| Objective1、To observe the expression of Protein interacting with Ca kineasel(PICK1) in the nocarcinoma tissues, colorectal polyp, colorectal adenoma and colorectal adenocarcinoma. And to understand its significance in tumors development procession.2、To explore the effect of siRNA targeting PICK1on cell proliferation and the cell invasive potential of HT-29.MethodsThe expression of PICK1in40nocarcinoma tissues,36colorectal polyp,36colorectal adenoma and102colorectal adenocarcinoma were detected by immunohistochemical technique. Western blot was used to detect protein expression of the PICK1in20nocarcinoma tissues and20colorectal adenocarcinoma. And the correlation between PICK1and clinical pathological data was explored. PICK1-siRNA was tansfected into HT-29cell. And we observed the transfection efficiency using fluorescence microscopy. Western Blot and Immunocytochemistry were used to detected protein respectively. With the use of Transwell invasion assay, the invasive property of HT-29cell was detected.Results1A total of74(72.5%) patients had varying expression of PICK1, of whom31(30.4%) had overexpression in colorectal adenocarcinoma. The ratios of high expression level of PICK1in colorectal adenoma tissues (22.2%,8/36) were significantly higher than that in the adjacent tissues (0.0%,0/40)(P<0.05). The ratios of high expression level of PICK1in colorectal polyp tissues(5.6%,2/36) were no statistically difference with the adjacent tissues(P>0.05).In adenoma canceration organization, expression of PICK1protein in cancer area was higher than adenoma area.2In colorectal adenocarcinoma tissues, expression of PICK1protein in infiltration front of tumor budding was significantly higher than in tumor body. Expression of PICK1in the same colorectal adenocarcinoma transferred to the lymph nodes was higher than the primary site of cancer cells.3It showed significant correlation with the tumor location, the degree of differentiation, depth of invasion, TNM stages and lymph metastasis(P<0.05). However, no correlation was revealed between PICK1expression and the age, gender(P>0.05).4Western Blot analysis revealed that the expression of PICK1in colorectal adenocarcinoma (1.10±0.04) was significantly higher than that in the adjacent tissues(0.75±0.07)(P<0.05).5Under the fluorescence microscope, the green fluorescence with the cells could be seen after transfection, suggesting the transfection was successful. The transfection efficiency was90percent.6After48hours of transfection, Western Blot analysis showed that the expression of PICK1in PICK1-siRNA group(0.11±0.04) was significantly lower than that in the negative control group(0.34±0.03) and blank group(0.36±0.02). The inhibition rate PICK1protein was69.4percent(P<0.05). Immunocytochemical assay showed that the expression of PICK1in the negative control group was higher than the PICK1-siRNA group, suggesting PICK1-siRNA could decreased the expression of PICK1protein. 7MTT showed that the cell proliferation was reduced significantly in PICK1-siRNA group(0.302±0.01,0.312±0.01,0.508±0.02,0.786±0.01), compared with the negative control(0.374±0.02,0.436±0.01,0.603±0.02,0.895±0.03) and blank(0.385±0.05,0.463±0.04,0.628±0.02,0.901±0.03)(P<0.05) in24h,48h,72h,96h respectively after transfection.8Transwell experiment showed that the number of cells which crossed the membrane was38±7.1in PICKl-siRNA transfection group,90±10.2in blank group and82±16.6in negative control group(P<0.05).Conclusion1PICK1plays an important role in the occurrence of human colorectal adenocarcinoma.2PICK1may be the diagnosis target for the adenocarcinoma differentiation, invasion and metastasis.3PICK1may be the prognosis target of colorectal adenocarcinoma.4siRNA targeting PICK1can inhibit cell proliferation and decrease the cell invasive potential of HT-29. |
PDF Full Text Request