| Research Aims and Background: Breast cancer is one of the most frequent malignant tumors for women,raned at number for its tumor-related death rate.With its incidence increasing steadily since the 1970’s,it has become a serious harm to the life and health of women.With its multi-stage occurrence and development,breast cancer has complex mechanism resulting from various factors.Recent years have seen considerable progress in the research of tumor markers related with breast cancer,and hence appreciable value has been contributed to the recognition,understanding,diagnosis and treatment of this cancer.With the rapid development of the society and the economy,the number of cases without symptoms discovered through screening has constantly increased,leading to higher proportion of early breast cancer and significant increase of five-year survival rate.However,due to the unsound screening system and insufficient awareness of prevention,the proportion of terminal breast cancer still remains high,unfortunately due to delayed diagnosis,so that there is still a gap between the breast cancer survival rate of our country and that of developed countries.Thus,it is of great significance to search for breast cancer tumor marker with high specificity and sensitivity.Genomics reveals the basic characters of human genetic maps,but it cannot reflect all the genetic functions and their mechanisms.Since most of these genetic functions depend on the coding protein,protein is the bearer of vital movement and the direct performer of biological functions.Hence,research in integrated level on the cell or body protein can help us reveal relevant metabolic process and life.Using high-flux proteomics,combined with bio-informatics,researchers in this program starts from differential protein in conducting breast cancer research,hence broadening the width of protein research and contributing to the more efficient,overall and accurate screening of bio-markers for breast cancer.Proceeding from the proteomics comparison of differential proteins,proteins worthy of further research in the occurrence and development of breast cancer have been screened so that the theoretical foundation and basis can be provided for the discovery of markers for diagnosis and treatment of breast cancer.Research Methods: Part One: identification of serum differential proteins from patients of stage II breast cancer and those from healthy women 1、abstraction of serum samples from stage II breast cancer patients and from healthy women 2、proteomics analysis for the screening of differential proteins 3、GO annotation analysis and functional cluster analysis on these differential proteins in Gene Co Dis3;classification and analysis on them in Uniprot database;analysis on the interaction among differential proteins combining the gating factor and the on-line tool STRING to screen the protein worthy of further research 4、verification of screened protein using Western Blot and q RT-PCR Part Two: the identification of serum differential protein from breast cancer patients with/without axillary lymph node metastasis(ALNM)1、 abstraction of serum from breast cancer patients with/without axillary lymph node metastasis(ALNM)2、proteomics analysis for the screening of differential proteins 3、GO annotation analysis and functional cluster analysis on these differential proteins in Gene Co Dis3;classification and analysis on them in Uniprot database;analysis on the interaction among differential proteins combining the gating factor and the on-line tool STRING to screen the protein worthy of further research 4、Verification of the screened differential proteins with Western Blot and q RT-PCR Part Three: the identification of serum differential protein from patients of ductal carcinoma in situ(DCIS)and from those of DCIS with invasion 1、abstraction of serum from patients of ductal carcinoma in situ(DCIS)and from those of DCIS with invasion 2、proteomics analysis for the screening of differential proteins 3、GO annotation analysis and functional cluster analysis on these differential proteins in Gene Co Dis3;classification and analysis on them in Uniprot database;analysis on the interaction among differential proteins combining the gating factor and the on-line tool STRING to screen the protein worthy of further research 4、Verification of the screened differential proteins with Western Blot and q RT-PCR5、perception of the influence of screened markers on the biological behaviors of cells like their proliferation and migration using MTT methods,scratch tests and Transwell cell migration assay.Experimental Results: Part One 1、359 proteins have been quantified in the serum proteomics analysis on stage II breast cancer patients and healthy women 2、GO annotation and significant enrichment analysis have been conducted on differential proteins,combined with analysis of gating factors and protein interaction,to find RAF1、VIM proteins that deserve further research.3、According to Western Blot,the expression level of VIM in the serum of stage II breast cancer patients is obviously higher than that in the control group,while the expression of RAF1 is obviously lower.According to the q RT-PCR probe method detection,the expression of VIM in the serum of stage II breast cancer patients demonstrates extremely significant up-regulation(P<0.01),while the expression of RAF1 demonstrates extremely significant down-regulation(P<0.01).As verified by Western Blot and q RT-PCR,the expressions of RAF1 and VIM in breast cancer correspond with those shown in the results of proteomics research.Part Two 1、346 proteins have been quantified in the serum proteomics analysis on breast cancer patients with/without axillary lymph node metastasis(ALNM),with 6 showing up-regulation of expression and 113 showing down-regulation of expression.2、GO annotation and significance enrichment analysis have been conducted on differential proteins,combined with analysis of gating factors and protein interaction,to find K1C19 and PSME2 proteins worthy of further research.3、According to Western Blot,the expression level of PSME2 in the serum of breast cancer patients with lymph node metastasis is obviously lower than that in the control group,while the expression of K1C19 is obviously higher.According to the q RT-PCR probe method detection,the expression of PSME2 in the serum of stage II breast cancer patients demonstrates extremely significant up-regulation(P<0.01),while the expression of KRT19 demonstrates extremely significant down-regulation(P<0.01).As verified by Western Blot and q RT-PCR,the expressions of K1C19 and KRT19 in breast cancer correspond with those shown in the results of proteomics research.Part Three 1、524 proteins have been quantified in proteomics analysis research on patients of ductal carcinoma in situ(DCIS)and on those of DCIS with invasion,with 11 showing up-regulation of expression and 43 showing down-regulation of expression.2 、GO annotation and significance enrichment analysis have been conducted on differential proteins,combined with analysis of gating factors and protein interaction,to find ACTC1 the protein worthy of further research.3 、According to Western Blot,the expression level of ACTC1 in tissues of patients with ductal carcinoma in situ(DCIS)with invasion is obviously higher than that in the control group.According to the q RT-PCR probe method detection,the expression of ACTC1 in tissues of patients with ductal carcinoma in situ(DCIS)with invasion demonstrates extremely significant up-regulation(P<0.01).As verified by Western Blot and q RT-PCR,the expressions of ACTC1 in breast cancer correspond with those shown in the results of proteomics research.4、The effects of over-expression of ACTC1 on the proliferation of MCF7 in its cell line are tested using MTT colorimetric method.Absorbency at 490 nm of the group with ACTC1 over-expression and that of the control group are tested respectively to find the former uniformly higher than the latter.Hence is proven that ACTC1 can promote the proliferation of MCF7 cells.5、According to Transwell cell migration experiment,MCF7 cell line with ACTC1 over-expression treatment demonstrates higher migration capacity than the control group,showing that ACTC1 can promote the migration of MCF7 cells.Conclusions: In the above three experiments,breast cancer proteins of different groups have been compared,with differentially expressed proteins screened and verified to perfectly match the results of proteomics research.Since no common differential proteins have been discovered in these three experiments,the occurrence and development of breast cancer demonstrate very complex causes: breast cancer is a combined result of many stages and many factors.The third part is the emphasized part in terms of elaboration,focusing on the in-depth research of the verified ACTC1.In order to study the effects of ACTC1 on the mammary carcinoma,by testing the effects of over-expression of ACTC1 on its proliferation,we have concluded that ACTC1 could promote the proliferation of MCF7 cells.Through Transwell cell migration assay,we have also concluded that ACTC1 could promote the migration of MCF7 cells.Till now,research reports on the effects of ACTC1 have been rare,being a similar case to those on the effects of ACTC1 on glioma.Hence,we believe proteomics technology has proven a powerful approach for the discovery and recognition of potential bio-markers of breast cancer. |
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