LOXL2介导胆管癌细胞Snai1蛋白苏素化的机制研究

发布时间：2018-03-22 16:54

本文选题：胆管癌细胞　切入点：LOXL2　出处：《昆明理工大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:(1)构建出稳定过表达LOXL2基因的胆管癌细胞系QBC939,以期进一步深入探索LOXL2蛋白的功能及作用机制。(2)在稳定细胞系QBC939-EX-LOXL2中转入SUMO-2/3干扰基因,观察沉默SUMO-2/3对QBC939细胞Snail蛋白表达量的影响。(3)在稳定细胞系QBC939-EX-LOXL2中转入过表达SENP1基因,观察过表达SENP1对QBC939细胞Snail蛋白表达量的影响。方法:(1)设计以G418为研究目的的剂量——反应分析实验,摸索G418对于QBC939细胞系的最低致死浓度。(2)通过脂质体转染的方法构建稳定过表达LOXL2基因的QBC939细胞系,利用实验得到的G418最低致死浓度筛选QBC939细胞以获取阳性克隆,扩增培养阳性细胞,并以普通QBC939细胞和QBC939-vector-LOXL2细胞作为对照组,运用荧光定量PCR实验和蛋白免疫印迹Western Blot技术检测LOXL2的mRNA的表达以及LOXL2的蛋白翻译情况,从而确保过表达LOXL2基因转染的QBC939细胞模型最终构建成功。(3)以稳定过表达LOXL2的QBC939细胞作为对照组,QBC939-EX-LOXL2+Control si RNA 细胞作为空载组,QBC939-EX-LOXL2+SUMO-2/3 siRNA作为实验组,运用蛋白免疫印迹Western Blot技术检测在干扰SUMO-2/3蛋白的影响下,Snail蛋白的表达变化量。(4)以稳定过表达LOXL2的QBC939细胞作为对照组,QBC939-EX-LOXL2+Vector-SENP1 1 胞作为空载组,QBC939-EX-LOXL2+EX-SENP1作为实验组,运用蛋白免疫印迹Western Blot技术检测在SENP1蛋白过表达的影响下,Snail蛋白的表达变化量。结果:(1)G418剂量-反应分析实验结果表明,浓度为600-1000ug/ml的G418将培养的QBC939细胞全部杀死,而600ug.ml以下均有细胞存活,故600ug/ml为G418筛选QBC939细胞的最低致死浓度。(2)600ug/ml的G418筛选转染过表达LOXL2基因的QBC939细胞7-11天后,可见阳性细胞的克隆,以普通QBC939细胞和Vector-LOXL2细胞作为对照组,运用荧光定量PCR实验检测LOXL2的mRNA的表达,蛋白免疫印迹Western Blot技术检测有LOXL2蛋白的翻译。(3)以稳定过表达LOXL2的QBC939细胞作为对照组,QBC939-EX-LOXL2+Control si RNA 细胞作为空载组,QBC939-EX-LOXL2+SUMO-2/3 siRNA作为实验组,运用蛋白免疫印迹Western Blot技术检测在干扰SUMO-2/3蛋白的影响下,Snail蛋白的表达量在实验组下降。(4)以稳定过表达LOXL2的QBC939细胞作为对照组,QBC939-EX-LOXL2+Vector-SENP1 1 胞作为空载组,QBC939-EX-LOXL2+EX-SENP1作为实验组,运用蛋白免疫印迹Western Blot技术检测在SENP1蛋白过表达的影响下,Snail蛋白的表达量下降。结论:(1)稳定过表达LOXL2基因的QBC939细胞系构建成功,为进一步探索LOXL2蛋白的功能及作用机制及其与胆管癌细胞侵袭转移的关系奠定了深厚的基础。(2)进一步证明LOXL2蛋白通过介导SUMOylation提高胆管癌细胞Snail蛋白的稳定性,为预防和治疗胆管癌细胞的侵袭和转移提供了新的靶点。
[Abstract]:Objective to construct a stable overexpression of LOXL2 gene in cholangiocarcinoma cell line QBC939, in order to further explore the function and mechanism of LOXL2 protein. To observe the effect of silencing SUMO-2/3 on the expression of Snail protein in QBC939 cells. To observe the effect of overexpression of SENP1 on the expression of Snail protein in QBC939 cells. To explore the lowest lethal concentration (LLC) of G418 on QBC939 cell line, we constructed a stable QBC939 cell line with overexpression of LOXL2 gene by liposome transfection, and screened QBC939 cell line by G418 lethality concentration to obtain positive clones. The expression of mRNA in LOXL2 and the protein translation of LOXL2 were detected by fluorescence quantitative PCR assay and Western blot Western Blot technique, and the normal QBC939 cells and QBC939-vector-LOXL2 cells were used as control group. Thus, the model of QBC939 cells transfected with overexpression of LOXL2 gene was successfully constructed.) QBC939 cells stably expressing LOXL2 were used as control group QBC939-EX-LOXL2 Control si RNA cells as control group, QBC939-EX-XL2 SUMO-2/3 siRNA was used as experimental group, QBC939-EX-XL2 SUMO-2/3 siRNA was used as experimental group, and QBC939-EX-XL2 SUMO-2/3 siRNA was used as experimental group. Western blotting Western Blot technique was used to detect the expression of Snail protein under the influence of interfering SUMO-2/3 protein. The QBC939-EX-LOXL2 Vector-SENP1 1 cell was used as the control group, QBC939-EX-LOXL2 EX-SENP1 was used as the experimental group, and QBC939 cells stably expressing LOXL2 were used as the control group, QBC939-EX-LOXL2 EX-SENP1 was used as the experimental group, QBC939-EX-LOXL2 EX-SENP1 was used as the experimental group. Western blot Western Blot technique was used to detect the expression of SENP1 protein under the influence of overexpression of SENP1 protein. Results the results of dose-response analysis of G418 showed that G418 at the concentration of 600-1000ug/ml killed all of the cultured QBC939 cells. However, all the cells below 600ug.ml survived, so 600ug/ml was the lowest lethal concentration of G418 to screen QBC939 cells. G418 was used to screen QBC939 cells transfected with LOXL2 gene for 7-11 days. The positive cells were cloned into normal QBC939 cells and Vector-LOXL2 cells as control group. Fluorescence quantitative PCR assay was used to detect the expression of LOXL2 mRNA, and Western blot Western Blot technique was used to detect the translation of LOXL2 protein.) QBC939-EX-LOXL2 Control si RNA cells were used as the control group, QBC939-EX-LOXL2 Control si RNA cells were used as the experimental group, QBC939-EX-LOXL2 SUMO-2/3 siRNA was used as the experimental group, and the QBC939-EX-LOXL2 SUMO-2/3 siRNA was used as the experimental group. Western blot Western Blot technique was used to detect the expression of Snail protein in the experimental group under the influence of interfering SUMO-2/3 protein. QBC939 cells with stable LOXL2 expression were used as the control group, QBC939-EX-LOXL2 Vector-SENP1 1 cell as the empty load group, QBC939-EX-LOXL2 EX-SENP1 as the experimental group, and QBC939-EX-LOXL2 EX-SENP1 as the experimental group. Western blot Western Blot technique was used to detect the decrease of SENP1 protein expression under the influence of overexpression of SENP1 protein. Conclusion the QBC939 cell line with stable overexpression of LOXL2 gene is successfully constructed. In order to further explore the function and mechanism of LOXL2 protein and its relationship with invasion and metastasis of cholangiocarcinoma cells, it is further proved that LOXL2 protein enhances the stability of Snail protein in cholangiocarcinoma cells by mediating SUMOylation. It provides a new target for the prevention and treatment of invasion and metastasis of cholangiocarcinoma cells.
【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.8

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