Effects Of P140Cap On Proliferation, Invasion And Metastasis In Colorectal Cancer

Background and Aimp140Cap 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.Methods1. 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.Results1. 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.