A Study Of The Role Of MiR-203、Notch1 And JAG1 Gene Methylation In Development Of Breast Cancer

Part 1 Notchl and its ligand of JAGl gene methylation role of onset in mammary gland carcinomaobjective Notchl and its ligand JAG1 gene methylation may play a significant part in the tumorigenesis and the development. The study that to exploring Notchl and JAG1 gene methylation role of onset in mammary gland carcinoma. Methods The methylation of Notchl and JAG1 gene in human breast invasive ductal carcinoma （IDC; n=96）,ductal carcinoma in situ（DCIS; n=33）, atypical ductal hyperplasia（ADH; n=33）and usual ductal hyperplasia （UDH; n=33）were quantitative detection by MassARRY.Results （1） Our results showed that the positive rates of Notchl protein in breast IDC and DCIS were higher than that of ADH and UDH group 24.2%, and the difference was statistically significant （P<0.05）. In the IDC group, the higher the Notchl protein expression, the higher the lymph node metastasis of breast cancer, the higher the pathological grade and the later TNM stage, the relationship was positive correlation （P<0.05）. The mean methylation levels of Notchl gene except CpG₃、CpG₄.5、 CpG₈ in samples,and others CpG sites in UDH was significant higher than else three group （ADH、DCIS、IDC） （P<0.05）.The methylation states of CpG₄.5、CpG₁0.11、 CpG₁3.14、CpG₁6 sites between axillary node metastasis and without axillary node metastasis breast caner, and CpG₇、CpG₈、CpG 13.、CpG₁8 sites in different clinical stage of TNM,and CpG₃、CpG₈、Cp G₁1.12 sites in different grades of pathohistology, and CpG₃、CpG₈、CpG₁3.14、CpG₁6 sites between positive expression of ER、PR、C-erbB-2 and negative expression of ER、PR、C-erbB-2 had significant statistics difference （P<0.05）.（2） The mean methylation of promoter region of JAG1 gene CpG₂、CpG₆、CpG₁3、CpG₂0.21.22、CpG₂3.24、CpG₂6 sites in UDH was higher than other three group （ADH,DCIS,IDC）.The methylation levels of CpG₅、CpG₁5、CpG₁8 sites between axillary node metastasis and without axillary node metastasis in breast caner, and CpG₃、CpG₆、CpG₁0、CpG₂9.30 sites in different clinical stage of TNM, and CpG₁1.12、CpG₁4、CpG₂7 sites in different grades of pathohistology.（2） Positive expression rates of JAGlprotein in IDC and DCIS were 86.5% and 75.8%, respectively, which were significantly higher than those of ADH 45.5%and UDH 27.3%（P<0.05）. JAG1 protein expression was significant positive correlation with lymph node metastasis, pathological grades and TNM stages of IDC. The mean methylation levels of JAG1 gene at CpG₂、CpG₆、Cp G₁3、CpG₂0.21.22、 CpG₂3.24.25、CpG₂6 significantly decreased in IDC group compared with those of DCIS, ADH and UDH groups（P<0.0083）. In IDC, the average methylation rates of JAG1 gene at CpG₅、CpG₁5、CpG₁8 loci in the samples with axillary node metastasis were significantly lower than those in the samples without axillary node metastasis （P<0.05）. And the methylation rates at CpG₃、CpG₆、CpG₁0、CpG₂9.30 loci in in TNM stage, the stage Ⅰ were higher than that in stage Ⅱ, further higher than that in stageⅢ（P<0.05）; There was no significant difference between the grade Ⅱ and Ⅲ period（P> 0.05）. and that in CpG₁1.12、CpG₁4、CpG₂7 loci in grade Ⅰ were higher than that in grade Ⅱ and gradeⅢ（P<0.05）; and the methylation rates at CpG₈.9、CpG₁7、CpG₂7 loci in ER+PR+HER2+group were higher than that in ER-PR-HER2-group（P<0.05）.Conclusions There is an overall hypomethylation alterations of Notchl and JAG gene in IDC, with corresponding over-expression of Notch1 and JAG1 protein. This inversecor-relation show that the alteration of protein expression result from hypomethylation oncogene Notch1 and JAG1, and this change may have play an important role in occurrence and progression of breast cancer. Specific hypomethylation at CpG₃CpG₄.5、CpG₈ loci of Notchl gene and CpG₂、CpG₆、Cp G₁3、 CpG₂0.21、CpG₂6、CpG₂9 loci of JAG1 gene may play rols in the pathogenesis of breast carcinoma,suggesting that the hypomethylation of specific CpG loci of the gene may be involved the progression and/or malignant transformation from benign glandular lesions of the breastPart2 Detect the miR-203\Notchl gene protein expression in mammary gland carcinoma and their relationships with clinical indicatorsObjective:To detect the miR-203\Notchl gene miRNA and protein expression in breast cancers and hyperplasia lesions, and their relationships with clinical indicators of breast cancers and hyperplasia lesions were analyzed.Methods:miR-203 and Notch 1 miRNA of 60 breast IDC and 60 normal breast tissues were amplified by one-step RT-PCR method,and the expression of miR-203and Notchl protein was detected by immunohistochemical Streptavidin-Biotin Complex （SP） stain in 96 breast IDC, 99breast hyperplasia lesions and 22 normal gland samples for which formalin-fixed, paraffin-embedded （FFPE）. All data were analyzed using the SPSS 17.0 software package.Results:（1）RT-PCR showed:（1）The expression rate and standardized coefficient of miR-203 in breast cancers were significantly lower than those of normal breast tissues. The expression rate of miR-203 in breast cancers were significantly higher than those of normal breast tissues. （2）The standardized coefficient of miR-203 in cases with axillary node metastasis was significantly lower than that in cases without axillary node matastasis, N0>N1> N2. The standardized coefficient of Notchl in cases with axillary node metastasis was significantly lowwer than that in cases without axillary node matastasis, N0<N1<N2. （3）The standardized coefficient of miR-203at stage Ⅰ was significantly higher than that at stage Ⅱ, and stage Ⅱ was significantly higher than stage Ⅲ. stage Ⅰ> stage Ⅱ> stage Ⅲ. The standardized coefficient of Notchl at stage Ⅰ was significantly lower than that at stage Ⅱ, and stage Ⅱ was significantly lower than stage Ⅲ. stage I<stage Ⅱ< stage Ⅲ.（4） The standardized coefficient of miR-203 at grade Ⅰ was significantly higher than that at grade Ⅱ, and grade Ⅱ was significantly higher than grade Ⅲ, grade Ⅰ> grade Ⅱ> grade Ⅲ. The standardized coefficient of Notchl at grade Ⅰ was significantly higher than that at grade Ⅱ, and grade Ⅱ was significantly higher than grade Ⅲ, grade Ⅰ< grade Ⅱ< grade Ⅲ. （5）The standardized coefficient of miR-203 at ER,PR,C-erbB-2 （-） was significantly higher than ER,PR,C-erbB-2 （++）,（+++）. The standardized coefficient of Notchl at ER,PR,C-erbB-2 （-） was significantly higher than ER,PR,C-erbB-2 （++）、（+++）2. IHC showed:The expression of Notch protein in breast cancer and hyperplasia lesions in both different frequencies. The expression rate of Notchl were gradually higher in normal breast tissues、UDH、ADH、DCIS and IDC.3. The standardized coefficient of miR-203 were gradually lower in normal breast tissues、UDH、ADH、DCIS and IDC. The standardized coefficient of Notchl were gradually higher in normal breast tissues、UDH、ADH、DCIS and IDC4. These result indicate that the alteration of the expression of of Notchl and JAG1 protein in breast cancer was inverse correlation.Conclusions （1）Abnormal expressing miR-203and Notchl in the formation of breast cancer. miR-203 and Notchl functions differently in different progression in breast cancer. （2） miR-203 and Notchlprotein expression in breast cancer and hyperplasia lesions have different levels. （3） the alteration of the expression of ofNotchl and JAG1 protein in breast cancer was inverse correlation; miR-203 and Notchl may play a important role in breast cancer.Part 3 miR-203 and Notchl gene methylation role of onset in mammary gland carcinomaobjective miR-203 and Notchl gene methylation may play a significant part in the tumorigenesis and the development. The study that to exploring miR-203 and Notchl gene methylation role of onset in mammary gland carcinoma.Methods The methylation of miR-203 and Notchl gene in human breast invasive ductal carcinoma （IDC; n=89）,ductal carcinoma in situ（DCIS; n=20）, atypical ductal hyperplasia（ADH; n=11）and usual ductal hyperplasia （UDH; n=20）were quantitative detection by MassARRY. miR-203 and Notchl miRNA of breast tissues were amplified by one-step RT-PCR method,and the expression of miR-203and Notchl protein was detected by immunohistochemical Streptavidin-Biotin Complex （SP） stain.Results （1） Our results showed that the mean methylation levels of miR-203 gene except CpG₁.2、CpG₃、CpG₈、CpG₉、CpG₁0.11、CpG₁4.15.16、 CpG₁9、CpG₂3、CpG₂4、CpG₂5.26、CpG₂7、CpG₂8.29、CpG 30.31 sites in 4 samples were were gradually lower in normal breast tissues、UDH、ADH、 DCIS and IDC. （P<0.05）. the mean methylation levels of miR-203 gene except CpG₅.6、CpG₂5.26were higher in DCIS and IDC. The methylation states of CpG₈、CpG₁0.11 sites of axillary node metastasis were lower without axillary node metastasis in breast caner, and CpG₈.和 CpG₁9.20 sites in different clinical stage of TNM, stage Ⅰ> stage Ⅱ> stage Ⅲ. CpG₄.5、CpG₈、CpG₁0.11、CpG₂0.21 sites in different grades of pathohistology.and CpG₃、5、CpG₉、CpG₁4.15、CpG 22.23 sites between positive expression of ER、PR、C-erbB-2 and negative expression of ER、PR、 C-erbB-2 had significant statistics difference （P<0.05）. （2） Our results showed that the mean methylation levels of Notchl gene except CpG₃、CpG₄.5、CpG₈ sites were gradually lower in normal breast tissues、UDH、ADH、DCIS and IDC. （P<0.05）.The methylation states of CpG₄.5、CpG₈、CpG₁0、CpG₁5、CpG₁8 sites of without axillary node metastasis were higher axillary node metastasis in breast caner, and CpG₄.5、CpG₁9 sites in different clinical stage of TNM, stage Ⅰ> stage Ⅱ> stage Ⅲ. CpG₁.2、Cp G₁2.13 sites in different grades of pathohistology.and CpG₃、CpG₈、CpG 14.15.16 sites between positive expression of ER、PR、 C-erbB-2 and negative expression of ER、PR、C-erbB-2 had significant statistics difference （P<0.05）. （3） The methylation states of all CpG sites of miR-203 and Notch1 in 4 samples were significant statistics difference. The methylation states of all CpG sites of miR-203 were higher than that of Notchl. The methylation states of CpG₄.5、 CpG₁0.11、CpG₂1.22 sites of miR-203 and the methylation states of CpG₃、 CpG₄.5、CpG₉ sites of Notchl in IDC were lower than DCIS. ADH and UDH. The methylation states of all CpG sites of MiR-203 was positive correlation with the expression of miR-203. The methylation states of all CpG sites of Notchl was negatively correlation with the expression of Notchl.Conclusions These result indicate that the alteration generally hypomethylation of MiR-203 and Notchl gene in breast cancer, and the expression of miR-203 was low, the expression of Notchl was high. This correlation show that the alteration of protein expression result from hypomethylation oncogene miR-203 and Notch1,and this change may have important significance in breast tumorigenesis and the development. The methylation states of CpG₄.5、CpG₁0.11、CpG₂1.22 sites of MiR-203 and the methylation states of CpG₃、CpG₄、CpG₉ sites of Notchl in IDC were lower than DCIS、ADH and UDH,which might be involved in the development and evolution of breast cancer from benign breast lesions to breast cancer.