| Background Beast cancer is not only one of the most malignant cancer, but also a highly heterogeneous cancer in women. It was divided into at least four subtypes, which were Lumina A, Lumina B, HER-2 overexpression and basal-like subtype according to the St Gallen International Expert Consensus hold in 2011. Basal-like breast cancer(BLBC) was also referred to tripe negative breast cancer due to negative expressions of estrogen receptor, progesterone receptor and Erb-B2, so endocrine therapy and anti-HER2 therapy were proved useless in clinic, and the main therapy for BLBC or TNBC was chemotherapy which was of high toxicity. Hence it is one direction of high importance of searching specific target for treating TNBC personalized. Epithelial-mesenchymal transition(EMT) is a biologic process which enables ploarized polarized epithelial cell to undergo multiple biochemical changes and then acquire the mesenchymal cell phenotype. From the initiation to the completion of EMT, a number of biochemistry processes are involved, for example, transcription factors are activated, cytoskeletal proteins are reorganized and expressed, ECM-degrading enzymes are producted, and the expression of specific micro RNAs are changes. The all changes mentioned above lead to a different inter- and intra-cellular characteristcis, which mainly are the lost of epithelial polarization, the degradation of basement membrane, and migration of mesenchymal to other sites. Normally, EMT is a critical process that participates in the embryonic development and repair of damaged tissues. Pathologically, EMT is an important biologic process that malignant tumor cells of epithelial source assume the migratory and invasive ability. Slug is an important transcript factor for regulating the progression of epithelial-mesenchymal transition(EMT) through multiple approch. E-cadherin is a kind of cell adhesion molecules that could prevent the migration, invasion and metastasis of cancer cells, which decrease the occurrence of EMT. It is well clear that the loss of E-cadherin is the most vital hallmark of EMT. Meanwhile, E-cadherin is directly down-regulated through the binding of E-box by Slug, and then relieves the inhibition of EMT. Micro RNA(mi RNA) is a kind of non-coding RNA with 22-25 nt in length and negatively regulate messenger RNA(m RNA) by partially or totally binding to the 3’untranslated regions(3’UTR) of the target m RNA. Mi R-200 c is a member of mi R-200 family(mi R-200 a, mi R-200 b, mi R-200 c, mi R-141 and mi R-429) and plays an role in inhibiting EMT and reducing the metastatic ability of cancer cells by directly binding transcript factors such as ZEB1, ZEB2, HMGB1, Pin1 and so on. Plenty of studies have proved mi R-200 c to be an essential factor in regulating cell infiltrating, invasion and metastasis of breast cancer cells. And it currently remains the hot spot in breast cancer research. Liu and Fu et al. found that mi R-200 c could inhibites EMT process by down-regulating Slug expression in human AC3 cell line and malignant glioblastoma, respectively. Later on we predicted that there were complementary site between mi R-200 c and the 3’UTR of Slug by analyzing the databases such like microrna.org and targetscan.org. We further conducted a series of experiments and found that in TNBC cell line BT549, mi R-200 c could also inhibit EMT process by up-regulating E-cadherin expression via the downregulation of Slug expression. However, it was pointed out by St Gallen International Expert Consensus in 2015 that there was still huge heterogeneity among the TNBC. For example, a proportion of triple negative cancer showed exquisite sensitivity to chemotherapy, while another part of triple negative cancers were highly resistant to the same chemotherapy regimen. So far, the research of relationship between mi R-200 c and Slug in breast cancer does not go farther, and whether or not mi R-200 c could regulate EMT process via the mentioned mechanism remains unknown.Objective In this study, we employed two TNBC cell lines(BT549 and MDA-MB-231) and two non-TNBC cell lines(MCF-7 and SK-BR-3) to detect the expressions of Slug and E-cadherin at both protein level and RNA level after being transfected with mi R-200 c mimics and corresponding negative control. Then we intended to found what the influences of mi R-200 c were on the migratory ability of breast cancer cell lines, and to provide a new efficient target for treating triple negative cancers.Materials and Methods 1 Materials and groups BT549, MDA-MB-231, MCF-7 and SK-BR-3 were all cultured in RPMI-1640 culture medium with 1% Penicillin-Streptomycin and 10% Fetal Bovine Serum(FBS) in incubator at 37℃ and 5% carbon dioxide. Each cell lines were divded into five groups, Group A, mi R-200 c mimics/Lipo 2000; Group B, mi R-200c-control/ Lipo2000; Group C, mi RNA-control/Lipo 2000; Group D, Lipo2000 and Group E: blank control. 2 Methods 2.1 Four cell lines were cultured with RPMI-1640 complete culture under the moist condition of 37℃,5%CO2. When cells reached the exponential phase, they were inoculated into six-well plates with a fixed concentration for regular culture. Then the cells were transfected with mi R-200 c mimics and corresponding negative control. Sequent experiments were carried out when cells reached about 95% confluence. 2.2 After cells being transfected with mi R-200 c, total RNA from different groups were extracted and reverse transcribed into c DNA. We then detected the expressions of Slug and E-cadherin with specific forward- and backward- primers by RT-PCR. 2.3 After cells being transfected with mi R-200 c, total protein from different groups were extracted, we then used western blot to detect the expressions of Slug and E-cadherin at protein level. 2.4 BT549 and MDA-MB-231 were seeded into six-well plates, after a 24-h culture, the cells were cultured with serum-free medium for another 12 h, then two parallel lines were drawn with a 200 μl tip to reflect the change of migratory ability of breast cancer in four different groups. 3 Statistics analysis SPSS 19.0 were adopted to analyze the experimental data. Measurement data were expressed as mean±SD, differences among different groups were compared using ANOVA. LSD-t test was used to compare the difference between any two groups. P<0.05 was considered statistically significant.Results 1 Only when cells reached exponential phase were they detatched from culture bottle and inoculated into six-well plates. For BT549 and MDA-MB-231, a total of 1×105 cells were used to be inoculated into each well; for MCF-7 and SK-BR-3, a total of 3×105 cells were used to be inoculated into each well. 2 There were significant differences in the expressions of Slug and E-cadherin at both m RNA and protein levels in blank control groups of TNBC and non-TNBC cell lines(F=65.65 and 6.57,P<0.001 and 0.015;P=92.72 and 255.64,P<0.001) 3 In TNBC cells BT549 and MDA-MB-231, the cells transfected with mi R-200 c showed significant down-regulation of Slug(F=39.29 and 35.73;41.79 and 38.31,P<0.001), up-regulation of E-cadherin(F=78.44 and 81.34;262.2 and 63.54, P<0.001) 4 In the cells transfected with mi R-200 c mimics, the wound recovery rate was lower than the rest groups as time passed by in particular at 36 h(F=8.23 和 12.99, P<0.001)Conclusion mi R-200 c could inhibit EMT of TNBC cell lines by up-regulating the expression of E-cadherin via down-regulating the expression of Slug. |
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