PAK1在大肠癌演进和转移中的作用
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摘要
背景与目的
大肠癌(colorectal carcinoma，CRC)是常见的恶性肿瘤，全球每年新增病例近1000000例，492000例死亡。随着经济的进步，生活方式和饮食结构的变化，我国大肠癌每年发病率也快速递增，特别是大城市上升趋势更为显著。大肠癌男女发病率等同，在40岁以后发病危险度随着年龄增加而递增；资料显示我国大肠癌男性发病率约为16.2／100000，女性约为14.5／100000，大肠癌已经成为我国致死性肿瘤中最常见的死亡原因之一。然而，大肠癌的治疗效果并不理想，在所有的病人中约有半数患者治疗失败。大肠癌传统治疗方法，即外科手术治疗、放射治疗和化学药物治疗方法的不断发展和完善，在延长患者生存期和提高患者生存质量方面取得了长足的进步；但是手术和放疗是局部治疗，难以控制肿瘤的复发和转移，化疗药物往往缺乏组织选择性和特异性，对骨髓等正常人体重要器官和组织产生严重的毒副作用，在过去几十年，大肠癌的五年生存率一直徘徊在40～50％左右。因此，探寻大肠上皮细胞癌变，演化和转移的机制，特别是寻找早期肿瘤标志物，以期早期发现和干预大肠癌的生物学进程，对大肠癌的治疗具有重要的意义。
p21-activated kinase-1(PAK1)即p21小GTP酶活化激酶1，是第一个被克隆和验证的的丝氨酸／苏氨酸蛋白质磷酸化激酶家族成员。PAK是分子量为21KD、Rho家族的小GTP酶(small GTPases)，即Cdc42和Rac的下游效应分子。在分子结构上，PAK N-末端为激酶的调节区，C-末端为激酶的底物结合区，具有激酶活性。PAK1-3构成第一组PAK，分子量为62-64 KD，其激活需要Cdc42和Rac，除调节胚胎发育、正常的细胞生长，分化和凋亡外，PAK家族成员还在人类疾病中起重要作用。PAK1是PAK家族中与人类肿瘤关系密切的成员，其在细胞骨架修复、延长正常细胞寿命和正向调节一些正常生理代谢等方面都起重要作用。PAK1只在脑、肌肉和脾脏正常组织表达，其他组织表达很少，但在很多人类肿瘤细胞都高表达PAK1，尤其是在乳腺癌和卵巢癌细胞中PAK1的表达远高于其它肿瘤细胞，而且PAK1表达与乳腺癌和卵巢癌细胞的增殖和侵袭转移呈正相关。但PAK1在大肠癌的发生发展、演化和转移中的作用机制尚未明确，因此本课题在系统地检测PAK1在大肠癌旁正常组织—息肉—腺瘤—原发性大肠癌组织—大肠癌转移癌瘤组织中的表达差异和分布特点基础上，同时构建PAK1真核表达载体和PAK1 shRNA真核表达载体，转染大肠癌SW480细胞，观察上调和下调大肠癌细胞PAK1表达后对大肠癌细胞增殖、凋亡，细胞粘附、运动、侵袭和致瘤等表型的改变，以期深入了解PAK1在大肠癌细胞演化和转移中的作用和机制，并探讨PAK1作为大肠癌转移标志和作为潜在治疗靶点的可能性。
材料与方法
一、PAK1在良、恶性大肠病变中的表达及其临床意义
用免疫组织化学S-P法检测PAK1在大肠癌癌旁正常粘膜、大肠息肉、大肠良性腺瘤、大肠癌和大肠癌转移瘤组织中的表达，并分析PAK1表达与大肠癌临床病理特征的关系。
二、PAK1对大肠癌细胞表型的影响
为检测PAK1对大肠癌细胞增殖、粘附、移动和侵袭等表型的影响，利用基因重组技术分别构建PAK1真核表达载体和PAK1 shRNA真核表达载体(RNAinterference，RNAi)，并以大肠癌细胞SW480作为模型，转染重组载体后观测对大肠癌SW480细胞表型的影响，并分析PAK1在大肠癌发生、发展和转移中的作用及其可能机制。
1、PAK1真核表达重组质粒的构建按照Genbank上公布的PAK1 mRNA序列(NM_002576)，在其编码区(CDS)设计两端带有EcoRI和BamHⅠ酶切位点的引物，利用聚合酶链式反应(Polymerase chain reaction，PCR)扩增PAK1 CDS区，EcoRI和BamHⅠ双酶切纯化的PCR产物和真核表达载体pEGFP／C1，T_4连接酶连接酶切纯化后的PCR产物和酶切纯化后的pEGFP／C1，转化到大肠杆菌DH5a中，挑单菌落培养扩增，提取重组质粒后行EcoRI和BamHⅠ双酶切鉴定，选择插入片段正确的克隆测序鉴定。
2、PAK1 shRNA重组载体的构建根据siRNA设计的原则，在PAK1 mRNA(NM_002576)CDS设计三对siRNA核苷酸序列，并设计一对非相关核苷酸序列作为对照，经Genbank在线BLAST确定确定与其它人类基因无高度同源后，分布在所设计的siRNA两端分别加上带有BamHⅠ和EcoRI酶切位点的shRNAs(short hairpin RNAs)单链送公司合成，配对单链核苷酸退火复性合成双链，按常规操作把shRNA序列克隆到pRNAT-U6.1／Neo载体中，重组质粒酶切和测序鉴定。
3、转染细胞和建立稳定转染细胞克隆采用Invintrogen公司的Lipofectamine2000~(TM)脂质体进行细胞转染。细胞转染48h后用分别用800μg／ml G418筛选阳性细胞克隆，挑取单克隆细胞扩增培养。
4、PAK1表达调控效应检测逆转录聚合酶链式反应(Reverse transcriptionpolymerase chain reaction，RT-PCR)和免疫印迹、免疫细胞化学与激光共聚焦显微镜检测PAK1 mRNA和蛋白质水平表达改变。
5、细胞增殖与凋亡MTT法检测PAK1对大肠癌细胞SW480增殖的影响；采用DNA片段法检测PAK1对5-Fu诱导的细胞凋亡的影响。
6、细胞基质粘附实验参照文献的方法研究PAK1对大肠癌细胞SW480粘附的影响。
7、单层细胞划痕愈伤实验分析单层细胞划痕愈伤实验分析PAK1对大肠癌细胞移行的影响，参照文献操作。
8、侵袭实验观察改变PAK1表达对大肠癌细胞侵袭能力的影响。
9、软琼脂糖集落形成实验观察RNAi沉默PAK1表达对SW480细胞锚着独立性生长的影响。
10．明胶酶谱实验观测PAK1对SW480细胞基质金属蛋白分泌和活性的影响。
11、裸鼠成瘤实验观察PAK1表达对SW480细胞裸鼠成瘤的影响，包括成瘤瘤体大小、侵袭转移部位差异。
三、统计学分析所得数据用SPSS12.0 for Windows软件，免疫组织化学采用等级资料多个独立样本和两个独立样本非参数检验；其它数据行单向方差分析(One-Way ANOVA)，组间多重比较用LSD方法进行分析，以均数±标准差(mean±SD)表示，以P≤0.05为有显著性差异。
结果
一、PAK1在良、恶性大肠病变中的表达及其临床意义
免疫组织化学检测结果表明PAK1主要在细胞胞浆表达，胞核偶有少量表达，而在大肠上皮细胞外基质中基本不表达(见图1-1和图1-2)；大肠正常粘膜和大肠癌癌旁正常组织粘膜中PAK1的表达极少，阴性、弱阳性、阳性和强阳性表达率为别为52.5％、49.1％与0.03％和0、48.7％与27％；大肠息肉PAK1的表达阴性、弱阳性、阳性和强阳性表达率分别为66.7％、22.2％与0和11.1％；大肠腺瘤组织PAK1的表达阴性、弱阳性、阳性和强阳性表达率为别为18.1％、45.4％与18.1％和27.2％；Dukes’A期大肠癌组织中阴性、弱阳性、阳性和强阳性率分别为8.4％、33.3％、25.0％和33.3％；Dukes’B期阴性、弱阳性、阳性和强阳性率分别为13.3％、13.3％、23.3％和50.0％；Dukes’C期阴性、弱阳性、阳性和强阳性率分别为16.7％、11.1％、16.7％和55.5％；Dukes’D期阴性、弱阳性、阳性和强阳性率分别为0、0、33.3％和66.7％。经等级资料多个独立样本非参数检验(Kruskal-Wallis检验)显示，大肠正常粘膜、大肠息肉、大肠腺瘤、大肠癌组织和大肠癌淋巴结和肝脏转移癌组织中的PAK1表达有显著性差异(P=0.000)，而且大肠正常粘膜、大肠息肉、大肠腺瘤、大肠癌组织和转移癌中的PAK1表达总体阳性率依次升高；不同Dukes’分期癌组织PAK1的表达不同，各组Dukes’分期癌组织PAK1的表达见图1-2和表1-4图，但各组PAK1表达无显著差异性(P=0.103)，分析原因主要可能与标本量少有关，其中A期和D期临床标本相对较少，收集较为困难。大肠癌高中低分化各组的PAK1表达的总体阳性率不同(74例大肠癌纳入研究)，有依次升高趋势，但各组PAK1表达无显著性差异(P=0.357)。
二、PAK1对大肠癌细胞表型的影响
1、PAK1真核表达载体pEGFP-C1／PAK1经酶切鉴定，所插入的片段与预期目的片段大小一致，并经测序证实，BLAST序列对比证实是PAK1基因mRNA CDS序列。
2．PAK1 shRNA真核表达载体经酶切和测序证实插入序列与设计序列完全一致；
3、重组质粒转染SW480经G418抗性筛选得到稳定细胞克隆株；
4、与对照细胞比较，RT-PCR和Western blotting从mRNA和蛋白水平证实SW480内源性PAK1被PAK1 shRNA抑制；
5、选取PAK1 shRNA中一个克隆细胞株用作后续实验，并把其细胞克隆命名为SW480~(shRNA1)，非相关序列细胞克隆命名为SW480~(shRNA-N)；PAK1真核载体细胞克隆株命名为SW480~(PAK1)，pEGFP／C1质粒对照细胞克隆命名为SW480~(svector)；
6、细胞增殖实验显示，在各组细胞孵育24h后，各组细胞间增殖有显著性差异，SW480、SW480~(vector)，SW480~(PAK1)，SW480~(shRNA-N)和SW480~(shRNA1)单因素方差分析结果F=11.006，P=0.000＜0.001；SW480~(shRNA1)细胞与SW480和SW480~(shRNA-N)比较结果分布是0.400±0.004 vs 0.532±0.0696，P=0.016＜0.05和0.400±0.004vs0.533±0.038，P=0.015＜0.05；SW480~(PAK1)与SW480和SW480~(vector)相比较的结果是0.719±0.125 vs 0.532±0.070，P=0.002＜0.01和0.719±0.125 vs0.530±0.039，P=0.001＜0.01)，结果说明下调SW480细胞PAK1表达，细胞增殖受到抑制，而上调SW480细胞的表达则促进细胞增殖。
7、细胞凋亡实验显示，未用5-FU处理的各组细胞培养8h均未检测到凋亡DNAladder；细胞分别用15.0μmol／ml 5-FU处理8h后，SW480~(shRNA1)可以见到明显的凋亡DNA ladder，而SW480、SW480~(vector)、SW480~(PAK1)和SW480~(shRNA-N)未检测到凋亡DNA ladder；在用30.0μmol／ml 5-FU处理8h后，SW480和SW480~(vector)可检测到DNA碎片，但SW480~(PAK1)未检测到凋亡，提示PAK1有抑制细胞凋亡的作用，PAK1可以增加大肠癌细胞对化疗药物5-Fu的敏感性；
8、细胞-基质粘附实验结果显示各组细胞之间粘附细胞明显不同，SW480~(shRNA1)细胞显著减少，与SW480细胞相比(P=0.015)和与SW480~(shRNA-N)相比较P=0.018)，而SW480细胞与SW480~(shRNA-N)细胞之间比较无显著性差异；SW480~(PAK1)与SW480相比(P=0.004)和与SW480~(vector)相比(P=0.004)，细胞粘附显著增加。
9通过软琼脂糖克隆形成实验可以观察细胞锚定独立性生长的能力，从而反映细胞的恶性程度。我们发现SW480细胞转染PAK1基因后，克隆形成率明显高于亲本细胞，表明细胞恶生程度增高，预示细胞侵袭能力的增加；而转染shRNA沉默PAK1表达后，SW480细胞在体外双层琼脂克隆形成能力减弱。
10、SW480、SW480~(vector)、SW480~(PAK1)、SW480~(shRNA-N)和SW480~(shRNA1)在划痕损伤后0h未有明显差异；在24后SW480、SW480~(vector)、SW480~(PAK1)和SW480~(shRNA-N)细胞划痕边缘有细胞移动，而SW480~(shRNA1)细胞划痕边缘整齐未见明显细胞移动；在24后，SW480~(PAK1)细胞较SW480、SW480~(vector)和SW480~(shRNA-N)细胞划痕面积明显缩小，而SW480~(shRNA1)细胞划痕边缘整齐，划痕面内未见明显细胞移动；在72h后，SW480、SW480~(vector)、SW480~(PAK1)和SW480~(shRNA-N)细胞划痕面内细胞都增多，SW480~(PAK1)细胞划痕面内被细胞铺满，而下调PAK1表达的SW480~(shRNA1)细胞划痕边缘稍有模糊，划痕面内几乎见不到细胞。
11、细胞侵袭实验显示SW480~(shRNA1)细胞显著降低。而SW480~(PAK1)细胞的侵袭能力增加。
12明胶酶谱实验显示，与对照细胞相比，SW480~(PAK1)细胞分泌活性MMP2增加，而SW480~(shRNA)细胞分泌活性MMP2减少，而MMP9活性未见有明显改变
13裸鼠皮下成瘤实验发现接种SW480~(shRNA)细胞和SW480~(shRNA-N)细胞后，裸鼠移植瘤体重量分别为(0.1075±0.06571)g和(0.9517±0.72323)g，RNA沉默组瘤体重量显著低于对照组(P＜0.001)。
结论
1、免疫组织化学检测显示PAK1表达从腺瘤到肿瘤、从原发大肠癌到转移大肠癌依次增加，提示PAK1在大肠癌发生、发展和转移中起重要作用。
2、上调PAK1表达可以促进大肠癌细胞SW480增殖，下调PAK1表达则可以抑制大肠癌细胞SW480的增殖；
3、PAK1可以增强大肠癌细胞的异质粘附，下调PAK1表达则抑制SW480细胞的异质粘附；
4、PAK1增强大肠癌细胞的非锚定式依赖式生长，下调PAK1表达则抑制大肠癌细胞的集落形成能力；
5、PAK1增强大肠癌细胞移行能力，下调PAK1表达则明显抑制大肠癌细胞的移行能力；
6、PAK1增强大肠癌细胞对基质的侵袭能力，下调PAK1表达则可以减弱大肠癌细胞的侵袭力；
7、PAK1可以抗5-Fu诱导的大肠癌凋亡，下调PAK1表达则有促进凋亡作用，抑制PAK1表达可以增强大肠癌细胞对5-Fu的敏感性；
8、PAK1可以增强大肠癌细胞基质金属蛋白酶2的活性，提示PAK1增加大肠癌细胞的转移潜能；
综上，通过临床病理标本、细胞模型到动物模型实验结果显示PAK1在大肠癌病程演进中发挥正向调控作用，而且正向调控大肠癌细胞粘附、侵袭和移动能力，PAK1正向调控大餐癌细胞转移潜能与其影响MMP-2活性有关，下调PAK1表达可以增加大肠癌细胞对化疗药物的敏感性，所以PAK1有希望成为大肠癌治疗的分子靶点，有必要继续深入研究其在大肠癌演进和转移中具体作用机制。
Background and objectives
Colorectal cancer(CRC) is one of the major malignancies in the world. The prognosis of CRCs is poor, due to frequent metastasis and tumor recurrence. Worldwide almost one million new cases occur annually, amounting to 492000 related deaths~[1-3]. With the many changes having taken place in people's diet and lifestyle, CRCs has become the third most common type of digestive tumor in China, and the number of new cases arising each year is still increasing. The overall incidence is identical in men and women, with the risk beginning at age 40 and increasing with age. Thus colorectal cancer ranks as the frequent cause of cancer deaths among China. Despite the rate of improvements in surgery, radiotherapy and chemotherapy, unfortunately, the prognosis of CRCs has not been gained progress over the past decades, with an overall five-year survival rate of around 40%- 50%~[4]. Therefore, novels diagnose and treatments need to be developing in order to enrich the therapeutic armamentarium. In recent years, molecular biology has applied to the study of colonic carcinoma, both in the human and in the experimental animal. The data obtained have enriched our understanding of colonic carcinogenesis and are of potential interest for CRCs diagnosis and prevention.
PAK1 (P21-activated kinase-1), a member of a family of serine/threonine protein kinase, was the first PAK to be cloned~[5] . Moreover, PAK1, which plays an essential role in embryonic development and tissue growth, and is also necessary for the spread and growth of tumor cells, such as breast and ovarian cancers~[6]. PAK1 is a direct target of the small GTPases Cdc42 and Racl, and binding of GTPases to PAK1 stimulates its kinase activity via autophosphorylation[7]. PAK1 contains an N-terminal regulatory domain and a C-terminal kinase domain, its N-terminal regulatory domain contains GTPase binding domain to mediate the binding of PAK1 to Rac/Cdc42~[8]. Among normal tissues, PAK1 is highly expressed in the brain, muscle, and spleen. Accumulating evidence indicates that PAK1 is important for a variety of cellular functions including cell morphogenesis, motility, survival, mitosis, cell cycle and angiogenesis. PAK1 are involved in the regulation of cellular function via phosphorylating a number of downstream target protein. Many evidences showed that Pakl activation occurs during the process of tumorigenesis, and PAK1 is likely to provide insight into the role of PAKs in human cancers. Despite the recent reports of the involvement of PAK1 in signaling cascades in human cancer cells and the fact of PAK1 is downstream of the small GTPases, Cdc42 and Rac1, the relationship of PAK1 to malignant progression of CRCs and the role of the PAK1 pathway in the biology of human colorectal cancer cells remain unknown. In this study, detect PAK1 expression in colon cancer patients and colorectal cancer cell lines and its molecular mechanism of action in transfected colorectal cancer cell to provide basis for further underscoring the link between PAK1 expression and CRCs progression..
Material and methods
The potential role of PAK1 protein expression in colorectal carcinomas
Immunohistochemistry of normal, benign colon polypus, colon adenoma, primary, and metastatic human tumor specimens was to detect the relationship of PAK1 expression with the pathological features. The study-included patients with colorectal carcinoma, according to the Dukes classification of malignant tumors, were divided as Dukes A, Dukes B, Dukes C, and Dukes D. Formalin-fixed paraffin sections were stained for PAK1 (1: 200 dilutions) using the Streptavidin-preoxidase (SP) technique. Antigen retrieval was achieved by microwave treatment with citrate buffer at pH 6.0 at 95℃for10 min. The immunohistochemical staining was scored in the following grades according to the percentage of positive cells:0,＜5% positive; 1, 5% to 25% positive; 2, 26% to 50% positive; and 3,＞51% positive. Morphological and immunohistochemical results were to correlate with clinicopathologic parameters.
The influences on the phenotypes of SW480 cell mediated by changing PAK1 expression
1. Cell Culture SW480(ATCC) cells were maintained in RPMI-1640(Gibco) supplemented with 10% fetal bovine serum (FBS) (Hycone), penicillin (100units/ml), and streptomycin(100mg/ml)). Cells were passaged using 0.25%Trypsin-EDTA and maintained in culture for 48h before performing experiments.
2. Construction recombinant eukaryotic PAK1 expression vector Primers, targeted to PAK1 (GenBank: NM_002576) code sequence(CDS) and containing EcoRⅠand BamHⅠsites at the ends, were designed. The polymerase chain reaction(PCR) products of CDS and pEGFP-C1 were both digested with EcoRⅠand BamHⅠand subsequently ligated for 4h at 16℃using T4 ligase. The recombinant plasmids was digested with EcoRⅠand BamHⅠfollowed by electrophoresis with 1% agarose and sequence analysis to identify.
3. Construction of recombinant PAKI shRNA expression vector Designing three pairs of small interfering RNAs(siRNAs) of PAK1 mRNA(GenBank: NM_002576), and six corresponding single-strand short hairpin RNAs(shRNAs), containing BamHⅠand HindⅢsites and 9nt hairpin structure, were synthesized and annealed. The annealed products and the linear pRNAT6.1/Neo plasmid, digested with BamHⅠand HindⅢ, were ligated for 4h at 16℃using T4 ligase. The recombinant plasmids were digested with BamHⅠand HindⅢfollowed by electrophoresis with agarose and sequence analysis to identify.
4. Transfection and selection of stably transfected cell clones The recombinant plasmids and control plasmids were transfected into SW480 cells using lipofectamine2000~(TM) reagent (Invitrogen) according to the manufacturer's protocol. Forty-eight hours after the addition of DNA, the transfected cells were selected in growth medium containing 0.8 mg/ml Geneticin (G418; Life Technologies, Inc.). Colonies resistant to G418 were isolated. After 3-4 wk of culture, visible colonies were picked up and expanded. The stably transfected clone cells were observed to show green fluorescence under fluorescent microscope and the clones without expression of the transfected gene did not show green fluorescence
5. PAK1 exprssion detecting The expression changes of PAK1 were detected using reverse transcription polymerase chain reaction (RT-PCR), western-blotting, and immunofluorescence staining at mRNA and protein levels.
6. Cell proliferation and apoptosis To observe the changes of cell growth after regulating expression of PAK1, cell proliferation was analyzed with MTT assay and 5-Fu induced cell apoptosis was detected by DNA fragment assays.
7. Cell-matrix adhesion analysis Cell-matrix adhesion assays were employed, as previously described~[9, 10]
8. Cell migration assays Migration of SW480, PAK1-overexpressng SW480(SW480~(PAK1)) and PAK1-lowexpressing SW480(SW480~(shRNA1)) cells was studied using 6.5mm Transwell chambers with 8μl pores (Costar) as previously described~[11].
9. Cell culture wound healing assay~[12]. Wounds were created in confluent cells using a pipette tip. The cells were then washed with medium to remove any free-floating cells and debris, and culture plates were incubated at 37℃. Wound healing was observed at 0, 24, 48, and 72 hours within the scrape line, and representa- tive scrape lines for each cell line were photographed.
10. Cell invasion assays Cell invasion assays were performed as described for the cell migration assays, except that the Transwell filters were additionally coated on the upper side with 30 mg of Matrigel.
11 Anchorage-independent growth Assays Anchorage-independent growth in soft agar was performed as described previously~[13]. In brief, the cells (10~5) were plated on 60-mm dishes. After 21 days, colonies were scored after staining the dishes with 0.5 mg/ml 3-(4, 5-dirnethylthiazol-2-y1)-2, 5-diphenyltetrazolium bromide (Sigma) overnight at 37℃.
12. Gelatin zymography~[14] Cells were plated at a density of 2×10~5 in 24-well plates. After 16 hours, cells washed with 0.01M PBS and incubated in 500μl of serum-free medium for 24 hours, were performed as described previously.
13. Tumorigenesis in nude mice Cells were suspended at the density of 2×10~7 cells in 200μl of RPMI-1640, and injected subcutaneously into the flank region of athymic nude mice. Twelve mice were to distribute to PAK1-shRNA group, vector control group, 6 per group at random. The animals were to inspecte at regular intervals for the appearance of visible tumors to measure the time of first appearance. All animals were observed for up to 30 days following the injection, the mice were sacrificed and the tumors were carefully removed by blunt dissection. The tumors were to weigh and their average growth rates were measured.
Statistical analysis
For immunohistochemistry results, Kruskal-Wallis Test was used, and the results of RT-PCR, Western blotting and MTT assays were expressed as mean±SD of at least three separate experiments, were analyzed by one-way analysis of variance (ANOVA). P values of=0.05 was considered statistically significant.
Results
1. PAK1 expression increases during human eolorectal cancer progression and metastasis. Immunohistochemistry was performed to examine PAK1 expression levels in paraffin-embeded tissue from normal colon mucosa, benign polypi and adenomas to primary colon cancers and metastasis colon cancers, which is a typical progression pathway during colon carcinogenensis. Representative shows that PAK1 expression is negative in benign polypi, begins to increase in adenoma but is still weak compared to the carcinoma, and over-expressed in primary colon adenocarcinoma but is absent or barely detectable in paired normal mucosa beside the cancer (P＜0.001). Furthermore, PAK1 expression is extremely much higher in lymph node metastasis and liver metastasis in contrast to primary colon carcinoma. However, the expression of PAK1 was not correlated with the histological differentiation(P.＞0.05) and Dukes' classification (P＞0.05).
2. The effects of changing PAK1 expression on cellular biological activity of SW480 cell.
(1) Constructed human PAK1 eukaryotic expression vector and human PAK1 hairpin siRNA eukaryotic expression vectors successfully.
The DNA oligonucleotides encoding PAK1 mRNA and short haipin siRNA against PAK1 were synthesized, and cloned to construct recombinant plasmid respectively, which were identified by restriction enzyme digestion analysis and DNA sequencing.
(2) Geneticin-resistant cell lines were screened after the recombinant plasmids were transfected into SW480 cells.
Following to the instruction of lipofectamine~(TM)2000 the transfection were performed, plasmids served as negative control, SW480 as blank control. The RT-PCR results showed that mRNA level of PAK1 in screened transfected cell lines SW480~(shnRNA1)was lowest, mRNA of PAK1 in transfected cell line SW480~(shRNA2-3)was significantly decreased also, its were confirmed in protein level by Western Blotting.
Six kinds of selected Geneticin-resistant colonies were respectively named as: SW480/pEGFP/C1-PAK1 (to abbreviate SW480~(PAK1)), SW480/pEGFP/C1(to abbreviate SW480~(Vector)), SW480/pRNAT-U6.1/Neo-PAK1~(shRNA1-3)(to abbreviate non specific 8W480 ~(shRNA1-3)), and SW480/pRNAT-U6.1/Neo-PAK1~(non-specific) construct (to abbreviate SW480~(shRNA-N)) of PAK1. This laid sound basis for the following research of PAK1.
(3)After 24h incubation, there were notable differences compared the growth velocity of SW480 cells in the group of 5W480~(vector), 5W480~(PAK1), SW480~(shRNA-N)and 8W480~(shRNA1) (F=11.006, P=O.O00＜0.001). The growth velocity of SW480~(shRNA1) was reduced obviously to contrast with SW480和SW480~(shRNA-N)(0.400±0.004 vs 0.532±0.0696, P= 0.016＜0.05; 0.400±0.004 vs0.533±0.038, P= 0.015＜0.05)., and 5W480~(PAK1) was increased significantly to compare with SW480 and SW480~(vector)(0.719±0.125 vs 0.532±0.070, P=0.002＜0.01; 0.719±0.125 vs0.530±0.039, P=0.001＜0.01).
(4) Compared with SW480和SW480~(shRNA-N), treatment with 5-Fu(15μg/ml) for 12h resulted in a significant increase of apoptosis in SW480~(shRNA1) cells, no significant apoptosis was observed in SW480~(PAK1) cells induced with 5-Fu (30μg/ml) compared with with SW480 and 8W480~(vector) cells.
(5) Alloplasm adhesiveness (cell-matrix adhesion) tests showed that there was a significant difference between the adhesive cell numbers of these three cell lines. The adhesive cell number of SW480~(shRNA1) was significantly less than that of SW480 (P＜0.05) and SW480~(shRNA-N) (P＜0.001), while the last two groups had no significant difference, suggesting that compared with SW480 and SW480~(shRNA-N), the cell-matrix adhesiveness of 5W480~(shRNA1) was decreased notably. As well as the adhesive cell number of SW480~(PAK1) was significantly more than that of SW480 (P＜0.001) and SW480~(vector) (P＜0.001),
(6) A monolayer wound-healing assay revealed almost no migration in SW480~(shRNA) cells compared with SW480~(control) cells, but is not of SW480~(PAK1), and Cell migration assays showed homoioplastic result too.
(7) Using a Boyden chamber invasion assay, we observed a significant decrease in the invasive capacity of SW480~(shRNA) cells, but increace of SW480~(PAK1).
(8)A gelatin zymogram for MMP activation demonstrated a decrease in MMP-2 activity in SW480~(shRNA1) compared with SW480 ~(shRNA-N) cells. However, a significant decrease of MMP- 9 activity was not observed.
(9)Tumorigenicity assay showed the tumor weight of SW480~(shRNA) group(0.1075±0.06571)g were less than control group (0.9517±0.72323) (P＜0.001). No lymph node and distant organ metastasis were found in all tumors.
Conclusions
Taken to gether, the results of immunohistochemistry show that PAK1 expression is increased with progression through the adenoma to carcinoma sequence, with the most dramatic increases in invasive and metastatic CRCs, suggesting PAK1 might play an important role in colorectal tumorigenesis. With molecular biology technology, cell biology technology, and RNA interference technique, the role of PAK1 in cell-matrix adhesion, cell migration, cell proliferation and cell apoptosis was studied in the model colorectal cancer SW480 cells. Experimental results indicate that PAK1 plays an important role in regulation of cell-matrix adhesion, cell migration and cell apoptosis in colorectal cancer cells and further has an effect on tumor invasion and metastasis. Increased expression of PAK1 mainly helps colorectal cancer metastasis in the distance, enhances the ability of anti-apoptosis in colorectal cancer SW480 cells and has significant effects to promote proliferation of SW480 cells. It not only provide the basis for the further study in the mechanisms of colorectal cancer invasion and metastasis but also suggest that signal pathway mediated by PAK1- targeted for therpeutic intervention in colorectal cancer. These data implicate PAK1 as an exciting target for therapy of colorectal carcinoma.
大肠癌(colorectal carcinoma，CRC)是常见的恶性肿瘤，全球每年新增病例近1000000例，492000例死亡。随着经济的进步，生活方式和饮食结构的变化，我国大肠癌每年发病率也快速递增，特别是大城市上升趋势更为显著。大肠癌男女发病率等同，在40岁以后发病危险度随着年龄增加而递增；资料显示我国大肠癌男性发病率约为16.2／100000，女性约为14.5／100000，大肠癌已经成为我国致死性肿瘤中最常见的死亡原因之一。然而，大肠癌的治疗效果并不理想，在所有的病人中约有半数患者治疗失败。大肠癌传统治疗方法，即外科手术治疗、放射治疗和化学药物治疗方法的不断发展和完善，在延长患者生存期和提高患者生存质量方面取得了长足的进步；但是手术和放疗是局部治疗，难以控制肿瘤的复发和转移，化疗药物往往缺乏组织选择性和特异性，对骨髓等正常人体重要器官和组织产生严重的毒副作用，在过去几十年，大肠癌的五年生存率一直徘徊在40～50％左右。因此，探寻大肠上皮细胞癌变，演化和转移的机制，特别是寻找早期肿瘤标志物，以期早期发现和干预大肠癌的生物学进程，对大肠癌的治疗具有重要的意义。
p21-activated kinase-1(PAK1)即p21小GTP酶活化激酶1，是第一个被克隆和验证的的丝氨酸／苏氨酸蛋白质磷酸化激酶家族成员。PAK是分子量为21KD、Rho家族的小GTP酶(small GTPases)，即Cdc42和Rac的下游效应分子。在分子结构上，PAK N-末端为激酶的调节区，C-末端为激酶的底物结合区，具有激酶活性。PAK1-3构成第一组PAK，分子量为62-64 KD，其激活需要Cdc42和Rac，除调节胚胎发育、正常的细胞生长，分化和凋亡外，PAK家族成员还在人类疾病中起重要作用。PAK1是PAK家族中与人类肿瘤关系密切的成员，其在细胞骨架修复、延长正常细胞寿命和正向调节一些正常生理代谢等方面都起重要作用。PAK1只在脑、肌肉和脾脏正常组织表达，其他组织表达很少，但在很多人类肿瘤细胞都高表达PAK1，尤其是在乳腺癌和卵巢癌细胞中PAK1的表达远高于其它肿瘤细胞，而且PAK1表达与乳腺癌和卵巢癌细胞的增殖和侵袭转移呈正相关。但PAK1在大肠癌的发生发展、演化和转移中的作用机制尚未明确，因此本课题在系统地检测PAK1在大肠癌旁正常组织—息肉—腺瘤—原发性大肠癌组织—大肠癌转移癌瘤组织中的表达差异和分布特点基础上，同时构建PAK1真核表达载体和PAK1 shRNA真核表达载体，转染大肠癌SW480细胞，观察上调和下调大肠癌细胞PAK1表达后对大肠癌细胞增殖、凋亡，细胞粘附、运动、侵袭和致瘤等表型的改变，以期深入了解PAK1在大肠癌细胞演化和转移中的作用和机制，并探讨PAK1作为大肠癌转移标志和作为潜在治疗靶点的可能性。
材料与方法
一、PAK1在良、恶性大肠病变中的表达及其临床意义
用免疫组织化学S-P法检测PAK1在大肠癌癌旁正常粘膜、大肠息肉、大肠良性腺瘤、大肠癌和大肠癌转移瘤组织中的表达，并分析PAK1表达与大肠癌临床病理特征的关系。
二、PAK1对大肠癌细胞表型的影响
为检测PAK1对大肠癌细胞增殖、粘附、移动和侵袭等表型的影响，利用基因重组技术分别构建PAK1真核表达载体和PAK1 shRNA真核表达载体(RNAinterference，RNAi)，并以大肠癌细胞SW480作为模型，转染重组载体后观测对大肠癌SW480细胞表型的影响，并分析PAK1在大肠癌发生、发展和转移中的作用及其可能机制。
1、PAK1真核表达重组质粒的构建按照Genbank上公布的PAK1 mRNA序列(NM_002576)，在其编码区(CDS)设计两端带有EcoRI和BamHⅠ酶切位点的引物，利用聚合酶链式反应(Polymerase chain reaction，PCR)扩增PAK1 CDS区，EcoRI和BamHⅠ双酶切纯化的PCR产物和真核表达载体pEGFP／C1，T_4连接酶连接酶切纯化后的PCR产物和酶切纯化后的pEGFP／C1，转化到大肠杆菌DH5a中，挑单菌落培养扩增，提取重组质粒后行EcoRI和BamHⅠ双酶切鉴定，选择插入片段正确的克隆测序鉴定。
2、PAK1 shRNA重组载体的构建根据siRNA设计的原则，在PAK1 mRNA(NM_002576)CDS设计三对siRNA核苷酸序列，并设计一对非相关核苷酸序列作为对照，经Genbank在线BLAST确定确定与其它人类基因无高度同源后，分布在所设计的siRNA两端分别加上带有BamHⅠ和EcoRI酶切位点的shRNAs(short hairpin RNAs)单链送公司合成，配对单链核苷酸退火复性合成双链，按常规操作把shRNA序列克隆到pRNAT-U6.1／Neo载体中，重组质粒酶切和测序鉴定。
3、转染细胞和建立稳定转染细胞克隆采用Invintrogen公司的Lipofectamine2000~(TM)脂质体进行细胞转染。细胞转染48h后用分别用800μg／ml G418筛选阳性细胞克隆，挑取单克隆细胞扩增培养。
4、PAK1表达调控效应检测逆转录聚合酶链式反应(Reverse transcriptionpolymerase chain reaction，RT-PCR)和免疫印迹、免疫细胞化学与激光共聚焦显微镜检测PAK1 mRNA和蛋白质水平表达改变。
5、细胞增殖与凋亡MTT法检测PAK1对大肠癌细胞SW480增殖的影响；采用DNA片段法检测PAK1对5-Fu诱导的细胞凋亡的影响。
6、细胞基质粘附实验参照文献的方法研究PAK1对大肠癌细胞SW480粘附的影响。
7、单层细胞划痕愈伤实验分析单层细胞划痕愈伤实验分析PAK1对大肠癌细胞移行的影响，参照文献操作。
8、侵袭实验观察改变PAK1表达对大肠癌细胞侵袭能力的影响。
9、软琼脂糖集落形成实验观察RNAi沉默PAK1表达对SW480细胞锚着独立性生长的影响。
10．明胶酶谱实验观测PAK1对SW480细胞基质金属蛋白分泌和活性的影响。
11、裸鼠成瘤实验观察PAK1表达对SW480细胞裸鼠成瘤的影响，包括成瘤瘤体大小、侵袭转移部位差异。
三、统计学分析所得数据用SPSS12.0 for Windows软件，免疫组织化学采用等级资料多个独立样本和两个独立样本非参数检验；其它数据行单向方差分析(One-Way ANOVA)，组间多重比较用LSD方法进行分析，以均数±标准差(mean±SD)表示，以P≤0.05为有显著性差异。
结果
一、PAK1在良、恶性大肠病变中的表达及其临床意义
免疫组织化学检测结果表明PAK1主要在细胞胞浆表达，胞核偶有少量表达，而在大肠上皮细胞外基质中基本不表达(见图1-1和图1-2)；大肠正常粘膜和大肠癌癌旁正常组织粘膜中PAK1的表达极少，阴性、弱阳性、阳性和强阳性表达率为别为52.5％、49.1％与0.03％和0、48.7％与27％；大肠息肉PAK1的表达阴性、弱阳性、阳性和强阳性表达率分别为66.7％、22.2％与0和11.1％；大肠腺瘤组织PAK1的表达阴性、弱阳性、阳性和强阳性表达率为别为18.1％、45.4％与18.1％和27.2％；Dukes’A期大肠癌组织中阴性、弱阳性、阳性和强阳性率分别为8.4％、33.3％、25.0％和33.3％；Dukes’B期阴性、弱阳性、阳性和强阳性率分别为13.3％、13.3％、23.3％和50.0％；Dukes’C期阴性、弱阳性、阳性和强阳性率分别为16.7％、11.1％、16.7％和55.5％；Dukes’D期阴性、弱阳性、阳性和强阳性率分别为0、0、33.3％和66.7％。经等级资料多个独立样本非参数检验(Kruskal-Wallis检验)显示，大肠正常粘膜、大肠息肉、大肠腺瘤、大肠癌组织和大肠癌淋巴结和肝脏转移癌组织中的PAK1表达有显著性差异(P=0.000)，而且大肠正常粘膜、大肠息肉、大肠腺瘤、大肠癌组织和转移癌中的PAK1表达总体阳性率依次升高；不同Dukes’分期癌组织PAK1的表达不同，各组Dukes’分期癌组织PAK1的表达见图1-2和表1-4图，但各组PAK1表达无显著差异性(P=0.103)，分析原因主要可能与标本量少有关，其中A期和D期临床标本相对较少，收集较为困难。大肠癌高中低分化各组的PAK1表达的总体阳性率不同(74例大肠癌纳入研究)，有依次升高趋势，但各组PAK1表达无显著性差异(P=0.357)。
二、PAK1对大肠癌细胞表型的影响
1、PAK1真核表达载体pEGFP-C1／PAK1经酶切鉴定，所插入的片段与预期目的片段大小一致，并经测序证实，BLAST序列对比证实是PAK1基因mRNA CDS序列。
2．PAK1 shRNA真核表达载体经酶切和测序证实插入序列与设计序列完全一致；
3、重组质粒转染SW480经G418抗性筛选得到稳定细胞克隆株；
4、与对照细胞比较，RT-PCR和Western blotting从mRNA和蛋白水平证实SW480内源性PAK1被PAK1 shRNA抑制；
5、选取PAK1 shRNA中一个克隆细胞株用作后续实验，并把其细胞克隆命名为SW480~(shRNA1)，非相关序列细胞克隆命名为SW480~(shRNA-N)；PAK1真核载体细胞克隆株命名为SW480~(PAK1)，pEGFP／C1质粒对照细胞克隆命名为SW480~(svector)；
6、细胞增殖实验显示，在各组细胞孵育24h后，各组细胞间增殖有显著性差异，SW480、SW480~(vector)，SW480~(PAK1)，SW480~(shRNA-N)和SW480~(shRNA1)单因素方差分析结果F=11.006，P=0.000＜0.001；SW480~(shRNA1)细胞与SW480和SW480~(shRNA-N)比较结果分布是0.400±0.004 vs 0.532±0.0696，P=0.016＜0.05和0.400±0.004vs0.533±0.038，P=0.015＜0.05；SW480~(PAK1)与SW480和SW480~(vector)相比较的结果是0.719±0.125 vs 0.532±0.070，P=0.002＜0.01和0.719±0.125 vs0.530±0.039，P=0.001＜0.01)，结果说明下调SW480细胞PAK1表达，细胞增殖受到抑制，而上调SW480细胞的表达则促进细胞增殖。
7、细胞凋亡实验显示，未用5-FU处理的各组细胞培养8h均未检测到凋亡DNAladder；细胞分别用15.0μmol／ml 5-FU处理8h后，SW480~(shRNA1)可以见到明显的凋亡DNA ladder，而SW480、SW480~(vector)、SW480~(PAK1)和SW480~(shRNA-N)未检测到凋亡DNA ladder；在用30.0μmol／ml 5-FU处理8h后，SW480和SW480~(vector)可检测到DNA碎片，但SW480~(PAK1)未检测到凋亡，提示PAK1有抑制细胞凋亡的作用，PAK1可以增加大肠癌细胞对化疗药物5-Fu的敏感性；
8、细胞-基质粘附实验结果显示各组细胞之间粘附细胞明显不同，SW480~(shRNA1)细胞显著减少，与SW480细胞相比(P=0.015)和与SW480~(shRNA-N)相比较P=0.018)，而SW480细胞与SW480~(shRNA-N)细胞之间比较无显著性差异；SW480~(PAK1)与SW480相比(P=0.004)和与SW480~(vector)相比(P=0.004)，细胞粘附显著增加。
9通过软琼脂糖克隆形成实验可以观察细胞锚定独立性生长的能力，从而反映细胞的恶性程度。我们发现SW480细胞转染PAK1基因后，克隆形成率明显高于亲本细胞，表明细胞恶生程度增高，预示细胞侵袭能力的增加；而转染shRNA沉默PAK1表达后，SW480细胞在体外双层琼脂克隆形成能力减弱。
10、SW480、SW480~(vector)、SW480~(PAK1)、SW480~(shRNA-N)和SW480~(shRNA1)在划痕损伤后0h未有明显差异；在24后SW480、SW480~(vector)、SW480~(PAK1)和SW480~(shRNA-N)细胞划痕边缘有细胞移动，而SW480~(shRNA1)细胞划痕边缘整齐未见明显细胞移动；在24后，SW480~(PAK1)细胞较SW480、SW480~(vector)和SW480~(shRNA-N)细胞划痕面积明显缩小，而SW480~(shRNA1)细胞划痕边缘整齐，划痕面内未见明显细胞移动；在72h后，SW480、SW480~(vector)、SW480~(PAK1)和SW480~(shRNA-N)细胞划痕面内细胞都增多，SW480~(PAK1)细胞划痕面内被细胞铺满，而下调PAK1表达的SW480~(shRNA1)细胞划痕边缘稍有模糊，划痕面内几乎见不到细胞。
11、细胞侵袭实验显示SW480~(shRNA1)细胞显著降低。而SW480~(PAK1)细胞的侵袭能力增加。
12明胶酶谱实验显示，与对照细胞相比，SW480~(PAK1)细胞分泌活性MMP2增加，而SW480~(shRNA)细胞分泌活性MMP2减少，而MMP9活性未见有明显改变
13裸鼠皮下成瘤实验发现接种SW480~(shRNA)细胞和SW480~(shRNA-N)细胞后，裸鼠移植瘤体重量分别为(0.1075±0.06571)g和(0.9517±0.72323)g，RNA沉默组瘤体重量显著低于对照组(P＜0.001)。
结论
1、免疫组织化学检测显示PAK1表达从腺瘤到肿瘤、从原发大肠癌到转移大肠癌依次增加，提示PAK1在大肠癌发生、发展和转移中起重要作用。
2、上调PAK1表达可以促进大肠癌细胞SW480增殖，下调PAK1表达则可以抑制大肠癌细胞SW480的增殖；
3、PAK1可以增强大肠癌细胞的异质粘附，下调PAK1表达则抑制SW480细胞的异质粘附；
4、PAK1增强大肠癌细胞的非锚定式依赖式生长，下调PAK1表达则抑制大肠癌细胞的集落形成能力；
5、PAK1增强大肠癌细胞移行能力，下调PAK1表达则明显抑制大肠癌细胞的移行能力；
6、PAK1增强大肠癌细胞对基质的侵袭能力，下调PAK1表达则可以减弱大肠癌细胞的侵袭力；
7、PAK1可以抗5-Fu诱导的大肠癌凋亡，下调PAK1表达则有促进凋亡作用，抑制PAK1表达可以增强大肠癌细胞对5-Fu的敏感性；
8、PAK1可以增强大肠癌细胞基质金属蛋白酶2的活性，提示PAK1增加大肠癌细胞的转移潜能；
综上，通过临床病理标本、细胞模型到动物模型实验结果显示PAK1在大肠癌病程演进中发挥正向调控作用，而且正向调控大肠癌细胞粘附、侵袭和移动能力，PAK1正向调控大餐癌细胞转移潜能与其影响MMP-2活性有关，下调PAK1表达可以增加大肠癌细胞对化疗药物的敏感性，所以PAK1有希望成为大肠癌治疗的分子靶点，有必要继续深入研究其在大肠癌演进和转移中具体作用机制。
Background and objectives
Colorectal cancer(CRC) is one of the major malignancies in the world. The prognosis of CRCs is poor, due to frequent metastasis and tumor recurrence. Worldwide almost one million new cases occur annually, amounting to 492000 related deaths~[1-3]. With the many changes having taken place in people's diet and lifestyle, CRCs has become the third most common type of digestive tumor in China, and the number of new cases arising each year is still increasing. The overall incidence is identical in men and women, with the risk beginning at age 40 and increasing with age. Thus colorectal cancer ranks as the frequent cause of cancer deaths among China. Despite the rate of improvements in surgery, radiotherapy and chemotherapy, unfortunately, the prognosis of CRCs has not been gained progress over the past decades, with an overall five-year survival rate of around 40%- 50%~[4]. Therefore, novels diagnose and treatments need to be developing in order to enrich the therapeutic armamentarium. In recent years, molecular biology has applied to the study of colonic carcinoma, both in the human and in the experimental animal. The data obtained have enriched our understanding of colonic carcinogenesis and are of potential interest for CRCs diagnosis and prevention.
PAK1 (P21-activated kinase-1), a member of a family of serine/threonine protein kinase, was the first PAK to be cloned~[5] . Moreover, PAK1, which plays an essential role in embryonic development and tissue growth, and is also necessary for the spread and growth of tumor cells, such as breast and ovarian cancers~[6]. PAK1 is a direct target of the small GTPases Cdc42 and Racl, and binding of GTPases to PAK1 stimulates its kinase activity via autophosphorylation[7]. PAK1 contains an N-terminal regulatory domain and a C-terminal kinase domain, its N-terminal regulatory domain contains GTPase binding domain to mediate the binding of PAK1 to Rac/Cdc42~[8]. Among normal tissues, PAK1 is highly expressed in the brain, muscle, and spleen. Accumulating evidence indicates that PAK1 is important for a variety of cellular functions including cell morphogenesis, motility, survival, mitosis, cell cycle and angiogenesis. PAK1 are involved in the regulation of cellular function via phosphorylating a number of downstream target protein. Many evidences showed that Pakl activation occurs during the process of tumorigenesis, and PAK1 is likely to provide insight into the role of PAKs in human cancers. Despite the recent reports of the involvement of PAK1 in signaling cascades in human cancer cells and the fact of PAK1 is downstream of the small GTPases, Cdc42 and Rac1, the relationship of PAK1 to malignant progression of CRCs and the role of the PAK1 pathway in the biology of human colorectal cancer cells remain unknown. In this study, detect PAK1 expression in colon cancer patients and colorectal cancer cell lines and its molecular mechanism of action in transfected colorectal cancer cell to provide basis for further underscoring the link between PAK1 expression and CRCs progression..
Material and methods
The potential role of PAK1 protein expression in colorectal carcinomas
Immunohistochemistry of normal, benign colon polypus, colon adenoma, primary, and metastatic human tumor specimens was to detect the relationship of PAK1 expression with the pathological features. The study-included patients with colorectal carcinoma, according to the Dukes classification of malignant tumors, were divided as Dukes A, Dukes B, Dukes C, and Dukes D. Formalin-fixed paraffin sections were stained for PAK1 (1: 200 dilutions) using the Streptavidin-preoxidase (SP) technique. Antigen retrieval was achieved by microwave treatment with citrate buffer at pH 6.0 at 95℃for10 min. The immunohistochemical staining was scored in the following grades according to the percentage of positive cells:0,＜5% positive; 1, 5% to 25% positive; 2, 26% to 50% positive; and 3,＞51% positive. Morphological and immunohistochemical results were to correlate with clinicopathologic parameters.
The influences on the phenotypes of SW480 cell mediated by changing PAK1 expression
1. Cell Culture SW480(ATCC) cells were maintained in RPMI-1640(Gibco) supplemented with 10% fetal bovine serum (FBS) (Hycone), penicillin (100units/ml), and streptomycin(100mg/ml)). Cells were passaged using 0.25%Trypsin-EDTA and maintained in culture for 48h before performing experiments.
2. Construction recombinant eukaryotic PAK1 expression vector Primers, targeted to PAK1 (GenBank: NM_002576) code sequence(CDS) and containing EcoRⅠand BamHⅠsites at the ends, were designed. The polymerase chain reaction(PCR) products of CDS and pEGFP-C1 were both digested with EcoRⅠand BamHⅠand subsequently ligated for 4h at 16℃using T4 ligase. The recombinant plasmids was digested with EcoRⅠand BamHⅠfollowed by electrophoresis with 1% agarose and sequence analysis to identify.
3. Construction of recombinant PAKI shRNA expression vector Designing three pairs of small interfering RNAs(siRNAs) of PAK1 mRNA(GenBank: NM_002576), and six corresponding single-strand short hairpin RNAs(shRNAs), containing BamHⅠand HindⅢsites and 9nt hairpin structure, were synthesized and annealed. The annealed products and the linear pRNAT6.1/Neo plasmid, digested with BamHⅠand HindⅢ, were ligated for 4h at 16℃using T4 ligase. The recombinant plasmids were digested with BamHⅠand HindⅢfollowed by electrophoresis with agarose and sequence analysis to identify.
4. Transfection and selection of stably transfected cell clones The recombinant plasmids and control plasmids were transfected into SW480 cells using lipofectamine2000~(TM) reagent (Invitrogen) according to the manufacturer's protocol. Forty-eight hours after the addition of DNA, the transfected cells were selected in growth medium containing 0.8 mg/ml Geneticin (G418; Life Technologies, Inc.). Colonies resistant to G418 were isolated. After 3-4 wk of culture, visible colonies were picked up and expanded. The stably transfected clone cells were observed to show green fluorescence under fluorescent microscope and the clones without expression of the transfected gene did not show green fluorescence
5. PAK1 exprssion detecting The expression changes of PAK1 were detected using reverse transcription polymerase chain reaction (RT-PCR), western-blotting, and immunofluorescence staining at mRNA and protein levels.
6. Cell proliferation and apoptosis To observe the changes of cell growth after regulating expression of PAK1, cell proliferation was analyzed with MTT assay and 5-Fu induced cell apoptosis was detected by DNA fragment assays.
7. Cell-matrix adhesion analysis Cell-matrix adhesion assays were employed, as previously described~[9, 10]
8. Cell migration assays Migration of SW480, PAK1-overexpressng SW480(SW480~(PAK1)) and PAK1-lowexpressing SW480(SW480~(shRNA1)) cells was studied using 6.5mm Transwell chambers with 8μl pores (Costar) as previously described~[11].
9. Cell culture wound healing assay~[12]. Wounds were created in confluent cells using a pipette tip. The cells were then washed with medium to remove any free-floating cells and debris, and culture plates were incubated at 37℃. Wound healing was observed at 0, 24, 48, and 72 hours within the scrape line, and representa- tive scrape lines for each cell line were photographed.
10. Cell invasion assays Cell invasion assays were performed as described for the cell migration assays, except that the Transwell filters were additionally coated on the upper side with 30 mg of Matrigel.
11 Anchorage-independent growth Assays Anchorage-independent growth in soft agar was performed as described previously~[13]. In brief, the cells (10~5) were plated on 60-mm dishes. After 21 days, colonies were scored after staining the dishes with 0.5 mg/ml 3-(4, 5-dirnethylthiazol-2-y1)-2, 5-diphenyltetrazolium bromide (Sigma) overnight at 37℃.
12. Gelatin zymography~[14] Cells were plated at a density of 2×10~5 in 24-well plates. After 16 hours, cells washed with 0.01M PBS and incubated in 500μl of serum-free medium for 24 hours, were performed as described previously.
13. Tumorigenesis in nude mice Cells were suspended at the density of 2×10~7 cells in 200μl of RPMI-1640, and injected subcutaneously into the flank region of athymic nude mice. Twelve mice were to distribute to PAK1-shRNA group, vector control group, 6 per group at random. The animals were to inspecte at regular intervals for the appearance of visible tumors to measure the time of first appearance. All animals were observed for up to 30 days following the injection, the mice were sacrificed and the tumors were carefully removed by blunt dissection. The tumors were to weigh and their average growth rates were measured.
Statistical analysis
For immunohistochemistry results, Kruskal-Wallis Test was used, and the results of RT-PCR, Western blotting and MTT assays were expressed as mean±SD of at least three separate experiments, were analyzed by one-way analysis of variance (ANOVA). P values of=0.05 was considered statistically significant.
Results
1. PAK1 expression increases during human eolorectal cancer progression and metastasis. Immunohistochemistry was performed to examine PAK1 expression levels in paraffin-embeded tissue from normal colon mucosa, benign polypi and adenomas to primary colon cancers and metastasis colon cancers, which is a typical progression pathway during colon carcinogenensis. Representative shows that PAK1 expression is negative in benign polypi, begins to increase in adenoma but is still weak compared to the carcinoma, and over-expressed in primary colon adenocarcinoma but is absent or barely detectable in paired normal mucosa beside the cancer (P＜0.001). Furthermore, PAK1 expression is extremely much higher in lymph node metastasis and liver metastasis in contrast to primary colon carcinoma. However, the expression of PAK1 was not correlated with the histological differentiation(P.＞0.05) and Dukes' classification (P＞0.05).
2. The effects of changing PAK1 expression on cellular biological activity of SW480 cell.
(1) Constructed human PAK1 eukaryotic expression vector and human PAK1 hairpin siRNA eukaryotic expression vectors successfully.
The DNA oligonucleotides encoding PAK1 mRNA and short haipin siRNA against PAK1 were synthesized, and cloned to construct recombinant plasmid respectively, which were identified by restriction enzyme digestion analysis and DNA sequencing.
(2) Geneticin-resistant cell lines were screened after the recombinant plasmids were transfected into SW480 cells.
Following to the instruction of lipofectamine~(TM)2000 the transfection were performed, plasmids served as negative control, SW480 as blank control. The RT-PCR results showed that mRNA level of PAK1 in screened transfected cell lines SW480~(shnRNA1)was lowest, mRNA of PAK1 in transfected cell line SW480~(shRNA2-3)was significantly decreased also, its were confirmed in protein level by Western Blotting.
Six kinds of selected Geneticin-resistant colonies were respectively named as: SW480/pEGFP/C1-PAK1 (to abbreviate SW480~(PAK1)), SW480/pEGFP/C1(to abbreviate SW480~(Vector)), SW480/pRNAT-U6.1/Neo-PAK1~(shRNA1-3)(to abbreviate non specific 8W480 ~(shRNA1-3)), and SW480/pRNAT-U6.1/Neo-PAK1~(non-specific) construct (to abbreviate SW480~(shRNA-N)) of PAK1. This laid sound basis for the following research of PAK1.
(3)After 24h incubation, there were notable differences compared the growth velocity of SW480 cells in the group of 5W480~(vector), 5W480~(PAK1), SW480~(shRNA-N)and 8W480~(shRNA1) (F=11.006, P=O.O00＜0.001). The growth velocity of SW480~(shRNA1) was reduced obviously to contrast with SW480和SW480~(shRNA-N)(0.400±0.004 vs 0.532±0.0696, P= 0.016＜0.05; 0.400±0.004 vs0.533±0.038, P= 0.015＜0.05)., and 5W480~(PAK1) was increased significantly to compare with SW480 and SW480~(vector)(0.719±0.125 vs 0.532±0.070, P=0.002＜0.01; 0.719±0.125 vs0.530±0.039, P=0.001＜0.01).
(4) Compared with SW480和SW480~(shRNA-N), treatment with 5-Fu(15μg/ml) for 12h resulted in a significant increase of apoptosis in SW480~(shRNA1) cells, no significant apoptosis was observed in SW480~(PAK1) cells induced with 5-Fu (30μg/ml) compared with with SW480 and 8W480~(vector) cells.
(5) Alloplasm adhesiveness (cell-matrix adhesion) tests showed that there was a significant difference between the adhesive cell numbers of these three cell lines. The adhesive cell number of SW480~(shRNA1) was significantly less than that of SW480 (P＜0.05) and SW480~(shRNA-N) (P＜0.001), while the last two groups had no significant difference, suggesting that compared with SW480 and SW480~(shRNA-N), the cell-matrix adhesiveness of 5W480~(shRNA1) was decreased notably. As well as the adhesive cell number of SW480~(PAK1) was significantly more than that of SW480 (P＜0.001) and SW480~(vector) (P＜0.001),
(6) A monolayer wound-healing assay revealed almost no migration in SW480~(shRNA) cells compared with SW480~(control) cells, but is not of SW480~(PAK1), and Cell migration assays showed homoioplastic result too.
(7) Using a Boyden chamber invasion assay, we observed a significant decrease in the invasive capacity of SW480~(shRNA) cells, but increace of SW480~(PAK1).
(8)A gelatin zymogram for MMP activation demonstrated a decrease in MMP-2 activity in SW480~(shRNA1) compared with SW480 ~(shRNA-N) cells. However, a significant decrease of MMP- 9 activity was not observed.
(9)Tumorigenicity assay showed the tumor weight of SW480~(shRNA) group(0.1075±0.06571)g were less than control group (0.9517±0.72323) (P＜0.001). No lymph node and distant organ metastasis were found in all tumors.
Conclusions
Taken to gether, the results of immunohistochemistry show that PAK1 expression is increased with progression through the adenoma to carcinoma sequence, with the most dramatic increases in invasive and metastatic CRCs, suggesting PAK1 might play an important role in colorectal tumorigenesis. With molecular biology technology, cell biology technology, and RNA interference technique, the role of PAK1 in cell-matrix adhesion, cell migration, cell proliferation and cell apoptosis was studied in the model colorectal cancer SW480 cells. Experimental results indicate that PAK1 plays an important role in regulation of cell-matrix adhesion, cell migration and cell apoptosis in colorectal cancer cells and further has an effect on tumor invasion and metastasis. Increased expression of PAK1 mainly helps colorectal cancer metastasis in the distance, enhances the ability of anti-apoptosis in colorectal cancer SW480 cells and has significant effects to promote proliferation of SW480 cells. It not only provide the basis for the further study in the mechanisms of colorectal cancer invasion and metastasis but also suggest that signal pathway mediated by PAK1- targeted for therpeutic intervention in colorectal cancer. These data implicate PAK1 as an exciting target for therapy of colorectal carcinoma.
引文
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