miR-183介导EMT在肺腺癌细胞放射抗拒中的作用探讨

发布时间：2018-06-21 08:11

本文选题：非小细胞肺癌 + 微小RNA-183　；参考：《遵义医学院》2017年硕士论文

【摘要】：目的:放疗是非小细胞肺癌治疗的主要手段之一,放射抵抗是影响NSCLC放疗疗效的重要原因,EMT是放射抵抗产生的主要原因。研究表明肺腺癌放射抗拒细胞发生了EMT表型的改变,且miR-183在肺腺癌放射抗拒细胞株中表达增高,提示miR-183可能介导EMT并在肺腺癌细胞放射抗拒中发挥作用。因此对miR-183调控机制的深入探讨,将有望阐明其与EMT的关系及二者在肺腺癌细胞放射抵抗中的作用,为临床上寻找提高NSCLC放射治疗疗效的靶点提供新的思路。方法:倒置显微镜观察细胞形态学的改变;CCK-8细胞增殖实验比较不同细胞接受相同剂量照射后细胞生存率的差异;克隆形成实验比较细胞放射抗拒性能之间的差异;划痕愈合实验比较细胞迁移能力的改变;qPCR法检测细胞中miR-183的表达,qPCR及Western Blot分别检测ZEB1及EMT相关分子标志物mRNA及蛋白表达水平的改变;慢病毒转染实验实现对H1299及H1299R细胞中mi R-183基因的上/下调。结果:1.X射线持续照射后,肺腺癌细胞的形态发生了变化,由原本紧密连接的上皮细胞形态变成连接疏松、形态狭长并伸出伪足的类似成纤维细胞形态;在课题组前期的研究中:克隆形成实验表明,相比于H1299细胞,H1299R细胞的存活肩区更宽,放射抗拒性更强(P0.05);CCK-8细胞增殖实验表明,相比于H1299细胞,H1299R细胞在接受亚致死剂量(6Gy)X射线照射后,表现出更高的生存率(P0.05);qPCR检测发现,mi R-183、ZEB1、Vimentin在H1299R细胞中高表达(P0.05),E-cadherin表达变化无差异(P0.05);Western Blot检测发现,ZEB1、Vimentin在H1299R细胞中高表达,E-cadherin表达降低(P0.05)。2.成功构建干扰miR-183表达的慢病毒载体,qPCR验证miR-183在H1299R-shRNA-miR183细胞中表达降低(P0.05)。进一步克隆形成实验表明,相比于阴性对照H1299R-shRNA-NC细胞,干扰miR-183表达后的H1299R-shRNA-miR183细胞的存活肩区变窄,放射抗拒性减弱(P0.05);CCK-8细胞增殖实验表明,相比于H1299R-shRNA-NC细胞,H1299R-shRNA-mi R183细胞在接受4Gy X射线照射后,生存率降低(P0.05);划痕愈合实验发现,H1299R-shRNA-miR183细胞的划痕愈合时间相对延长,细胞迁移能力减弱(P0.05);qPCR及Western Blot检测发现在基因及蛋白水平ZEB1、Vimentin在H1299R-shRNA-miR183细胞中表达均降低,E-cadherin表达均相对增高(P0.05)。3.成功构建miR-183上调表达的慢病毒载体,qPCR验证miR-183在H1299-EGFP-miR183细胞中表达增高(P0.05)。进一步克隆形成实验表明,相比于阴性对照H1299-EGFP-NC细胞,mi R-183上调表达后的H1299-EGFP-miR183细胞的存活肩区变宽,放射抗拒性增强(P0.05);CCK-8细胞增殖实验表明,相比H1299-EGFP-NC细胞,H1299-EGFP-miR183细胞在接受4Gy X射线照射后,生存率增高(P0.05);划痕愈合实验发现,H1299-EGFP-miR183细胞的划痕愈合时间相对缩短,细胞迁移能力增强(P0.05);qPCR及Western Blot检测发现在基因及蛋白水平ZEB1、Vimentin在H1299-EGFP-miR183细胞中表达均增高,而E-cadherin表达均相对降低(P0.05)。结论:1.持续X射线照射过程中,H1299细胞发生了EMT,其增殖活性增强,放射抗拒能力增加;2.miR-183在H1299细胞发生放射抗拒的过程中表达增高,并在其中发挥重要作用:过表达miR-183的表达,可能会促进H1299细胞发生EMT,且增强其增殖、迁移及放射抗拒的能力;下调miR-183的表达,可能会逆转H1299R细胞的EMT过程,且减弱其增殖、迁移及放射抗拒的能力。
[Abstract]:Objective: radiotherapy is one of the main means of the treatment of non-small cell lung cancer, radiation resistance is an important factor affecting NSCLC radiotherapy, EMT is the main reason for radiation resistance generated. Studies have shown that the EMT phenotype changes of lung adenocarcinoma radioresistant cell, and miR-183 in lung adenocarcinoma cell lines in radioresistant expression increased, suggesting that miR-183 Can mediate EMT and play a role in lung adenocarcinoma cells. So the radioresistant miR-183 regulation mechanism deeply, is expected to clarify its relationship with EMT and the two in the lung cancer cell radiation resistance in vitro, to improve the targeting of NSCLC treatment to provide new ideas for clinical methods: cells were observed under inverted microscope. The morphological changes of CCK-8 cell proliferation; experimental comparison of different cell difference cell survival rate after irradiation at the same dosage; radiation cell clone formation experiment to resist performance differences between; wound healing migration ability of experimental cells; the expression of miR-183 in qPCR cell was detected by qPCR, Western and Blot were detected in Z EB1 and EMT molecular marker mRNA expression and protein level changes; lentiviral transfection experiments achieve the down-regulation of the H1299 and H1299R cells in MI / R-183 gene. Results: 1.X ray continued after irradiation, changes in lung adenocarcinoma cell morphology, from the original tight junction on the skin cells into loose connections the elongated shape and extending. A fibroblast morphology similar to actin; research group in the colony forming experiments show that, compared with H1299 cells, H1299R cells survival shoulder region wider, stronger radiation resistance (P0.05); CCK-8 showed that cell proliferation experiment, compared with H1299 cells, H1299R cells at sublethal dose (6Gy X) after irradiation, showing Higher survival rates (P0.05); qPCR mi R-183, detected ZEB1, Vimentin expression in H1299R cells (P0.05), the expression of E-cadherin had no difference (P0.05); Western Blot ZEB1, detected the high expression of Vimentin in H1299R cells, the expression of E-cadherin decreased (P0.05) lentiviral vector.2. the successful construction of miR-183 interference expression, qPCR verification mi The decreased expression of R-183 protein in H1299R-shRNA-miR183 cells (P0.05). Further cloning experiments show that, compared to the negative control H1299R-shRNA-NC cells, narrow interference miR-183 expression of H1299R-shRNA-miR183 cells after the survival of shoulder region, radiation resistance decreased; (P0.05) showed that CCK-8 cell proliferation experiment, compared with H1299R-shRNA-NC cells, H129 9R-shRNA-mi R183 4Gy X in cells after irradiation, the survival rate decreased (P0.05); wound healing experiment found that relatively prolonged H1299R-shRNA-miR183 cell wound healing time, cell migration ability decreased (P0.05); qPCR and Western Blot detected ZEB1 in gene and protein level, Vimentin expression in H1299R-shRNA-miR183 cells Reduced expression of E-cadherin was relatively higher (P0.05).3. successfully constructed lentiviral vector miR-183 expression, qPCR expression in H1299-EGFP-miR183 cells increased in the verification of miR-183 (P0.05). Further cloning experiments show that, compared to the negative control of H1299-EGFP-NC cells, the expression of MI R-183 increased after the survival of H1299-EGFP-miR183 cells Shoulder width, radiation resistance enhancement; (P0.05) showed that CCK-8 cell proliferation experiment, compared with H1299-EGFP-NC cells, H1299-EGFP-miR183 cells in 4Gy X after irradiation, the survival rate increased (P0.05); wound healing experiment found that H1299-EGFP-miR183 cells relative to shorten the wound healing time, enhance the ability of cell migration (P0.05) and qPCR; West Ern Blot detected ZEB1 in gene and protein level, the expression of Vimentin in H1299-EGFP-miR183 cells was increased, and the expression of E-cadherin was decreased (P0.05). Conclusion: 1. continuous X irradiation process, the EMT H1299 cells, enhance the proliferation activity, anti radiation ability to increase; 2.miR-183 radiation resistance in H1299 cell The expression process, and plays an important role in the overexpression of miR-183 may promote H1299 expression, EMT cells, and enhance the ability of proliferation, migration and radioresistant; down regulating the expression of miR-183 and EMT may reverse H1299R cell, and decrease the proliferation, migration and the ability of radiation resistance.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2

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