| BACKGROUND:Mammary carcinoma is one of the most common malignant tumor in women. The incidence of mammary carcinoma is not the same around the world, its etiology is not clear, may be related to susceptibility genes and environmental factors, lifestyle and other factors. Presenting the incidence of mammary carcinoma in the world, European and American countries’ women are the high incidence of mammary carcinoma, which is given priority to with North America and northern Europe, and the incidence in most countries of Asia, Africa and Latin America are low compare with them. According to the American Cancer Society (the American Cancer Society, ACS) released data show that breast Cancer each year more than1.3million people worldwide, about a third of the new female Cancer patients, breast Cancer kills about450000people, its first in women of various kinds of malignant tumor mortality. Our country female breast cancer accounts for7%～10%of whole body all kinds of malignant tumor, second only to cervical cancer, what’s more there is an upward trend and have a tendency to over cervical cancer in recent years, It’s become one of the most threat to women’s health and life. At present, people mainly through pathology and imaging of morphological changes to evaluate breast cancer degree, then according to the mass size, lymph node, the presence of distant metastasis after surgery for TNM staging, combined with the pathological type, immunohistochemistry case its efficacy and prognosis. One of the most widely used international classification of breast cancer is a grade of B-R, also known as the Nottingham grading system, the rating method to tumor cells morphological and cytological characteristics for evaluation basis, breast cancer can be divided into three malignant degree as an important predictor of breast cancer recurrence and death. However, due to the presence of subjective grading and the operation is cumbersome, its application has some limitations.Therefore, it is necessary to develop a simple, sensitive and accurate evaluation method to improve and perfect the evaluation system of existing. With the deepening of the research, the breast cancer research has entered to the genetic level from cell level. Gene chip (also called DNA chips, biochip), and its prototype is put forward by the middle of1980s, its sequencing principle is hybridization sequencing method, namely with a set of known sequence of nucleic acid probe hybridization method for determination of nucleic acid sequences. Specific method is:the probe of known target nucleotide sequence is fixed on a substrate surface, when the sequences of nucleic acids with fluorescent tags in the solution and the corresponding location on gene chip nucleic acid probe produce complementary matching, by determining the fluorescence intensity of the strongest probe position, to obtain a set of completely complementary probe sequences, thus can restructure the target nucleic acid sequence. With the development of gene chip technology, more and more molecular defects in breast cancer have been revealed, when application of the new technology in the early diagnosis of breast cancer such as molecular biology and molecular epidemiology, the research of breast cancer is already enter into the molecular biology from cytology. Nowdays molecular diagnosis has now become an important means of early diagnosis of breast cancer. At present, people have been found someone related to the prognosis of breast cancer genes such as EGFR, HER-2, nm23etc. These genes from various aspects for the prognosis of breast cancer provides a more accurate judgment method. But due to breast cancer gene expression profile of individual difference is very big, people need to find more genes associated with breast cancer metastasis and prognosis. The past be used segments to complete the entire genetic analysis method and aimed at one or several genes of low flux research methods have been was replaced by the method of gene chip technology with high-throughput and mass detection.At home and abroad, according to several studies have shown that gene expression can be used as a tumor marker to predict the prognosis of breast cancer. Zajchowsik DA by using containing588genes such as Atlas (7740-1) and cancer (7742-1) cDNA chip analyzed nine low invasive and four high invasive breast cancer cell line gene expression profile, found that there are24genes in the invasive ability of different expression differences exist in the breast cancer cell lines; Using these differentially expressed genes to predicted nine known its invasive breast cancer cell lines and that the results are consistent with known infiltrating extent. In1999, the Lander according to the result of expression profile chip chose70genes and made into low density gene chips, the chip made of70function specific genes get a lot of attention, because it was the first clinical diagnostic products by the chip technology based in Europe and American. Belgian Lacroix, also was prepared in2002, including250with breast cancer classification and guidelines related to the low density expression profile chip. In recent years, domestic use of biochip in the prognosis of breast cancer expression spectrum research also has carried out some of the work. Ye Li hong etc with chip, containing the329genes in human breast cancer cell line MCF-7and transfer the clone LM-MCF-7gene expression spectrum analysis,35of67differentially expressed genes is associated with tumor, including9is associated with breast cancer metastasis,6May be involved in tumor invasion and metastasis process.This study was to apply the advantages of high-throughput microarray technology, for a breast cancer samples simultaneously in a single experiment to detect the expression of different genes, and according to the situation have been screened for breast cancer gene expression meaningful combinations of genes, molecular typing of breast cancer research and treatment guidance gene chip for detecting early breast cancer patients needle biopsy tissue, fresh tissue samples stored in liquid nitrogen or minus80degrees refrigerator retrospective sample, the establishment of follow-up, according to whether the patient actually recurrence establishing individualized treatment of early breast cancer molecular diagnostic criteria, in order to achieve a timely, efficient, accurate and sensitive type of breast cancer diagnosis, provide reference and guidance for individualized patient treatment and improve patient outcomes.OBJECT:To screen molecular markers in early breast cancer and establish gene subtyping-based diagnostic criteria for predicting the prognosis of early breast cancers.METHODS:(1).Specimen collection: all specimens collected from March2007to June2008, zhujiang hospital, southern medical university which department of general surgery patients with modified radical mastectomy tumor tissue samples. Patients admitted to hospital after biological security checks. Routine detection of HIV, HBV, HCV infections, HIV, HBV and HCV infections are not included in the scope of collecting specimens. We are based in breast tumor resection after ten minutes, and quickly put into liquid nitrogen tank preservation, at the same time label for each sample.(2).Chip preparation: the gene expression made by national engineering research center of Shanghai biochip preparation at biological technology co., LTD.(Shanghai), USES the Agilent customized8x15000chip,8refers to one chip can do eight samples, each sample can detect1500015000genes. Sequence information used in the design of the product from the RefSeq, Goldenpath, in-depth study of famous Ensembl and Unigene database, With the human genome and stitching(3).Sample genetic screening: extracting total RNA in breast cancer samples, using QIAGEN RNeasy Mini Kit purification of total RNA, using the method of reverse transcription cDNA synthesis and purification of cRNA synthesis using aaUTP tag and purification, detection of the concentration and qualified quality and chip hybridization, using Agilent scanner scan and analysis results.(4).Statistical methods:gene chip experiments according to the ratio of1.4or0.7or less or more times as the standard, and Screening of differentially expressed genes between prognosis and the prognosis is good, using SPSS13.0statistical software processing data, to analyze t-test between groups.RESULTS:Gene microarray analysis found that the experimental group with poor prognosis sample than the prognosis is good sample132differentially expressed genes,44genes significantly up-regulated and88genes were significantly down-regulated (the differences are more than2times). Two groups of differentially expressed genes Pathway enrichment function analysis of the differences are mainly concentrated in the cell cycle, P53signaling pathways, amino acid metabolism, ECM receptor interactions, etc. The largest concentration of differentially expressed genes pathway is the cell cycle. CONCLUSION:The gene expression profiles differ in early breast cancer tissues of the same pathological type and clinical stages but with different prognoses, and CD44, MKI67, NTRK2, Nek2, BRCA1,C16orf60, TOP2A, CCNB2, ANCCA and RRM2genes can be used as the prognostic markers for early breast cancer. |
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