A Research On The Relationship Between The Expression Of Tumor Suppressor Gene FRY And The Malignant Degree Of Breast Cancer

Research BackgroundBreast cancer is the most frequent cancer among women,whose formation and development are related to various factors.The pathogenesis and susceptibility gene of breast cancer has always been a research focus.In recent years,scientists had found a lot of tumor biomarkers related to the susceptibility of breast cancer,which including tissue markers,serum markers and genetic markers.BRAC1 and BRAC2 were two most typical genetic marlers,whose mutant detection had been a powerful tool in breast cancer prediction,but the number of breast cancer patients caused by mutantion of BRAC1 or BRAC2 only occupied 5%～10%of all breast cancer patients.At present,there are few discovered breast cancer biomarkers that can be practically and extensively applied to clinical diagnosis and treatment.Still,it has been lack a sensitive and specific tumor biomarker that can be used to early diagnosis so far.By candidate gene strategy and Genome-wide association study(GWAS),scientists had found a lot of breast cancer susceptibility genes like BRAC1 and BRAC2 discovered by genetic linkage studies of high-risk families.In contrast to high penetrance susceptibility genes like BRAC1 and BRAC2,low penetrance susceptibility genes had a high distribution rate but with weak effections.Some low penetrance susceptibility genes even gave rise to breast cancer combined with other genetic and environmental factors instead of directly acting on tumorgenesis.To solve the problem of human genetic linkage analysis,researchers started to study the mechanism of tumor development by breast carcinoma models of rat inbred strains.Taking advantage of genetic linkage analysis,the sequence change of FRY gene was proved to be a reason of the high susceptibility to breast carcinomas of Fischer 344 strain rat.FRY gene is a homologous gene of Drosophila’s furry gene in mammal.In primary eukaryote,the homologous gene of FRY(furry in drosophila、tao3 in yeast)could regulate the polarization of epithelial cells、cellular morphology、cell proliferation and division.In previous research,FRY was found to be reduced in most rats breast tumors as a breast cancer suppressor gene,and compared with untumorigenic MCF1OA human breast epithelial cell,the expression of FRY activity decreased in human breast cancer cells.Based on the previous study,our research aimed to detect the expression of FRY in human breast cancer samples with specific FRY monoclonal antibody,and combine with in vivo tests of breast cancer cell lines that stably expressing different FRY genes to further study the relationship between FRY gene and the development and malignant transformation of breast cancer,which would provide a theoretical basis for finally application of FRY gene to be a biomarker of diagnosis,treatment and prognosis in breast cancer and to be a target site in breast cancer treatment.ObjectiveTo study the relationship between FRY gene and the malignant degree of breast cancer.Method1.Identification of specific FRY monoclonal antibodyThe titers of obtained FRY polyclonal and monoclonal antibodies were identified by Enzyme-linked Immunosorbent Assay(ELISA).FRY protein in breast cancer cells was detected with specific monoclonal antibody by Western Blot.The application of FRY monoclonal antibody which was identified as specificity was tested in immunohistochemistry method to finally figure out a monoclonal antibody that could specifically recognize the FRY protein.2.Expression and significance of FRY protein in human breast cancer tissuesBenign breast neoplasms and malignant breast neoplasems with different degrees were collected.Immunohistochemistry method was used to detect the expression of FRY protein in these tissue with pathological analyse.The percentage of positive cells together with the staining intensity of positive cells were scored to analyze the differential expression of FRY protein in benign and malignant breast neoplasms.The differential expression of FRY protein among various breast carcinoma histological grades,TNM stages,molecular typing,tumor size,lymph node metastasis status and the difference between FRY and the expression of known related biomarkers of breast cancer was investigated.3.In vivo tests using breast cancer cell lines with stable expression of FRY geneTo investigate the tumorigenicity of different expression of FRY gene in cell lines on nude mice,breast cancer cell line MDA-MB-231 that had been transfected the wild type and mutant type FRY alleles separately were inoculated to BALB/c nude mice to establish transplanted tumor models.45 female BALB/c nude mice aged 4～5 weeks were divided into three groups,named as MDA-MB-231-293 cell group(M-293)、MDA-MB-231-MCF7 cell group(M-M)、MDA-MB-231-ctrl cell group(M-C).The three breast cancer cell lines in exponential phase were made into cell suspensions of 1×107/mL,then given a subcutaneous inoculation to the left back of nude mice,each animal 0.2 mL with a cell amount of 2×106.Animal weight and the long diameter and short diameter of tumor were measured 3 times every week to picture the tumor growth curves.As for mice whose long diameters of tumor had reached 1 cm before the specified observation period of 35 days,the tumor would be removed via operation and the mice be observed for another 30 days before put to death,while other mice were put to death after 35 days’observation.The tumor tissues、lung and axillary lymph nodes were cut at the end of the experiment for pathological HE staining and Masson’s Trichrome staining tests.Results1.Identification of specific FRY monoclonal antibodyELISA showed that the titers of one FRY polyclonal antibody and five monoclonal antibodies could all reach 1:128000.Western Blot showed that FRY polyclonal antibody detected a specific band over 250 kD but with some unspecific bands appeared.FRY-13010252 monoclonal antibody could specifically identify the FRY protein in breast cancer cells,which detected a specific band over 250 kD with no other unspecific bands,which the rest four monoclonal antibodies didn’t.Immunohistochemistry method showed that compared with the FRY polyclonal antibody produced by SIGMA.FRY-13010252 could specifically identify the FRY protein in human breast cancer tissues.2.Expression and significance of FRY protein in human breast cancer casesOne hundred and four cases of breast neoplasms had been collected.among which 11 cases were from fibrocystic breast disease and 93 cases were from breast invasive ductal carcinomas.In 93 cases of breast cancer,according to the histological grades information,there were 7 cases of stage Ⅰ.47 cases of stage Ⅱ and 39 cases of stageⅢ.According to the TNM stage standards of American National Comprehensive Cancer Network(NCCN).there were 27 cases of stage Ⅰ,33 cases of stage Ⅱ and 22 cases of stage Ⅲ.According to classification of the breast cancer molecular typing.there were 23 cases of Luminal A subtype,47 cases of Luminal B subtypes,13 cases of HER2 over-expression subtypes and 10 cases of triple negative breast cancer.Immunohistochemistry test showed that,there was a significant difference in the expression level of FRY between fibrocystic breast disease cases and breast invasive ductal carcinoma cases(positive rates of 90.9%vs 54.8%,P<0.05).In breast invasive ductal carcinomas,the positive rates of FRY protein expression in Ⅰ、Ⅱ、Ⅲhistological grades were 57.1%,72.3%and 33.3%,respectively(P<0.01).According to TNM stages,the positive rates of FRY protein expression in Ⅰ、Ⅱ plus Ⅲ stages were 74.1%,47.3%,respectively(P<0.05).There was a significant difference in the expression level of FRY between tumor diameter sizes ≤3 cm and>3 cm(positive rates of 67.9%vs 35.7%,P<0.01).The positive rates of FRY protein between PR positive and PR negative cases were 63.5%and 35.7%,which was significant different from each other(P<0.05).There were no significant differences between the expression level of FRY in age,lymph node metastasis,molecular typing,ER,HER2,and P53 expression level(P>0.05).3.Nude mice tumorigenic assays using breast cancer cell lines with stable expression of FRY geneIn all the three MDA-MB-231 cells transfected,MDA-MB-231-ctrl cell line,which had been transfected an empty plasmid with no FRY allele,was observed to achieve a significant higher tumor growth rate than the MDA-MB-231-MCF7 cell line that transfected the mutant FRY allele and the MDA-MB-231-293 cell line that transfected the wild FRY allele(P<0.01).In the twenty-fifth day when it started to have a tumor resection,the average tumor volume of MDA-MB-231-ctrl cell group was(347.80±286.17)mm3,which was significantly bigger than the other two groups(P<0.01).The average tumor volume of MDA-MB-231-MCF7 cell group was(134.70±63.38)mm3,almost half bigger than the MDA-MB-231-293 cell group(75.37±58.55)mm3.but with no significant difference(P>0.05).There was no significant difference between the tumor weight in three groups(P>0.05).HE staining showed that the tumor of three groups all had typical pathological features,all the axillary lymph nodes could see metastasis of cancer cells.There were two cases of lung metastasis in MDA-MB-231-ctrl cell group,which hadn’t be seen in the other two groups.In Masson’s Trichrome staining,compared with MDA-MB-231-ctrl cell group.the MDA-MB-231-293 cell group that stablely expressed wild type FRY allele showed a more organized morphology and a clearer tumor border with the underlying tissue.Testing the blue collagen in tumor,the ratio of collagen content within tumors in MDAMB-231-ctrl cell group was(20.07±5.46)%,which was significantly higher than the ratios in MDA-MB-231-MCF7 cell group and MDA-MB-231-293 cell group which were(14.63±6.54)%and(2.96±4.84)%,respectively(P<0.01).ConclusionThe expression of FRY gene had a negative correlation with the maglinant degrees of breast cancer.1.Monoclonal antibody FRY-13010252 that could specifically detect FRY protein was successfully identified.2.The expression levels of FRY protein in tissues are strongly negatively correlated with the malignant degrees of breast carcinoma.3.FRY gene could inhibit the tumor generation、growth and distant metastasis of breast cancer cells in vivo to some extent.