Ouabain通过MKK6调控SOX9基因表达抑制食管腺癌细胞增殖作用的研究

发布时间：2018-06-14 19:29

本文选题：食管腺癌 + Ouabain　；参考：《福建医科大学》2016年硕士论文

【摘要】：目的研究Ouabain对于食管腺癌细胞的生长抑制作用,探索其作用的基因靶点MKK6及其下游基因SOX9对食管腺癌细胞体外生长及体内成瘤状况的影响。方法1考察1043种药物对食管腺癌细胞体外生长的抑制情况,获得候选药物Ouabain,并利用肿瘤细胞体外生长曲线和体内成瘤实验来验证该药物对的食管腺癌细胞生长的干扰作用。2通过RNA测序技术分析经Ouabain作用过的食管腺癌细胞内基因表达量的变化,获得潜在靶标基因MKK6,并运用实时荧光定量PCR和Western Blot检验该基因在Ouabain作用的食管腺癌细胞及肿瘤中的表达情况。3应用慢病毒在食管腺癌细胞导入可介导MKK6基因敲落的sh RNA,并利用实时荧光定量PCR和Western Blot检验该基因在肿瘤细胞中的敲落效率。进一步分析MKK6基因敲除对食管腺癌细胞体外生长状况的影响,以及小鼠体内成瘤情况的作用。4基于相关信号通路理论,利用实时荧光定量PCR甄选出受MKK6调控的下游靶标基因SOX9,进而验证Ouabain作用及MKK6敲落对SOX9基因表达水平及蛋白翻译水平的影响。5利用CRISPR-Cas9技术于食管腺癌细胞内敲除SOX9,通过对比空白对照组与SOX9基因敲除组在肿瘤细胞体外生长曲线和体内成瘤实验中的差别,探求SOX9在食管腺癌细胞增殖中发挥的作用。结果1 Ouabain对食管腺癌细胞的抑制作用:药物筛选获得的候选药物Ouabain,可抑制食管腺癌细胞OE33及OE19的体外增殖情况,腹腔注射给药可限制NSC小鼠体内成瘤的重量及体积大小。2 Ouabain对MKK6基因转录与翻译水平的影响:RNA测序显示经Ouabain作用的OE33细胞内MKK6基因表达水平下降明显,蛋白翻译水平相较空白对照组也有所下降,OE19细胞内影响亦同。3体外敲落MKK6基因对食管腺癌细胞的抑制作用:sh RNA介导的食管腺癌细胞内MKK6基因敲落,可抑制食管腺癌细胞OE33及OE19的体外增殖情况,减小细胞在裸鼠体内的成瘤的重量及体积大小。4食管腺癌细胞内MKK6对SOX9的调控作用:基因敲落MKK6可降低OE33及0E19细胞内转录因子SOX9的基因表达水平,和蛋白质翻译水平;5 SOX9对于食管腺癌细胞增殖的影响:食管腺癌细胞OE19内敲除SOX9基因,可抑制其在体外的增殖和体内的成瘤。结论1 Ouabain可抑制食管腺癌细胞体外增殖及体内成瘤;2 Ouabain可降低食管腺癌细胞及小鼠肿瘤内MKK6基因表达与蛋白翻译水平;3基因敲落MKK6可抑制食管腺癌细胞体外增殖及体内成瘤;4基因敲落MKK6可降低下游基因SOX9的基因表达与蛋白翻译水平;5基因敲除SOX9可影响食管腺癌细胞体外增殖及体内成瘤。
[Abstract]:Objective to study the inhibitory effect of Ouabain on the growth of esophageal adenocarcinoma cells, and to explore the effect of MKK6 and its downstream gene SOX9 on the growth of esophageal adenocarcinoma cells in vitro and tumor formation in vivo. Methods 1 the inhibition of 1043 drugs on the growth of esophageal adenocarcinoma cells in vitro was investigated. A candidate drug Ouabain was obtained, and the tumor cell growth curve in vitro and in vivo tumorigenesis test were used to verify the interference effect of the drug on the growth of esophageal adenocarcinoma cells. 2. Analysis of Ouabain treated esophageal adenocarcinoma cells by RNA sequencing technique Changes in the amount of internal gene expression, The potential target gene MKK6 was obtained, and the expression of MKK6 gene in Ouabain treated esophageal adenocarcinoma cells and tumors was detected by real-time fluorescent quantitative PCR and Western blot. 3. 3 Lentivirus transfection into esophageal adenocarcinoma cells can mediate MKK6 gene knockout. The knockout efficiency of the gene in tumor cells was detected by real-time fluorescent quantitative PCR and Western Blot. To further analyze the effect of MKK6 knockout on the growth of esophageal adenocarcinoma cells in vitro, and the role of tumorigenesis in mice based on the theory of related signal pathway. The downstream target gene SOX9 regulated by MKK6 was selected by real-time fluorescent quantitative PCR, and the effect of Ouabain and MKK6 knockout on SOX9 gene expression and protein translation was verified. 5 the CRISPR-Cas9 technique was used to knockout SOX9 in esophageal adenocarcinoma cells. By comparing the difference between blank control group and SOX9 gene knockout group in tumor cell growth curve in vitro and in vivo tumorigenesis test, To explore the role of SOX 9 in the proliferation of esophageal adenocarcinoma cells. Results 1 the inhibitory effect of Ouabain on esophageal adenocarcinoma cells: Ouabain, a candidate drug obtained by drug screening, could inhibit the proliferation of esophageal adenocarcinoma cells OE33 and OE19 in vitro. The effect of intraperitoneal injection on the transcription and translation of MKK6 gene in NSC mice by limiting the weight and volume of tumor. 2. The expression of MKK6 gene in OE33 cells induced by Ouabain was significantly decreased. The effect of MKK6 gene knockout on esophageal adenocarcinoma cells was also similar to that of the control group. The inhibitory effect of MKK6 gene knockout on esophageal adenocarcinoma cells was also similar to that of the control group. The effect of MKK6 gene knockout on esophageal adenocarcinoma cells mediated by 20% sh RNA was also similar to that of MKK6 gene knockout in vitro. Can inhibit the proliferation of esophageal adenocarcinoma cells OE33 and OE19 in vitro, Decrease the weight and volume of tumor in nude mice. 4 the regulation of SOX9 by MKK6 in esophageal adenocarcinoma cells: knockout MKK6 can decrease the expression of SOX9 in OE33 and 0E19 cells. Effects of 5SOX9 on the proliferation of esophageal adenocarcinoma cells: knockout of SOX9 gene in esophageal adenocarcinoma OE19 can inhibit its proliferation in vitro and tumorigenesis in vivo. Conclusion 1 Ouabain can inhibit the proliferation of esophageal adenocarcinoma cells in vitro and tumorigenesis in vivo. 2. Ouabain can reduce the expression of MKK6 gene and the level of protein translation in esophageal adenocarcinoma cells and mouse tumors. The knockout of MKK6 gene can inhibit the proliferation of esophageal adenocarcinoma cells in vitro. The expression of SOX9 gene and protein translation of downstream gene SOX9 can be reduced by knockout MKK6 of tumor-forming 4 gene in vivo. The knockout of SOX9 gene may affect the proliferation of esophageal adenocarcinoma cells in vitro and tumorigenesis in vivo.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.1

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