi-Ku70(a) si-Ku70(b)120100806040200PANC-1120100806040200sh-NCsh-Ku70(a) sh-Ku70(b) sh-Ku70(c)Ku70Ku801.96

16、0.11 0.0970KD-Ku701.51 1.52 1.54Tublin操作驗(yàn)證WB PCR0.70 0.10 0.05 0.06Ku8086KD-0.41 0.46 0.49 0.51Tublin55KD-CtrlGemcitabine(100nM)72-hrsCtrlGemcitabine(100nM)72-hrs細(xì)胞表型敲減實(shí)驗(yàn)Fig.1 Ku70 knockdown sensitizes gemcitabine-induced killing of pancreatic cancer cells.解 螺 旋|Ku-70 mRNA(% vs.”Parental” ）Cell viab

17、ility(% vs.”Ctrl” ）Cell viability(% vs.”Ctrl” ）Ku-70 mRNA(% vs.”Parental” ）Cell viability(% vs.”Ctrl” ）Colonies (per well）數(shù)據(jù)泛讀案例二AMIA-PaCa-2MIA-PaCa-225IA-PaCa-250B1.2C40200.9DNA損傷30凋亡凋亡150.62010100.3500Ctrl10501005000CtrlGemcitabine(100nM) 48-hrsGemcitabine(nM),36-hrsGemcitabine(100nM),24-hrsDPrima

18、ry pancreatic cancer cellsPrimary pancreatic cancer cellsE1.2NC-1F16050403020100si-SCsi-Ku70(a)si-Ku70(b)0.8DNA損傷凋亡0.8凋亡0.60.40.4細(xì)胞表型敲減0.200CtrlGemcitabine(100nM) 36-hrsCtrlGemcitabine(100nM)36-hrsGemcitabine(100nM),24-hrsFig.2 Ku70 knockdown facilies gemcitabine-induced DNA damage and pancreatic ca

19、ncer cell apoptosis.解 螺 旋|Apoptosis ELISA OD-H2AX(%)Apoptosis ELISA OD-H2AX(%)Apoptosis ELISA ODTUNEL(%)PAsh-NCsh-Ku70(a) sh-Ku70(b)sh-Ku70(c)Msh-NCsh-Ku70(a) sh-Ku70(b) sh-Ku70(c)sh-NCsh-Ku70(a) sh-Ku70(b) sh-Ku70(c)數(shù)據(jù)泛讀案例二MIA-PaCa-2AB635Emp Vector wt-Ku-70-a wt-Ku-70-b5Ku70-flag Ku700.24 0.69 0.66

20、Ku802870KD4DNA損傷操作驗(yàn)證WB PCR2186KD30.99 1.01 1.07Tublin55KD1427100CtrlGemcitabine(100nM), 24-hrsCEDEmp Vector wt-Ku-70-a wt-Ku-70-b0.51201008060402035Emp Vector wt-Ku-70-a wt-Ku-70-b0.428增殖表型克隆形成增殖表型MTT凋亡0.321140.270.1細(xì)胞表型過(guò)表達(dá)000CtrlGemcitabine(100nM), 7-daysCtrlGemcitabine(100nM), 72-hrsCtrlGemcitabin

21、e(100nM), 36-hrsFig.3 Forced overexpresof Ku70 deceases gemcitabines sensitivity in pancreatic cancer cells.解 螺 旋|Cell viability(% vs.”Ctrl” ）Ku-70 mRNA(fold vs.”Parental” ）Colonies (per well）-H2AX(%)ssDNA ELISA OD數(shù)據(jù)泛讀案例二ACsh-NC+Saline sh-NC+Gemsh-Ku70(a)+Gemsh-Ku70(b)+Gem1200100080060023腫瘤大小小鼠體重211

22、9400200017n=1015D0 D7 D14 D21 D28 D35 D42D0D7D14D21 D28 D35 D42BD25Tumor lysates (at Day-14)Tumor lysates (at Day-7)蛋白表達(dá)腫瘤生長(zhǎng)20151070KD-70KD-Ku70Ku7050.36 0.37 0.01 0.010.86 0.890.02 0.0486KD-86KD-Ku800Ku80動(dòng)物敲減0.53 0.520.51 0.620.75 0.740.69 0.7742KD-42KD-Erk1/2Erk1/2Fig.4 Ku70 knockdown sensitizes g

23、emcitabine-induced anti-tumor activity in vivo.解 螺 旋|Estimated tumor size (cubic millimeter）Eroswtitmhated tum(ecrudbaicy millimet）Mice body weigt (grams）n=10sh-NC+Saline sh-NC+Gemsh-Ku70(a)+Gemsh-Ku70(b)+Gem套路總結(jié)案例二恒量變量疾型模法標(biāo)分子in vitro MIA, PANC-1PrimaryPancreaticcancerDNAdamageqRT-PCRWB-Ku70in vivoS

24、CID miceMTTclone表達(dá)差異正反回復(fù)細(xì)胞動(dòng)物Apoptosis-in vitro MIA, PANC-1PrimaryProliferation-H2AX-shRNATUNELELISAin vivoSCID miceover- expres-解 螺 旋|題目要素案例三分子表型YKL-40regulatedthelial-mesenchymaltransitionand migration/inva g cancer.enhancement in non-small cellJefri M et al. 2015 15:590. IF=3.27. PMID: 26275425疾病表

25、型科學(xué)假設(shè)：在【疾病】中，【分子】經(jīng)由【機(jī)制】介導(dǎo)了【表型】。在【肺癌】中，【 YKL-40 】介導(dǎo)【EMT、遷移、侵襲】（無(wú)機(jī)制）。解 螺 旋|假設(shè)邏輯案例三需證明已知分子表型疾病肺癌EMT遷移侵襲YKL-40解 螺 旋|細(xì)胞動(dòng)物細(xì)胞實(shí)驗(yàn)動(dòng)物模型正反回復(fù)加減YKL-40回復(fù)YKL-40表達(dá)差異組織標(biāo)本細(xì)胞培養(yǎng)數(shù)據(jù)泛讀案例三組織表達(dá)A. Western Blot12345678910NTTNTTNTTNTTNTTNTTNTTNTTNTTNTTYKL-40-actin組織表達(dá)B. Immunohistochemistry Staining數(shù)據(jù)庫(kù)預(yù)后分析D. PrognoScanKaplan-Me

26、iC. TCGA databaseysislotHigh n=104 Low n=1008000600040002000基礎(chǔ)表達(dá)TumorNormal0LuadLuadLuscLuscnormaltumornormal tumor0 500 1000 1500 2000 2500 3000 3500Fig.1 Western Blotysis comparison of YKL-40 gene express in normalg ties and non-small cellg cancer (NSCLC)解 螺 旋|Probability0.00.4 0.8數(shù)據(jù)泛讀案例三A. Transw

27、ell Assay侵襲遷移表達(dá)水平B. YKL-40 ExpresRT-PCRLow metastasisLevelInvaCL1-1AssayH23H838CL1-5H2009High metastasis60005000400030002000100000 hrCL1-1H23H838CL1-5H2009YKL-40 GAPDH18 hrCL1-1 H23 H838 CL1-5 H2009Migration AssayCL1-1H23H838CL1-5H2009Western BlotLow metastasisHigh metastasis350030002500200015001000

28、00 hrCL1-1H23H838CL1-5H2009YKL-40-actin6 hrCL1-1 H23 H838 CL1-5 H2009細(xì)胞表型Fig.2 The effect of YKL-40 expreslevel on the invaand migration abilities of the NSCLC cell lines.解 螺 旋|Migrated cell numberInvaded cell number數(shù)據(jù)泛讀案例三lossrescueC. InvaAssay(II) CL1-5Vec(I) CL1-5wt(III) CL1-5 (IV) CL1-5Gainrescu

29、eKDKD.YKL-406000500040003000200010000* *A. Inva(I) CL1-1wtAssay(II) CL1-1Vec(III) CL1-1 YKL-40(IV) CL1-1 YKL-40KD40003000200010000 *III III IVD. Migration Assay(I) CL1-5wt(II) CL1-5Vec(III) CL1-5 (IV) CL1-5KDKD.YKL-40III III IV6000500040003000200010000* *B. Migration Assay(I) CL1-1(II) CL1-1(III) CL

30、1-1 YKL-40(IV) CL1-1 YKL-40KDwtVecE. In vivo Assay1410840Wild typeIII III IV6000500040003000200010000* *動(dòng)物實(shí)驗(yàn)細(xì)胞表型Vector CL1-1 only YKL-40CL1-1 YKL-40.KDIII III IVFig.3 a and b CL1-1 cells (I) were transfected with the green fluorescent protein (GFP) expresplasmid (pEGFP-C1) plasmid, encodingempty vector (II) or full-length YKL-40 (III). After stable expres, cells were knockdowned via shRNA against human YKL-40 (IV).解 螺 旋|Migrated cell num