【作者】 李亚勇；

【导师】 陈道瑾；

【作者基本信息】 中南大学 ， 临床医学， 2013， 博士

【摘要】 在世界范围内,结直肠癌(colorectal carcinoma,CRC)的发病率以及其死亡率在恶性肿瘤中均位居于第三位。研究发现,CRC在亚洲地区中的经济较发达的地区,其发生率也呈现逐年增长的趋势。CRC的发病率在我国也呈现出逐步上升之趋势。CRC已经严重威胁到我国人民的健康,因而深入的探索CRC发病的机制以及治疗手段就成了研究热点。作为一种非编码小RNA,microRNA即miRNA在基因的调控、表达等方面有着重要的作用。多项研究表明miRNA的表达失调和CRC的发生及发展存在密切的关联关系。已有的研究表明,miR-143可能参与调节CRC细胞生长,细胞凋亡和某些化疗反应关键蛋白,但其具体的作用机制、调控途径、与CRC遗传易感性等有待于进一步深入的研究和阐明。本研究从miR-143对结直肠癌细胞生物学行为的影响、miR-143对结直肠癌细胞中Survivi、C-myc表达的影响、miR-143联合奥沙利铂对结直肠癌细胞的作用影响、miR-143基因启动子区多态性与结直肠癌遗传易感性的关系等四个方面对miR-143在结直肠癌发生发展中的作用机制做了初步的探讨和研究。miR-143对结直肠癌细胞生物学行为的影响目的：研究过表达miR-143在结直肠癌细胞中对其生物学行为的影响。方法：构建miR-143真核表达载体,用lipofectamine2000脂质体转染SW-480细胞,建立稳定高表达的miR-143细胞系。采用Realtime-PCR技术鉴定转染效率。并分别运用流式细胞、transwell以及MTT检测细胞的凋亡、侵袭和增殖等特征。结果：两组载体转染组均有明显的GFP表达。其中1μgpGenesil-1-miR-143载体组和2μg pGenesil-1-miR-143载体组的miR-143的相对表达量分别为10.61±0.36和12.83±0.38,而对照组的miR-143的相对表达量为2.53±0.83。1μg pGenesil-1载体组细胞,2μgpGenesil-1载体组细胞穿过基底膜细胞明显少于对照组细胞。两组实验组与对照组相比能使细胞凋亡增加,细胞周期较多停滞在G2期。MTT结果显示两组实验组细胞生长活力较对照组均明显降低,并且的MTT值在48h、72h、96h及120h均有显著性差异(P<0.05)。结论：成功构建miR-143真核表达载体。转染SW-480构建了稳定高表达mir-143的CRC细胞系；miR-143对CRC SW480肿瘤细胞的凋亡、生长活力和侵袭等细胞生物学行为有明显的影响作用；miR-143在CRC细胞中起到抑癌基因的作用。miR-143对结直肠癌细胞中Survivin、C-myc表达的影响目的：研究miR-143对结直肠癌细胞中Survivin、C-myc表达的影响,从而探讨miR-143在结直肠癌中可能的靶基因。方法：将结肠癌细胞SW-480细胞分为4组,未处理对照组,1μgpGenesil-1-miR143载体组,5μg pGenesil-1-miR143载体组和10μgpGenesil-l-miR143载体组。在转染实验完成后通过realtime PCR检测不同组别的miR-143和survivin。c-myc表达的变化,并采用统计学方法分析miR-143和survivin、miR-143和c-myc表达的关联。通过western-blot检测Akt信号通路的变化。结果：3组实验组miR143的表达水平均显著性高于对照组(P<0.05)。其中,5μg pGenesil-1-miR143组的miR-143表达量明显高于其它组,且有显著性差异(P<0.05)；3组实验组c-myc基因的表达水平较对照组无显著性差异(P>0.05)。相关性分析证明miR-143和C-myc基因的表达量成无相关性。其相关系数为-0.06；3组实验组Survivin基因的表达水平均显著性低于对照组(P<0.05)。相关性分析证实miR-143和Survivin基因的表达量成显著性负相关。其相关系数达-0.95; western-blot蛋白印迹检测结果显示在miR-143转染过表达后,3组实验组的AKT磷酸化水平较对照组明显降低。结论：C-myc的表达和miR-143的表达相关性不明显；Survivin的表达和miR-143的表达有显著负相关性。Survivin的表达受到miRNA-143的负性调控,是其靶基因之一；通过降低Akt磷酸化水平下调Survivin基因的表达是miR-143影响Survivin表达的可能途径之一。miR-143联合奥沙利铂作用对结直肠癌细胞的作用影响目的：研究miR-143联合奥沙利铂作用对结直肠癌细胞的作用影响。方法：将结肠癌细胞系分为4组,包括未处理对照组、奥沙利铂处理组、mir-143过表达组、奥沙利铂+mir-143过表达组。实验处理2天后,采用TUNEL技术分析对细胞凋亡的影响；利用流式细胞技术检测对细胞凋亡、细胞周期的影响；MTT实验检测对细胞生长活力的影响；然后采用realtime PCR, Western-blot分析miR-143对DNMT3A、 DNMT3B、DNMT1、Bcl-2、caspase-3等肿瘤因子的表达的影响。结果：TUNEL技术检测结果：在4个组别中,mir-143过表达组和奥沙利铂+mir-143过表达组对SW-480细胞有较明显的凋亡作用,并且以奥沙利铂+mir-143过表达联合作用组对SW-480细胞的凋亡作用为最明显；流式细胞技术检测结果：3组实验组和未处理对照组相比能使细胞凋亡增加,而且细胞周期较多停滞在G2、G2/M期。而且,奥沙利铂处理组对细胞凋亡的作用又低于mir-143过表达组和奥沙利铂+mir-143过表达组。但是在mir-143过表达组和奥沙利铂+mir-143过表达组见对比则无显著性差异；MTT实验检测结果：mir-143过表达组和奥沙利铂+mir-143过表达组细胞生长活力较对照组明显降低,并且其MTT值在72h、96h及120h均有显著性差异(P<0.05)。且奥沙利铂+mir-143过表达组与奥沙利铂组比较MTT值在96h、120h有明显下降；在Realtime PCR检测中,对于DNMT3A和DNMT3B基因,mir-143过表达组和奥沙利铂+mir-143过表达组的这个两个基因表达明显低于未处理对照组和奥沙利铂处理组(P<0.05)。但是未处理对照组和奥沙利铂处理组的表达未见有显著性差异(P>0.05)。而DNMT1在3组实验组中表达量均显著性低于未处理对照组(P<0.05)。Western blot的结果也证实了蛋白水平的表达差异：DNMT3A、 DNMT3B、DNMT1这3个基因在mir-143过表达组、奥沙利铂+miR-143组都有明显的表达水平降低；3组实验组的Bcl-2基因表达明显低于未处理对照组(P<0.05),Bcl-2基因在奥沙利铂+mir-143过表达组的表达量最低并且明显低于奥沙利铂处理组.、miR-143过表达组的表达(P<0.05)。caspase-3基因的RNA水平则在未处理对照组的表达量最低,在奥沙利铂+mir-143过表达组的表达量最高；而且奥沙利铂处理组的表达明显低于奥沙利铂+mir-143过表达组(P<0.05)。Western blot的结果也证实了蛋白水平的表达差异：与对照组相比,在3组实验组中Bcl-2均有不同程度的表达下降而caspase-3则均有不同程度的表达上升。结论：miR-143在CRC细胞中过表达对奥沙利铂的敏感性有明显的增强作用；奥沙利铂+miR-143联合作用能抑制DNMT1,DNMT3a和DNMT3b基因的表达；奥沙利铂+miR-143联合作用在CRC细胞中对Bcl-2和caspase-3的表达影响明显强于仅用奥沙利铂处理,提示Bcl-2、caspase-3信号通路是miR-143增加CRC细胞对奥沙利铂敏感性的可能通路。miR-143启动子区多态性与结直肠癌遗传易感性研究目的：初步探讨和研究miR-143启动子区rs4705342T>C多态性与结直肠癌遗传易感性的关系。方法：采用HRM技术,对miR-143启动子区的rs4705342T>C位点进行了基因多态性分析,检测结直肠癌病例组和正常对照组的位点差异。通过Hardy-Weinberg平衡检验,检测病例组与对照组rs4705342T>C多态性的基因型频率；各基因型在病例组与对照组之间分布的差异及等位基因频率的比较采用χ2检验。结果：HRM快速检测了rs4705342T>C各基因型结果显示：TT,TC和CC共3个基因型被检测到。各SNP位点的测序结果与HRM法比较,HRM检测基因多态性的符合率为100%；病例组与对照组rs4705342T>C多态性的基因型频率经Hardy-Weinberg遗传平衡检验,分别为1.0635和0.929,P值分别为0.3024和0.3351(P>0.05),表明其基因频率达到遗传平衡,具有群体代表性；病例组和对照组中TT、TC. CC基因型的分布总体有统计学差异,P<0.05。C等位基因频率在病例组与对照组中分别为43.60%、57.30%,两者间有显著性差异(P<0.05)。结论：miR-143基因启动子区rs4705342T>C多态性可影响CRC的遗传易感性；rs4705342T>C可作为潜在的CRC易感性生物标志物。图22幅,表4个,参考文献139篇。更多还原

【Abstract】 Colorectal carcinoma (CRC) is the third morbidity and mortality cancer around the world. According to new research, in economically developed regions of Asia, the incidence of CRC is increasing year by year and gradually approaching the level of Western developed countries. In China, the incidence of CRC shows a sharp rise in trend. Thus, CRC has been a serious threat to the health of our people. Deeply looking for the pathogenesis and treatment of CRC is became a research focus.As a non-coding RNAs, miRNA plays an important role in the regulation of gene expression. With the deepening of the understanding to miRNA’s regulation mechanism, abundant researches have demonstrated that the expression of miRNA or biological function could change CRC occurrence and development. The corresponding miRNA researches provide the basis for the screening of molecular markers in CRC and reveal the pathogenesis of CRC to provide a theoretical basis. Previous studies have shown that miR-143may be used as a regulatory gene in cell growth, apoptosis, and key protein in response to chemotherapy. Nevertheless, mechanism of action, the specific regulation pathways, and genetic susceptibility of CRC need to be further studied and clarified. Regulation approach Regulation approach.To study and research the role of miR-143in the occurrence and development of CRC, we observed the effect of miR-143on CRC cells in vitro, the effect of miR-143on the expression of survivin and c-myc in CRC cells. In this study, we also observed the combined effect of miR-143and Oxaliplatin on CRC cells, the relationship between polymorphism in the promoter region of microRNA-143gene and genetic susceptibility of CRC Effect of miR-143on CRC cells in vitroObjective:To explore the effect of over-expression miR-143on biological behavior of CRC cells.Methods:To construct eukaryotic expression vector of miR-143. Then we used lipofectamine2000to transfect it into SW-480cells to establish stable SW-480cell lines of high expression miR-143. We used Realtime-PCR to identified transfection efficiency. And we used flow cytometry, transwell and MTT to detect apoptosis, invasion and proliferation respectively.Results:1μg pGenesil-1-miR-143vector and2μg pGenesil-1-miR-143vector were transfected into colon cancer cells SW-480. Results showed a clear expression of GFP in transfected group. The expression of miR-143in1μg pGenesil-1-miR-143vector group and2μg pGenesil-1-miR-143vector group were10.61±0.36and12.83±0.38, respectively. Meanwhile, the expression of miR-143in control group was2.53±0.83. The cells which could through the basement membrane were significantly more in1μg pGenesil-1vector group and2μg pGenesil-1vector group than compare control. Meanwhile,1μg pGenesil-1-miR-143vector group induced more apoptosis than1μg pGenesil-1-miR-143vector group. The cells were stayed in G2stage. MTT result showed after transfection, the cell growth activity. The significant differences were found in48h,72h,96hand120h.Conclusion:1. The eukaryotic expression vector of miR-143was successfully constructed. And stable SW-480cell lines with high expression of miR-143were obtained.2. miR-143had significant effect on biological behavior of SW-480tumor cell,including apoptosis, proliferation and invasion. miR-143played the role of tumor suppressor genes on CRC cell. The effect of miR-143on the expression of survivin and c-myc in CRC cellsObjective:To study the effect of miR-143on the expression of survivin and c-myc in CRC cells, and investigate the potential target gene.of miR-143.Methods:In this research we divided SW-480cell into4groups:the control group,1μg pGenesil-1-miR-143vector group,5μg pGenesil-1-miR-143vector group and10μg pGenesil-1-miR-143vector group. After transfection experiment, we used realtime-PCR analysis to detect the change of gene expression of miR-143, survivin and c-myc. Furthermore, with statistical method we made the correlation analysis between miR-143and survivin. And the same to miR-143and c-myc.At last we studied the change of Akt signal path by western-blot analysis.Results:1.The gene expression levels of miR-143in the other three experimental group were higher than the control group significantly (P <0.05). It was highest in5μg pGenesil-l-miR143group, and there was significant difference compared with the other three groups.2. The gene expression levels of c-myc had no significant difference among four groups(p>0.05). Correlation analysis showed that no correlation was found between miR-143and c-myc. And the correlation coefficient was-0.06.3. The gene expression levels of survivin in the other three experimental group were lower significantly than the control group (P <0.05). Correlation analysis showed that the relativity about miR-143and survivin was negative significantly. And the correlation coefficient was-0.95.4. Western-blot analysis showed that the phosphorylation of Akt in the other three experimental group were lower than the control group.Conclusion:1. No correlation was found between miR-143and c-myc.2. The relativity about miR-143and survivin was negative significantly. miR-143could modulate the expression of survivin negatively and survivin was one of target genes of miR-143.3. miR-143could down-regulate gene expression of survivin through inhibiting phosphorylation of Akt. It maybe one of the possible regulation pathway by which miR-143modulated the expression of survivin. The combined effect of miR-143and Oxaliplatin on CRC cellsObjective:To observe the combined effect of miR-143and Oxaliplatin on CRC cells.Methods:Colon cancer cell lines were divided into4groups, including untreated control group, Oxaliplatin treatment group, mir-143over-expression group and Oxaliplatin+mir-143over-expression group. After two days treatment, we employed TUNEL to detect apoptosis which was induced by over-expression of miR-143, and used flow cytometric to analyze the effects of high expression of miR-143on cell apoptosis and cell cycles. We also used MTT to detect cell proliferation. Subsequently, we used realtime PCR and Western-blot to assay the effect of miR43to some tumor factors, including DNMT3A, DNMT3B, DNMT1, Bcl-2and caspase-3.Results:1.TUNEL experiment showed that SW-480cells in mir-143over-expression group and Oxaliplatin+mir-143over-expression group appeared obvious apoptosis among four experimental groups. Besides, in Oxaliplatin+mir-143over-expression group, tumor cell apoptosis were observed most significantly.most significant2. Flow cytometric experiment showed that apoptosis of tumor cells in Oxaliplatin group, miR-143over-expression group and Oxaliplatin+miR-143group were more obvious than the control group, and the cell cycle was arrested in G2and G2/M phase. The role of apoptosis in Oxaliplatin treatment group was lower than miR-143over-expression group and Oxaliplatin+miR-143over-expression group. However, there was no significant difference between miR-143over-expression group and Oxaliplatin+miR-143over-expression group.3.MTT results showed that cell growth activity decreased more significantly in miR-143over-expression group and Oxaliplatin+miR-143over-expression group than in the control group. The significant differences were found in72h,96h and120h. Besides, contrasted with Oxaliplatin treatment group, the significant decline in Oxaliplatin+miR-143over-expression group was found in96h and120h.4.By realtime PCR analysis, gene expression of DNMT3A and DNMT3B in miR-143over-expression group and Oxaliplatin+miR-143over-expression group was significantly lower than the control group and Oxaliplatin treatment group (P<0.05). But between the control group and Oxaliplatin treatment group, there was no significant difference (P>0.05). The DNMT1expression in the three groups were significantly lower than in the control group (P<0.05). Furthermore, differences in gene expression were approved by western-blot analysis. Both in miR-143over-expression group and Oxaliplatin+miR-143over-expression group, gene expression levels of DNMT3A,DNMT3B and DNMT1declined significantly.5.By realtime PCR analysis, gene expression of Bcl-2in miR-143over-expression group and Oxaliplatin+miR-143over-expression group was significantly lower than the control group (P<0.05).And gene expression of Bcl-2in Oxaliplatin+miR-143over-expression group was lowest. Gene expression level of caspase-3was lowest in the control group while it was highest in the Oxaliplatin+miR-143over-expression group. And there was significant difference between the control group and the Oxaliplatin+miR-143over-expression group. Besides, differences in gene expression were approved by western-blot analysis. Compared with the control group, gene expression level of Bcl-2decreased differently while gene expression level of caspase-3raised respectively in the other three groups.Conclusion:1.miR-143over-expression could enhance the chemosensitivity of Oxaliplatin in CRC tumor cells.2.The combined effect of miR-143and Oxaliplatin could inhibit gene expression of DNMT3A,DNMT3B and DNMT1.3.The combined effect of miR-143and Oxaliplatin could influence gene expression of Bcl-2and caspase-3more obviously than Oxaliplatin solely. It indicated that Bcl-2/caspase-3path maybe the possible signal path by which miR-143over-expression enhanced the chemosensitivity of Oxaliplatin in CRC cells. Polymorphism in the promoter region of microRNA-143gene and genetic susceptibility of CRC.Objective:To preliminarily discuss the relationship between the rs4705342T>C polymorphism in microRNA-143promoter region and genetic susceptibility of CRC.Methods:We used HRM technology to analysis the gene polymorphism of rs4705342T>C site in microRNA-143promoter region, to detect the difference of sites between colorectal cancer patients group and normal control group. By Hardy-Weinberg analysis,we detected the genotypic frequencies of the two groups, and we also tested the difference of genotype distribution frequencies and alleles frequencies between colorectal cancer patients group and normal control group with x2-test.Results:1.Three genotypes including TT, TC and CC were detected through HRM.analysis, and the coincidence of gene polymorphism by HRM analysis was100%.2.The genotypic frequency of rs4705342T>C polymorphism in colorectal cancer patients group and normal control group was1.0635and0.929respectively through Hardy-Weinberg analysis. P value was0.3024and0.3351individually(P>0.05).It indicated that genotypic frequencies of the two groups attained genetic balance and had group representation.3. The genotype distribution frequencies of TT, TC and CC had statistical difference between colorectal cancer patients group and normal control group(P<0.05). C alleles frequency was43.60%and57.30%in colorectal cancer patients group and normal control group. There was significant difference (P<0.05).Conclusion:The gene polymorphism of rs4705342T>C site in microRNA-143promoter region may alter the genetic susceptibility of CRC. It could be used as a potential CRC susceptibility biomarker.更多还原