p140Cap在结直肠癌增殖、侵袭和转移的作用
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摘要
背景和目的
p140Cap主要表达于大脑、睾丸以及富含上皮的组织如乳腺、肺、肾和大肠。关于p140Cap在人类肿瘤组织中表达的报道还比较少。近期一项乳腺癌的筛查中发现,在伴有淋巴结转移和高度增殖的肿瘤组织中,p140Cap的阴性表达率高达70%,说明p140Cap表达与肿瘤的恶性程度成负相关。另外一项研究结果显示,乳腺癌中Srcinl基因的mRNA表达水平与预后不良因素有一定的相关关系。目前对p140Cap对大肠癌的影响的研究甚少,本实验于基于临床标本,质粒过表达、RNAi干扰细胞株,裸鼠原位移植瘤,从组织水平、细胞水平以及体内水平研究pl40Cap对大肠癌增殖、凋亡、转移侵袭的影响。
材料和方法
1. p140Cap在癌旁和结肠癌组织中的表达：南方医院胃肠外科手术室收集23对肠癌组织和对应的癌旁组织,应用Western blot法检测p140Cap蛋白表达,GAPDH为内参。
2. p140Cap在癌旁和结肠癌组织中的表达：南方医院消化科内镜室收集126对以上同一患者结肠癌组织及癌旁正常组织(>5cm)标本,制作组织切片,应用免疫组化染色(IHC)检测p140Cap表达,判断阳性表达,并收集年龄、性别、肿瘤大小、肿瘤淋巴结转移、浆膜浸润、组织病理学分型、TNM分期等临床病理资料,Pearson χ2检验。
3. p140Cap在结肠癌转移淋巴结组织的表达：南方医院胃肠外科手术室收集25例结肠癌转移淋巴结组织,应用免疫组化染色(IHC)检测p140Cap表达。
4. p140Cap在结肠息肉表达：南方医院消化科内镜室收集38例大肠增生性息肉、42例腺瘤性息肉,制作石蜡切片,免疫组织p140Cap的表达,观察p140Cap在细胞表达部位和强度的差异。进行了p140Cap在大肠组织中表达分布差异分析,并与结肠癌阳性表达进行比较。
5. p140Cap在肠癌细胞系中的表达：收集南方医院实验室保存的8个LS174T, SW620, SW1116, LoVo, SW480, Caco-2, DLD1和HT29等肠癌细胞株,提取细胞总蛋白,应用Western blot方法检测p140Cap蛋白表达。
6.建立p140Cap稳定过表达细胞株(p140Cap-pcDNA3.1, pcDNA3.1为其阴性对照株)：化学合成全基因,酶切pcDNA3.1质粒载体,重组目的质粒p140Cap-pcDNA3.1, PCR验证并测序。lipofectamine2000稳定转染p140Cap-pcDNA3.1质粒到LoVo细胞,G418筛选,Western Blot验证。
7.建立瞬时敲低p140Cap表达细胞株(p140Cap-siRNA, siRNA为其阴性对照株)：lipofectamine2000瞬时转染p140Cap-siRNA到LoVo细胞,G418筛选,Western Blot验证。
8. p140Cap对结肠癌细胞侵袭转移的影响：划痕实验、侵袭小室实验检测过表达或敲低p140Cap细胞对侵袭转移能力的作用。
9.建立稳定敲低p140Cap表达细胞株(p140Cap-shRNA, shRNA为其阴性对照株)：以5MOI、10MOI、20MOI不同滴度的p140Cap-shRNA漫病毒分别感染LoVo细胞,荧光显微镜下观判断察感染效率,Western blot验证。
10. p140Cap对细胞增殖影响：WST-1实验和平板细胞克隆形成实验检测稳定过表达或敲低p140Cap细胞对细胞增长速度的作用。
11. p140Cap对细胞凋亡影响：流式细胞技术检测敲低p140Cap细胞对细胞凋亡作用,分析凋亡细胞比例。
12.建立裸鼠原位移植瘤模型以及评估5-Fu对原位移植瘤生长影响：分别将shRNA, p140Cap-shRNA两组细胞(5×106/0.1mL)皮下接种到5周龄雄性BALB/c-nu/nu裸鼠的右侧背部各10只,2周后原位移植瘤长至长径5mm左右时,予注射有感染p140Cap-siRNA或siRNA细胞的裸鼠腹腔注射5-Fu(20μg/g,每2天1次,共两周)或生理盐水(100μl/只,每2天1次,共3周)各五只。每两天观察生长情况,每周测量肿瘤体积,计算肿瘤生长抑制率,5周后取肿瘤组织HE染色。
13. p140Cap与Raf-MEK-ERK信号转导通路：Western blot分别检测p140Cap-shRNA或shRNA两组细胞ERK1/2、phospho-ERK1/2、MEK1、 phospho-MEK1的表达,并进一步检测凋亡相关蛋白Capase3、8、9、Bcl2,以及增殖相关蛋白CyclinD1、p21、p27的表达。
14.统计学分析：数据以X±s表示,采用SPSS16.0统计软件,率的比较采用Pearson x2或fisher检验,细胞增殖、凋亡、侵袭转移采用独立实验t检验,裸鼠肿瘤体积的比较采用单因素方差分析(one-way ANOVA)进行统计学处理,以P<0.05为差异具显著性。
结果
1. Western blot证实73.9%肠癌组织p140Cap表达高于对应癌旁组织(17/23)。
2. p140Cap在癌旁、结肠腺瘤和结肠癌组织以及结肠癌转移淋巴结中的表达：经免疫组化SP法染色的阳性物质为黄色颗粒,主要定位于细胞浆中。结肠癌组织p140Cap阳性表达率(评分大于4分,++/+++)在为85.7%(108/126)；癌旁正常组织p140Cap阳性表达率为12%(6/50),,结肠腺瘤p140Cap阳性率为38.1%(16/42),非肿瘤性息肉p140Cap阳性率为15.8%(6/38),结肠癌转移淋巴结p140Cap阳性率为100%(25/25)。经统计分析,结肠癌组织p140Cap表达阳性表达率高于癌旁组织(P<0.001)。
3.结肠癌组织中p140Cap表达与临床病理特征的关系：结肠癌p140Cap阳性表达率与性别、年龄、肿瘤位置无显著性差异(P<0.05)；与肿瘤大小、肿瘤淋巴结转移、浆膜浸润、组织病理学分型及TMN有显著性差异(P<0.05)。
4. p140Cap在肠癌细胞系中的表达：8株结直肠癌细胞系中p140Cap蛋白均有高或较高的表达水平,而293T不表达。
5.成功构建p140Cap-pcDNA质粒：化学合成的p140Cap基因片段重组到pcDNA3.1质粒,转化到感受态细胞,扩增后提取质粒,琼脂糖凝胶电泳验证分子量符合,基因测序测通成功。
6.成功建立p140Cap过表达细胞株：经过Western Blot验证筛选出的第7个单克隆LoVo (Clone7) p140Cap表达最高,命名为p140Cap-PcDNA3.1细胞株,其阴性对照pcDNA3.1质粒转染LoVo命名为pcDNA3.1细胞株,选择p140Cap表达最高的p140Cap-PcDNA3.1株,进行下一步研究。
7. siRNA干扰敲低LoVo细胞p140Cap表达：Western Blot检测发现,由3条p140Cap-siRNA瞬转的3株LoVo细胞p140Cap均被敲低,p140Cap-siRNA2感染株p140Cap最低,命名为p140Cap-siRNA株。
8. p140Cap促进对结肠癌细胞侵袭转移：划痕实验发现,p140Cap上调的细胞划痕48h后显微镜下观察划痕宽度变窄(P<0.01,三次独立试验)；侵袭小室实验发现,36h后p140Cap上调的细胞穿过小室的细胞增多(P<0.01,三次独立试验),同理p140Cap下调则相反。
9.建立稳定敲低p140Cap表达细胞株：利用慢病毒敲低p140Cap, p140Cap-shRNA感染LoVo,48h荧光显微镜下观查20MOI滴度p140Cap-shRNA感染率达到90%以上,Western blot证实p140Cap表达下调,命名为p140Cap-shRNA株。
10. p140Cap促进结肠癌细胞增殖：WST-1实验证实p140Cap上调促进细胞生长(第3,5,7天),平板细胞克隆形成实验显示p140Cap上调克隆数量增加P<0.01,三次独立试验)。p140Cap下调则相反。
11. p140Cap抑制结肠癌细胞凋亡：流式细胞技术检测显示,p140Cap-shRNA组的细胞凋亡率(Q2+Q3)高于对照组shRNA组(P<0.01,三次重复试验)。
12. p140Cap下调抑制裸鼠原位移植瘤生长,增加裸鼠对5-Fu化疗敏感性：接种细胞5周后,20只裸鼠无死亡,均有肿瘤生长,解剖后未发现其它肺、肝组织有转移结节,HE证实原位移植瘤为恶性肿瘤组织,显示敲低组较之对照组肿瘤坏死面积小。接种细胞5周后,测得各组种植瘤体积shRNA+生理盐水组(1485.2±224.7)、shRNA+5-FU组(595.5±117.3)、p140Cap-shRNA组+生理盐水组(581.4±139.7)(mm3)、p140Cap-shRNA+5-FU组(240.6±78.3)(mm3),经统计学分析,p140Cap-shRNA+生理盐水组,p140Cap-shRNA组+生理盐水组、p140Cap-shRNA+5-FU组三组肿瘤体积与shRNA+生理盐水组比差异有显著性(P<0.05, One-Way ANOVA)。
13. p140Cap参与Raf-MEK-ERK信号转导通路影响结肠癌细胞增殖和凋亡：p140Cap-shRNA组phospho-ERK1/2、phospho-MEK1的表达shRNA组细胞ERK1/2,MEK1的表达。p140Cap-shRNA组shRNA组细胞较的活性Capase3、8、9,p21、p27表达增高,Bcl2, CyclinD1表达降低。
结论
1.p140Cap在大肠癌组织高表达,并且与某些预后相关的大肠癌临床病理分型存在显著差异,可能影响大肠癌不良预后。
2. p140Cap在大部分大肠癌细胞浆高表达,少部分大肠腺瘤表达,正常组织极少表达,可能在大肠癌发展的过程中发挥促癌作用。
3. p140Cap在结肠癌细胞高表达,p140Cap促进肠癌细胞生长、转移和侵袭,抑制凋亡。
4. p140Cap下调表达抑制裸鼠原位移植瘤的生长,增加裸鼠对5-Fu药物敏感性。
5. p140Cap参与Raf-MEK-ERK信号转导通路,从而影响细胞增殖与凋亡。
6. p140Cap在在大肠癌的发生发展中表现为癌基因的特性,为预测大肠癌预后的一个新的分子标志物。
Background and Aim
p140Cap is mainly expressed in the brain, testis, and rich in the epithelial tissue, such as breast, lung, kidney and intestine. So far, there are few report about p140Cap, a recent report found it negatively associated with malignant degree of tumor, p140Cap negatively expression as high as70%in lymph node metastasis and high proliferated tumor tissues. In another study, Srcin1(p140Cap) mRNA expression level have a positively certain correlationand to poor prognosis. As soon as noww, therr is no research about p140Cap effecting on colorectal cancer. This paper based on clinical specimens, cell lines with plasmid transfection or RNAi interfere, nude mouse transplantation tumor in situ, we try to investigate proliferation, invasion. metastasis and invasiont effection with p140Cap on colorectal cancer at the level from tissue, cell and animal.
Methods
1. The expressions of p140Cap in the cancer tissues and perinunor normal colon tissues:23pairs of cancer tissues and perinunor normal colon tissues were the collected from operating room, department of gastrointestinal surgery, Nanfang hospital, the total protein of tissues were extracted, the expressions of p140Cap were detected by Western blot, GAPDH as the internal control.
2. The expressions of p140Cap in the cancer tissues and perinunor normal colon tissues:126pairs of cancer tissues and perinunor normal colon tissues (>5cm),25CRC lymph node metastasis tissues were the collected from endoscopy operating room, department of gastroenterology, Nanfang hospital. The samples were made to paraffin slices. The expressions of p140Cap were detected by immunohistochemical staining (IHC). Relationship between the expression and the clinical pathological data, such as age, gender, tumor location, tumor size, pathological grading, serosal invasion, lymph node metastasis and AJCC TNM stages were analysed by Pearson x2test (P<0.05was considered significant).
3. The expressions of p140Cap in colorectal metastasis lymph node tissues:20colorectal carcinoma metastasis lymph node were the collected from operating room, department of gastrointestinal surgery, Nanfang hospital. IHC staining was used to detect the expression of p140Cap in these samples.
4. The expressions of p140Cap in colon polyps:38non-adenomatous polyp and42adenomatous polyps were the collected from endoscopy operating room, department of gastroenterology, Nanfang hospital. The samples were made to paraffin slices and detected the expressions of p140Cap by IHC. cell area and the intensity d of Stained were recorded. The expressions of p140Cap distribution and variance in CRC and colon polyps were analyzed,
5. The expressions of p140Cap in colorectal cancer cell lines:eight CRC cells preserved in our laboratory, LS174T,, SW1116, LoVo, SW480SW620, Caco-2, DLD1, HT29were collected, the total protein of cells were extracted, the expressions of p140Cap were detected by Western blot.
6. Develop a stablyover-expression p140Cap cell lines:The aim gene synthesis was accomplished though chemical analysis. Recombinant aim plasmid was constructed by enzyme digestion of pcDNA3.1plasmid. over-expression p140Cap plasmid was identified by agarose gel electrophoresis and Gene sequencing. Restructured p140Cap-pcDNA3.1plasmid was stablely transfected into LoVo with with lipofectamine2000, and screened with G418. p140Cap was detected in transfected LoVo by Western Blot.
7. Establish a Instantaneously low-expression of p140Cap cell lines (p140Cap-siRNA, siRNA as negativ econtrol cells):p140Cap-siRNA was instantaneously transfected into LoVo cells with with lipofectamine2000, and screened with G418. p140Cap was detected in transfected LoVo by Western Blot.
8. p140Cap effects on cell invasion and metastasis:cell Wound Healing assay, Matrigel invasion chamber detection was applied to test cell invasion and metastasis.
9. Construction a stablely low-expression of p140Cap cell lines:(p140Cap-shRNA, shRNA as negative control) LoVo were transfected with viral titers of5MOI,10MOI,20MOI respectively. The efficiency of p140Cap-shRNA infection was judged by fluorescence microscope observation, also verificaed by Western blot.
10. p140Cap effects on cell proliferation:the growth of four groups of p140Cap-pcDNA3.1, pcDNA3.1, siRNA, siRNA cells was checked by WST-1experiment and flat cell clone forming test.
11. p140Cap Effects cell apoptosis:p140Cap-siRNA and siRNA cells were ananlysed by flow cytometry. The percent of apoptotic cells were compared.
12. Establish the nude mice orthotopic transplantation tumor model and to evaluate the effect of5-Furespectively, LoVo with shRNAor p140Cap-shRNA (5×106/0.2mL) were inoculated subcutaneously into the right neck in5week old male BALB/c-nu/nu nude mice. When the length diameter of orthotopic transplantation tumor reach to5mm in2weeks of transplantation, the nude mice was injected with5-Fu(20μg/g,1, every2days for3weeks) or saline (100μ, every2days for3weeks) by intraperitoneal injection. Weekly measurements of tumor volume, tumor growth inhibitory rate was calculated. Tunor tissues were stained HE after5weeks.
13. P140Cap with Raf-MEK-ERK signal transduction pathways:the expression ERK1/2, phospho-ERK1/2, MEK1and phospho-MEK1were detected by western blot in p140Cap-shRNA or shRNA cells. Further, the expression of cell apoptosis related proteins Capase3,8,9, Bcl2, and CyclinD1, p21and p27protein involved in cell proliferation were also detected.
14. Statistical assay:The data was demonstrated as x±s. SPSS16.0stastistic soft was used to analyse statistically these data. The comparison of the rate was analysed by Pearson x2or fisher test, cell proliferation, apoptosis, invasion and metastasis related independent experiment measured data was analysed by two-sample t test. Random unit group design data analysis of variance of the volume of nude mouse tumor was analysed by one-way ANOVA. There was significant difference as P<0.05.
Results
1. Western blot confirmed expression of p140Cap in73.9%colorectal tissue higher than the corresponding normal tissues adjacent to colorectal cancer (17/23).
2. he expressions of p140Cap in normal tissues adjacent to colorectal cancer, non-adenomatous colon polyps, colon adenoma and colon carcinoma:positive substance in the immunohistochemical SP staining showed yellow particles, mainly located in the cytoplasm, the p140Cap positive expression rate (score>4,++/+++) in colon cancer tissue was85.7%(108/126), the p140Cap positive expression rate in normal tissues adjacent to colorectal cancer was12%(6/50), the p140Cap positive expression rate in colonic adenoma was38.1%(16/42), the p140Cap positive expression rate in non-adenomatous colon polyps was15.8%(6/38), the p140Cap positive expression rate in colon cancer metastasis lymph nodes p140Cap positive rate was100%(25/25). The expression of p140Cap in colorectal cancer tissues and normal tissues adjacent to colorectal cancer was statistically significant (p<0.05).
3. The expression of pl40Cap relate to ccolorectal cancer linicopathological features: There is no significant difference there between p140Cap expression and age, gender, tumor location (p>0.05). There are statistical significanc between p140Cap expression and tumor size, pathological grading, serosal invasion, lymph node metastasis and AJCC TNM stages (p<0.05).
4. The expression of p140Cap in colon cancer cell lines:8strains colorectal cancer cell had a highexpression of140cap protein, and no expression in293T.
5. Obtain a plasmid with high-expression of p140Cap:the140cap gene fragment from chemical synthesis was linked to pcDNA3.1vector, then was transformed to competence cell. Plasmid was extracted after amplification, molecular weight was tested by the agarose gel electrophoresis, gene sequencing measured successfully.
6. Harvest a over-expression p140Cap cell line:a LoVo monoclonal cell line (Clone7) with a highest expression p140Cap was screened from seven clones, with a name of p140Cap-pcDNA3.1cell, and negtive control with a name of pcDNAS.1cell, cells transfected with recombinant p140Cap-pcDNA3.1plasmid were named as LoVo-p140Cap-PcDNA3.1cell line.
7. p140Cap knockdowned by siRNA interference:p140Cap was obviously knocked down instantaneously by one of three p140Cap-siRNA. Cell tranfected with siRNA2with a lowest expression of p140Cap was selected for research in the next steps.
8. p140Cap promoted the invasion and metastasis of colon cancer cell:in p140cap-pcDNA3.1cell compare to pcDNA3.1cell, scratch width (P<0.01, three independent experimets) was decreased in48h, and also cell numbers (P<0.01, three independent experimets) increased though the well of Matrigel invasion chambe in in36h under microscope obeservation. Similarly, of p140Cap-siRNA cell lines, we obtained the opposite results.
9. The establishment of stable p140Cap knockdown cell lines:Above90%LoVo transfected with viral titers p140Cap-shRNA of20MOI with a blue vision under fluorescence microscope. A downreglation of p140Cap was also verificaed by Western blot.
10. p140Cap boosted the proliferation of colon cancer cells:in the p140Cap-pcDNA3.1cells, WST-1experiment showesd the cell growth speed was accelerated (Day3, Day5, Day7), cell clones increased in cell clone formation experiment (P<0.01, three independent experimets). on the contrary, in p140Cap-shRNA cells, the opposite results were gained.
11. P140Cap inhibition of colon cancer cell apoptosis:flow cytometry technique test showed that p140Cap-shRNA group of apoptotic cells (Q2+Q3) rate is higher than the control shRNA group (P<0.01, three independent experimets).
12. p140Cap下调抑制裸鼠原位移植瘤生长,增加裸鼠对5-Fu化疗敏感性：接种细胞5周后,20只裸鼠无死亡,均有肿瘤生长,解剖后未发现其它肺、肝组织有转移结节,HE证实原位移植瘤为恶性肿瘤组织,显示敲低组较之对照组肿瘤坏死面积小。接种细胞5周后,测得各组种植瘤体积shRNA+生理盐水组(1485.2±224.7)、shRNA+5-FU组(595.5±117.3)、p140Cap-shRNA组+生理盐水组(581.4±139.7)(mm3)、p140Cap-shRNA+5-FU组(240.6±78.3)(mm3),经统计学分析,p140Cap-shRNA+生理盐水组,p140Cap-shRNA组+生理盐水组、p140Cap-shRNA+5-FU组三组肿瘤体积与shRNA+生理盐水组比差异有显著性(P<0.05, One-Way ANOVA)。
12. p140Cap Knockdown inhibits the growth of orthotopic transplantation tumo, increased5-Fu chemotherapy sensitivity in nude mice:All20nude mice grew with tumor. The autopsy showed no metastatic nodules in lung, liver or other. HE confirmed the orthotopic transplantation tumor was malignant tumor tissue, and showed that knockdown group had less tumor necrosis aress than in the control group. Five weeks after vaccination cells, Volume of tumor were measured, respectively, shRNA+saline(1485.2±224.7), shRNA+5-FU(595.5±117.3), p140Cap-shRNA+saline(581.4±139.7), p140Cap-shRNA+5-FU (240.6±78.3)(mm3). With the statistical analysis, there were significant difference there between shRNA+5-FU, p140Cap-shRNA+salin, p140Cap-shRNA+5-FU and shRNA+saline (as control group)(P<0.05, One-Way ANOVA).
13. p140Cap play a role in the Raf-EK-ERK signal transduction pathway affect cell proliferation and apoptosis of colon cancer cells:downregulaition of p140Cap has a higher expression of phospho-ERK1/2, phospho-MEK1shRNA ERK1/2, MEK1. Active Capase3,8,9, p21and p27expression was increased in p140Cap-shRNA cells, Bcl2and CyclinDl were reduced.
Discusion
1. The high expression of p140Cap in colorectal cancer, and high expression of p140Cap a is closely related to clinical pathology classification of colorectal carcinoma, may affect the prognosis of CRC.
2. High expression of p140Cap in most CRCs, weaker expression in fewer colorectal adenoma, rarely expressed in nomal tissues, p140Cap may be a promoted role in thr development of CRC.
3. High expression of p140Cap in colon cancer cells, p140Cap upregulation can promote cancer cell growth, apoptosis, invasion and metastasis.
4. Down-regulated expression of p140Cap inhibits orthotopic tumor growth in nude mice,140Cap also increases the sensitivity of5-Fu.
5. p140Cap acs on the Raf-EK-ERK signal transduction pathway promting cell proliferation and inhabitingcell apoptosis:
6. p140Cap palys a role of a cancer genes in the development of colorectal carcinoma, may zct as a new molecular marker for predicting the prognosis of CRC.
p140Cap主要表达于大脑、睾丸以及富含上皮的组织如乳腺、肺、肾和大肠。关于p140Cap在人类肿瘤组织中表达的报道还比较少。近期一项乳腺癌的筛查中发现,在伴有淋巴结转移和高度增殖的肿瘤组织中,p140Cap的阴性表达率高达70%,说明p140Cap表达与肿瘤的恶性程度成负相关。另外一项研究结果显示,乳腺癌中Srcinl基因的mRNA表达水平与预后不良因素有一定的相关关系。目前对p140Cap对大肠癌的影响的研究甚少,本实验于基于临床标本,质粒过表达、RNAi干扰细胞株,裸鼠原位移植瘤,从组织水平、细胞水平以及体内水平研究pl40Cap对大肠癌增殖、凋亡、转移侵袭的影响。
材料和方法
1. p140Cap在癌旁和结肠癌组织中的表达：南方医院胃肠外科手术室收集23对肠癌组织和对应的癌旁组织,应用Western blot法检测p140Cap蛋白表达,GAPDH为内参。
2. p140Cap在癌旁和结肠癌组织中的表达：南方医院消化科内镜室收集126对以上同一患者结肠癌组织及癌旁正常组织(>5cm)标本,制作组织切片,应用免疫组化染色(IHC)检测p140Cap表达,判断阳性表达,并收集年龄、性别、肿瘤大小、肿瘤淋巴结转移、浆膜浸润、组织病理学分型、TNM分期等临床病理资料,Pearson χ2检验。
3. p140Cap在结肠癌转移淋巴结组织的表达：南方医院胃肠外科手术室收集25例结肠癌转移淋巴结组织,应用免疫组化染色(IHC)检测p140Cap表达。
4. p140Cap在结肠息肉表达：南方医院消化科内镜室收集38例大肠增生性息肉、42例腺瘤性息肉,制作石蜡切片,免疫组织p140Cap的表达,观察p140Cap在细胞表达部位和强度的差异。进行了p140Cap在大肠组织中表达分布差异分析,并与结肠癌阳性表达进行比较。
5. p140Cap在肠癌细胞系中的表达：收集南方医院实验室保存的8个LS174T, SW620, SW1116, LoVo, SW480, Caco-2, DLD1和HT29等肠癌细胞株,提取细胞总蛋白,应用Western blot方法检测p140Cap蛋白表达。
6.建立p140Cap稳定过表达细胞株(p140Cap-pcDNA3.1, pcDNA3.1为其阴性对照株)：化学合成全基因,酶切pcDNA3.1质粒载体,重组目的质粒p140Cap-pcDNA3.1, PCR验证并测序。lipofectamine2000稳定转染p140Cap-pcDNA3.1质粒到LoVo细胞,G418筛选,Western Blot验证。
7.建立瞬时敲低p140Cap表达细胞株(p140Cap-siRNA, siRNA为其阴性对照株)：lipofectamine2000瞬时转染p140Cap-siRNA到LoVo细胞,G418筛选,Western Blot验证。
8. p140Cap对结肠癌细胞侵袭转移的影响：划痕实验、侵袭小室实验检测过表达或敲低p140Cap细胞对侵袭转移能力的作用。
9.建立稳定敲低p140Cap表达细胞株(p140Cap-shRNA, shRNA为其阴性对照株)：以5MOI、10MOI、20MOI不同滴度的p140Cap-shRNA漫病毒分别感染LoVo细胞,荧光显微镜下观判断察感染效率,Western blot验证。
10. p140Cap对细胞增殖影响：WST-1实验和平板细胞克隆形成实验检测稳定过表达或敲低p140Cap细胞对细胞增长速度的作用。
11. p140Cap对细胞凋亡影响：流式细胞技术检测敲低p140Cap细胞对细胞凋亡作用,分析凋亡细胞比例。
12.建立裸鼠原位移植瘤模型以及评估5-Fu对原位移植瘤生长影响：分别将shRNA, p140Cap-shRNA两组细胞(5×106/0.1mL)皮下接种到5周龄雄性BALB/c-nu/nu裸鼠的右侧背部各10只,2周后原位移植瘤长至长径5mm左右时,予注射有感染p140Cap-siRNA或siRNA细胞的裸鼠腹腔注射5-Fu(20μg/g,每2天1次,共两周)或生理盐水(100μl/只,每2天1次,共3周)各五只。每两天观察生长情况,每周测量肿瘤体积,计算肿瘤生长抑制率,5周后取肿瘤组织HE染色。
13. p140Cap与Raf-MEK-ERK信号转导通路：Western blot分别检测p140Cap-shRNA或shRNA两组细胞ERK1/2、phospho-ERK1/2、MEK1、 phospho-MEK1的表达,并进一步检测凋亡相关蛋白Capase3、8、9、Bcl2,以及增殖相关蛋白CyclinD1、p21、p27的表达。
14.统计学分析：数据以X±s表示,采用SPSS16.0统计软件,率的比较采用Pearson x2或fisher检验,细胞增殖、凋亡、侵袭转移采用独立实验t检验,裸鼠肿瘤体积的比较采用单因素方差分析(one-way ANOVA)进行统计学处理,以P<0.05为差异具显著性。
结果
1. Western blot证实73.9%肠癌组织p140Cap表达高于对应癌旁组织(17/23)。
2. p140Cap在癌旁、结肠腺瘤和结肠癌组织以及结肠癌转移淋巴结中的表达：经免疫组化SP法染色的阳性物质为黄色颗粒,主要定位于细胞浆中。结肠癌组织p140Cap阳性表达率(评分大于4分,++/+++)在为85.7%(108/126)；癌旁正常组织p140Cap阳性表达率为12%(6/50),,结肠腺瘤p140Cap阳性率为38.1%(16/42),非肿瘤性息肉p140Cap阳性率为15.8%(6/38),结肠癌转移淋巴结p140Cap阳性率为100%(25/25)。经统计分析,结肠癌组织p140Cap表达阳性表达率高于癌旁组织(P<0.001)。
3.结肠癌组织中p140Cap表达与临床病理特征的关系：结肠癌p140Cap阳性表达率与性别、年龄、肿瘤位置无显著性差异(P<0.05)；与肿瘤大小、肿瘤淋巴结转移、浆膜浸润、组织病理学分型及TMN有显著性差异(P<0.05)。
4. p140Cap在肠癌细胞系中的表达：8株结直肠癌细胞系中p140Cap蛋白均有高或较高的表达水平,而293T不表达。
5.成功构建p140Cap-pcDNA质粒：化学合成的p140Cap基因片段重组到pcDNA3.1质粒,转化到感受态细胞,扩增后提取质粒,琼脂糖凝胶电泳验证分子量符合,基因测序测通成功。
6.成功建立p140Cap过表达细胞株：经过Western Blot验证筛选出的第7个单克隆LoVo (Clone7) p140Cap表达最高,命名为p140Cap-PcDNA3.1细胞株,其阴性对照pcDNA3.1质粒转染LoVo命名为pcDNA3.1细胞株,选择p140Cap表达最高的p140Cap-PcDNA3.1株,进行下一步研究。
7. siRNA干扰敲低LoVo细胞p140Cap表达：Western Blot检测发现,由3条p140Cap-siRNA瞬转的3株LoVo细胞p140Cap均被敲低,p140Cap-siRNA2感染株p140Cap最低,命名为p140Cap-siRNA株。
8. p140Cap促进对结肠癌细胞侵袭转移：划痕实验发现,p140Cap上调的细胞划痕48h后显微镜下观察划痕宽度变窄(P<0.01,三次独立试验)；侵袭小室实验发现,36h后p140Cap上调的细胞穿过小室的细胞增多(P<0.01,三次独立试验),同理p140Cap下调则相反。
9.建立稳定敲低p140Cap表达细胞株：利用慢病毒敲低p140Cap, p140Cap-shRNA感染LoVo,48h荧光显微镜下观查20MOI滴度p140Cap-shRNA感染率达到90%以上,Western blot证实p140Cap表达下调,命名为p140Cap-shRNA株。
10. p140Cap促进结肠癌细胞增殖：WST-1实验证实p140Cap上调促进细胞生长(第3,5,7天),平板细胞克隆形成实验显示p140Cap上调克隆数量增加P<0.01,三次独立试验)。p140Cap下调则相反。
11. p140Cap抑制结肠癌细胞凋亡：流式细胞技术检测显示,p140Cap-shRNA组的细胞凋亡率(Q2+Q3)高于对照组shRNA组(P<0.01,三次重复试验)。
12. p140Cap下调抑制裸鼠原位移植瘤生长,增加裸鼠对5-Fu化疗敏感性：接种细胞5周后,20只裸鼠无死亡,均有肿瘤生长,解剖后未发现其它肺、肝组织有转移结节,HE证实原位移植瘤为恶性肿瘤组织,显示敲低组较之对照组肿瘤坏死面积小。接种细胞5周后,测得各组种植瘤体积shRNA+生理盐水组(1485.2±224.7)、shRNA+5-FU组(595.5±117.3)、p140Cap-shRNA组+生理盐水组(581.4±139.7)(mm3)、p140Cap-shRNA+5-FU组(240.6±78.3)(mm3),经统计学分析,p140Cap-shRNA+生理盐水组,p140Cap-shRNA组+生理盐水组、p140Cap-shRNA+5-FU组三组肿瘤体积与shRNA+生理盐水组比差异有显著性(P<0.05, One-Way ANOVA)。
13. p140Cap参与Raf-MEK-ERK信号转导通路影响结肠癌细胞增殖和凋亡：p140Cap-shRNA组phospho-ERK1/2、phospho-MEK1的表达shRNA组细胞ERK1/2,MEK1的表达。p140Cap-shRNA组shRNA组细胞较的活性Capase3、8、9,p21、p27表达增高,Bcl2, CyclinD1表达降低。
结论
1.p140Cap在大肠癌组织高表达,并且与某些预后相关的大肠癌临床病理分型存在显著差异,可能影响大肠癌不良预后。
2. p140Cap在大部分大肠癌细胞浆高表达,少部分大肠腺瘤表达,正常组织极少表达,可能在大肠癌发展的过程中发挥促癌作用。
3. p140Cap在结肠癌细胞高表达,p140Cap促进肠癌细胞生长、转移和侵袭,抑制凋亡。
4. p140Cap下调表达抑制裸鼠原位移植瘤的生长,增加裸鼠对5-Fu药物敏感性。
5. p140Cap参与Raf-MEK-ERK信号转导通路,从而影响细胞增殖与凋亡。
6. p140Cap在在大肠癌的发生发展中表现为癌基因的特性,为预测大肠癌预后的一个新的分子标志物。
Background and Aim
p140Cap is mainly expressed in the brain, testis, and rich in the epithelial tissue, such as breast, lung, kidney and intestine. So far, there are few report about p140Cap, a recent report found it negatively associated with malignant degree of tumor, p140Cap negatively expression as high as70%in lymph node metastasis and high proliferated tumor tissues. In another study, Srcin1(p140Cap) mRNA expression level have a positively certain correlationand to poor prognosis. As soon as noww, therr is no research about p140Cap effecting on colorectal cancer. This paper based on clinical specimens, cell lines with plasmid transfection or RNAi interfere, nude mouse transplantation tumor in situ, we try to investigate proliferation, invasion. metastasis and invasiont effection with p140Cap on colorectal cancer at the level from tissue, cell and animal.
Methods
1. The expressions of p140Cap in the cancer tissues and perinunor normal colon tissues:23pairs of cancer tissues and perinunor normal colon tissues were the collected from operating room, department of gastrointestinal surgery, Nanfang hospital, the total protein of tissues were extracted, the expressions of p140Cap were detected by Western blot, GAPDH as the internal control.
2. The expressions of p140Cap in the cancer tissues and perinunor normal colon tissues:126pairs of cancer tissues and perinunor normal colon tissues (>5cm),25CRC lymph node metastasis tissues were the collected from endoscopy operating room, department of gastroenterology, Nanfang hospital. The samples were made to paraffin slices. The expressions of p140Cap were detected by immunohistochemical staining (IHC). Relationship between the expression and the clinical pathological data, such as age, gender, tumor location, tumor size, pathological grading, serosal invasion, lymph node metastasis and AJCC TNM stages were analysed by Pearson x2test (P<0.05was considered significant).
3. The expressions of p140Cap in colorectal metastasis lymph node tissues:20colorectal carcinoma metastasis lymph node were the collected from operating room, department of gastrointestinal surgery, Nanfang hospital. IHC staining was used to detect the expression of p140Cap in these samples.
4. The expressions of p140Cap in colon polyps:38non-adenomatous polyp and42adenomatous polyps were the collected from endoscopy operating room, department of gastroenterology, Nanfang hospital. The samples were made to paraffin slices and detected the expressions of p140Cap by IHC. cell area and the intensity d of Stained were recorded. The expressions of p140Cap distribution and variance in CRC and colon polyps were analyzed,
5. The expressions of p140Cap in colorectal cancer cell lines:eight CRC cells preserved in our laboratory, LS174T,, SW1116, LoVo, SW480SW620, Caco-2, DLD1, HT29were collected, the total protein of cells were extracted, the expressions of p140Cap were detected by Western blot.
6. Develop a stablyover-expression p140Cap cell lines:The aim gene synthesis was accomplished though chemical analysis. Recombinant aim plasmid was constructed by enzyme digestion of pcDNA3.1plasmid. over-expression p140Cap plasmid was identified by agarose gel electrophoresis and Gene sequencing. Restructured p140Cap-pcDNA3.1plasmid was stablely transfected into LoVo with with lipofectamine2000, and screened with G418. p140Cap was detected in transfected LoVo by Western Blot.
7. Establish a Instantaneously low-expression of p140Cap cell lines (p140Cap-siRNA, siRNA as negativ econtrol cells):p140Cap-siRNA was instantaneously transfected into LoVo cells with with lipofectamine2000, and screened with G418. p140Cap was detected in transfected LoVo by Western Blot.
8. p140Cap effects on cell invasion and metastasis:cell Wound Healing assay, Matrigel invasion chamber detection was applied to test cell invasion and metastasis.
9. Construction a stablely low-expression of p140Cap cell lines:(p140Cap-shRNA, shRNA as negative control) LoVo were transfected with viral titers of5MOI,10MOI,20MOI respectively. The efficiency of p140Cap-shRNA infection was judged by fluorescence microscope observation, also verificaed by Western blot.
10. p140Cap effects on cell proliferation:the growth of four groups of p140Cap-pcDNA3.1, pcDNA3.1, siRNA, siRNA cells was checked by WST-1experiment and flat cell clone forming test.
11. p140Cap Effects cell apoptosis:p140Cap-siRNA and siRNA cells were ananlysed by flow cytometry. The percent of apoptotic cells were compared.
12. Establish the nude mice orthotopic transplantation tumor model and to evaluate the effect of5-Furespectively, LoVo with shRNAor p140Cap-shRNA (5×106/0.2mL) were inoculated subcutaneously into the right neck in5week old male BALB/c-nu/nu nude mice. When the length diameter of orthotopic transplantation tumor reach to5mm in2weeks of transplantation, the nude mice was injected with5-Fu(20μg/g,1, every2days for3weeks) or saline (100μ, every2days for3weeks) by intraperitoneal injection. Weekly measurements of tumor volume, tumor growth inhibitory rate was calculated. Tunor tissues were stained HE after5weeks.
13. P140Cap with Raf-MEK-ERK signal transduction pathways:the expression ERK1/2, phospho-ERK1/2, MEK1and phospho-MEK1were detected by western blot in p140Cap-shRNA or shRNA cells. Further, the expression of cell apoptosis related proteins Capase3,8,9, Bcl2, and CyclinD1, p21and p27protein involved in cell proliferation were also detected.
14. Statistical assay:The data was demonstrated as x±s. SPSS16.0stastistic soft was used to analyse statistically these data. The comparison of the rate was analysed by Pearson x2or fisher test, cell proliferation, apoptosis, invasion and metastasis related independent experiment measured data was analysed by two-sample t test. Random unit group design data analysis of variance of the volume of nude mouse tumor was analysed by one-way ANOVA. There was significant difference as P<0.05.
Results
1. Western blot confirmed expression of p140Cap in73.9%colorectal tissue higher than the corresponding normal tissues adjacent to colorectal cancer (17/23).
2. he expressions of p140Cap in normal tissues adjacent to colorectal cancer, non-adenomatous colon polyps, colon adenoma and colon carcinoma:positive substance in the immunohistochemical SP staining showed yellow particles, mainly located in the cytoplasm, the p140Cap positive expression rate (score>4,++/+++) in colon cancer tissue was85.7%(108/126), the p140Cap positive expression rate in normal tissues adjacent to colorectal cancer was12%(6/50), the p140Cap positive expression rate in colonic adenoma was38.1%(16/42), the p140Cap positive expression rate in non-adenomatous colon polyps was15.8%(6/38), the p140Cap positive expression rate in colon cancer metastasis lymph nodes p140Cap positive rate was100%(25/25). The expression of p140Cap in colorectal cancer tissues and normal tissues adjacent to colorectal cancer was statistically significant (p<0.05).
3. The expression of pl40Cap relate to ccolorectal cancer linicopathological features: There is no significant difference there between p140Cap expression and age, gender, tumor location (p>0.05). There are statistical significanc between p140Cap expression and tumor size, pathological grading, serosal invasion, lymph node metastasis and AJCC TNM stages (p<0.05).
4. The expression of p140Cap in colon cancer cell lines:8strains colorectal cancer cell had a highexpression of140cap protein, and no expression in293T.
5. Obtain a plasmid with high-expression of p140Cap:the140cap gene fragment from chemical synthesis was linked to pcDNA3.1vector, then was transformed to competence cell. Plasmid was extracted after amplification, molecular weight was tested by the agarose gel electrophoresis, gene sequencing measured successfully.
6. Harvest a over-expression p140Cap cell line:a LoVo monoclonal cell line (Clone7) with a highest expression p140Cap was screened from seven clones, with a name of p140Cap-pcDNA3.1cell, and negtive control with a name of pcDNAS.1cell, cells transfected with recombinant p140Cap-pcDNA3.1plasmid were named as LoVo-p140Cap-PcDNA3.1cell line.
7. p140Cap knockdowned by siRNA interference:p140Cap was obviously knocked down instantaneously by one of three p140Cap-siRNA. Cell tranfected with siRNA2with a lowest expression of p140Cap was selected for research in the next steps.
8. p140Cap promoted the invasion and metastasis of colon cancer cell:in p140cap-pcDNA3.1cell compare to pcDNA3.1cell, scratch width (P<0.01, three independent experimets) was decreased in48h, and also cell numbers (P<0.01, three independent experimets) increased though the well of Matrigel invasion chambe in in36h under microscope obeservation. Similarly, of p140Cap-siRNA cell lines, we obtained the opposite results.
9. The establishment of stable p140Cap knockdown cell lines:Above90%LoVo transfected with viral titers p140Cap-shRNA of20MOI with a blue vision under fluorescence microscope. A downreglation of p140Cap was also verificaed by Western blot.
10. p140Cap boosted the proliferation of colon cancer cells:in the p140Cap-pcDNA3.1cells, WST-1experiment showesd the cell growth speed was accelerated (Day3, Day5, Day7), cell clones increased in cell clone formation experiment (P<0.01, three independent experimets). on the contrary, in p140Cap-shRNA cells, the opposite results were gained.
11. P140Cap inhibition of colon cancer cell apoptosis:flow cytometry technique test showed that p140Cap-shRNA group of apoptotic cells (Q2+Q3) rate is higher than the control shRNA group (P<0.01, three independent experimets).
12. p140Cap下调抑制裸鼠原位移植瘤生长,增加裸鼠对5-Fu化疗敏感性：接种细胞5周后,20只裸鼠无死亡,均有肿瘤生长,解剖后未发现其它肺、肝组织有转移结节,HE证实原位移植瘤为恶性肿瘤组织,显示敲低组较之对照组肿瘤坏死面积小。接种细胞5周后,测得各组种植瘤体积shRNA+生理盐水组(1485.2±224.7)、shRNA+5-FU组(595.5±117.3)、p140Cap-shRNA组+生理盐水组(581.4±139.7)(mm3)、p140Cap-shRNA+5-FU组(240.6±78.3)(mm3),经统计学分析,p140Cap-shRNA+生理盐水组,p140Cap-shRNA组+生理盐水组、p140Cap-shRNA+5-FU组三组肿瘤体积与shRNA+生理盐水组比差异有显著性(P<0.05, One-Way ANOVA)。
12. p140Cap Knockdown inhibits the growth of orthotopic transplantation tumo, increased5-Fu chemotherapy sensitivity in nude mice:All20nude mice grew with tumor. The autopsy showed no metastatic nodules in lung, liver or other. HE confirmed the orthotopic transplantation tumor was malignant tumor tissue, and showed that knockdown group had less tumor necrosis aress than in the control group. Five weeks after vaccination cells, Volume of tumor were measured, respectively, shRNA+saline(1485.2±224.7), shRNA+5-FU(595.5±117.3), p140Cap-shRNA+saline(581.4±139.7), p140Cap-shRNA+5-FU (240.6±78.3)(mm3). With the statistical analysis, there were significant difference there between shRNA+5-FU, p140Cap-shRNA+salin, p140Cap-shRNA+5-FU and shRNA+saline (as control group)(P<0.05, One-Way ANOVA).
13. p140Cap play a role in the Raf-EK-ERK signal transduction pathway affect cell proliferation and apoptosis of colon cancer cells:downregulaition of p140Cap has a higher expression of phospho-ERK1/2, phospho-MEK1shRNA ERK1/2, MEK1. Active Capase3,8,9, p21and p27expression was increased in p140Cap-shRNA cells, Bcl2and CyclinDl were reduced.
Discusion
1. The high expression of p140Cap in colorectal cancer, and high expression of p140Cap a is closely related to clinical pathology classification of colorectal carcinoma, may affect the prognosis of CRC.
2. High expression of p140Cap in most CRCs, weaker expression in fewer colorectal adenoma, rarely expressed in nomal tissues, p140Cap may be a promoted role in thr development of CRC.
3. High expression of p140Cap in colon cancer cells, p140Cap upregulation can promote cancer cell growth, apoptosis, invasion and metastasis.
4. Down-regulated expression of p140Cap inhibits orthotopic tumor growth in nude mice,140Cap also increases the sensitivity of5-Fu.
5. p140Cap acs on the Raf-EK-ERK signal transduction pathway promting cell proliferation and inhabitingcell apoptosis:
6. p140Cap palys a role of a cancer genes in the development of colorectal carcinoma, may zct as a new molecular marker for predicting the prognosis of CRC.
引文
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[34]Klemke RL, Leng J, Molander R, Brooks PC, Vuori K, Cheresh DA. CAS/Crk Coupling Serves as a "Molecular Switch"for Induction of Cell Migration. J of Cell Biol,1998; 140(4):962
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