治疗人肺腺癌细胞出现耐药发生EMT转化与MiRNA-181a作用于PTEN的关系研究

发布时间：2018-05-30 06:11

本文选题：肺腺癌 + 耐药　；参考：《山东大学》2016年博士论文

【摘要】：第一部分一线化疗药物(紫杉醇、顺铂)治疗肺腺癌细胞产生耐药并发生EMT转化目的：1.一线化疗药物治疗肺腺癌细胞出现耐药；2.评价肺腺癌细胞耐药；3.细胞学水平模拟临床化学药物治疗肺腺癌肿瘤,成功建立肺腺癌耐药模型；4.比较肺腺癌耐药细胞株与亲代敏感细胞侵袭迁移能力差别；5.观察肺腺癌耐药细胞株与亲代敏感细胞EMT(上皮-间质转化)分子标志物E-钙粘蛋白、β-连环蛋白、波形蛋白、纤维连接蛋白、MMP-2/9、Snail、Slug、 Twist、ZEB1不同表达,探讨EMT与耐药肺腺癌侵袭和迁移的关系。方法：1.一线化疗药物(紫杉醇、顺铂)治疗肺腺癌细胞诱导耐药。逐渐增加药物浓度治疗肺腺癌A549细胞,历时较长约12个月。每一个化疗周期存活的细胞约70%时,用胰酶消化分解与提高浓度的紫杉醇和顺铂共同培育。直到达到耐受目标化疗浓度：紫杉醇0.23μmol/ml和顺铂33μmol/ml。然后用这样的高浓度药物再培养3个月使细胞状态稳定。2.MTT法评价化疗药物对肺腺癌耐药细胞和其亲代敏感株的增殖抑制作用的差别。(1)检测不同浓度的紫杉醇PTX(0、0.2、0.4、0.8、1.6、3.2μmol·l-1)在24、48、72h不同时段对人肺腺癌细胞A549/PTX和A549增殖抑制作用比较,用剂量-效应曲线图表示。(2)检测不同浓度的顺铂DDP(0、4、8、16、32.6μmol·l-1作用于肺腺癌细胞A549/DDP和A549在24h.48h.72h后,进行增殖抑制作用比较(用剂量-效应曲线图表示),评价耐药特性和离体肺腺癌模型。3.采用细胞划痕实验检测肺腺癌敏感细胞和耐药细胞A549.A549/PTX和A549/DDP的迁移能力；细胞迁移实验采用Transwell小室法与聚碳酸酯膜模拟细胞外基质进行侵袭实验检测肺腺癌细胞A549/PTX.A549/DDP和A549体外侵袭迁移能力。4.×200放大倍数显微镜下观察肺腺癌敏感细胞A549和耐药细胞A549/PTX和A549/DDP形态特征：细胞极性,形态和排列方式等；5.应用Western blot检测肺腺癌敏感细胞A549和耐药细胞A549/PTX、A549/DDP中相关蛋白分子表达：上皮细胞标志物包括E-cadherin(E-钙粘蛋白)和β-catenin(B-连环蛋白)；间质细胞标志物包括Vimentin(波形蛋白)和降解基膜使细胞侵入细胞外基质中的基质金属蛋白酶MMP-2、MMP-9;转录因子包括Snail.Slug.Twist和ZEBl;6.应用RT-PCR荧光定量检测亲代细胞和化疗后出现耐药的肺腺癌细胞A549和A549/PTX.A549/DDP中上皮细胞标志物E-cadherin和B-catenin的mRNA变化；间质细胞标志物Vimentin、MMP-2和MMP-9的mRNA变化；另外检测转录因子Snail、Slug、Twist和ZEB1的mRNA变化。结果：1.建立对紫杉醇耐药(A549/PTX)和对顺铂耐药(A549/DDP)肺腺癌细胞(1)化疗药物筛选并培养出2株耐药细胞：A549/PTX耐紫杉醇0.23μmol/ml和A549/DDP顺铂33μmol/ml.(2)A549/PTX耐紫杉醇0.23μmol/ml和A549/DDP顺铂33μmol/ml再培养3个月,细胞状态稳定后液氮冻存以备实验使用。2.检测肺腺癌细胞A549/PTX和A549/DDP耐药的特性(1)不同浓度的紫杉醇PTX(0、0.2μmol·l-1、0.4μmol·l-1、0.8μmol·l-1、1.6μmol·l-1、3.2μmol·l-1)作用于肺腺癌细胞A549/PTX和 A549,在 24h、48h、72h不同时段对人肺腺癌细胞A549/PTX和A549增殖抑制作用比较剂量-效应曲线图表明表明紫杉醇对人肺腺癌细胞A549/PTX不敏感,A549/PTX细胞发生耐药。(2)不同浓度的顺铂DDP(0、4μmol·-1、8μmol·l-1、16μmol·l-1、1.32μmol·l-1、 64μmol·l-1、)作用于肺腺癌细胞A549/DDP和A549,在24h、48h、72h不同时段对人肺腺癌细胞A549/DDP在A549增殖抑制作用比较剂量-效应曲线图表明表明顺铂对人肺腺癌细胞A549/DDP不敏感,A549/DDP细胞发生耐药。3. A549/PTX和A549/DDP耐药细胞较亲代A549细胞增加侵袭和迁移的潜能(1)细胞划痕实验结果显示,A549/PTX、A549/DDP细胞与A549细胞相比迁移能力增强具有统计学意义。(2) Transwell、室法结果显示,A549/PTX、A549/DDP细胞相对于亲代体外迁移和侵袭能力增强明显具有统计学意义。4.A549/PTX和A549/DDP耐药细胞发生EMT转化(1)显微镜下观察细胞形态特征,亲代A549细胞形态类似于上皮样呈类圆形没有伪足、细胞与细胞连接、集落样生长；相对而言,耐药细胞A549/PTX、 A549/DDP细胞类似于间质样,可观察到细胞形态狭长有伪足形成,细胞与基质连接、分散性生长。(2) Western blot和荧光定量RT-PCR结果显示,耐药A549/PTX、A549/DDP细胞中上皮标志分子E-钙粘蛋白和β-连环蛋白的表达显著低于亲代敏感A549细胞(p0.05)；细胞间质标志分子、/imentin、MMP-2/9的表达以及转录因子Snail、 Slug、Twist和ZEB1高于亲代A549细胞具有统计学意义(p0.05)。结论：1.长期一线化疗药物紫杉醇和顺铂单药治疗人肺腺癌细胞A549出现耐药；2.耐药细胞A549/PTX和A549/DDP较亲代A549细胞侵袭和迁移能力增加；3. A549/PTX和A549/DDP发生EMT转化导致耐药。第二部分MiRNA-181a-inhibitor逆转EMT减弱人肺腺癌耐药细胞侵袭和转移能力目的：1.检测肺腺癌耐药细胞中A549/PTX和A549/DDP中 miRNA-181a与亲代敏感细胞A549表达差异；2.探讨niRNA-181a调控EMT影响肺腺癌耐药细胞侵袭和迁移的机制；3.抑制肺腺癌耐药细胞A549/PTX和A549/DDP侵袭迁移能力,逆转耐药。方法：1.荧光定量RT-PCR检测并验证miRNA-181a的差异表达。2.将肺腺癌耐药细胞A549/PTX和A549/DDPA转染miRNA-181a的抑制物miRNA-18la-inhibitor,亲代敏感细胞A549转染miRNA-181a的模拟物miRN A-181 a-mimic,再用荧光定量RT-PCR检测miRNA-181a的改变,明确原高表达niRNA-181a的细胞低表达,原低表达niRNA-181a的细胞高表达。3.进一步研究转染后细胞EMT相关特征的变化：a.细胞形态学方面变化；b.细胞生物学行为侵袭和迁移能力变化；c.上皮-间质转化EMT相关标志蛋白表达;d. EMT相关蛋白分子的mRNA变化。3.根据MicroCosm和miRBase数据库,PTEN是miRNA-181 a的靶点,利用Western blot和荧光定量RT-PCR检测肺腺癌耐药细胞A549/PTX和A549/DDPA转染miRNA-181a的抑制物miRN A-181a-inhibitor,亲代敏感细胞A549转染miRNA-181a的模拟物niRNA-181a-mimic前后PTEN的蛋白和mRNA的变化。4.MTT法评价miRNA-181a-inhibitor治疗耐药肺腺癌耐药细胞A549/PTX和A549/DDP的耐药情况。结果：1.肺腺癌细胞中miRNA的表达(1)在A549/PTX和A549/DDP细胞与A549细胞相比中,荧光定量RT-PCR结果表明miR-181a是最具有差异表达的miRNA。2. MiR-181a调控EMT及其对肺腺癌细胞侵袭和迁移的影响(1)荧光定量RT-PCR评价转染后miR-181a表达量结果显示,miR-181a模拟物能升高miR-181a的表达,而miR-181a抑制剂能抑制miR-181a的表达：亲代敏感细胞A549转染miR-181 a-mimic后niR-181a高表达,耐药细胞A549/PTX和A549/DDP转染miRNA-181 a-inhibitor使miR-181a低表达。(2)细胞形态方面miRNA-181 a-inhibitor能使A549/PTX和A549/DDP细胞从间质样梭形细胞转化为上皮样细胞,而miR-181a-mimic使A549细胞出现更多的间质样细胞。(3)细胞生物学方面细胞划痕实验表明miRNA-181 a-inhibitor减弱耐药细胞A549/PTX和A549/DDP细胞迁移能力,miR-181 a-mimic增强A549细胞迁移能力具有统计学意义(p0.005)。并且, Transwell小室实验表明miRNA-181 a-inhibitor减少耐药A549/PTX和A549/DDP细胞的侵袭迁移的细胞数,miR-181a模拟物能增加A549细胞的侵袭迁移的细胞数具有统计学意义(p0.005)。(4)转染后EMT相关分子的蛋白表达和mRNA含量的检测Western blot和荧光定量RT-PCR结果显示,niR-181a模拟物能使A549细胞中E-cadherin减少和Vimentn、MMP-9增加,miR-181a抑制剂能使A549/PTX和A549/DDP细胞中E-cadherin增加和Vimentin、MMP-9减少。3. miR-181a通过介导PTEN对肺腺癌细胞EMT的调控作用(1)采用miRTarBase软件预测miR-181a的靶基因,其中PTEN选作进一步研究。(2)肺腺癌耐药细胞A549/PTX和A549/DDPA与亲代敏感细胞A549比较PTEN的表达差异Western blot口荧光定量RT-PCR结果表明A549/PTX和A549/DDP细胞中PTEN的表达明显低于A549细胞。(2)肺腺癌耐药细胞A549/PTX和A549/DDPA转染niRNA-181 a-inhibitor,亲代敏感细胞A549转染miRNA-181a-mimic后,Western blot结果表明,miRNA-181a-inhibitor能使肺腺癌耐药细胞中PTEN增加,miRNA-181a-mimic使A549细胞中PTEN减少。但荧光定量RT-PCR结果显示,miR-181a的过表达或抑制对PTEN没有影响。4.MiRNA-181a-inhibitor治疗耐药肺腺癌细胞A549/PTX和A549/DDP后耐药特性改变,侵袭转移缓解(1)MTT测量A549/PTX、A549/DDP细胞转染抑制剂：miR-181 a-inhibitor紫杉醇(02,0.4,0.8,1.6 and 3.2μmol/l)、顺铂(4,8,16,32 and 64μmol/1)单药治疗48小时后细胞存活率较前减少,具有统计学意义。结论：1.miR-181a在耐药细胞株中高表达,在敏感株中低表达,调控miR-181a可以使上皮-间质相互转化EMT的发生。2.PTEN是miR-181a的靶点,在肺腺癌细胞中可能通过PTEN调控EMT。3.miRNA-181 a-inhibitor治疗耐药肺腺癌细胞A549/PTX和A549/DDP后耐药缓解。
[Abstract]:The first line of first - line chemotherapy drugs ( paclitaxel , cisplatin ) is resistant to lung adenocarcinoma cells and EMT is transformed into 1 . First - line chemotherapy drugs are resistant to lung adenocarcinoma cells .
2 . To evaluate the resistance of lung adenocarcinoma cells .
3 . The lung adenocarcinoma tumor was successfully established by cytological level simulation of clinical chemistry drugs , and lung adenocarcinoma drug resistance model was successfully established .
4 . Compare the difference of invasion and migration ability of lung adenocarcinoma resistant cell line and parent sensitive cell ;
5 . The expression of E - cadherin , 尾 - catenin , P - catenin , fibronectin , MMP - 2 / 9 in lung adenocarcinoma A549 / DDP and A549 cells was studied by MTT assay .
In vitro invasion and migration of lung adenocarcinoma cells A549 / PTX , A549 / DDP and A549 / DDP and A549 / DDP were investigated by using Transwell chamber method and polycarbonate membrane in vitro . The morphological characteristics of lung adenocarcinoma sensitive cells A549 and A549 / PTX and A549 / DDP were observed under a microscope of 4 . 脳 200 magnification : polarity , morphology and arrangement of cells .
5 . Western blot was used to detect the expression of related protein molecules in lung adenocarcinoma sensitive cells A549 and A549 / PTX , A549 / DDP : the epithelial markers include E - cadherin ( E - cadherin ) and 尾 - catenin ( B - catenin ) ;
The expression of E - cadherin and B - catenin in lung adenocarcinoma cells A549 and A549 / PTX , A549 / DDP , A549 / PTX , A549 / DDP were detected by RT - PCR .
The mRNA changes of vimentin , MMP - 2 and MMP - 9 were observed in the interstitial cell markers .
The results showed that A549 / PTX and A549 / DDP cells A549 / PTX and A549 / DDP were resistant to paclitaxel ( 0 , 0.2 渭mol 路 l - 1 , 0.4 渭mol 路 l - 1 , 16 渭mol 路 l - 1 , 1.32 渭mol 路 l - 1 , 64 渭mol 路 l - 1 ) . A549 / PTX and A549 / DDP cells had significant statistical significance compared with A549 / PTX , A549 / DDP cells compared with A549 / PTX , A549 / DDP cells and A549 / DDP cells .
( 2 ) Western blot and quantitative RT - PCR showed that the expression of E - cadherin and 尾 - catenin in A549 / DDP cells was significantly lower than that of sensitive A549 / PTX , A549 / DDP cells ( p < 0.05 ) .
Conclusion : 1 . Long - term first - line chemotherapy with paclitaxel and cisplatin alone can be used to treat human lung adenocarcinoma cells A549 .
2 . The invasion and migration ability of A549 / PTX and A549 / DDP cells was increased compared with the parent A549 / DDP cells .
The second part MiRNA - 181a - inhibitor reversed EMT to weaken the invasion and metastasis of human lung adenocarcinoma cells .
2 . To investigate the mechanism of niRNA - 181a regulating EMT to influence invasion and migration of lung adenocarcinoma drug - resistant cells ;
3 . To inhibit the invasion and migration ability of lung adenocarcinoma resistant cells A549 / PTX and A549 / DDP and to reverse the resistance . Methods : 1 . The expression of miRNA - 181a was detected by fluorescence quantitative RT - PCR .
b . Changes in cell biological behavior invasion and migration ability ;
3.According to MicroCosm and miRBase database , PTEN is the target of miRNA - 181 a . The expression of miRNA - 181a - inhibitor in lung adenocarcinoma cell A549 / PTX and A549 / DDPA is detected by Western blot and fluorescence quantitative RT - PCR . The expression of miR - 181a in A549 / PTX and A549 / DDP cells was significantly increased by miR - 181a - inhibitor . ( 2 ) The expression of miR - 181a - inhibitor in lung adenocarcinoma cell line A549 / PTX and A549 / DDP decreased . The expression of miR - 181a - inhibitor in lung adenocarcinoma cells A549 / PTX and A549 / DDP decreased .
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R734.2

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