重组质粒pcDNA3.1-Annexin A1对结直肠癌细胞SW480迁移和侵袭的影响
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摘要
膜联蛋白超家族由13个生物学上和结构上高度相似(40%-60%)的钙依赖的磷脂结合蛋白组成。膜联蛋白A1 (Annexin A1, ANXA1, lipocortinⅠ)是膜联蛋白超家族中第一个被发现的成员,参与膜转运及膜表面一系列依赖于钙调蛋白的活动,包括胞吞/胞吐作用、细胞分化和增殖、抑制花生四烯酸合成等多种重要的细胞生命活动。近年来的研究表明Annexin A1表达失调与肿瘤密切相关,并可能与肿瘤的侵袭转移相关,但机制不清。本实验旨在构建pcDN A3.1-Annexin A1真核表达载体,以转染低表达Annexin A1基因的结直肠癌细胞株SW480,使之上调该基因的表达,探讨Annexin A1对结直肠癌细胞迁移、侵袭能力的影响,为进一步研究Annexin A1在结直肠癌侵袭和转移等方面的角色增添基础。
第一部分人Annexin A1基因真核表达载体的构建
目的：克隆Annexin A1基因和构建Annexin A1真核表达质粒,为进一步的功能研究提供有利的工具。方法：提取正常人体胎盘组织总RNA, RT-PCR扩增人AnnexinA1基因编码区全长序列,通过DNA重组技术将该基因片段重组于pcDNA3.1真核表达载体上,构建pcDNA3.1-Annexin A1重组真核表达质粒,通过PCR扩增、酶切电泳分析及DNA测序的方法对重组DNA进行鉴定。结果：将Annexin A1基因克隆至真核表达载体pcDNA3.1 (+),酶切、PCR和测序鉴定结果表明,插入的序列与GenBank上的人的Annexin A1基因mRNA序列完全相符。结论：成功构建重组质粒pcDNA3.1-Annexin A1真核表达载体。
第二部分pcDN A3.1-Annexin A1转染SW480细胞及其生物学效应
目的：建立并鉴定稳定转染pcDNA3.1-Annexin A1的结直肠癌细胞株SW480,研究Annexin A1基因对结直肠癌细胞株SW480细胞迁移和侵袭能力的影响。方法：将Annexin A1基因的真核表达质粒pcDNA3.1-Annexin A1和空载体pcDNA3.1质粒应用脂质体介导的方法分别转染体外培养的结直肠癌细胞SW480,G418筛选稳定表达株,实时荧光定量PCR和Western blot方法检测Annexin A1基因的表达。以重组体pcDN A3.1-Annexin A1转染的SW480细胞为研究对象,以空载体转染组及未转染的SW480细胞作为对照,通过划痕修复实验、Transwell细胞侵袭实验对转染前后细胞的迁移和侵袭能力进行比较观察和分析。结果：pcDNA3.1-Annexin A1载体转染SW480细胞后,G418筛选获得稳定表达株；实时荧光定量PCR和Western blot检测结果显示,重组体pcDNA3.1-Annexin A1稳定转染细胞株中Annexin A1 mRNA水平和蛋白水平的表达明显高于空载体转染及未转染组细胞,而后两者间比较无统计学差异。对转染前后的细胞观察表明,重组体pcDNA3.1-Annexin A1转染组SW480细胞的迁移率(0.415±0.0022)明显高于空载体转染组(0.267±0.0025)及未转染组细胞(0.271±0.0021),差异具有统计学意义(P<0.05),而后两者间差异无统计学意义(P>0.05)；与空载体转染组(162.8±12.07)及未转染组(164.25±9.5)相比,重组体pcDNA3.1-Annexin A1转染组穿膜细胞数(221.75±12.07)显著增多,差异具有统计学意义(P<0.05).结论：成功构建稳定转染pcDNA3.1-Annexin A1的结直肠癌细胞系；Annexin A1基因表达上调能提高结直肠癌细胞迁移和侵袭能力。
The annexin superfamily consists of 13 calcium or calcium and phospholipid binding proteins with a significant degree of biological and structural homology (40-60%). Annexin A1 (ANXA1), the first characterized member of the annexin superfamily, is a member of multifunctional protein shown to regulate a variety of cellular processes in epithelial cells including endocytosis/exocytosis, proliferation, differentiation, as well as eicosanoid production. Recently, Annexin Al has been reported that it is associated with the development of metastasis in some invasive malignancies suggesting a role for this protein in regulating epithelial cell migration/invasion. However, mechanisms by which Annexin A1 regulates epithelial eel lmigration/invasion are not understood. In our study, we constructed the eukaryotic expression vector of pcDN A3.1-Annexin A1 and transfected it stably into the SW480 cell lines, to study the effects of Annexin A1 on migratory and invasive ability of colon cancer cell line, provides a fundamental tool to further study the role of Annexin A1 gene in invasion and metastasis of human colorectal carcinoma.
Part 1. Construction of eukaryotic expression vector of human Annexin A1 Gene
Objective:To construct the eukaryotic expression vector of pcDNA3.1-Annexin A1, provides a fundamental tool to further study the role of Annexin A1 gene in invasion and metastasis of human colorectal carcinoma.Methods:Fresh normal human placenta total RNA were extracted and the Annexin A1 gene was amplified by RT-PCR and construct the eukaryotic expression vector of pcDN A3.1-Annexin A1.Identification of the recombinant plasmid pcDNA3.1-Annexin A1 by PCR,restriction enzyme analysis and sequencing.
Results:Restriction enzyme analysis and sequencing result showed that the sequence of this gene fragment was identical with Annexin A1 gene sequence reported in GenBank.
Conclusion:The recombinant plasmid pcDNA3.1-Annexin Al was successfully constructed.
Part 2. The biological effects of recombinate vector in transfected colorectal carcinoma cell line SW480
Objective:pcDNA3.1-Annexin A1 was transfected into colorectal carcinoma cell line SW480,to observed and study the biological behaviors of SW480 before and after transfection in vitro.Methods:Two different plasmids including a recombinate pcDNA3.1-Annexin A1 and an empty pcDNA3.1 were reseparately transfered into colorectal carcinoma cell line SW480 cultured in vitro by liposome-mediated gene transferred method.After screening with G418,the stable transfectants were obtained.Realtime PCR and Western blot methods were used to analyze the expression of Annexin A1 mRNA and protein in SW480 cells before and after transfection to confirm whether the recombinant vector DNA integrated with the genomic DNA of SW480 cells. Wound-healing experiment and Transwell invasion assays were used in vitro to study the effects of Annexin A1 expression on the migratory movement and invasion of SW480 cells.Results:The results of real time PCR and Western blot showed that the Annexin A1 expression was higher in cells transfected with pcDNA3.1-Annexin A1 than in those un-transfected or empty vector-transfected cells and there is no significant difference between the latter two groups. Transfected with Annexin A1 eukaryotic expression vector could significantly increase migratory and invasive ability of SW480 cells compared with the untransfected cells.The migratory rate of SW480 cells in pcDNA3.1-Annexin A1 group was significantly higher than those in un-transfected cells or those transfected with empty vectors ([0.415±0.0022] vs [0.267±0.002] and [0.271±0.002], P<0.05), and there was no significant difference between the latter two groups.The migrating SW480 cells in pcDNA3.1-Annexin A1 group was significantly more than those in the other two groups ([221.75±12.07] vs [162.8±12.07] and [164.25±9.5], P<0.05).Conclusion:The eukaryotic vector was transfected successfully to SW480 cells with liposome, which integrated with SW480 cell genome and markedly enhanced the expression of SW480 mRNA and protein in SW480cells; overexpression of Annexin Al eukatyotic expression vector can incerease migratory and invasive ability of SW480 cell line.
第一部分人Annexin A1基因真核表达载体的构建
目的：克隆Annexin A1基因和构建Annexin A1真核表达质粒,为进一步的功能研究提供有利的工具。方法：提取正常人体胎盘组织总RNA, RT-PCR扩增人AnnexinA1基因编码区全长序列,通过DNA重组技术将该基因片段重组于pcDNA3.1真核表达载体上,构建pcDNA3.1-Annexin A1重组真核表达质粒,通过PCR扩增、酶切电泳分析及DNA测序的方法对重组DNA进行鉴定。结果：将Annexin A1基因克隆至真核表达载体pcDNA3.1 (+),酶切、PCR和测序鉴定结果表明,插入的序列与GenBank上的人的Annexin A1基因mRNA序列完全相符。结论：成功构建重组质粒pcDNA3.1-Annexin A1真核表达载体。
第二部分pcDN A3.1-Annexin A1转染SW480细胞及其生物学效应
目的：建立并鉴定稳定转染pcDNA3.1-Annexin A1的结直肠癌细胞株SW480,研究Annexin A1基因对结直肠癌细胞株SW480细胞迁移和侵袭能力的影响。方法：将Annexin A1基因的真核表达质粒pcDNA3.1-Annexin A1和空载体pcDNA3.1质粒应用脂质体介导的方法分别转染体外培养的结直肠癌细胞SW480,G418筛选稳定表达株,实时荧光定量PCR和Western blot方法检测Annexin A1基因的表达。以重组体pcDN A3.1-Annexin A1转染的SW480细胞为研究对象,以空载体转染组及未转染的SW480细胞作为对照,通过划痕修复实验、Transwell细胞侵袭实验对转染前后细胞的迁移和侵袭能力进行比较观察和分析。结果：pcDNA3.1-Annexin A1载体转染SW480细胞后,G418筛选获得稳定表达株；实时荧光定量PCR和Western blot检测结果显示,重组体pcDNA3.1-Annexin A1稳定转染细胞株中Annexin A1 mRNA水平和蛋白水平的表达明显高于空载体转染及未转染组细胞,而后两者间比较无统计学差异。对转染前后的细胞观察表明,重组体pcDNA3.1-Annexin A1转染组SW480细胞的迁移率(0.415±0.0022)明显高于空载体转染组(0.267±0.0025)及未转染组细胞(0.271±0.0021),差异具有统计学意义(P<0.05),而后两者间差异无统计学意义(P>0.05)；与空载体转染组(162.8±12.07)及未转染组(164.25±9.5)相比,重组体pcDNA3.1-Annexin A1转染组穿膜细胞数(221.75±12.07)显著增多,差异具有统计学意义(P<0.05).结论：成功构建稳定转染pcDNA3.1-Annexin A1的结直肠癌细胞系；Annexin A1基因表达上调能提高结直肠癌细胞迁移和侵袭能力。
The annexin superfamily consists of 13 calcium or calcium and phospholipid binding proteins with a significant degree of biological and structural homology (40-60%). Annexin A1 (ANXA1), the first characterized member of the annexin superfamily, is a member of multifunctional protein shown to regulate a variety of cellular processes in epithelial cells including endocytosis/exocytosis, proliferation, differentiation, as well as eicosanoid production. Recently, Annexin Al has been reported that it is associated with the development of metastasis in some invasive malignancies suggesting a role for this protein in regulating epithelial cell migration/invasion. However, mechanisms by which Annexin A1 regulates epithelial eel lmigration/invasion are not understood. In our study, we constructed the eukaryotic expression vector of pcDN A3.1-Annexin A1 and transfected it stably into the SW480 cell lines, to study the effects of Annexin A1 on migratory and invasive ability of colon cancer cell line, provides a fundamental tool to further study the role of Annexin A1 gene in invasion and metastasis of human colorectal carcinoma.
Part 1. Construction of eukaryotic expression vector of human Annexin A1 Gene
Objective:To construct the eukaryotic expression vector of pcDNA3.1-Annexin A1, provides a fundamental tool to further study the role of Annexin A1 gene in invasion and metastasis of human colorectal carcinoma.Methods:Fresh normal human placenta total RNA were extracted and the Annexin A1 gene was amplified by RT-PCR and construct the eukaryotic expression vector of pcDN A3.1-Annexin A1.Identification of the recombinant plasmid pcDNA3.1-Annexin A1 by PCR,restriction enzyme analysis and sequencing.
Results:Restriction enzyme analysis and sequencing result showed that the sequence of this gene fragment was identical with Annexin A1 gene sequence reported in GenBank.
Conclusion:The recombinant plasmid pcDNA3.1-Annexin Al was successfully constructed.
Part 2. The biological effects of recombinate vector in transfected colorectal carcinoma cell line SW480
Objective:pcDNA3.1-Annexin A1 was transfected into colorectal carcinoma cell line SW480,to observed and study the biological behaviors of SW480 before and after transfection in vitro.Methods:Two different plasmids including a recombinate pcDNA3.1-Annexin A1 and an empty pcDNA3.1 were reseparately transfered into colorectal carcinoma cell line SW480 cultured in vitro by liposome-mediated gene transferred method.After screening with G418,the stable transfectants were obtained.Realtime PCR and Western blot methods were used to analyze the expression of Annexin A1 mRNA and protein in SW480 cells before and after transfection to confirm whether the recombinant vector DNA integrated with the genomic DNA of SW480 cells. Wound-healing experiment and Transwell invasion assays were used in vitro to study the effects of Annexin A1 expression on the migratory movement and invasion of SW480 cells.Results:The results of real time PCR and Western blot showed that the Annexin A1 expression was higher in cells transfected with pcDNA3.1-Annexin A1 than in those un-transfected or empty vector-transfected cells and there is no significant difference between the latter two groups. Transfected with Annexin A1 eukaryotic expression vector could significantly increase migratory and invasive ability of SW480 cells compared with the untransfected cells.The migratory rate of SW480 cells in pcDNA3.1-Annexin A1 group was significantly higher than those in un-transfected cells or those transfected with empty vectors ([0.415±0.0022] vs [0.267±0.002] and [0.271±0.002], P<0.05), and there was no significant difference between the latter two groups.The migrating SW480 cells in pcDNA3.1-Annexin A1 group was significantly more than those in the other two groups ([221.75±12.07] vs [162.8±12.07] and [164.25±9.5], P<0.05).Conclusion:The eukaryotic vector was transfected successfully to SW480 cells with liposome, which integrated with SW480 cell genome and markedly enhanced the expression of SW480 mRNA and protein in SW480cells; overexpression of Annexin Al eukatyotic expression vector can incerease migratory and invasive ability of SW480 cell line.
引文
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[1]Sun M, Liu Y, Gibb W. Distribution of annexin I and II in term human fetal membranes,decidua and placenta[J]. Placenta,1996,17(2-3):181-184.
[2]Markoff A,Gerke V. Expression and functions of annexins in the kidney[J]. Am J Physiol Renal Physiol,2005,289:949-956.
[3]Gerke V, Creutz CE, Moss SE. Annexins:linking Ca2+ signalling to membrane dynamics[J]. Nat Rev Mol Cell Biol,2005,6:449-461.
[4]Lim LH, Pervaiz S Annexin 1:the new face of an old molecule[J].FASEB J,2007,21 (4):968-975.
[5]Kreunin P, Yoo C, Urquidi V, Goodison S.Proteomic profiling identifies breast tumor metastasis-associated factors in an isogenic model[J].Proteomics,2007,7(2):299-312.
[6]Yu QWang J,Chen Y, Wang X, Pan J,Li Q,et al.Tissue microarray analysis reveals strong clinical evidence for a close association between loss of annexin Al expression and nodal metastasis in gastric cancer[J].Clin Exp Metastasis,2008,25(7):695-702.
[7]Dai Z,Liu YK,Cui JF,Shen HL,Chen J,Sun RX,et al.Identification and analysis of altered alphal,6-fucosylated glycoproteins associated with hepatocellular carcinoma metastasis[J].Proteomics,2006,6 (21):5857-5867.
[8]Rondepierre F,Bouchon B,Papon J,Bonnet-Duquennoy M,Kintossou R,Moins N,et al. Proteomic studies of B16 lines:Involvement of Annexin A1 in melanoma dissemination[J].Biochim Biophysica Acta,2009,1794(1):61-69.
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[10]Wu W,Tang X, Hu W, Lotan R, Hong WK, Mao LIdentification and validation of metastasis-associated protein in head and neck cancer cell line by two-dimensional electrophoresis and mass spectrometry[J].Clin Exp Metastasis,2002,19(4):319-326.
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[1]Bai XF, Ni XG, Zhao P,Liu SM,Wang HX,Guo B,et al.Overexpression of annexin 1 in pancreatic cancer and its clinical significance[J]. World J Gastroenterol,2004,10(10):1466-1470.
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[8]Koike H,Uzawa K,Nakashima D,et al.Identification of differentially expressed proteins in oral squamous cell carcinoma using a global proteomic approach[J].Int J Oncol,2005,27(1):59-67.
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