FOXQ1基因沉默型大肠癌细胞的构建及大肠癌FOXQ1与EGFR基因的相关性研究
发布时间：2019-02-24 00:00
【摘要】：第一部分：慢病毒表达载体的构建及沉默FOXQ1基因在大肠癌细胞系DLD-1中的表达目的：构建FOXQ1基因shRNA慢病毒干扰系统,沉默FOXQ1基因在大肠癌细胞系DLD-1中的表达。方法：根据FOXQ1基因的序列,设计合成3对shRNA干扰序列,退火后连接到载体质粒PLKO.1-puro,将重组质粒转化至STB13感受态中,涂板培养挑取单菌落,摇菌后小提质粒,选取酶切及测序鉴定正确的重组质粒去内毒素大提,将质粒与辅助包装质粒pRSV-rev, pMDlg-pRRE, pCMV-VSV-G利用磷酸钙法共转染293-T细胞,收集病毒上清,感染目的细胞DLD-1,嘌呤霉素筛选FOXQ1沉默细胞,经荧光定量PCR及Western blot检测干扰效果。结果：酶切及测序结果显示shRNA成功插入载体PLKO.1-puro中,共转染293-T细胞成功获取病毒上清,感染DLD1细胞,经嘌呤霉素筛选出FOXQ1沉默细胞,荧光定量PCR及Western blot鉴定FOXQ1基因表达最高抑制率为90.4%。结论：通过构建FOXQ1基因shRNA慢病毒干扰系统,成功获取FOXQ1基因沉默细胞。为后续FOXQ1基因在肿瘤发生发展的研究奠定实验基础。第二部分：大肠癌FOXQ1与EGFR基因的相关性研究目的：探讨大肠癌FOXQ1与EGFR基因间的相关性,为今后研究大肠癌中FOXQ1基因在EGFR-PI3K-Akt通路中的作用机制奠定基础。方法：应用荧光定量PCR检测大肠癌细胞系DLD1, HT29, HCT116, LOVO中FOXQ1及EGFR基因mRNA相对表达量；荧光定量检测经shRNA-FOXQ1慢病毒干扰后的DLD1细胞(命名为DLD1-shRNA-FOXQ1)中EGFR的相对表达量改变；DLD1-shRNA-FOXQ1经EGFR酪氨酸激酶抑制剂Erlotinib HC1和siRNA-EGFR处理后,荧光定量PCR分别检测FOXQ1和EGFR基因mRNA相对表达量。结果：(1)大肠癌细胞系中FOXQ1与EGFR基因的表达水平：FOXQ1在DLD1、HT29、LOVO、HCT116中的相对表达量分别为83.09、59.58、0.06、0.03,EGFR在DLD1、HT29、LOVO、HCT116中的相对表达量分别为4.95、3.67、2.08、1.36；(2)经shRNA-FOXQ1干扰的DLD1细胞EGFR表达量随FOXQ1表达量的降低而增高；(3)细胞DLD1-shRNA-FOXQ1、DLD1-shRNA-Control分别经siRNA-EGFR处理抑制EGFR的表达后,FOXQ1表达量随EGFR表达量的降低而增高,经Erlotinib HC1阻断EGFR酪氨酸激酶后,FOXQ1表达量增高。结论：大肠癌细胞系中FOXQ1与EGFR基因的表达趋势基本一致；大肠癌中FOXQ1的表达被抑制后,EGFR表达升高,而EGFR的表达或其蛋白激酶活性被抑制后,FOXQ1表达升高；推测二者间可能存在相互负反馈调节机制,从而维持大肠癌细胞中FOXQ1与EGFR高表达的状态。
[Abstract]:Part I: construction of lentivirus expression vector and expression of silencing FOXQ1 gene in colorectal cancer cell line DLD-1 objective: to construct FOXQ1 gene shRNA lentivirus interference system and silence FOXQ1 gene expression in colorectal cancer cell line DLD-1. Methods: according to the sequence of FOXQ1 gene, three pairs of shRNA interference sequences were designed and synthesized. After annealing, three pairs of shRNA interference sequences were designed and synthesized. After annealing, the recombinant plasmids were transformed into STB13 receptive states. The recombinant plasmid was digested by enzyme digestion and sequenced. The recombinant plasmid pRSV-rev, pMDlg-pRRE, pCMV-VSV-G was co-transfected into 293-T cells by calcium phosphate method to collect the virus supernatant. DLD-1, purine mycin was used to screen FOXQ1 silencing cells, and the interference effect was detected by fluorescence quantitative PCR and Western blot. Results: restriction endonuclease digestion and sequencing showed that shRNA was successfully inserted into the vector PLKO.1-puro. The virus supernatant was successfully obtained by co-transfection of 293-T cells and infected with DLD1 cells. FOXQ1 silencing cells were screened out by purine mycin. Fluorescence quantitative PCR and Western blot showed that the highest inhibitory rate of FOXQ1 gene expression was 90.4. Conclusion: FOXQ1 gene silencing cells were successfully obtained by constructing FOXQ1 gene shRNA lentivirus interference system. To lay an experimental foundation for the further study of FOXQ1 gene in tumorigenesis and development. Part two: study on the relationship between FOXQ1 and EGFR genes in colorectal carcinoma objective: to explore the relationship between FOXQ1 and EGFR genes in colorectal cancer, and to lay a foundation for the study of the role of FOXQ1 gene in EGFR-PI3K-Akt pathway in colorectal cancer. Methods: the relative expression of FOXQ1 and EGFR gene mRNA in colorectal cancer cell line DLD1, HT29, HCT116, LOVO was detected by fluorescence quantitative PCR. The relative expression of EGFR in DLD1 cells (named DLD1-shRNA-FOXQ1) after shRNA-FOXQ1 lentivirus interference was detected by fluorescence quantitative analysis. After DLD1-shRNA-FOXQ1 was treated with EGFR tyrosine kinase inhibitor Erlotinib HC1 and siRNA-EGFR, the relative expression of FOXQ1 and EGFR gene mRNA was detected by fluorescence quantitative PCR. Results: (1) the expression level of FOXQ1 and EGFR gene in colorectal cancer cell line: the relative expression of FOXQ1 in DLD1,HT29,LOVO,HCT116 was 83.09% 59.59.59.56% and 0.03% in DLD1,HT29,LOVO, respectively. The relative expression of HCT116 was 4.95 ~ 3.67 ~ 2.08 ~ 1.36, respectively. (2) the EGFR expression of DLD1 cells interfered with shRNA-FOXQ1 increased with the decrease of FOXQ1 expression. (3) after DLD1-shRNA-FOXQ1,DLD1-shRNA-Control was treated with siRNA-EGFR to inhibit the expression of EGFR, the expression of FOXQ1 increased with the decrease of EGFR expression, and the expression of FOXQ1 increased after Erlotinib HC1 blocked EGFR tyrosine kinase. Conclusion: the expression trend of FOXQ1 and EGFR gene in colorectal cancer cell line is basically the same, the expression of EGFR is increased after the expression of FOXQ1 is inhibited, but the expression of EGFR or its protein kinase activity is inhibited, the expression of FOXQ1 is increased. It is speculated that there may be a negative feedback regulation mechanism between the two, so as to maintain the high expression of FOXQ1 and EGFR in colorectal cancer cells.
【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】：R735.34
本文编号：2429359
[Abstract]:Part I: construction of lentivirus expression vector and expression of silencing FOXQ1 gene in colorectal cancer cell line DLD-1 objective: to construct FOXQ1 gene shRNA lentivirus interference system and silence FOXQ1 gene expression in colorectal cancer cell line DLD-1. Methods: according to the sequence of FOXQ1 gene, three pairs of shRNA interference sequences were designed and synthesized. After annealing, three pairs of shRNA interference sequences were designed and synthesized. After annealing, the recombinant plasmids were transformed into STB13 receptive states. The recombinant plasmid was digested by enzyme digestion and sequenced. The recombinant plasmid pRSV-rev, pMDlg-pRRE, pCMV-VSV-G was co-transfected into 293-T cells by calcium phosphate method to collect the virus supernatant. DLD-1, purine mycin was used to screen FOXQ1 silencing cells, and the interference effect was detected by fluorescence quantitative PCR and Western blot. Results: restriction endonuclease digestion and sequencing showed that shRNA was successfully inserted into the vector PLKO.1-puro. The virus supernatant was successfully obtained by co-transfection of 293-T cells and infected with DLD1 cells. FOXQ1 silencing cells were screened out by purine mycin. Fluorescence quantitative PCR and Western blot showed that the highest inhibitory rate of FOXQ1 gene expression was 90.4. Conclusion: FOXQ1 gene silencing cells were successfully obtained by constructing FOXQ1 gene shRNA lentivirus interference system. To lay an experimental foundation for the further study of FOXQ1 gene in tumorigenesis and development. Part two: study on the relationship between FOXQ1 and EGFR genes in colorectal carcinoma objective: to explore the relationship between FOXQ1 and EGFR genes in colorectal cancer, and to lay a foundation for the study of the role of FOXQ1 gene in EGFR-PI3K-Akt pathway in colorectal cancer. Methods: the relative expression of FOXQ1 and EGFR gene mRNA in colorectal cancer cell line DLD1, HT29, HCT116, LOVO was detected by fluorescence quantitative PCR. The relative expression of EGFR in DLD1 cells (named DLD1-shRNA-FOXQ1) after shRNA-FOXQ1 lentivirus interference was detected by fluorescence quantitative analysis. After DLD1-shRNA-FOXQ1 was treated with EGFR tyrosine kinase inhibitor Erlotinib HC1 and siRNA-EGFR, the relative expression of FOXQ1 and EGFR gene mRNA was detected by fluorescence quantitative PCR. Results: (1) the expression level of FOXQ1 and EGFR gene in colorectal cancer cell line: the relative expression of FOXQ1 in DLD1,HT29,LOVO,HCT116 was 83.09% 59.59.59.56% and 0.03% in DLD1,HT29,LOVO, respectively. The relative expression of HCT116 was 4.95 ~ 3.67 ~ 2.08 ~ 1.36, respectively. (2) the EGFR expression of DLD1 cells interfered with shRNA-FOXQ1 increased with the decrease of FOXQ1 expression. (3) after DLD1-shRNA-FOXQ1,DLD1-shRNA-Control was treated with siRNA-EGFR to inhibit the expression of EGFR, the expression of FOXQ1 increased with the decrease of EGFR expression, and the expression of FOXQ1 increased after Erlotinib HC1 blocked EGFR tyrosine kinase. Conclusion: the expression trend of FOXQ1 and EGFR gene in colorectal cancer cell line is basically the same, the expression of EGFR is increased after the expression of FOXQ1 is inhibited, but the expression of EGFR or its protein kinase activity is inhibited, the expression of FOXQ1 is increased. It is speculated that there may be a negative feedback regulation mechanism between the two, so as to maintain the high expression of FOXQ1 and EGFR in colorectal cancer cells.
【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】：R735.34
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