CDK2AP1对肺癌A549细胞影响的实验研究
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摘要
肺癌已经成为全球癌症死亡的主要原因,是目前对人类健康和生命威胁最大的恶性肿瘤之一。在20世纪初期肺癌还是比较少见的疾病。可是到了20世纪末,据统计肺癌已经成为因恶性肿瘤原因而死亡之首根据世界卫生组织(WHO)定期公布的资料显示,尤其是在工业发达的国家,肺癌的发病率和死亡率在世界都呈明显的上升趋势。已有28个发达国家公布的调查资料显示肺癌已经成为恶性肿瘤最常见的死亡原因。2005年统计资料显示,世界上每年有1,350,000肺癌新发病例,而有约1,180,000肺癌死亡病例。也就是说世界上平均不到30秒就有一人死于肺癌。预计到2020年,全世界癌症发病率将比现在增加50%,全球每年新增癌症患者将达到1500万例,死亡1000万例。在许多发达国家肺癌是最常见的恶性肿瘤之一,在男性常见恶性肿瘤中居第一位,位于女性常见恶性肿瘤的第二位。许多国家的研究人员对不同人种,不同地域,不同职业等人群进行大规模的流行病学调查,获得许多宝贵资料,为我们进一步了解肺癌的发生和发展奠定了基础。
我国近年来城乡前10位恶性肿瘤构成来看,肺癌已代替肝癌成为我国首位恶性肿瘤死亡原因,占全部恶性肿瘤死亡的22.7%。且发病率和死亡率仍在继续迅速上升。根据卫生部全国肿瘤防治办公室提供的资料显示,自2000～2005年间,中国肺癌的发病人数估计增加12万人,其中,男性肺癌病人从2000年的26万人增加到2005年的33万人,同期女性肺癌患者从12万人增加到17万人。目前我国肺癌发病率每年增长26.9%,如不及时采取有效控制措施,预计到2025年,我国肺癌病人将达到100万,成为世界第一肺癌大国。
传统的治疗方法并没有使肺癌的生存率有大幅度的提升。随着人们对基因治疗认识的逐渐深入,抑癌基因的肺癌治疗,为人们打开了一个新的思路。在细胞周期中存在着各种周期蛋白和周期蛋白依赖激酶,它们互相作用,并有一套复杂的机制调控着细胞的运转。CDK2AP1是Todd等于1995年利用仓鼠口腔癌模型分离和证实的一个候选抑癌基因,能在正常的仓鼠所有组织体内表达,在所有正常人体组织中也是高表达的,是从正常角质化细胞中分离出来的生长抑制因子。它是一种细胞生长抑制因子,被定位在人染色体12q24,分子量为12.4Kda,该基因的cDNA全长为1.6kbp,编码115个氨基酸,具有4个外显子,三个连接位点分别在cDNA序列(GenBankAF006468)的第577和588；675和676；802和803,较小的编码外显子区为第55至127bp。该基因在癌细胞中异常的表达,这也许可能会给我们对肺癌的发生发展起着一些提示。
CDK2AP1是一种对细胞周期蛋白依赖的CDK2活化产生负调节作用的生长抑制因子,仅仅表达于正常组织,未发现肿瘤组织或者变化的细胞株表达。其细胞周期负性调节与CDK2相关。有人通过体外实验筛选了CDK家族的7个成员,证明了CDK2AP1与CDK2的紧密联系,认为CDK2AP1与CDK2的分子结合。而经基因突变实验分析证实该蛋白通过第109-111位氨基酸与CDK2非磷酸化单体形式(p34)发生相互作用。而CDK2可具有非活性形式p34和活性形式p33两种单体形式,p12蛋白的表达可降低p33的水平而提高p34的水平,即降低了活性CDK2的水平,从而降低其介导的生物活性。具体地说,其异位表达导致细胞CDK2减少和CDK2相关激酶活性降低,并伴随细胞周期中CDK2AP1转染子位置的移动(G1 and S)。细胞DNA pol-a:primase (DNA polymerase alpha/primase)通过磷酸化和去磷酸化调节其活性,它的磷酸化形式可以激活DNA的复制,cyclinsA和cyclinsE与CDK2结合的复合物是DNA pol-a:primase的亚单位p180、p70磷酸化的关键酶。由于CDK2AP1可以降低CDK2表达水平和活性,使DNA pol-a:primase的活性降低。Mats等证实：CDK2AP1对CDK2介导的DNA pol-a:primase的磷酸化抑制具有特异性,CDK2AP1与DNA pol-a:primase结合位点可能位于其N端的六个氨基酸提示两者间可能还有直接的作用。CDK2AP1与DNA pol-a:primase的作用机理既有可能通过直接抑制DNA聚合酶的活性也有可能通过CDK2介导来完成。
氨基酸109-111在这个突变体中的突变,消除了它和CDK2的结合,使CDK2AP1和CDK2之间的关联增加了非活化形式的CDK2。这种关联可能早于cyclin E和A作用于CDK2活化的生化通路。CDK2AP1和CDK2结合,并抑制DNA的α螺旋酶,调节DNA的复制。有实验表明,CDK2AP1在293细胞中的异位表达是通过蛋白酶体依赖的蛋白水解,导致了CDK2的下降。这两种机制(隔绝p34CDK2和标靶p34CDK2的水解),可能单独或同时发生作用,以降低p34CDK2的细胞水平和相关的CDK2介导的生物活性。CDK2AP1可通过调节DNA合成期促使细胞周期停滞并可能导致细胞编程性凋亡。
对CDK2AP1的研究,在国际学术界已经成为一种热门话题。现在已有对该基因与头颈部癌,食管癌,胃癌,结肠癌,前列腺癌的相关报到。但是,直到现在仍然没有任何关于人类肺癌中CDK2AP1功能研究的报道。本实验中,我们构建了CDK2AP1的慢病毒载体,将其转染到肺癌A549细胞中,通过细胞功能体外实验及裸鼠肿瘤移植实验,来研究该基因对肺癌细胞的在体内及体外的作用。
目的：
构建CDK2AP1慢病毒过表达载体和siRNA-CDK2AP1慢病毒载体,分别对照的进行人肺癌细胞的体内、外功能影响的实验,以来明确基因CDK2AP1对肺癌细胞作用的影响。
实验方法：
(1)慢病毒表达载体的构建：根据genebank中CDK2AP1基因mRNA的已知序列,确定合适的靶位点,合成编码siRNA的DNA模板,连接退火的siRNA模板寡核苷酸到线性化的pGCSIL-GFP表达载体,构建siRNA-CDK2AP1的慢病毒表达载体,经测序及酶切鉴定；
(2)CDK2AP1过表达载体的构建：经逆转录,钓取目的基因,PCR扩增功能基因,酶切后与线性化的真核过表达载体连接,构建目的过表达载体,经测序及酶切鉴定；
(3) Western blot外源筛选靶点：siRNA-CDK2AP1慢病毒表达载体与CDK2AP1过表达载体共转染293T细胞,Western blot外源筛选靶点。
(4)慢病毒载体的包装及滴度测定及检测目的基因的转录及蛋白表达情况：将最佳靶点的慢病毒表达载体与pHelper 1.0载体和pHelper 2.0载体,三种质粒载体在293T细胞内包装,并标定病毒滴度。将CDK2AP1慢病毒过表达载体及si-CDK2AP1载体转染人肺癌A549细胞株,应用Real-time PCR和Western blot分别检测目的基因的转录水平及蛋白表达；
(5)肺癌细胞体外功能实验：分别应用流式细胞术检测细胞周期；应用MTT比色法、BrdU方法,transwell实验、细胞克隆形成实验,观察CDK2AP1对细胞周期、增殖、侵袭能力及细胞凋亡,并作出比较。
(6)体内实验：裸鼠移植人肺癌模型,分别将siRNA-CDK2AP1慢病毒表达载体及CDK2AP1过表达载体转染的人肺癌A549细胞株,细胞培养后移植在裸鼠皮下；测量肿瘤组织体积及重量,绘制成瘤曲线,比较说明CDK2AP1对肺癌细胞的抑制作用。
结果：
(1)经测序及酶切鉴定,成功构建了siRNA-CDK2AP1慢病毒表达载体。
(2)经测序及酶切鉴定,成功构建了CDK2AP1过表达载体。
(3) (1)与(2)两种载体共转染293T细胞,荧光表达率大于90%；Western blot外源筛选,Target 1#对目的基因的表达有较为显著的敲减作用,因而是最佳靶点。
(4)通过CDK2AP1过表达及目的基因去除反向的对比,细胞功能体外实验说明CDK2AP1过表达载体对人肺癌细胞具有抑制增殖、侵袭及促进凋亡的作用。流式细胞术、MTT实验、BrdU实验、transwell实验、细胞克隆形成实验检测显示：CDK2AP1能抑制细胞增殖、侵袭,具有统计学差异。
(5)成功建立裸鼠移植人肺癌模型,通过对肿瘤体积及重量的测量,绘制成瘤曲线,证明CDK2AP1对肺癌细胞的生长具有明显的抑制。
结论：
(1)成功构建了CDK2AP1过表达载体
(2)应用CDK2AP1过表达载体干扰人肺癌A549细胞,在体外能高效地抑制肺癌细胞的增殖、侵袭。
(3)通过裸鼠移植瘤模型说明CDK2AP1基因能够在体内起到抑制人肺癌A549细胞产生抑制作用。
Lung cancer is a malignant tumor which seriously threat human health in these years. The morbidity of lung cancer, one of the most common primary malignant carcinomas in the world, has increased annually. There is a prevalence of more than 1.5 million cases worldwide per year, with high incidence especially in China.The aging population, the progression of urban industrialization as well as the contamination and destruction of human life environment result in the rising incidence. Furthermore, to date, the therapeutic efficacy for this malignancy has not improved due to current high mortality and poor long-term survival rates.
The developing knowledge of molecular biology of tumor progression has revealed changes of gene expression during lung cancer progression. And the elucidation of molecular mechanism under carcinogenesis would be of great importance in the prevention and treatment of cancers. CDK2AP1 (cyclin-dependent kinase 2-associated protein 1), also known as DOC1, ST19, DORC1, doc-1, or p12DOC-1, was first isolated from normal keratinocytes as growth inhibitory factor by Todd et al in 1995. Located on human chromosome 12q24.31, the full length of CDK2AP1 gene is 1.6 kb, and its encoded protein is 12.4 KD(115 amino acids). CDK2AP1 interacted with DNA polymerase a, regulating the phosphorylation of the large subunit p180 and negatively modulating DNA replication in S phase of cell cycle.CDK2AP1 could also arrest cell cycle at DNA synthesis phase, leading to programmed cell apoptosis. Some study confirmed that CDK2AP1 played an important role in TGF-β1-modulated growth inhibitory system. Recently, it is revealed that the defective expression of CDK2AP1 was accompanied by the abnormal TGF-β-smad pathway, which ultimately resulted in the resistance of human oral squamous cell carcinoma cells to TGF-β-induced growth inhibitory effect. CDK2AP1 could regulate the activity of CDK2, the phosphorylation of pRB and p180, and TGF-β-Smad signaling pathway so as to play its inhibitory effect on cell growth.
Cyclin-dependent kinase 2-associated protein 1 (CDK2AP1), a cell growth inhibitory factor, is abnormally expressed in cancer cells, and might be implicated in the development of lung cancer. However, to date, no study on the function of CDK2AP1 in human lung cancer has ever been reported. In this study, overexpressing and lentiviral vectors containing CDK2AP1 cDNA and CDK2AP1 shRNA (short hairpin RNA) were constructed, and up-regulated and down-regulated CDK2AP1 expression in human lung cancer A549 cells was found, and explored the function of CDK2AP1 in lung cancer cell's development and progression.
Study objectives:
(1) CDK2AP1 siRNA design and pGCSIL-GFP-CDK2AP1 shRNAs construction.
(2) Construction of overexpressing vector pGC-FU-CDK2AP1 cDNA.
(3) To investigate the effect of CDK2AP1 on the growth in the cells in vitro and in vivo.
Methods:
(1) The human CDK2AP1 cDNA sequence was searched for suitable siRNA target sequences, and three CDK2AP1 siRNA sequences were designed by the Ambion siRNA Target Finder. DNA oligos containing the target sequences were chemically synthesized, annealed, and inserted into the expression vector pGCSIL-GFP by double digestion and ligation in accordance with the manufacturer's guidelines. The ligation product was then transformed into competent Escherichia coli DH5acells.As a control for CDK2AP1 siRNA, a corresponding random siRNA sequence was used. The targeting sequence of the short hairpin RNA (shRNA) was then confirmed by sequencing.
(2) CDK2AP1 cDNA was obtained the CDK2AP1-mRNA of lung tissue,by the RT-PCR assay, and then cloned into pGC-FU by double digestion, and in accordance with the manufacturer's guidelines. The ligation product was transformed into competent Escherichia coli DH5a cells. The recombinant was identified by PCR and confirmed by sequencing.
(3) The selection of the most effective CDK2AP1 shRNA with the Western blot assay.
(4) Lentiviral particle production with the vectors pHelper 1.0 and pHelper 2.0, and the detection of knockdown efficiency of CDK2AP1 shRNA with TR-PCR and Western blot assay.
(5) In vitro studies:The Lewis lung cancer cell lines were infected by the both plasmid vectors. The cycle phase distribution of the cells were analyzed with the flow cytometry. The effective function of CDK2AP1 in the infected the lung cancer cells was revealed with the MTT assay, the BrdU assay, the Cell Invasion Assay Kit (The transwell assay) and the Colony formation.
(6)In vivo studies:we builded a BALB/c nude mouse tumor xenograft model. The mice were transplanted with the both infected(CDK2AP1 and si-CDK2AP1) Lewis lung cancer cells into the right flank. Then, we got the results of the volume and weight of the tumors.
Statistical analysis
Comparison between groups was performed with the Student's t test and X2analysis using a SigmaStat statistical software package(SPSS, Chicago,IL). P<0.05 was taken as showing significance.
Results:
(1)The plasmids which were designed for expression of CDK2AP1 siRNA was constructed, and confirmed by restriction enzyme digest and sequence analysis.
(2) The plasmids were designed for over-expression of CDK2AP1 was constructed, and confirmed by restriction enzyme digest and sequence analysis.
(3) The both vectors were transfecting into the 293T cells. The most effective CDK2AP1 shRNA, the target 1, was founded with the westen blot assay.
(4) The study in vitro:The cell cycle phase distribution analyzed with a flow cytometry,the MTT assay, the BrdU assay, the Cell Invasion Assay Kit (The transwell) and the Colony formation showed that the over-expression of CDK2AP1 vector can inhibit the proliferation, invasion, and promote apoptosis of human lung cancer A549 cells.
(5) The study in vivo:The BALB/c nude mouse tumor xenograft model was builded. The results of the volume and weight of the the Lewis lung cance cells revealed that CDK2AP1 inhibited the growth of the cells.
Conclusions
1.The vectors of Lentivirus-mediated RNA interference and over-expression of CDK2AP1 were constructed successfully.
2.The study revealed that CDK2AP1 could inhibit the proliferation, invasion, and promote apoptosis of human lung cancer A549 cells in both vitro and vivo.
我国近年来城乡前10位恶性肿瘤构成来看,肺癌已代替肝癌成为我国首位恶性肿瘤死亡原因,占全部恶性肿瘤死亡的22.7%。且发病率和死亡率仍在继续迅速上升。根据卫生部全国肿瘤防治办公室提供的资料显示,自2000～2005年间,中国肺癌的发病人数估计增加12万人,其中,男性肺癌病人从2000年的26万人增加到2005年的33万人,同期女性肺癌患者从12万人增加到17万人。目前我国肺癌发病率每年增长26.9%,如不及时采取有效控制措施,预计到2025年,我国肺癌病人将达到100万,成为世界第一肺癌大国。
传统的治疗方法并没有使肺癌的生存率有大幅度的提升。随着人们对基因治疗认识的逐渐深入,抑癌基因的肺癌治疗,为人们打开了一个新的思路。在细胞周期中存在着各种周期蛋白和周期蛋白依赖激酶,它们互相作用,并有一套复杂的机制调控着细胞的运转。CDK2AP1是Todd等于1995年利用仓鼠口腔癌模型分离和证实的一个候选抑癌基因,能在正常的仓鼠所有组织体内表达,在所有正常人体组织中也是高表达的,是从正常角质化细胞中分离出来的生长抑制因子。它是一种细胞生长抑制因子,被定位在人染色体12q24,分子量为12.4Kda,该基因的cDNA全长为1.6kbp,编码115个氨基酸,具有4个外显子,三个连接位点分别在cDNA序列(GenBankAF006468)的第577和588；675和676；802和803,较小的编码外显子区为第55至127bp。该基因在癌细胞中异常的表达,这也许可能会给我们对肺癌的发生发展起着一些提示。
CDK2AP1是一种对细胞周期蛋白依赖的CDK2活化产生负调节作用的生长抑制因子,仅仅表达于正常组织,未发现肿瘤组织或者变化的细胞株表达。其细胞周期负性调节与CDK2相关。有人通过体外实验筛选了CDK家族的7个成员,证明了CDK2AP1与CDK2的紧密联系,认为CDK2AP1与CDK2的分子结合。而经基因突变实验分析证实该蛋白通过第109-111位氨基酸与CDK2非磷酸化单体形式(p34)发生相互作用。而CDK2可具有非活性形式p34和活性形式p33两种单体形式,p12蛋白的表达可降低p33的水平而提高p34的水平,即降低了活性CDK2的水平,从而降低其介导的生物活性。具体地说,其异位表达导致细胞CDK2减少和CDK2相关激酶活性降低,并伴随细胞周期中CDK2AP1转染子位置的移动(G1 and S)。细胞DNA pol-a:primase (DNA polymerase alpha/primase)通过磷酸化和去磷酸化调节其活性,它的磷酸化形式可以激活DNA的复制,cyclinsA和cyclinsE与CDK2结合的复合物是DNA pol-a:primase的亚单位p180、p70磷酸化的关键酶。由于CDK2AP1可以降低CDK2表达水平和活性,使DNA pol-a:primase的活性降低。Mats等证实：CDK2AP1对CDK2介导的DNA pol-a:primase的磷酸化抑制具有特异性,CDK2AP1与DNA pol-a:primase结合位点可能位于其N端的六个氨基酸提示两者间可能还有直接的作用。CDK2AP1与DNA pol-a:primase的作用机理既有可能通过直接抑制DNA聚合酶的活性也有可能通过CDK2介导来完成。
氨基酸109-111在这个突变体中的突变,消除了它和CDK2的结合,使CDK2AP1和CDK2之间的关联增加了非活化形式的CDK2。这种关联可能早于cyclin E和A作用于CDK2活化的生化通路。CDK2AP1和CDK2结合,并抑制DNA的α螺旋酶,调节DNA的复制。有实验表明,CDK2AP1在293细胞中的异位表达是通过蛋白酶体依赖的蛋白水解,导致了CDK2的下降。这两种机制(隔绝p34CDK2和标靶p34CDK2的水解),可能单独或同时发生作用,以降低p34CDK2的细胞水平和相关的CDK2介导的生物活性。CDK2AP1可通过调节DNA合成期促使细胞周期停滞并可能导致细胞编程性凋亡。
对CDK2AP1的研究,在国际学术界已经成为一种热门话题。现在已有对该基因与头颈部癌,食管癌,胃癌,结肠癌,前列腺癌的相关报到。但是,直到现在仍然没有任何关于人类肺癌中CDK2AP1功能研究的报道。本实验中,我们构建了CDK2AP1的慢病毒载体,将其转染到肺癌A549细胞中,通过细胞功能体外实验及裸鼠肿瘤移植实验,来研究该基因对肺癌细胞的在体内及体外的作用。
目的：
构建CDK2AP1慢病毒过表达载体和siRNA-CDK2AP1慢病毒载体,分别对照的进行人肺癌细胞的体内、外功能影响的实验,以来明确基因CDK2AP1对肺癌细胞作用的影响。
实验方法：
(1)慢病毒表达载体的构建：根据genebank中CDK2AP1基因mRNA的已知序列,确定合适的靶位点,合成编码siRNA的DNA模板,连接退火的siRNA模板寡核苷酸到线性化的pGCSIL-GFP表达载体,构建siRNA-CDK2AP1的慢病毒表达载体,经测序及酶切鉴定；
(2)CDK2AP1过表达载体的构建：经逆转录,钓取目的基因,PCR扩增功能基因,酶切后与线性化的真核过表达载体连接,构建目的过表达载体,经测序及酶切鉴定；
(3) Western blot外源筛选靶点：siRNA-CDK2AP1慢病毒表达载体与CDK2AP1过表达载体共转染293T细胞,Western blot外源筛选靶点。
(4)慢病毒载体的包装及滴度测定及检测目的基因的转录及蛋白表达情况：将最佳靶点的慢病毒表达载体与pHelper 1.0载体和pHelper 2.0载体,三种质粒载体在293T细胞内包装,并标定病毒滴度。将CDK2AP1慢病毒过表达载体及si-CDK2AP1载体转染人肺癌A549细胞株,应用Real-time PCR和Western blot分别检测目的基因的转录水平及蛋白表达；
(5)肺癌细胞体外功能实验：分别应用流式细胞术检测细胞周期；应用MTT比色法、BrdU方法,transwell实验、细胞克隆形成实验,观察CDK2AP1对细胞周期、增殖、侵袭能力及细胞凋亡,并作出比较。
(6)体内实验：裸鼠移植人肺癌模型,分别将siRNA-CDK2AP1慢病毒表达载体及CDK2AP1过表达载体转染的人肺癌A549细胞株,细胞培养后移植在裸鼠皮下；测量肿瘤组织体积及重量,绘制成瘤曲线,比较说明CDK2AP1对肺癌细胞的抑制作用。
结果：
(1)经测序及酶切鉴定,成功构建了siRNA-CDK2AP1慢病毒表达载体。
(2)经测序及酶切鉴定,成功构建了CDK2AP1过表达载体。
(3) (1)与(2)两种载体共转染293T细胞,荧光表达率大于90%；Western blot外源筛选,Target 1#对目的基因的表达有较为显著的敲减作用,因而是最佳靶点。
(4)通过CDK2AP1过表达及目的基因去除反向的对比,细胞功能体外实验说明CDK2AP1过表达载体对人肺癌细胞具有抑制增殖、侵袭及促进凋亡的作用。流式细胞术、MTT实验、BrdU实验、transwell实验、细胞克隆形成实验检测显示：CDK2AP1能抑制细胞增殖、侵袭,具有统计学差异。
(5)成功建立裸鼠移植人肺癌模型,通过对肿瘤体积及重量的测量,绘制成瘤曲线,证明CDK2AP1对肺癌细胞的生长具有明显的抑制。
结论：
(1)成功构建了CDK2AP1过表达载体
(2)应用CDK2AP1过表达载体干扰人肺癌A549细胞,在体外能高效地抑制肺癌细胞的增殖、侵袭。
(3)通过裸鼠移植瘤模型说明CDK2AP1基因能够在体内起到抑制人肺癌A549细胞产生抑制作用。
Lung cancer is a malignant tumor which seriously threat human health in these years. The morbidity of lung cancer, one of the most common primary malignant carcinomas in the world, has increased annually. There is a prevalence of more than 1.5 million cases worldwide per year, with high incidence especially in China.The aging population, the progression of urban industrialization as well as the contamination and destruction of human life environment result in the rising incidence. Furthermore, to date, the therapeutic efficacy for this malignancy has not improved due to current high mortality and poor long-term survival rates.
The developing knowledge of molecular biology of tumor progression has revealed changes of gene expression during lung cancer progression. And the elucidation of molecular mechanism under carcinogenesis would be of great importance in the prevention and treatment of cancers. CDK2AP1 (cyclin-dependent kinase 2-associated protein 1), also known as DOC1, ST19, DORC1, doc-1, or p12DOC-1, was first isolated from normal keratinocytes as growth inhibitory factor by Todd et al in 1995. Located on human chromosome 12q24.31, the full length of CDK2AP1 gene is 1.6 kb, and its encoded protein is 12.4 KD(115 amino acids). CDK2AP1 interacted with DNA polymerase a, regulating the phosphorylation of the large subunit p180 and negatively modulating DNA replication in S phase of cell cycle.CDK2AP1 could also arrest cell cycle at DNA synthesis phase, leading to programmed cell apoptosis. Some study confirmed that CDK2AP1 played an important role in TGF-β1-modulated growth inhibitory system. Recently, it is revealed that the defective expression of CDK2AP1 was accompanied by the abnormal TGF-β-smad pathway, which ultimately resulted in the resistance of human oral squamous cell carcinoma cells to TGF-β-induced growth inhibitory effect. CDK2AP1 could regulate the activity of CDK2, the phosphorylation of pRB and p180, and TGF-β-Smad signaling pathway so as to play its inhibitory effect on cell growth.
Cyclin-dependent kinase 2-associated protein 1 (CDK2AP1), a cell growth inhibitory factor, is abnormally expressed in cancer cells, and might be implicated in the development of lung cancer. However, to date, no study on the function of CDK2AP1 in human lung cancer has ever been reported. In this study, overexpressing and lentiviral vectors containing CDK2AP1 cDNA and CDK2AP1 shRNA (short hairpin RNA) were constructed, and up-regulated and down-regulated CDK2AP1 expression in human lung cancer A549 cells was found, and explored the function of CDK2AP1 in lung cancer cell's development and progression.
Study objectives:
(1) CDK2AP1 siRNA design and pGCSIL-GFP-CDK2AP1 shRNAs construction.
(2) Construction of overexpressing vector pGC-FU-CDK2AP1 cDNA.
(3) To investigate the effect of CDK2AP1 on the growth in the cells in vitro and in vivo.
Methods:
(1) The human CDK2AP1 cDNA sequence was searched for suitable siRNA target sequences, and three CDK2AP1 siRNA sequences were designed by the Ambion siRNA Target Finder. DNA oligos containing the target sequences were chemically synthesized, annealed, and inserted into the expression vector pGCSIL-GFP by double digestion and ligation in accordance with the manufacturer's guidelines. The ligation product was then transformed into competent Escherichia coli DH5acells.As a control for CDK2AP1 siRNA, a corresponding random siRNA sequence was used. The targeting sequence of the short hairpin RNA (shRNA) was then confirmed by sequencing.
(2) CDK2AP1 cDNA was obtained the CDK2AP1-mRNA of lung tissue,by the RT-PCR assay, and then cloned into pGC-FU by double digestion, and in accordance with the manufacturer's guidelines. The ligation product was transformed into competent Escherichia coli DH5a cells. The recombinant was identified by PCR and confirmed by sequencing.
(3) The selection of the most effective CDK2AP1 shRNA with the Western blot assay.
(4) Lentiviral particle production with the vectors pHelper 1.0 and pHelper 2.0, and the detection of knockdown efficiency of CDK2AP1 shRNA with TR-PCR and Western blot assay.
(5) In vitro studies:The Lewis lung cancer cell lines were infected by the both plasmid vectors. The cycle phase distribution of the cells were analyzed with the flow cytometry. The effective function of CDK2AP1 in the infected the lung cancer cells was revealed with the MTT assay, the BrdU assay, the Cell Invasion Assay Kit (The transwell assay) and the Colony formation.
(6)In vivo studies:we builded a BALB/c nude mouse tumor xenograft model. The mice were transplanted with the both infected(CDK2AP1 and si-CDK2AP1) Lewis lung cancer cells into the right flank. Then, we got the results of the volume and weight of the tumors.
Statistical analysis
Comparison between groups was performed with the Student's t test and X2analysis using a SigmaStat statistical software package(SPSS, Chicago,IL). P<0.05 was taken as showing significance.
Results:
(1)The plasmids which were designed for expression of CDK2AP1 siRNA was constructed, and confirmed by restriction enzyme digest and sequence analysis.
(2) The plasmids were designed for over-expression of CDK2AP1 was constructed, and confirmed by restriction enzyme digest and sequence analysis.
(3) The both vectors were transfecting into the 293T cells. The most effective CDK2AP1 shRNA, the target 1, was founded with the westen blot assay.
(4) The study in vitro:The cell cycle phase distribution analyzed with a flow cytometry,the MTT assay, the BrdU assay, the Cell Invasion Assay Kit (The transwell) and the Colony formation showed that the over-expression of CDK2AP1 vector can inhibit the proliferation, invasion, and promote apoptosis of human lung cancer A549 cells.
(5) The study in vivo:The BALB/c nude mouse tumor xenograft model was builded. The results of the volume and weight of the the Lewis lung cance cells revealed that CDK2AP1 inhibited the growth of the cells.
Conclusions
1.The vectors of Lentivirus-mediated RNA interference and over-expression of CDK2AP1 were constructed successfully.
2.The study revealed that CDK2AP1 could inhibit the proliferation, invasion, and promote apoptosis of human lung cancer A549 cells in both vitro and vivo.
引文
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