Primary Study On The Effect Of MicroRNA-10b By Targeting CDH1 On Promoting Metastasis Of Breast Cancer

Part 1 The expression analysis of miR-10b and its downstreamregulated gene in breast cancer tissues and nomal breast tissues Objective To study the expression and clinical implications of miR-10b, CDH1mRNA and Epithelial-cadherin (E-cad) protein in breast cancer tissues and nomal breast tissues. And to analyse the correlation between the expression of miR-lOb and CDH1mRNA and E-cad. Methods The expressions of miR-10b and CDH1mRNA were detected in 16 normal breast tissues,21 primary breast cancer tissues and 23 metastatic breast cancer tissues by Real-time fluorescent quantitative reverse transcription-polymerase chain reaction. Western blot was used to examine the expression of E-cad in three different types of breast tissues. Results 1. The expression of miR-10b was mildly lower by 18.8 per cent in primary breast cancer group than in normal breast group (p=0.132); the expression levels of miR-10b in metastatic breast cancer group was significantly higher than that in normal breast tissues and primary breast cancer tissues with 5.0 and 6.1 folds, respectively (p=0.015).2. The expression of CDH1mRNA was mildly higher by 11.5 per cent in primary breast cancer group than in normal breast group (p=0.270); the expression levels of CDH1mRNA in metastatic breast cancer group was significantly lower than that in normal breast tissues and primary breast cancer tissues with 5 and 6 folds, respectively(p=0.021).3. The expression of E-cad was mildly higher by 20 per cent in primary breast cancer group than in normal breast group (p=0.386); the expression levels of E-cad in metastatic breast cancer group was significantly lower than that in normal breast tissues and primary breast cancer tissues with 5.2 and 6.3 folds, respectively (p=0.007).4. The expression level of miR-10b was positively correlated with with tumor size, histological grade, lymph node metastasis, human epidermal growth factor receptor-2, ki-67 and clinical stage, and was negatively with estrogen receptor and progesterone receptor (p<0.05), while miR-10b expression, histological type of breast cancer and age were not correlated (p>0.05).5. In breast cancer, significantly correlation was found between the expression of CDH1mRNA, E-cad and that of miR-10b.Conclusion 1. The expression level of miR-10b is significantly increased in metastatic breast cancer, while the expression level of CDH1mRNA and E-cad are significantly reduced, there is a significantly negative correlation between CDH1mRNA, E-cad and miR-10b.2. MicroRNA-10b promotes metastasis of breast cancer by targeting the expression of CDH1mRNA.3. MicroRNA-10b can be a predictor of metastasis and prognosis of breast cancer, and it is closely related to poor prognostic factors of breast cancer. Part 2 The effect of down-regulation miR-10b on cell invasion ability of human breast cancer cell line MDA-MB-231Objective To analyze the changes of expression level of CDH1mRNA and E-cad, and the effect on cell invasion ability for MDA-MB-231 human breast cancer cell after down-regulation of miR-10b. To explore the function and pathway of miR-10b in metastasis of breast cancer.Methods MDA-MB-231 human breast cancer cells were transiently transfected with miR-10b inhibitor by means of liposomes media methods. Transient transfection were evaluated by fluorescent microscope. After 48h, the expression of miR-10b and CDH1mRNA was detected by Real-time fluorescent quantitative reverse transcription-polymerase chain reaction (FQ-RT-PCR) and the expression of E-cad was tested by Western blot. Mock group was used as control. Transwell invasion assay was used to examine metastatic ability of MDA-MB-231 cell transfected by miR-10b inhibitor.Results 1. Green fluorescence was observed between four and six hours after transfection. 2. The expression of miR-10b was significantly down-regulated in MDA-MB-231 cells transfected with inhibitor as compared with that in mock group (p=0.009), and the expression of CDH1mRNA was significantly increased as compared with that in mock group (p=0.009). E-cad was significantly increased (p=0.037).3. Down-regulation of miR-10b significantly reduces the number of cell penetrated through the membrane in miR-10b inhibitor group (p=0.033).Conclusion 1. Down-regulation of miR-10b can significantly increase the expression of CDH1mRNA and E-cad, and then result in the decrease of metastatic ability of MDA-MB-231.2.This study of cell function preliminarily verified that the miR-10b/ CDH1mRNA pathways promoted metastasis in breast cancer. Part 3 The effect of over-expression miR-10b on cell invasion ability of human breast cancer cell line MDA-MB-231Objective To analyze the changes of expression level of CDH1mRNA and E-cad, and the effect on cell invasion ability for MDA-MB-231 human breast cancer cell after over-expression of miR-10b. To explore the function and pathway of miR-10b in metastasis of breast cancer.Methods MDA-MB-231 human breast cancer cells were transiently transfected with the stimulant (mimics) of mature homo miR-10b by means of liposomes media methods. Transient transfection were evaluated by fluorescent microscope. After 24h and 48h, the expression of miR-10b and CDH1mRNA was detected by Real-time fluorescent quantitative reverse transcription-polymerase chain reaction (FQ-RT-PCR) and the expression of E-cad was tested by Western blot. Mock group was used as control. Transwell invasion assay was used to examine metastatic ability of MDA-MB-231 cell transfected by miR-10b mimics.Results 1. Green fluorescence was observed between four and six hours after transfection. 2. The expression of miR-10b was significantly up-regulated in MDA-MB-231 cells transfected with mimics as compared with that in mock group (p=0.001), and the expression of CDH1mRNA was significantly decreased as compared with that in mock group (p=0.002). E-cad was decreased up to 12.5 folds (p=0.006).3. Over-expression of miR-10b significantly increases the number of cell penetrated through the membrane in miR-10b inhibitor group (p=0.018).Conclusion 1. Over-expression of miR-10b can significantly decrease the expression of CDH1mRNA and E-cad, and then conduce to the enhancement of metastatic ability of MDA-MB-231.2. This study of cell function verified again that the miR-10b/ CDH1mRNA pathways promoted metastasis in breast cancer.