STK33基因对非小细胞肺癌相关信号通路Ras-MAPK的调控研究

发布时间：2018-09-19 09:21

【摘要】：目的：探讨STK33在非小细胞肺癌(NSCLC)中对肿瘤相关信号通路Ras-MAPK的调控作用,从而为明确其在促进NSCLC转移及侵袭能力的发生机制方面提供一定的理论依据,进而为NSCLC的靶向治疗寻找新靶点提供理论支持。方法：用已经建立稳定的高转移性人大细胞肺癌细胞L9981和低转移性人大细胞肺癌细胞NL9980,通过RT-PCR技术检测L9981、NL9980中STK33的mRNA表达量。构建STK33瞬时低表达质粒并转染细胞L9981,记为L9981(-),构建STK33瞬时高表达质粒并转染细胞NL9980,记为NL9980(+),采用RT-PCR鉴定转染结果。转染成功后,通过Western-blot技术检测4组细胞中STK33和Ras-MAPK信号通路中重要蛋白PAK1、p38MAPK的蛋白表达量；通过免疫共沉淀,分析STK33和PAK1-p、p38MAPK-p之间的相互作用关系。结果：(1) STK33 在 NL9980中的mRNA表达量低于L9981(P0.001)；(2)成功构建出STK33瞬时高表达质粒和STK33瞬时低表达质粒并转染成功。(3)L9981(-)中STK33的蛋白表达量较L9981降低,NL9980(+)中STK33的蛋白表达量较NL9980增加；(4)当STK33蛋白表达量变化时,Ras-MAPK信号通路的重要蛋白PAK1,p38MAPK的非磷酸化蛋白表达水平变化不大,磷酸化蛋白在L9981(-)中的表达量低于L9981,在NL9980(+)中的表达量高于NL9980细胞。(5)用免疫共沉淀的方法检测STK33与Ras-MAPK信号通路中磷酸化蛋白PAK1-p和p38MAPK-p的相互作用。实验结果证实STK33与PAK1-p无直接作用,但是与p38MAPK-p可能有直接作用。结论：STK33在不同转移性肺癌细胞中的差异性表达,提示它可能与肺癌的分期、分化程度有关。STK33表达量的变化能够引起Ras-MAPK信号通路中磷酸化蛋白PAK1-p 和 p38MAPK-p表达量的变化,STK33高表达时,磷酸化蛋白PAK1-p和p38MAPK-p表达量增加,并可能促进上皮细胞—间充质转化(EMT),降低细胞凋亡,提示STK33可能能够促进NSCLC的转移和侵袭能力；抑制STK33在肺癌细胞中的表达,可能会使得NSCLC的转移能力下降。STK33可能成为治疗NSCLC的新靶点。
[Abstract]:Objective: to investigate the role of STK33 in the regulation of tumor related signal pathway (Ras-MAPK) in non-small cell lung cancer (NSCLC) (NSCLC), so as to provide a theoretical basis for the mechanism of NSCLC metastasis and invasion. Thus, it provides theoretical support for finding new targets for targeted therapy of NSCLC. Methods: the expression of STK33 mRNA in L9981NL9980 cells was detected by RT-PCR technique with the established stable high metastatic human lung cancer cell line L9981 and low metastatic human lung cancer cell line NL9980,. The transient low expression plasmid of STK33 was constructed and transfected into L9981 (-) cell line L9981 (-). The transient high expression plasmid of STK33 was constructed and the transfection cell NL9980, was recorded as NL9980 (),. The transfection results were identified by RT-PCR. After transfection, the expression of PAK1,p38MAPK, an important protein in STK33 and Ras-MAPK signaling pathway, was detected by Western-blot technique, and the interaction between STK33 and PAK1-p,p38MAPK-p was analyzed by co-immunoprecipitation. Results: (1) the mRNA expression of STK33 in NL9980 was lower than that in L9981 (P0.001); (2). The transient high expression plasmid of STK33 and the transient low expression plasmid of STK33 were successfully constructed and transfected successfully. (3) the expression of STK33 in L9981 (-) was lower than that in L9981 (-) and the expression of STK33 in L9980 (-) was higher than that in NL9980. (4) when the expression of STK33 protein changed, the expression level of non-phosphorylated protein of PAK1,p38MAPK, an important protein in Ras-MAPK signaling pathway, did not change significantly. The expression of phosphorylated protein in L9981 (-) was lower than that in L9981 (-) and was higher in NL9980 () than in NL9980 cells. (5) the interaction between STK33 and PAK1-p and p38MAPK-p in Ras-MAPK signaling pathway was detected by immunoprecipitation. The results show that STK33 has no direct effect on PAK1-p, but may have direct effect on p38MAPK-p. Conclusion the differential expression of TK33 in different metastatic lung cancer cells suggests that it may be related to the stage of lung cancer. The expression of phosphorylated protein PAK1-p and p38MAPK-p in Ras-MAPK signaling pathway increased when the expression of phosphorylated protein PAK1-p and p38MAPK-p increased when the expression level of STK33 was related to the degree of differentiation. It is suggested that STK33 can promote the metastasis and invasion of NSCLC and inhibit the expression of STK33 in lung cancer cells. STK 33 may be a new target for the treatment of NSCLC.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R734.2

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