TGF-β1 Suppresses MiRNA-196a-3p Expression To Promote Triple Negative Breast Cancer Metastasis

【Objective】To investigate the biological functions and molecular mechanisms of mi RNAs in the malignant progression of triple negative breast cancer(TNBC) by TGF-β1 and provide potential predictive diagnostic or prognostic biomarkers in TNBC and promising molecular therapeutic targets for TNBC patients.【Methods】Part I: 1. The expression of mi RNAs by TGF-β1 was detected by Real-time quantitative PCR assays in MCF-7 and MDA-MB-231 cells. 2. Using Real-time quantitative PCR assays is for confirming whether invasive potential of five different breast cancer cells stimulated with TGF-β1 was relevant to mi R-196a-3p expression. 3. The changes of migratory and invasive potential in MDA-MB-231 cells transfected mi R-196a-3p mimics were detected by Transwell assays. 4. The changes of proliferative potential in MDA-MB-231 cells transfected mi R-196a-3p mimics were detected by MTT assays.Part II: 1. Using Real-time quantitative PCR assays, we investigated whether the 4 potential candidate genes of mi R-196a-3p were up-regulated in response to TGF-β1. 2. Using Real-time quantitative PCR assays and Western blot assays, we examined the TGF-β1 response on their respective m RNA and protein levels in MDA-MB-231 cells transfected or not with the mi R-196a-3p mimic. 3. Using Real-time quantitative PCR assays, we examined the expression of NRP2 in MDA-MB-231 cells transfected with increasing concentrations of mi R-196a-3p mimics. 4. Using dual luciferase reporter gene system, we investigated whether mi R-196a-3p specifically binds to the NRP2 3’-UTR sequence to control NRP2 gene expression.Part III: 1. Using Real-time quantitative PCR assays, we examined the efficiency of NRP2 si RNA silencing the NRP2 levels in MDA-MB-231 cells. 2. The changes of migratory and invasive potential in MDA-MB-231 cells which the expression of NRP2 was silenced were detected by Transwell assays. 3. The changes of proliferative potential in MDA-MB-231 cells which the expression of NRP2 was silenced were detected by MTT assays.Part IV: 1. Establishing xenograft tumor nude mice model, we investigated whether mi R-196a-3p could suppress the growth of TNBC in vivo. 2. Establishing lung metastasis models, we investigated whether mi R-196a-3p could suppress TNBC metastasis in vivo. 3. Using Real-time quantitative PCR assays, we examined the different expression of mi R-196a-3p in breast cancer tissues and normal breast tissues. 4. Using Real-time quantitative PCR assays, we examined the different expression of mi R-196a-3p in 4 kinds of molecular subtypes of breast cancer.【Results】Part I: 1. There was the most obvious difference of mi R-196a-3p levels between MCF-7 and MDA-MB-231 cells. 2. The magnitude of mi R-196a-3p expression stimulated by TGF-β1 correlated negatively with the metastatic potential of breast cancer cells. There was the lowest expression of mi R-196a-3p in MDA-MB-231 cells. 3. Overexpression of mi R-196a-3p inhibited the migratory and invasive properties of MDA-MB-231 cells. 4. Overexpression of mi R-196a-3p had no influence on the proliferative properties of MDA-MB-231 cells.Part II: 1. Two target genes ZNF280 B and NRP2 showed up-regulation in response to TGF-β1. 2. Mi R-196a-3p mimic expression did reverse the TGF-β1 effect on NRP2 m RNA and protein levels. 3. With MDA-MB-231 cells transfected increased concentration of mi R-196a-3p mimics, the expression of candidate gene NRP2 gradually decreased. 4. Mi R-196a-3p specifically binded to the NRP2 3’-UTR sequence to control NRP2 gene expression.Part III: 1. The expression of NRP2 was silenced using NRP2 si RNA in MDA-MB-231 cells. 2. Silence of NRP2 inhibited the migratory and invasive properties of MDA-MB-231 cells. 3. Silence of mi R-196a-3p had no influence on the proliferative properties of MDA-MB-231 cells.Part IV: 1. Overexpression of mi R-196a-3p had no effect on the growth of transplantation tumor. 2. Overexpression of mi R-196a-3p suppressed TNBC metastasis. 3. The expression of mi R-196a-3p was down-regulated in breast cancer. 4. There was the lowest expression of mi R-196a-3p in basal-like breast cancer.【Conclusions】In the development process of TNBC, with the concentration of TGF-β1 in the tumor microenvironment gradually increasing, the expression of mi R-196a-3p gradually occurs to decline, resulting in the disappearance of down-regulation of NRP2. The expression of NRP2 occurs to rise. Tumor cells ultimately obtain more invasive potential and occur to distant metastasis to lung.