Study On The Correlation Between CDK5RAP2 And Progression Of Breast Carcinoma

Partâ… Evaluated the expression of CDK5RAP2 in human breast cancerObjective:CDK5RAP2 was a pericentriolar protein that functions in microtubule organization of the centrosome. The aim of this study was to introduce breast cancer into the CDK5RAP2 research. Evaluate the difference of CDK5RAP2 expression between normal breast tissue and malignant breast cancer by preparing its monoclonal antibody. This work would make a foundation for further research on association between CDK5RAP2 and breast cancer.Method:Monoclonal antibody of CDK5RAP2 was created by the means of hybridoma technique (semi-solid medium containing methylcellulose) and its specificity was verified by immunoflurecence on the cell knockout CDK5RAP2. Use the technique of RT-PCR and IHC to detect the expression of CDK5RAP2 in tissue of breast cancer and its counterparts.Result:1. Following cell fusion,788 clones were transfered from methylcellulose medium into 96 well plates. Through repeated screening of ELISA,5 clones with higher titer were finally obtained. The antibody was purified from the ascites prepared by DG10 clone with IgGl subtype and its specificity was verified by immunoflurecence on the cell knockout CDK5RAP2.2. CDK5RAP2 immunoreactivity (66.7%) was detected in the breast carcinoma compared to its corresponding normal tissue. RT-PCR displayed the 73.3% positive ratio. CDK5RAP2 expression was detected in 6 breast cancer cell lines (T47D, MDA-MB-435, MDA-MB-231, MCF-7, SK-BR-3, ADR), and higher expression appeared into the two cells, MDA-MB-435 and MDA-MB-231, which were associated with cancer migration in several reports.Conclusion:1. Methylcellulose semi-solid medium was a useful and efficient method for screening the separate clone in preparing hybridomas.2. CDK5RAP2 expression was significantly enhanced in breast carcinoma, which implicated that CDK5RAP2 might play a potential role in breast cancer occurrence and progressing.3. Considered the previous in vitro research related CDK5RAP2 functions in microtubule organization of the centrosome, one hypothesis was proposed that CDK5RAP2 could prompt the tumor cell proliferation by its microtubule nucleation.Partâ…¡Alteration of CDK5RAP2 expression in breast carcinogenesisObjective: Evaluate the alteration of CDK5RAP2 expression in four group:normal breast tissue (N), precancerous lesion (per-Ca), ductal carcinoma in situ (DCIS) and primary infiltrative ductal carcinoma (IDC). Elucidate the role of CDK5RAP2 in different stages of malignancy of breast cancer.Method: Each group (N, pre-Ca, IDC, DCIS) consisted of 30 paraffin-embeded samples and 15 fresh surgical samples. Immunohistochemistry was performed on paraffin blocks to determine expression pattern of CDK5RAP2 in these four groups. RNA was extracted from fresh surgical samples and then Real time quantitative RT-PCR was performed to evaluate the level of CDK5RAP2 mRNA. The correlation between the expression of CDK5RAP2 and breast pathological changes was established by statistic calculation.Result:1. IHC result indicated that CDK5RAP2 was a cytoplasmic protein, with a very small portion existing in nucleus. CDK5RAP2 expression was observed in Pre-Ca, DCIS and IDC, which had a significant difference compared with normal breast tissue (p<0.001). The positive ratio of high CDK5RAP2 expression in Pre-Ca, DCIS and IDC was 57%,73%,83%., which displayed an increasingly enhancement.2. Real time RT-PCR revealed that CDK5RAP2 mRNA level in IDC group was the highest (P<0.001) in four groups. Overall expression in four groups tended to increase. Compared with normal tissue, the amount of CDK5RAP2 expression in pre-Ca, DCIS and IDC had 2.8,3.7 and 11.8 fold, respectively.Conclusion:1. CDK5RAP2 expression existed in Pre-Ca, DCIS and IDC as compared to normal tissue, which indicated that CDK5RAP2 might be a potential indicator of the development of breast cancer.2. CDK5RAP2 expression was increasingly enhanced in a course of breast cancer occurring and pathological progressing, which gave us an implication that a strong correlation exist between CDK5RAP2 expression and tumor pathological grade.3. The potential mechanism of CDK5RAP2 was followed:when the cell proliferated abnormally, always companied with centrosome abnormality, CDK5RAP2 could prompt the cell cleavage and passage by its microtubule nucleation, which leaded to the accumulation of cells with unstable genome. Ultimately, the cells transformed into atypical proliferation, DCIS and IDC.4. Because of high expression of CDK5RAP2 in breast cancer, one hypothesis that the enhancement of microtubule nucleation prompts carcinogenesis would be established.Partâ…¢Analysis of association between CDK5RAP2 and clinicopathologic parameters in breast invasive ductal carcinomaObjective:To study the association between CDK5RAP2 and clinicopathologic parameters in breast infiltrative ductal carcinoma.Method:CDK5RAP2 IHC performed on the tissue chip slide containing 127 cases of breast IDC. Evaluated the association between CDK5RAP2 and clinicopathologic parameters, as well as patient prognosis.Result:1. Patient positive ratio of CDK5RAP2 was 61.4%(78/127) in a large scale population. CDK5RAP2 expression was positively associated with lymph node status, not correlated with other clinicopathologic parameters, such as menopausal status, age, tumor size, clinical stage, histologic grade, ER and PR. The statistic values in PR and tumor size were on the terminal line,0.053 and 0.059 respectively.2. Survival analysis indicated CDK5RAP2 expression was negatively correlated with patient disease-free survival and overall survival, which meant CDK5RAP2 expression was significantly correlated with poor prognosis of breast cancer patients. Univariate and multivariate analysis of disease-free survival and overall survival for 127 breast cancer patients showed that CDK5RAP2 expression and tumor size was the independent marker.Conclusion:1. CDK5RAP2 expression was a potential prognosis factor in breast cancer patients because of its correlation with lymph node status.2. Considered overall research, the conclusion might be proposed as followed:in breast IDC, CDK5RAP2 was not only functioned as a tumor promoter by microtubule nucleation, but also as cell migration promoter by microtubule modulation.