Sipa1基因啟動子序列及其SNPs結(jié)構(gòu)和功能研究
發(fā)布時間：2019-01-29 20:07
【摘要】：SIPA1蛋白(signaling-induced proliferating-associating protein 1)即信號誘導(dǎo)增殖相關(guān)蛋白1,是一個Rap1GAP(Rap1 GTPase activating protein)蛋白,它可以催化Rap1蛋白從活化的Rap1GTP形式轉(zhuǎn)變?yōu)槭Щ頡ap1GDP形式,進(jìn)而實現(xiàn)對Rap1蛋白功能的調(diào)節(jié):細(xì)胞的粘附,細(xì)胞內(nèi)激酶的活性,細(xì)胞核內(nèi)基因的調(diào)控等。Sipa1基因位于第11號染色體上,由16個外顯子組成,其翻譯的蛋白由1042個氨基酸構(gòu)成。浸潤性乳腺癌是女性中最常被診斷的癌癥,早期診斷和精準(zhǔn)治療是提高生存率的關(guān)鍵。研究發(fā)現(xiàn)SIPA1蛋白與乳腺癌的發(fā)生、增殖、粘附、浸潤、轉(zhuǎn)移等密切相關(guān),可以促進(jìn)乳腺癌的發(fā)生、浸潤、轉(zhuǎn)移,同時也有報道發(fā)現(xiàn)SIPA1蛋白與結(jié)直腸癌、膀胱癌、前列腺癌、宮頸癌和黑色素瘤等癌癥的轉(zhuǎn)移浸潤相關(guān),可以調(diào)控前列腺癌的浸潤和轉(zhuǎn)移,還可以調(diào)節(jié)結(jié)直腸癌細(xì)胞的增殖和移動等。除了SIPA1蛋白與癌癥特征相關(guān)外,研究者們還發(fā)現(xiàn)Sipa1基因上面的兩個SNPs位點,即rs931127和rs3741378,與宮頸癌的轉(zhuǎn)移、非小細(xì)胞肺癌和乳腺癌的存活和發(fā)病也是密切相關(guān)的。SIPA1蛋白功能的多樣化是如何實現(xiàn)的呢?其調(diào)控機(jī)制的研究是比較少的,因此,本論文主要從Sipa1基因的結(jié)構(gòu)和功能進(jìn)行了探討,結(jié)果如下:1)預(yù)測并構(gòu)建了啟動子區(qū)域的不同序列、采用雙熒光素酶基因報告實驗,定位了Sipa1基因的啟動子位置,并且確定了Sipa1基因啟動子的核心區(qū)域。2)通過點突變構(gòu)建了Sipa1基因啟動子區(qū)的SNP rs931127(GA)突變子,并采用雙熒光素酶基因報告實驗,發(fā)現(xiàn)該SNP沒有顯著影響Sipa1基因啟動子的活性。3)通過點突變構(gòu)建了Sipa1基因第三個外顯子上的SNP rs3741378(CT)突變子(pcDNA3-flag-SIPA1 C和pcDNA3-flag-SIPA1 T),采用蛋白質(zhì)免疫印跡實驗證實該SNP,rs3741378(CT),可以引起SIPA1蛋白的表達(dá)下降,并且差異顯著。4)通過測序,檢測了不同腫瘤細(xì)胞系中Sipa1 gene SNP rs931127和rs374137,并研究了SIPA1蛋白在這些細(xì)胞中的SIPA1蛋白的細(xì)胞核亞定位,發(fā)現(xiàn)Sipa1基因的SNPs與SIPA1蛋白的細(xì)胞核亞定位沒有顯著的相關(guān)性�？偨Y(jié)以上實驗結(jié)果主要是成功定位了Sipa1基因的啟動子位置及其核心區(qū)域,不僅成功的揭示了Sipa1基因的結(jié)構(gòu),而且也為以后進(jìn)一步研究SIPA1蛋白的被調(diào)控機(jī)制和鑒定與Sipa1基因相互作用的轉(zhuǎn)錄因子奠定了基礎(chǔ);同時,Sipa1基因上面的兩個SNPs功能鑒定為以后的臨床診斷也提供了依據(jù)。
[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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：Q78
本文編號：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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：Q78
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，本文編號：2417818
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