A Study On The Expression Of SH2-B In Colon Cancer And The Molecular Mechanism

Objective:1) To investigate the expression profile of SH2-B in colon carcinogenesis, SH2-B expression was detected in colonic polyps, colonic adenomas and colon cancers tissues.2) To clarity the relationship of diseas staging and SH2-B expression, SH2-B was detected in colonal cancer tissues in stageⅠ,Ⅱ,ⅢandⅣ.3) To determine the association of colonal cancer and SH2-B expression, SH2-B was analyzed in colonal cancer tissues with metastasis and no metastasis.4) To determine whether SH2-B involves in the proliferation of colonal caner cells, MTT assay and flow cytometer were used to measure the cell proliferation and cell cycle of HT-29 with SH2-B gene transfection respectively.5) To determine whether SH2-B involves in metastasis of colonal caner cells, wound healing assay and Trans-well assay were used to measure the cell motility and metastasis prepectively with SH2-B gene transfection.Methods:1) Using immunohistochemistry assay, SH2-B expression was detected in 69 cases of colon cancers,27 cases of colonic adenomas,29 cases of colonic polyps and 38 cases of nasopharyngeal carcinoma. The expression profile of SH2-B in colon carcinogenesis was gained.2) SH2-B was detected in 69 cases of colonal caner including 11 case of stageⅠ,22 case of stageⅡ,19 case of stageⅢand 17 case of stage IV. The relationship of SH2-B expression and colonal cancer stages was clarified.3) SH2-B was detected in 69 cases of colonal caner including 34 caseS without metastasis and 35 cases with lymph nodes metastasis. The relationship of SH2-B expression and colonal cancer metastasis was clarified.4) At first, colonal cancer cell lines, LOVE1, LOVO, SW620 and HT-29, were tested SH2-B expression using immunoflurence assay and a cell line with low expressed-SH2-B was harvested. The second, pcDNA3.1-SH2-B was transfected into HT-29 cells using LipofecTAMINE 2000. After gene transfection, SH2-B expression was detected by Western blotting, and cell proliferation was measured by MTT assay, and cell cycle was analyzed by flowcytometer. SH2-B involving colonal carcinogenesis was investigated.5) pcDNA3.1-SH2-B was transfected into HT-29 cells using Lipofec-TAMINE 2000. Wound healing assay was used to measure HT-29 cell motility and Trans-well assay was used to test HT-29 cell metastasis.Results:1) The results of immunohistochemistry assay showed that the positive rate of SH2-B in colon polyps is 31% (9/29), colonic adenoma was 52% (14/27), and colon cancer tissue was 93%(27/29), while the cancer control, nasopharyngeal carcinoma tissue was 87%(33/38). SH2-B expressed in cytoplast and cytoplasm of colonic polyps, colonic adenoma, colon cancer and nasopharyngeal carcinoma tissue tissue, mainly located in cytoplasm. Image analysis revealed that the positive cell rate of SH2-B in colon cancer tissues (0.64+0.26) was significantly higher than that in colon polyps (0.12 +0.21) and colon adenoma tissues (0.26±0.27) (P<0.01), the positive cell rate of SH2-B in colonic adenomas was significantly higher than colon polyps (P<0.05), the positive cell rate of SH2-B in colon cancer (0.64±0.26) was lightly higher than that in nasopharyngeal carcinoma tissues (0.49±0.28, P<0.05). The score of staining intensity of SH2-B in colon cancer tissues (2.76±1.18) was significantly higher than that in colon polyps (0.72±1.16) and colon adenoma tissues (0.77±0.85, P<0.01), but there was no signifcatant between colonic adenomas and colon polyps (P>0.05). There was also no signifcatant between colon cancer (2.76±1.18) and nasopharyngeal carcinoma (3.03±1.30, P>0.05), it showed that SH2-B may involve in colon carcinogenesis.2) Immunohistochemistry results showed that positive rates of SH2-B of stageⅠ,Ⅱ,Ⅲ,Ⅳwere 90.9%(10/11),100%(22/22),100%(19/19), 100%(17/17) respectively, and there were no significant difference between of them. The strong positive rates of stageⅠ,Ⅱ,Ⅲ,Ⅳwere 18.2%(2/11), 45.5%(10/22),57.9%(11/19) and 82.4%(14/17), respectively. SH2-B gradually increased along with stageⅠ,Ⅱ,Ⅲ,Ⅳ. SH2-B expression was associated with clinic stages.3) 69 cases of colonal cancer included 35 cases with lymph nodes metastasis and 34 cases with no metastasis. The positive rates of metastasis and no metastasis were respectively 85.3%(29/24) and 100%(35/35), The strong positive rates of them were respectively 23.5%(8/34) and 45.7% (16/35), which the rate was higher in metastasis than no metastasis (p< 0.05).4) Immunofluerence assay results showed that SH2-B lowly expressed in HT-29 cells. HT-29 served cell model. In cell proliferation experiments, we transfected the plasmids of pcDNA3.1-SH2-B to HT-29 cells and used Western-blotting assay to analyze the SH2-B expression, the results showed that pcDNA3.1-SH2-B expression dramatically increased in HT-29 cells after gene tansfection. Using MTT assay to detected the cell proliferation after gene transfection, the cell proliferation was significantly strong (P <0.05). Cell cycle analysis showed that S stage cells increased in SH2-B after transfect SH2-B. (39.4±2.31)%vs, (18.6±4.25)%or (11.1±2.39) %. The results suggested that high expression of SH2-B in the process of colon carcinogenesis may promote abnormal proliferation of epithelial cells.5) Wound healing assay results showed that moving cell numbers of the transfect with SH2-B, the blank and the vector were respectively 867±187, 349±121,279±158, which moving cells was more in the transfect with SH2-B than the blank and the vector. Trans-well assay results showed that meatastatic cell numbers of the transfect with SH2-B, the blank and the vector were respectively 278±107,112±81,129±88, which metastatic cells was more in the transfect with SH2-B than the blank and the vector.Conclusions:1) SH2-B expression gradually increased in colonic polyps, colonic adenoma and colon cancer. This implys that SH2-B may involve in colonal carcinogensis, and SH2-B is a early molecular event of colonal carcinogensis. Overexpression of SH2-B plays an important role in colonal carcinogensis.2) The strong positive rates of SH2-B gradually increased in the clinic stage I, II and III, and this indicates that SH2-B expression is related with clinic stages of colonal cancer.3) The positive rates of SH2-B were higher in the metastatic group than no metastatic group. This indicats that SH2-B may involve in colonal cancer metastasis, and high expression of SH2-B was associated with colonal cancer metastasis。4) MTT data showed that the cell proliferation of HT-29 transfected with SH2-B was higher than that of the blank control and vector control. Flow cytometer results showed that S stage cell number was more in the transfect with SH2-B than that in the blank control and vector control. This implys that SH2-B may enhance cell proliferation through promoting cell cycle.5) Wound healing assay data showed that the moving cells were more in the transfect with SH2-B than that in the blank control and vector control. This implys that SH2-B may ehances colonal cancer moving. Trans-well assay results showed that the invasive cell number was more in the transfect with SH2-B than that in the blank control and vector control. This implys that SH2-B ehances colonal cancer invasion and metastasis.