【摘要】：第一部分：慢病毒表達載體的構建及沉默F(xiàn)OXQ1基因在大腸癌細胞系DLD-1中的表達目的：構建FOXQ1基因shRNA慢病毒干擾系統(tǒng),沉默F(xiàn)OXQ1基因在大腸癌細胞系DLD-1中的表達。方法：根據FOXQ1基因的序列,設計合成3對shRNA干擾序列,退火后連接到載體質粒PLKO.1-puro,將重組質粒轉化至STB13感受態(tài)中,涂板培養(yǎng)挑取單菌落,搖菌后小提質粒,選取酶切及測序鑒定正確的重組質粒去內毒素大提,將質粒與輔助包裝質粒pRSV-rev, pMDlg-pRRE, pCMV-VSV-G利用磷酸鈣法共轉染293-T細胞,收集病毒上清,感染目的細胞DLD-1,嘌呤霉素篩選FOXQ1沉默細胞,經熒光定量PCR及Western blot檢測干擾效果。結果：酶切及測序結果顯示shRNA成功插入載體PLKO.1-puro中,共轉染293-T細胞成功獲取病毒上清,感染DLD1細胞,經嘌呤霉素篩選出FOXQ1沉默細胞,熒光定量PCR及Western blot鑒定FOXQ1基因表達最高抑制率為90.4%。結論：通過構建FOXQ1基因shRNA慢病毒干擾系統(tǒng),成功獲取FOXQ1基因沉默細胞。為后續(xù)FOXQ1基因在腫瘤發(fā)生發(fā)展的研究奠定實驗基礎。第二部分：大腸癌FOXQ1與EGFR基因的相關性研究目的：探討大腸癌FOXQ1與EGFR基因間的相關性,為今后研究大腸癌中FOXQ1基因在EGFR-PI3K-Akt通路中的作用機制奠定基礎。方法：應用熒光定量PCR檢測大腸癌細胞系DLD1, HT29, HCT116, LOVO中FOXQ1及EGFR基因mRNA相對表達量；熒光定量檢測經shRNA-FOXQ1慢病毒干擾后的DLD1細胞(命名為DLD1-shRNA-FOXQ1)中EGFR的相對表達量改變；DLD1-shRNA-FOXQ1經EGFR酪氨酸激酶抑制劑Erlotinib HC1和siRNA-EGFR處理后,熒光定量PCR分別檢測FOXQ1和EGFR基因mRNA相對表達量。結果：(1)大腸癌細胞系中FOXQ1與EGFR基因的表達水平：FOXQ1在DLD1、HT29、LOVO、HCT116中的相對表達量分別為83.09、59.58、0.06、0.03,EGFR在DLD1、HT29、LOVO、HCT116中的相對表達量分別為4.95、3.67、2.08、1.36；(2)經shRNA-FOXQ1干擾的DLD1細胞EGFR表達量隨FOXQ1表達量的降低而增高；(3)細胞DLD1-shRNA-FOXQ1、DLD1-shRNA-Control分別經siRNA-EGFR處理抑制EGFR的表達后,FOXQ1表達量隨EGFR表達量的降低而增高,經Erlotinib HC1阻斷EGFR酪氨酸激酶后,FOXQ1表達量增高。結論：大腸癌細胞系中FOXQ1與EGFR基因的表達趨勢基本一致；大腸癌中FOXQ1的表達被抑制后,EGFR表達升高,而EGFR的表達或其蛋白激酶活性被抑制后,FOXQ1表達升高；推測二者間可能存在相互負反饋調節(jié)機制,從而維持大腸癌細胞中FOXQ1與EGFR高表達的狀態(tài)。
[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.
【學位授予單位】：昆明醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R735.34

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