APC蛋白不同功能区域真核表达载体的构建及其对人结直肠癌细胞中β-catenin表达的影响
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摘要
近年来的科学研究发现,超过90%的结直肠癌的发生与经典的Wnt/β-catenin信号通路的激活密切相关,该通路异常激活的标志是β-catenin的稳定和在细胞核内的积聚。在结直肠癌细胞中,Wnt/β-catenin信号的激活主要是因APC基因的突变所导致。超过70%的散发结直肠癌均存在APC基因突变。大多数的APC基因体细胞突变都发生在密码子1250至1500间,该区域的APC突变可导致15氨基酸重复序列和20氨基酸重复序列的部分缺失,使其失去正常调控β-catenin表达的功能。
多项研究表明,野生型的APC蛋白导入结直肠癌细胞内,可以降低β-catenin的表达水平。然而由于全长的野生型APC片段较大(大于10kb),直接用于基因治疗的操作性较困难。
目的:本研究旨在通过构建、验证含有APC蛋白不同功能区域的重组真核表达载体,研究其功能及其对结直肠癌细胞内β-catenin表达水平的影响,筛选到既可有效降低β-catenin表达水平同时片段长度较小的APC基因片段,为将来的基因治疗奠定一定的实验基础。
方法:1、根据APC基因的功能结构以及APC突变簇集区的特点,使用Primer premier 5.0软件设计7条引物,以含有APC全长cDNA的pBluescript质粒为模板,扩增APC基因特异性的功能区域片段。将扩增出的APC片段克隆到含有优化突变型绿色荧光蛋白的pEGFP-N3载体的N端,构建含有APC功能区域的重组真核表达载体。2、使用ORF查找器对重组质粒的测序结果进行分析,将返回的翻译蛋白序列在线protein BLAST,检测重组质粒表达APC功能区域和GFP能否融合表达。3、使用脂质体转染法将重组质粒转染结直肠癌细胞HCT 116和HT-29,通过观察细胞中绿色荧光蛋白的表达情况来验证APC功能区域在细胞内的表达。4、通过RT-PCR进一步验证重组质粒在HT-29中的表达及转染效率是否与插入片段的大小及细胞特性密切相关。5、利用Western blot技术检测重组载体对结直肠癌细胞中β-catenin表达的影响。以β-actin作内参,使用统计学软件SPSS13.0对western blot电泳条带的灰度值进行one-way ANOVA分析。
结果:扩增了5条APC基因特异性的功能区域片段。构建5个pEGFP-N3-APC结构区域的重组真核表达载体:pEGFP-N3-APC1(APC aa 6-767)、pEGFP-N3-APC2(APC aa 1020-1169)、pEGFP-N3-APC3(APC aa 1262-2033)、pEGFP-N3-APC4(APC aa1020-2033)、pEGFP-N3-APC5(APC aa 1020-1698)。ORF查找器对重组质粒的测序结果分析,重组质粒均可表达APC功能区域和GFP的融合蛋白,APC蛋白同源性为99%以上,GFP蛋白同源性为100%。通过观察细胞中绿色荧光蛋白的表达验证APC功能区域在细胞内的表达,结果显示:5个重组真核表达载体转染HCT 116细胞后,细胞中均可见绿色荧光蛋白的表达,荧光观察显示转染效率分别约为pEGFP-N3-APC1 50%、pEGFP-N3-APC2 50%、pEGFP-N3-APC3 30%、pEGFP-N3-APC4 30%、pEGFP-N3-APC5 50%,空载体pEGFP-N380%,证明重组载体构建成功,在细胞内均可表达相应的APC功能区域。RT-PCR进一步验证重组质粒在HT-29中的表达,RT-PCR结果显示,5个重组质粒在HCT 116和HT-29细胞中的表达基本一致。转染效率与插入片段的大小及细胞特性密切相关。Western blot技术检测重组载体对结直肠癌细胞中β-catenin表达,结果显示:在HCT116细胞中,条带灰度值间的差异无显著性(p>0.05),重组质粒对细胞内β-catenin的表达无明显影响。在HT-29细胞中,前四组条带(分别是未转染细胞、转染pEGFP-N3-APC1、转染pEGFP-N3-APC2和转染pEGFP-N3-APC3的细胞)灰度值间的差异无显著性(p>0.05),而后两组(转染pEGFP-N3-APC4和转染pEGFP-N3-APC5的细胞)的条带灰度值同前相比,差异有显著性(p<0.05)。重组质粒pEGFP-N3-APC4和pEGFP-N3-APC5转染入HT-29细胞后可明显降低细胞内β-catenin的表达。pEGFP-N3-APC4和pEGFP-N3-APC5相比较而言,pEGFP-N3-APC5的片段长度相对较小。
结论:本实验研究根据APC基因的功能结构以及APC突变簇集区的特点,使用Primer premier 5.0软件设计7条引物,以含有APC全长cDNA的pBluescript质粒为模板,扩增的5条APC基因特异性的功能区域片段中,经构建重组、ORF查找器对重组质粒的测序、转染、功能证实及Western blot技术检测重组载体对结直肠癌细胞中β-catenin表达的影响,证实5个重组载体构建成功,在细胞内均可表达相应的APC功能区域。APC功能区域在细胞内的成功表达,为进一步研究其在细胞内的功能提供了基础。在Western blot技术检测重组载体对结直肠癌细胞中β-catenin表达证实APC5基因片段是既可有效降低β-catenin的表达同时相对长度较短的最优片段。APC5基因片段在细胞内功能的进一步研究可为基因治疗奠定一定的分子理论与实验依据。
The remarkable thing about Colorectal cancer(CRC)-the most common cause of non-smoking-related cancer deaths in the world-is that the molecular mechanisms underlying virtually all of these cases are uniform.Recent studies on molecular carcinogenesis have highlighted that greater than 90%of all CRCs will have an activating mutation of the canonical Wnt/β-catenin signaling pathway,ultimately leading to the stabilization and accumulation ofβ-catenin in the hallmark of an active canonical Wnt pathway;the presence of nuclearβ-catenin is evident in even the smallest detectable lesions resulting from Wnt mutations.The consistency of mutations at some level of the Wnt signaling pathway in CRC makes this cancer an attractive model for molecular intervention.In CRC cells,Wnt/β-catenin signaling is activated mainly by mutations in the tumor suppressor gene APC.＞70%of sporadic CRCs contain APC gene mutations,which result in inefficientβ-catenin degradation and up-regulation ofβ-catenin-mediated transcription.The majority of somatic mutations in the APC gene occur within a segment called the mutation cluster region(codons 1,250-1,500) and almost all of the mutations result in truncated APC protein.APC mutations occurring in the mutation cluster region produce abnormal APC proteins that retain only the three 15-amino-acid repeats and the first one or two 20-amino-acid repeats while losing the other domains and failing to effectively down-regulateβ-catenin.
Recent research have highlighted that wild type APC could degradeβ-catenin level in colorectal cells,while using wild type APC for gene therapy is hard to obtain because the length of gene is longer than 10 kb, which couldn't be expressed stably in vivo and unwell to be used for gene therapy.
Objectives
The present study aimed to construct several recombinant eukaryotic expression vectors,containing various functional domains of APC protein, and investigated their functions and impact onβ-catenin level in human colorectal cancer cells.
Methods
1.APC gene fragments were amplified by PCR with pBluescript (involving full-length APC cDNA) as template and primers designed according to APC cDNA sequence and mutation cluster domain, containing BamH I and Xho I restriction site for directed cloning into pEGFP-N3 vector.2.The recombinant plasmids were identified by sequencing and analyzed by ORF finder and protein BLAST.3.The recombinant plasmids were transfected into colorectal cancer cells HCT 116 and HT-29 mediated by lipofectamine~(TM) 2000.Green fluorescence was observed via fluorescence microscope to investigate the expression of recombinant plasmids in cells.4.RT-PCR was employed to validate the expression of recombinant vectors in HT-29 cells.5.Western blot technique was applied to detect the influence onβ-catenin expression level of recombinant plasmids,gray scales of electrophoresis strips were analyzed by SPSS 13.0 via one-way ANOVA.
Results
1.Five recombinant expression vectors were constructed,named pEGFP-N3-APC1(including APC peptide from aa 6 to aa 767), pEGFP-N3-APC2(aa 1020-1169),pEGFP-N3-APC3(aa 1262-2033), pEGFP-N3-APC4(aa 1020-2033),pEGFP-N3-APC5(aa 1020-1698).
2.Bioinformatics analysis showed that the recombinant plasmid could express a fusion protein involving APC protein domains and GFP.
3.The stably transfected colorectal cancer cells were selected by neomycin resistance using 300μg/ml G418.Optimizing transfection parameters were determined by experiments.Results illustrated the five recombinant vectors could all be expressed in HCT 116 cells,green fluorescence detection showed transfection efficiency of pEGFP-N3-APC 1 was 50%,pEGFP-N3-APC2's was 50%, pEGFP-N3-APC3's was 30%,pEGFP-N3-APC4's was 30%, pEGFP-N3-APC5's was 50%,pEGFP-N3's was 80%,respectively, which certificated successful construction of recombinant vectors. Nevertheless,fluorescence intensity of GFP might be influenced by multiple factors such as property of cell itself,green fluorescence was hard to be observed in HT-29 cells.
4.Results of RT-PCR made clear that vectors constructed could be expressed in HT-29 cells,transfection efficiency in HT-29 was no less than that in HCT-116.
5.Results of Western blot showed,in HCT 116 cells,discrepancy of gray scales of electrophoresis strips wasn't significant(p＞0.05),that was to say,β-catenin level wasn't effected a change when recombinant plasmids were transfected;in HT-29 cells,the situation wasn't so, discrepancy of gray scales of electrophoresis strips wasn't significant between the anterior four ones,which were normal cells,cells transfected with pEGFP-N3-APC1,cells transfected with pEGFP-N3-APC1,cells transfected with pEGFP-N3-APC2 and cells transfected with pEGFP-N3-APC3,respectively,discrepancy of gray scales of electrophoresis strips was significant between the posterior two ones(cells transfected with pEGFP-N3-APC4 and cells transfected with pEGFP-N3-APC5) and the anterior four ones,that was to say, recombinant plasmid pEGFP-N3-APC4 and pEGFP-N3-APC5 could degradeβ-catenin level obviously.As for pEGFP-N3-APC4 and pEGFP-N3-APC5,the length of pEGFP-N3-APC5 was shorter than that of pEGFP-N3-APC4.
Conclusions
1.Five recombinant pEGFP-N3-APC vectors carrying various APC functional domains were succefully constructed
2.The recombinant plasmids were identified by sequencing and analyzed by ORF finder and protein BLAST,transfected into colorectal cancer cells HCT 116 and HT-29 mediated by lipofectamine~(TM) 2000.Detection of green fluorescence and RT-PCR illustrated the successful expression of recombinant plasmid in human colorectal cancer cells.
3.Recombinant plasmid pEGFP-N3-APC4 and pEGFP-N3-APC5 could degradeβ-catenin level obviously in HT-29 cell lines.APC fragment No.5 was just the target gene we need,which could degradeβ-catenin level and possess short length relatively.
多项研究表明,野生型的APC蛋白导入结直肠癌细胞内,可以降低β-catenin的表达水平。然而由于全长的野生型APC片段较大(大于10kb),直接用于基因治疗的操作性较困难。
目的:本研究旨在通过构建、验证含有APC蛋白不同功能区域的重组真核表达载体,研究其功能及其对结直肠癌细胞内β-catenin表达水平的影响,筛选到既可有效降低β-catenin表达水平同时片段长度较小的APC基因片段,为将来的基因治疗奠定一定的实验基础。
方法:1、根据APC基因的功能结构以及APC突变簇集区的特点,使用Primer premier 5.0软件设计7条引物,以含有APC全长cDNA的pBluescript质粒为模板,扩增APC基因特异性的功能区域片段。将扩增出的APC片段克隆到含有优化突变型绿色荧光蛋白的pEGFP-N3载体的N端,构建含有APC功能区域的重组真核表达载体。2、使用ORF查找器对重组质粒的测序结果进行分析,将返回的翻译蛋白序列在线protein BLAST,检测重组质粒表达APC功能区域和GFP能否融合表达。3、使用脂质体转染法将重组质粒转染结直肠癌细胞HCT 116和HT-29,通过观察细胞中绿色荧光蛋白的表达情况来验证APC功能区域在细胞内的表达。4、通过RT-PCR进一步验证重组质粒在HT-29中的表达及转染效率是否与插入片段的大小及细胞特性密切相关。5、利用Western blot技术检测重组载体对结直肠癌细胞中β-catenin表达的影响。以β-actin作内参,使用统计学软件SPSS13.0对western blot电泳条带的灰度值进行one-way ANOVA分析。
结果:扩增了5条APC基因特异性的功能区域片段。构建5个pEGFP-N3-APC结构区域的重组真核表达载体:pEGFP-N3-APC1(APC aa 6-767)、pEGFP-N3-APC2(APC aa 1020-1169)、pEGFP-N3-APC3(APC aa 1262-2033)、pEGFP-N3-APC4(APC aa1020-2033)、pEGFP-N3-APC5(APC aa 1020-1698)。ORF查找器对重组质粒的测序结果分析,重组质粒均可表达APC功能区域和GFP的融合蛋白,APC蛋白同源性为99%以上,GFP蛋白同源性为100%。通过观察细胞中绿色荧光蛋白的表达验证APC功能区域在细胞内的表达,结果显示:5个重组真核表达载体转染HCT 116细胞后,细胞中均可见绿色荧光蛋白的表达,荧光观察显示转染效率分别约为pEGFP-N3-APC1 50%、pEGFP-N3-APC2 50%、pEGFP-N3-APC3 30%、pEGFP-N3-APC4 30%、pEGFP-N3-APC5 50%,空载体pEGFP-N380%,证明重组载体构建成功,在细胞内均可表达相应的APC功能区域。RT-PCR进一步验证重组质粒在HT-29中的表达,RT-PCR结果显示,5个重组质粒在HCT 116和HT-29细胞中的表达基本一致。转染效率与插入片段的大小及细胞特性密切相关。Western blot技术检测重组载体对结直肠癌细胞中β-catenin表达,结果显示:在HCT116细胞中,条带灰度值间的差异无显著性(p>0.05),重组质粒对细胞内β-catenin的表达无明显影响。在HT-29细胞中,前四组条带(分别是未转染细胞、转染pEGFP-N3-APC1、转染pEGFP-N3-APC2和转染pEGFP-N3-APC3的细胞)灰度值间的差异无显著性(p>0.05),而后两组(转染pEGFP-N3-APC4和转染pEGFP-N3-APC5的细胞)的条带灰度值同前相比,差异有显著性(p<0.05)。重组质粒pEGFP-N3-APC4和pEGFP-N3-APC5转染入HT-29细胞后可明显降低细胞内β-catenin的表达。pEGFP-N3-APC4和pEGFP-N3-APC5相比较而言,pEGFP-N3-APC5的片段长度相对较小。
结论:本实验研究根据APC基因的功能结构以及APC突变簇集区的特点,使用Primer premier 5.0软件设计7条引物,以含有APC全长cDNA的pBluescript质粒为模板,扩增的5条APC基因特异性的功能区域片段中,经构建重组、ORF查找器对重组质粒的测序、转染、功能证实及Western blot技术检测重组载体对结直肠癌细胞中β-catenin表达的影响,证实5个重组载体构建成功,在细胞内均可表达相应的APC功能区域。APC功能区域在细胞内的成功表达,为进一步研究其在细胞内的功能提供了基础。在Western blot技术检测重组载体对结直肠癌细胞中β-catenin表达证实APC5基因片段是既可有效降低β-catenin的表达同时相对长度较短的最优片段。APC5基因片段在细胞内功能的进一步研究可为基因治疗奠定一定的分子理论与实验依据。
The remarkable thing about Colorectal cancer(CRC)-the most common cause of non-smoking-related cancer deaths in the world-is that the molecular mechanisms underlying virtually all of these cases are uniform.Recent studies on molecular carcinogenesis have highlighted that greater than 90%of all CRCs will have an activating mutation of the canonical Wnt/β-catenin signaling pathway,ultimately leading to the stabilization and accumulation ofβ-catenin in the hallmark of an active canonical Wnt pathway;the presence of nuclearβ-catenin is evident in even the smallest detectable lesions resulting from Wnt mutations.The consistency of mutations at some level of the Wnt signaling pathway in CRC makes this cancer an attractive model for molecular intervention.In CRC cells,Wnt/β-catenin signaling is activated mainly by mutations in the tumor suppressor gene APC.＞70%of sporadic CRCs contain APC gene mutations,which result in inefficientβ-catenin degradation and up-regulation ofβ-catenin-mediated transcription.The majority of somatic mutations in the APC gene occur within a segment called the mutation cluster region(codons 1,250-1,500) and almost all of the mutations result in truncated APC protein.APC mutations occurring in the mutation cluster region produce abnormal APC proteins that retain only the three 15-amino-acid repeats and the first one or two 20-amino-acid repeats while losing the other domains and failing to effectively down-regulateβ-catenin.
Recent research have highlighted that wild type APC could degradeβ-catenin level in colorectal cells,while using wild type APC for gene therapy is hard to obtain because the length of gene is longer than 10 kb, which couldn't be expressed stably in vivo and unwell to be used for gene therapy.
Objectives
The present study aimed to construct several recombinant eukaryotic expression vectors,containing various functional domains of APC protein, and investigated their functions and impact onβ-catenin level in human colorectal cancer cells.
Methods
1.APC gene fragments were amplified by PCR with pBluescript (involving full-length APC cDNA) as template and primers designed according to APC cDNA sequence and mutation cluster domain, containing BamH I and Xho I restriction site for directed cloning into pEGFP-N3 vector.2.The recombinant plasmids were identified by sequencing and analyzed by ORF finder and protein BLAST.3.The recombinant plasmids were transfected into colorectal cancer cells HCT 116 and HT-29 mediated by lipofectamine~(TM) 2000.Green fluorescence was observed via fluorescence microscope to investigate the expression of recombinant plasmids in cells.4.RT-PCR was employed to validate the expression of recombinant vectors in HT-29 cells.5.Western blot technique was applied to detect the influence onβ-catenin expression level of recombinant plasmids,gray scales of electrophoresis strips were analyzed by SPSS 13.0 via one-way ANOVA.
Results
1.Five recombinant expression vectors were constructed,named pEGFP-N3-APC1(including APC peptide from aa 6 to aa 767), pEGFP-N3-APC2(aa 1020-1169),pEGFP-N3-APC3(aa 1262-2033), pEGFP-N3-APC4(aa 1020-2033),pEGFP-N3-APC5(aa 1020-1698).
2.Bioinformatics analysis showed that the recombinant plasmid could express a fusion protein involving APC protein domains and GFP.
3.The stably transfected colorectal cancer cells were selected by neomycin resistance using 300μg/ml G418.Optimizing transfection parameters were determined by experiments.Results illustrated the five recombinant vectors could all be expressed in HCT 116 cells,green fluorescence detection showed transfection efficiency of pEGFP-N3-APC 1 was 50%,pEGFP-N3-APC2's was 50%, pEGFP-N3-APC3's was 30%,pEGFP-N3-APC4's was 30%, pEGFP-N3-APC5's was 50%,pEGFP-N3's was 80%,respectively, which certificated successful construction of recombinant vectors. Nevertheless,fluorescence intensity of GFP might be influenced by multiple factors such as property of cell itself,green fluorescence was hard to be observed in HT-29 cells.
4.Results of RT-PCR made clear that vectors constructed could be expressed in HT-29 cells,transfection efficiency in HT-29 was no less than that in HCT-116.
5.Results of Western blot showed,in HCT 116 cells,discrepancy of gray scales of electrophoresis strips wasn't significant(p＞0.05),that was to say,β-catenin level wasn't effected a change when recombinant plasmids were transfected;in HT-29 cells,the situation wasn't so, discrepancy of gray scales of electrophoresis strips wasn't significant between the anterior four ones,which were normal cells,cells transfected with pEGFP-N3-APC1,cells transfected with pEGFP-N3-APC1,cells transfected with pEGFP-N3-APC2 and cells transfected with pEGFP-N3-APC3,respectively,discrepancy of gray scales of electrophoresis strips was significant between the posterior two ones(cells transfected with pEGFP-N3-APC4 and cells transfected with pEGFP-N3-APC5) and the anterior four ones,that was to say, recombinant plasmid pEGFP-N3-APC4 and pEGFP-N3-APC5 could degradeβ-catenin level obviously.As for pEGFP-N3-APC4 and pEGFP-N3-APC5,the length of pEGFP-N3-APC5 was shorter than that of pEGFP-N3-APC4.
Conclusions
1.Five recombinant pEGFP-N3-APC vectors carrying various APC functional domains were succefully constructed
2.The recombinant plasmids were identified by sequencing and analyzed by ORF finder and protein BLAST,transfected into colorectal cancer cells HCT 116 and HT-29 mediated by lipofectamine~(TM) 2000.Detection of green fluorescence and RT-PCR illustrated the successful expression of recombinant plasmid in human colorectal cancer cells.
3.Recombinant plasmid pEGFP-N3-APC4 and pEGFP-N3-APC5 could degradeβ-catenin level obviously in HT-29 cell lines.APC fragment No.5 was just the target gene we need,which could degradeβ-catenin level and possess short length relatively.
引文
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