Expression And Promoter Methylation Of FEZ1/LZTS1 Gene In Breast Carcinoma

Breast carcinoma is a common malignant tumor; the morbidity is increasedcontinually and obviously. It is well known that tumor is a disease because of genealtered, so studying the key genes related to malignant tumor can not onlycomprehend clearly the mechanic of tumor development and progression, butestablish the theory foundation to overcome carcinoma finally.FEZ1/LZTS1 gene is a new anti-oncogene in 8p22 cloned by Ishii et al in 1999.FEZ1/LZTS1 gene is expressed in many normal tissues, but several studies haveshowed that its expression is altered in at least 50% tumors, so FEZ1/LZTS1 genemay be take effect in the development and progression of many tumors. It isnoteworthy that Ishii has reported that FEZ1/LZTS1 gene is not expressed in 15 casesbreast cancer cell lines and 10 cases primary breast carcinoma, which showed that thechange of this gene has more important association with breast carcinoma. But as ananti-oncogene, the mechanic of inactivation of FEZ1/LZTS1 gene is not elucidatednow. It is always considered that the inactivation of anti-oncogene is induced by genemutation, chromosome deletion, but several studies showed that mutation ofFEZ1/LZTS1 gene is rare in many tumors; it is evident that mutation could not theprimary mechanic of inactivation of FEZ1/LZTS1. Recently, DNA methylation as themost important event of epigenetic has become the focus of studing gene silence.Thus, our study focuses on the mRNA and protein expression of FEZ1/LZTS1 gene inbreast carcinoma tissue, and expounds the role of FEZ1/LZTS1 gene in thedevelopment and progression in breast carcinoma deeply, and then investigates thesituation of promoter methylation in FEZ1/LZTS1 gene.Part One: Expression of FEZ1/LZTS1 gene mRNA in breast carcinomaMaterial and Methods:Total RNA of fifty cases fresh frozen breast carcinoma tissues and fifty casesaccording normal breast tissues were extracted. RT-PCR was used to detect themRNA expression of FEZ1/LZTS1 gene.Results: 1. In 50 cases breast carcinoma tissues, the expression of FEZ1/LZTS1 genemRNA as follow: 7 cases (14%) were absent, 23 cases (46%) were down regulation,and 20 cases (40%) were normal expression. Accordingly, mRNA of 50 cases normalbreast tissues were all expressed, mRNA expression of breast carcinoma was moredecreased than that of normal breast tissues, difference has statistically significance(P=0.000).2. Absence/down regulation of FEZ1/LZTS1 gene mRNA was related to the lymphnode metastases in breast carcinoma; that is, the rate of lymph node metastases in theabsence/down regulation expression group was more higher than the normalexpression group significantly (P=0.032).Conclusion:1. FEZ1/LZTS1 gene is altered obviously in primary breast carcinoma, whichshows FEZ1/LZTS1 gene takes important effect in the development of breastcarcinoma.2. Absence/down regulation of FEZ1/LZTS1 gene mRNA could promote invasionand metastasis of breast carcinoma cells.Part Two: Expression of Fez1/Lzts1 protein in breast carcinoma and itsrelationship with FEZ1/LZTS1 gene mRNAExperiment One: Expression of Fez1/Lzts1 protein in breast carcinomaMaterial and Methods:100 cases paraffin embedded specimen were detected the expression of Fez1/Lzts1protein using immunohistochemistry (SP).Results:1. Fez1/Lzts1 protein were expressed in normal breast ductal epithelial, located atcell plasma and shows yellow-brown granular.2. In 100 cases breast carcinoma tissues, 72 cases (72%) were absent and/or downregulation of Fez1/Lzts1 protein expression; of all, 28 cases(+), 29 cases(+/-), 29 cases (-/+), and 14 cases (-). In 88 cases invasive ductal carcinoma (IDC), 65 cases(73.9%) were absent and/or down regulation of Fez1/Lzts1 protein expression; of all,23 cases(+), 25 cases (+/-), 26 cases (-/+), and 14 cases (-).3. Absence/down regulation of Fez1/Lzts1 protein was related to the lymph nodemetastases (P＜0.05); especially, the rate of lymph node metastases in the group of(-)and(-/+) were more higher than the group of(+) and (+/-) significantly (P＜0.05).4. Expression of FEZ1/LZTS1 mRNA is related to Fez1/Lzts1 protein expressionsignificantly(P=0.000).Experiment Two: Mutation detection of FEZ1/LZTS1 gene coding regionMaterial and Methods:DNA was extracted from 2 cases fresh frozen breast carcinoma tissues, andFEZ1/LZTS1 gene coding region mutation was detected by using PCR and followsequencing.Results:In two cases, 3 mutations were found: 1. G to A mutation at FEZ1/LZTS1 gene912bp; 2. A to G mutation at FEZ1/LZTS1 gene 1516bp; 3. T to C mutation atFEZ1/LZTS1 gene1992bp. And the mutations at 912bp and 1992bp are nonsensemutations, whereas the mutation at 1516bp leaded asparagines (N) to aspartic (D) at468 amino acid residue.Conclusion:1. Fez1/Lzts1 protein is altered obviously in primary breast carcinomas, whichmaybe happen in the progression phases.2. Absence/down regulation of Fez1/Lzts1 protein could promote invasion andmetastasis of breast carcinoma cells, especially when positive cells less than 50%.3. Expression of FEZ1/LZTS1 gene mRNA is strong correlated with expression ofFez1/Lzts1 protein.4. A to G mutation at 1516bp in FEZ1/LZTS1 gene coding region lead to aminoacid change, its significant need to be researched further. Part Three: Promoter methylation of FEZ1/LZTS1 gene in breast cancer celllinesMaterial and Methods:DNA was extracted from 11 cases breast cancer cells and treated by bisulfite. Andthen promoter methylation of FEZ1/LZTS1 gene was detected by using PCRamplification and sequencing.Results:In 11 cases breast cancer cell lines, promoter methylations of FEZ1/LZTS1 genewere found in10 cases (90.91%). In total 28 CpG islands, every 5' cytosine site hasbeen methylated at least in one cell line, and the rate of methlation is 0%～100%(average 65.26%).Conclusion:Promoter methylation level of FEZ1/LZTS1 gene in breast cancer cell lines is veryhigh; the average rate is up to 65.26%.Conclusion:1. FEZ1/LZTS1 gene takes important effect in the development and progression ofbreast carcinoma.2. Absence/down regulation of FEZ1/LZTS1 gene mRNA and Fez1/Lzts1 proteincould promote invasion and metastasis of breast carcinoma cells.3. Promoter methylation level of FEZ1/LZTS1 gene in breast cancer cell lines isvery high.