外分泌體中l(wèi)耐藥的分子機(jī)制研究

1、Exosome-Transmitted lncARSR Promotes SunitinibinRenal Cancer by Acting as aCompeting Endogenous RNA解螺旋李雷目前研究的幾點(diǎn)熱點(diǎn)腫瘤異質(zhì)性的一系列問題腫瘤耐藥 自噬長鏈非編碼外體腫瘤耐藥和免疫調(diào)控腫瘤耐藥和腸道菌群Exosome-Transmitted lncARSR Promotes SunitinibinRenal Cancer by Acting as aCompeting Endogenous RNA摘要Sunitinibis a major challenge for advanced

2、renal cellcarcinoma (RCC). Understanding the under- lying mechanisms anddeveloping effective strategies against sunitinibare highlydesired in theclinic. Here we identified an lncRNA, named lncARSR(lncRNA Activated in RCC with Sunitinib),whichcorrelated with clinically poor sunitinib response. lncARS

3、R promotedsunitinibvia compet-itively binding miR-34/miR-449 tofacilitate AXL and c-MET expression in RCC cells. Furthermore, bioactivelncARSR could be incorporated into exosomes and transmitted to sensitive cells, thus disseminating suni-tinib. Treatment of sunitinib-resistant RCC with lockednuclei

4、c acids targeting lncARSR or anAXL/c-MET inhibitor restored sunitinib response. Therefore, lncARSR may serve as a predictorand a poten-tial therapeutic target for sunitinib.lncARSR Is Highly Expressed in Sunitinib-ResistantRCC Cells(A) . (建立耐藥細(xì)胞系的方法）有兩大類三種方法(B).（差異性的lncrna)siRNA實(shí)驗(yàn)，確定的(C) Schematic a

5、nnotation of lncARSR genomic locus on chromosome9q82.120.717-82.185.824 in humans.?(D) Northern blot analysis of lncARSR with LNA probes in sunitinib-resistant and parental cells. b-Actin was used as a loading control.（race ,擴(kuò)增出，用探針的目的？）表達(dá)量比較低，提高敏感性lncARSR Is Highly Expressed in Sunitinib-ResistantR

6、CC Cells無進(jìn)展的組織樣本在復(fù)發(fā)性腎癌患者中，可以發(fā)到lncARSR的表達(dá)量是升高的。用的原位雜交的方法。第一部分的思考作者原文中說P-STAT3， P-AKT,P-EKR都明顯升高，這三個(gè)都和細(xì)胞的生存存在明顯的關(guān)系。在圖中P-AKT并不是最明顯的，我覺得P-STAT3反而是最明顯的？第二個(gè)問題：LNCRNA是長非后，沒有進(jìn)行濕實(shí)驗(yàn)驗(yàn)證？第三個(gè)不明白的問題：為什么作者不用最明顯的平板克隆的藥敏實(shí)驗(yàn)，選擇軟瓊脂克隆實(shí)驗(yàn)？lncARSR Is Modulated by AKT/FOXO Axis in Sunitinib- Resistant RCCCells的倍增和甲基化對lncARSR

7、表達(dá)的影響。根據(jù)通過實(shí)驗(yàn)，確定不是lncARSR的啟動子區(qū)域，發(fā)現(xiàn)FOXO1，F(xiàn)OX03A能夠結(jié)合。CHIP實(shí)驗(yàn)表明， 能夠調(diào)控lncARSR.lncARSR Is Modulated by AKT/FOXO Axis in Sunitinib- Resistant RCC Cells調(diào)控lncARSR表達(dá)乙酰化酶抑制劑， 作用后lncrasr的 表達(dá)量不受影響。耐藥細(xì) 胞系中， 細(xì)胞核 中的FOXO3A量下調(diào)lncARSR Is Modulated by AKT/FOXO Axis in Sunitinib-Resistant RCC Cells抑制AKT，降低lncARSR的表達(dá)量，升高f

8、oxo1,foxo3a的表達(dá)量。也就是想說明活化的Akt,通過降低FOXO1，F(xiàn)OXO3A的表達(dá)量，從而促進(jìn)lncRASR的表達(dá)量。第二部分小結(jié)第一個(gè)問題是作者如何找到FOXO1，F(xiàn)OXO3A？一般情況下，我們會想到一個(gè)正向調(diào)控的轉(zhuǎn)錄因子，一般是一個(gè)調(diào)控因子激活了lncARSR的表達(dá)。與AKT勾搭上。但是在本文中，作者找到一個(gè)負(fù)相關(guān)的轉(zhuǎn)錄因子。第二個(gè)問題，過表達(dá)FOXO1，或者FOXO3A后，腫瘤對靶向的耐藥性會不會增強(qiáng)？為什么這樣說呢，在本文中作者構(gòu)建了過表達(dá)FOXO1，F(xiàn)OXO3A1的質(zhì)粒，為什么沒有做藥敏實(shí)驗(yàn)?zāi)?？并且FOX和LncARSR在耐藥樣本中的相關(guān)性， 作者為什么沒有做上去？這些

9、問題讀文章的時(shí)候需要我們?nèi)ニ伎?。lncARSR Is Required for Sunitinibof RCC干擾掉lncARSR后，細(xì)胞的IC50值明顯降低，克隆細(xì)胞數(shù)目明顯減少。lncARSR Is Required for Sunitinibof RCCWb水平和動物模式試驗(yàn)中驗(yàn)證。lncARSR Levels in Plasma and Tumor Tissues Correlatewith Sunitinib Response in Ratients(A) qRT-PCR analysis of lncARSR in the plasma of R before and after

10、tumor resection.（血漿中）atients (n = 32)(B) qRT-PCR analysis of lncARSR in the CM of sunitinib-resistant and parental cells.(細(xì)胞培養(yǎng)的上清中）(C) qRT-PCR analysis of lncARSR in the plasma of mice xenografted with sunitinib-resistant and parental cells (n = 6 per group for 786-O and 7Su3rd, ACHN andACSu3rd cell

11、s, n = 3 per group for 771S and 771R cells).（老鼠模型中，血漿中的表達(dá)量）lncARSR Levels in Plasma and Tumor Tissues CorrelatewithSunitinib Response in Ratients臨床樣本(D) qRT-PCR analysis of lncARSR in the pre-therapy plasma of Ratientswith PD (n = 33) or non-PD (n = 38) during sunitinib therapy (p = 0.007).(E) Kapla

12、n-Meier analysis表明血漿中低表達(dá)lncrna的在服用靶向時(shí)間更長。后，生存(F) 治療過程中，lncrna表達(dá)量的升高和中復(fù)發(fā)存在明顯的相關(guān)性。lncARSR Levels in Plasma and Tumor Tissues Correlatewith SunitinibResponse in Ratients低表達(dá)的lncrna代表加入靶向更好的預(yù)后(G) Kaplan-Meier analysis of PFS in Ratients with and withoutsunitinib therapy. Left: all patients (p = 0.062); m

13、iddle: patients with low lncARSR expression(p = 0.028); right: patients with high lncARSR expression (p = 0.349). The median lncARSR expression was used as a cutoff. Results are presented as mean ± SD.Packaging of lncARSR into Exosomes Is Mediated by hnRNPA2B(C) qRT-PCR analysis of lncARSR in e

14、xosomes isolated from the CM of sunitinib-resistant and parental cells (n = 3).(D) Western blot analysis of indicated proteins in precipitates retrieved byin vitro-transcribed biotin-labeled lncARSRor antisenseRNA inthe cytoplasmic lysatesof 7Su3rd cells. RNA-PULL-DOWN實(shí)驗(yàn)(E) Upper: RNA pull-down anal

15、ysis using biotin-labeled RNAscorresponding to different fragments of lncARSR. （截短體試驗(yàn)，確定那個(gè)部位結(jié)合）Packaging of lncARSR into Exosomes Is Mediated by hnRNPA2B1(F) Left: RIP assay of the enrichment of hnRNPA2B1 on lncARSR relative to IgG in the nuclear, cytoplasmic, or exosomal lysates of sunitinib-resist

16、ant RCC cells(n = 3). Right: gel electrophoresis of PCR products from RIP assay. (G)Left:RIPassayoftheenrichmentofhnRNPA2B1onlncARSRrelativetoIgGinthec ytoplasmiclysatesofsunitinib-resistantandparentalcells(n=3).Right:gel electrophoresis of PCR products from RIP assay.Packaging of lncARSR into Exoso

17、mes Is Mediated by hnRNPA2B1(H) qRT-PCR analysis of lncARSR in exosomes derived from 7Su3rd or 771R cells 72 hr after transfection with control or hnRNPA2B1 siRNA (n = 3).(I) qRT-PCR analysis of lncARSR in exosomes derived from 786-O or 771S cells 72 hr after transfection with the indicated plasmid

18、(n = 3). Results are presented as mean ± SD. *p < 0.05, *p < 0.01, *p <0.001. See also Figure S3.Intercellular Transfer of lncARSR by Exosomes Disseminates SunitinibA) qRT-PCR analysis of lncARSR in 786-O cells 48 hr after incubation with indicated exosomes (or PBS as control). Normal

19、kidney cell HK-2 acted as negativecontrol (n = 3).(C) Colony-formation assays of 7Su3rd-RFP cells co-cultured with lncARSR-knockdown or control 786-O-GFP cells at a ratio of 1:1 with sunitinib treatment GFP-labeled parental cells became insensitive to sunitinibwhen co-cultured with RFP-labeled sunit

20、inib- resistant cells,whereas lncARSR knockdown in parental cells abolishedthis effectIntercellular Transfer of lncARSR by Exosomes Disseminates SunitinibNockdown of hnRNPA2B1 in resistant cells suppressed the ability of the co-cultured parental cells to acquireIntercellular Transfer of lncARSR by E

21、xosomesDisseminates Sunitinib的耐藥性可以逆轉(zhuǎn)播散耐藥性過表達(dá)lncRNA可以產(chǎn)生耐藥性(A) Western blot analysis of AXL and c-MET in lncARSR-knockdown and control cells.(B) LNA ISH of lncARSR and immunostaining of AXL and c-MET in consecutive tumor sections from matched human RCC taken before sunitinib therapy.復(fù)發(fā)中，三者的關(guān)系(C) Soft

22、 agar colony formation assay of lncARSR-knockdown and control 7Su3rd cells transfected with Lv-AXL and Lv-c-MET with sunitinib treatment (2 mM) in a12-well dish (5 3 10 3 cells per well) for 3 weeks (n = 3). Average number of colonies (upper) and representative images (lower) are shown.(D) Western b

23、lot analysis of indicated proteins in lncARSR-knockdown and control 7Su3rd cells transfected with Lv-AXL and Lv-c-MET with sunitinib treatment(2 mM) for 60 hr.(E) Soft agar colony formation assay of lncARSR-overexpressing and control 786-O cells transfected with indicated plasmid with sunitinib trea

24、tment (2 mM) in a12-well dish (5 3 10 3 cells per well) for 3 weeks (n = 3). Average number of colonies (upper) and representative images (lower) are shown.(F) Western blot analysis of indicated proteins in lncARSR-overexpressing and control 786-O cells transfected with indicated plasmid upon suniti

25、nib treatment(2 mM) for 60 hr.(G) Soft agar colony formation assay of lncARSR-overexpressing and control 786-O cells treated with sunitinib (2 mM) or BMS777607 (1 mM) in a 12-well dish(5 3 10 3 cells per well) for 3 weeks (n = 3). Average number of colonies (upper) and representative images (lower)

26、are shown.(H) Western blot analysis of indicated proteins in lncARSR-overexpressing and control 786-O cells treated with sunitinib (2 mM) or BMS777607 (1 mM) for 60 hr.lncARSR Functions as a ceRNA for miR-34 and miR-449(A) RIP assay of the enrichment of Ago2 on lncARSR, AXL, and c-MET transcripts re

27、lative to IgG in 786-O cells transfected with pcDNA3.1-Ctrl or pcDNA3.1-lncARSR (upper) and in 7Su3rd cells transfected with control or lncARSR siRNA (lower) (n = 3).lncARSR could compete with AXL and c-MET transcripts for the Ago2-based miRNA-induced repression complex(C) Luciferase activity of psi

28、CHECK2- lncARSR and psiCHECK2-lncARSRmt upon transfection of indicated miRNA mimics in 293T cells (n = 3). psiCHECK2-miR-34 (33) was used as positive control. Data are presented as the ratio of Renilla luciferase activity to Firefly luciferase activity.(D) Luciferase activity of psiCHECK2- miR-34 (3

29、3) upon transfection of indicated plasmids in 293T cells (n = 3).lncARSR contained functional miR-34 and miR-449 binding sites(G) Western blot analysis of AXL and c-MET in 7Su3rd cells transfected with control or lncARSR siRNA along with miR-34a sponge.(H) WesternblotanalysisofAXLandc-METin786- Ocel

30、lstransfectedwithpcDNA3.1-Ctrl,pcDNA3.1-lncARSR, orpcDNA3.1-lncARSRmt alongwithmiR-34andmiR-449 mimics. +, 2 mg of plasmid; +, 4 mg of plasmidlncARSR functions as a molecular sponge for miR-34 and miR-449lncARSR作為miR的ME海綿 to facilitate expression of AXL and c-(AC) Nude mice were orthotopically xenog

31、rafted with 771R-luc cells (1 3 10 6 cells) and treated intravenously with scramble or lncARSR LNA(50 nmol per mouse) twice a week and orally with vehicle or sunitinib daily (40 mg/kg) (n = 5 per group). Representative bioluminescent images (A), quantification of bioluminescent imaging signal intensities (B), and survival rate (C) of mice in indicated groups are shown.LncARSR的(鎖核酸)修飾的寡核苷酸(D and E) Nude mice were subcutaneously xeno- grafted with PDX R1 (5 mm in diameter) and treated intratumorally with scramble or lncARSR LNA (