Sipa1基因启动子序列及其SNPs结构和功能研究
发布时间：2019-01-29 20:07
【摘要】：SIPA1蛋白(signaling-induced proliferating-associating protein 1)即信号诱导增殖相关蛋白1,是一个Rap1GAP(Rap1 GTPase activating protein)蛋白,它可以催化Rap1蛋白从活化的Rap1GTP形式转变为失活Rap1GDP形式,进而实现对Rap1蛋白功能的调节:细胞的粘附,细胞内激酶的活性,细胞核内基因的调控等。Sipa1基因位于第11号染色体上,由16个外显子组成,其翻译的蛋白由1042个氨基酸构成。浸润性乳腺癌是女性中最常被诊断的癌症,早期诊断和精准治疗是提高生存率的关键。研究发现SIPA1蛋白与乳腺癌的发生、增殖、粘附、浸润、转移等密切相关,可以促进乳腺癌的发生、浸润、转移,同时也有报道发现SIPA1蛋白与结直肠癌、膀胱癌、前列腺癌、宫颈癌和黑色素瘤等癌症的转移浸润相关,可以调控前列腺癌的浸润和转移,还可以调节结直肠癌细胞的增殖和移动等。除了SIPA1蛋白与癌症特征相关外,研究者们还发现Sipa1基因上面的两个SNPs位点,即rs931127和rs3741378,与宫颈癌的转移、非小细胞肺癌和乳腺癌的存活和发病也是密切相关的。SIPA1蛋白功能的多样化是如何实现的呢?其调控机制的研究是比较少的,因此,本论文主要从Sipa1基因的结构和功能进行了探讨,结果如下:1)预测并构建了启动子区域的不同序列、采用双荧光素酶基因报告实验,定位了Sipa1基因的启动子位置,并且确定了Sipa1基因启动子的核心区域。2)通过点突变构建了Sipa1基因启动子区的SNP rs931127(GA)突变子,并采用双荧光素酶基因报告实验,发现该SNP没有显著影响Sipa1基因启动子的活性。3)通过点突变构建了Sipa1基因第三个外显子上的SNP rs3741378(CT)突变子(pcDNA3-flag-SIPA1 C和pcDNA3-flag-SIPA1 T),采用蛋白质免疫印迹实验证实该SNP,rs3741378(CT),可以引起SIPA1蛋白的表达下降,并且差异显著。4)通过测序,检测了不同肿瘤细胞系中Sipa1 gene SNP rs931127和rs374137,并研究了SIPA1蛋白在这些细胞中的SIPA1蛋白的细胞核亚定位,发现Sipa1基因的SNPs与SIPA1蛋白的细胞核亚定位没有显著的相关性。总结以上实验结果主要是成功定位了Sipa1基因的启动子位置及其核心区域,不仅成功的揭示了Sipa1基因的结构,而且也为以后进一步研究SIPA1蛋白的被调控机制和鉴定与Sipa1基因相互作用的转录因子奠定了基础;同时,Sipa1基因上面的两个SNPs功能鉴定为以后的临床诊断也提供了依据。
[Abstract]:SIPA1 protein (signaling-induced proliferating-associating protein 1), a signal inducible proliferation-associated protein 1, is a Rap1GAP (Rap1 GTPase activating protein) that catalyzes the transformation of Rap1 from an activated Rap1GTP to an inactive Rap1GDP. The function of Rap1 protein was regulated by cell adhesion, intracellular kinase activity and nuclear gene regulation. The Sipa1 gene was located on chromosome 11 and consisted of 16 exons, and the translated protein consisted of 1042 amino acids. Invasive breast cancer is the most commonly diagnosed cancer in women. Early diagnosis and accurate treatment are key to improving survival rate. The study found that SIPA1 protein is closely related to the occurrence, proliferation, adhesion, infiltration and metastasis of breast cancer, and can promote the occurrence, invasion and metastasis of breast cancer. There are also reports that SIPA1 protein is associated with colorectal cancer, bladder cancer, prostate cancer, etc. The metastasis and infiltration of cervical cancer and melanoma are related to the invasion and metastasis of prostate cancer, and can also regulate the proliferation and migration of colorectal cancer cells. In addition to the association between SIPA1 protein and cancer features, the researchers also found that two SNPs loci on the Sipa1 gene, rs931127 and rs3741378, were associated with cervical cancer metastasis. The survival and pathogenesis of non-small cell lung cancer and breast cancer are also closely related. How can the diversification of SIPA1 protein function be realized? Therefore, the structure and function of Sipa1 gene were discussed in this paper. The results are as follows: 1) the different sequences of promoter region were predicted and constructed, and the double luciferase gene report experiment was used. The promoter location of Sipa1 gene was located, and the core region of Sipa1 gene promoter was determined. 2) the SNP rs931127 (GA) mutants in the promoter region of Sipa1 gene were constructed by point mutation, and the double luciferase gene report experiment was used. It was found that the SNP did not significantly affect the activity of the Sipa1 promoter. 3) the SNP rs3741378 (CT) mutants (pcDNA3-flag-SIPA1 C and pcDNA3-flag-SIPA1 T),) on the third exon of the Sipa1 gene were constructed by point mutation. Western blot analysis showed that the SNP,rs3741378 (CT), could induce the decrease of SIPA1 protein expression, and the difference was significant. 4) Sipa1 gene SNP rs931127 and rs374137, in different tumor cell lines were detected by sequencing. The nuclear sublocalization of SIPA1 protein in these cells was studied. It was found that there was no significant correlation between SNPs of Sipa1 gene and nuclear sublocalization of SIPA1 protein. The main results of the above experiments are that the promoter position and core region of Sipa1 gene have been successfully located, and not only the structure of Sipa1 gene has been successfully revealed, but also the structure of Sipa1 gene has been successfully revealed. It also lays a foundation for further study on the regulatory mechanism of SIPA1 protein and identification of transcription factors interacting with Sipa1 gene. At the same time, the identification of the two SNPs functions above the Sipa1 gene also provides the basis for future clinical diagnosis.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q78
本文编号：2417818
[Abstract]:SIPA1 protein (signaling-induced proliferating-associating protein 1), a signal inducible proliferation-associated protein 1, is a Rap1GAP (Rap1 GTPase activating protein) that catalyzes the transformation of Rap1 from an activated Rap1GTP to an inactive Rap1GDP. The function of Rap1 protein was regulated by cell adhesion, intracellular kinase activity and nuclear gene regulation. The Sipa1 gene was located on chromosome 11 and consisted of 16 exons, and the translated protein consisted of 1042 amino acids. Invasive breast cancer is the most commonly diagnosed cancer in women. Early diagnosis and accurate treatment are key to improving survival rate. The study found that SIPA1 protein is closely related to the occurrence, proliferation, adhesion, infiltration and metastasis of breast cancer, and can promote the occurrence, invasion and metastasis of breast cancer. There are also reports that SIPA1 protein is associated with colorectal cancer, bladder cancer, prostate cancer, etc. The metastasis and infiltration of cervical cancer and melanoma are related to the invasion and metastasis of prostate cancer, and can also regulate the proliferation and migration of colorectal cancer cells. In addition to the association between SIPA1 protein and cancer features, the researchers also found that two SNPs loci on the Sipa1 gene, rs931127 and rs3741378, were associated with cervical cancer metastasis. The survival and pathogenesis of non-small cell lung cancer and breast cancer are also closely related. How can the diversification of SIPA1 protein function be realized? Therefore, the structure and function of Sipa1 gene were discussed in this paper. The results are as follows: 1) the different sequences of promoter region were predicted and constructed, and the double luciferase gene report experiment was used. The promoter location of Sipa1 gene was located, and the core region of Sipa1 gene promoter was determined. 2) the SNP rs931127 (GA) mutants in the promoter region of Sipa1 gene were constructed by point mutation, and the double luciferase gene report experiment was used. It was found that the SNP did not significantly affect the activity of the Sipa1 promoter. 3) the SNP rs3741378 (CT) mutants (pcDNA3-flag-SIPA1 C and pcDNA3-flag-SIPA1 T),) on the third exon of the Sipa1 gene were constructed by point mutation. Western blot analysis showed that the SNP,rs3741378 (CT), could induce the decrease of SIPA1 protein expression, and the difference was significant. 4) Sipa1 gene SNP rs931127 and rs374137, in different tumor cell lines were detected by sequencing. The nuclear sublocalization of SIPA1 protein in these cells was studied. It was found that there was no significant correlation between SNPs of Sipa1 gene and nuclear sublocalization of SIPA1 protein. The main results of the above experiments are that the promoter position and core region of Sipa1 gene have been successfully located, and not only the structure of Sipa1 gene has been successfully revealed, but also the structure of Sipa1 gene has been successfully revealed. It also lays a foundation for further study on the regulatory mechanism of SIPA1 protein and identification of transcription factors interacting with Sipa1 gene. At the same time, the identification of the two SNPs functions above the Sipa1 gene also provides the basis for future clinical diagnosis.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q78
【参考文献】
相关期刊论文 前3条
1 陈万青;郑荣寿;曾红梅;邹小农;张思维;赫捷;;2011年中国恶性肿瘤发病和死亡分析[J];中国肿瘤;2015年01期
2 杨晓娜;赵昶灵;李云;李会容;苏丽;周燕琼;;启动子序列克隆和功能分析方法的研究进展[J];云南农业大学学报(自然科学版);2010年02期
3 毋光明,徐怀逵;从西方释义学角度探究肿瘤定义[J];医学与哲学;1998年03期
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