The Role Of MiR-181a In ER Positive Breast Cancer Endocrine Resistance And Prediction Of Prognosis

BACKGROUNDIn China, breast cancer has become the highest incidence malignant tumors in women. Estrogen receptor (ER) and/or progesterone receptor (PR) is positive in about2/3of breast cancer, and its growth is estrogen-dependent. Anti-estrogen therapy is an effective, less toxic treatment, and specific estrogen receptor modulators tamoxifen (TAM), which blocks the binding of estrogen receptor, has been the golden standards in endocrine treatment. Especially, large-scale randomized clinical trials showed that postoperative tamoxifen therapy for five years, reducing the51%of recurrence risk and28%of death risk in the early ER positive breast cancer patients.Yet, not all patients with metastatic breast cancer respond to endocrine treatments and are considered to have primary (de novo) resistance. Furthermore, all patients who initially respond to endocrine treatment will eventually develop acquired resistance. Unfortunately, up to now the mechanism of endocrine resistance remains uncertain. MicroRNAs (miRNAs) is endogenous short-chain RNA about22nt length, and not encode proteins, which can be complementary to mRNA encoding the binding protein, silence the expression, and plays many roles including regulating cell differentiation, growth, proliferation, metabolism, apoptosis and other functions. Currently studies have found a variety of microRNA involved in development and treatment treatment resistance of breast cancer. We analyzed209cases of breast cancer by microRNA microarray, and found that5microRNAs were associated with the survival of ER positive breast cancer. RT-PCR was used to detect the difference of them between in the tamoxifen resistant breast cancer patients and non drug resistant patients, and found that only miR-181a was elevated in drug resistant patients. Therefore, we speculate that it plays a role in resistance to endocrine therapy in breast cancer. Currently clinical significance of miR-181a in ER positive breast cancer is unclear, its role in endocrine resistance requires further study. We intend in the following study to clarify the relationship between miR-181a and breast cancer endocrine resistant and explore its possible molecular mechanisms through in vitro studies, in order to find a method to predict endocrine resistance and to find potential therapeutic targets.Methods and results1.MiR-181a expression changes in endocrine resistant breast cancer cell lines resistant and impact to endocrine resistanceWe examined the the expression level of miR-181a in SK-BR-3、MDA231、T47D、 MCF-7/W、MCF-7/R-TAM and Bcap-37breast cancer cell lines, and found that miR-181a increased significantly in ER negative breast cancer cells, especially in Her-2positive cancer cells. Expression level of miR-181a was detected by qRT-PCR in breast cancer cells (wild-type tamoxifen sensitive CMF-7/W cell line, tamoxifen resistant CMF-7/R-TAM cell line). The results demonstrate that miR-181a was increased significantly in endocrine resistant CMF-7/R-TAM cells. Pre-miR-181a was transfect to CMF-7/W. The results showed that miR-181a level of CMF-7/W cells increased significantly after transfection, and they were lesser sensitive to tamoxifen. MiR-181a of tamoxifen resistant CMF-7/R-TAM cell lines was knockdown by siRNA interference, and CMF-7/R-TAMcells were more sensitive to tamoxifen correlation with miR-181a level by MTT detection. 2. Relationship between miR-181a and endocrine related pathway markers in breast cancerFulvestranta, elimination ER agent, was used to knockout ER in positive breast cancer cell lines, then expression of miR-181a increased significantly. ER gene was inducted into ER negative breast cancer cell Bcap-37to form Bcap-37(ER positive) with stable expression of ER. Then expression of miR-181a decreased significantly in Bcap-37(ER positive) cells. Above results show that ER is closely related to expression of miR-181a, and decrease miR-181a expression.After regulating of miR-181a expression in MCF-7/W and MCF-7/R-TAM cells respectively, we detect the expression of ER, Her-2, mTOR and PI3K protein. Results indicate that expression of mTOR is higher than before after increasing expression of miR-181a. Expression of mTOR is reduced following by decreasing expression of miR-181a. But the expression of ER, Her-2and PI3K is not influenced by miR-181a.3.Correlation analysis of expression of miR-181a with survival of ER positive breast cancer80patients of early stage breast cancer treated from1998to2005in our department were enrolled in this study. Total RNA was extracted from formalin-fixed paraffin embedded tissues of breast invasive carcinomas.76cases were got useful result. The expressions of miR-181a in specimens were detected by qRT-PCR. Low expression was defined as the value below the cutoff value of miR-181a, which got by ROC curve. High expression is defined as beyond cutoff value. Survival analysis demonstrated that patients with low expression of miR-181a are better in survival, with statistical significance. Further analysis showed that miR-181a cancer was a good predictors of survival in ER positive breast, with statistical significance. Multivariate COX regression analysis showed that miR-181a, endocrine therapy, tumor size and lymph node metastasis were correlated with prognosis of breast cancer, with statistical significance. It is suggested that miR-181a is the independent predictor of prognosis of breast cancer. Bivariate correlation analysis showed that the expression of miR-181a, ER and age, menstrual status were negatively correlated, but had no correlation with other factors, there are significant and statistically significant.ConclusionIn vitro studies show that the expression of miR-181a in tamoxifen resistant breast cancer cells increase significantly. Decreased expression of miR-181a in breast cancer cell line, then sensitivity to tamoxifen significantly increased. Further study show that miR-181a expression is influenced by ER expression; miR-181a can promote the expression of mTOR, but had no significant effect on ER, Her-2and PI3K expression. The expression levels of miR-181a are negatively correlated with the prognosis of the patients with breast cancer by detection of clinical sample. miR-181a is independent prognosis predictors in ER positive breast cancer. Data of our study show the expression levels of miR-181a are negatively correlated with ER and age, menstrual status. Therefore, miR-181a can be used as a potential prognostic factor for prediction of ER positive breast cancer.