| Backgroud: Breast cancer is a major cause of mortality among women. Paclitaxel is commonly used for chemotherapy of breast cancer, yet its efficacy is limited by chemoresistance. Generally, drug resistance is associated with acquisition of the epithelial-mesenchymal transition(EMT) in cancer. Targeting EMT could be a novel therapeutic strategy for treatment of breast cancer. mi RNA are master regulators of gene expression in many biological and pathological processes, including mammary gland development and breast cancer. Emerging evidence has demonstrated that micro RNAs(mi RNA) play a key role in chemotherapy-induced epithelial-mesenchymal transition(EMT) in breast cancer. Some of these mi RNA have been shown to control cellular plasticity through the suppression of EMT-inducers or to influence cellular phenotype through the suppression of genes involved in defining the epithelial and mesenchymal cell states.Objective: To study the regulation of mi R-125 b to acquisition of epithelial-mesenchymal transition in paclitaxel-resisitance breast cancer cells.Methods: ⑴ q RT-PCR was used to analyze the differential expression of mi R-125 b between breast cancer and adjacent normal tissue as well as the expression in PR(paclitaxel-resistant) cells and their parental cells. ⑵ The attachment and detachment assay, the wound-healing assay and the transwell assay were applied in detecting the biological activities of PR cells after mi R-125 mimics treatment and changes of cellular morphology were observated and recorded under the binocular inverted microscope. ⑶ q RT-PCR and Western blotting assay were used to detect the expression of markers of EMT such as E-cadherin,Snail, Slug, E-cadherin, Vimentin in PR cells after mi R-125 mimic treatment. ⑷ Bioinformatics technology, and q RT-PCR, the western blotting, Luciferase test were used to verify whether SEMA4Cis a target gene of mi R-125 b. ⑸ Immunohistochemical experiment testing SEMA4 C expression in breast cancer tissue. ⑹ Fisher’s exact probability method is used to analysis the expression of mi R-125 b and SEMA4 C in mammary infiltrating ductal carcinoma and the correlation between the patients with pathological features. ⑺ RNAi reduce the expression of SEMA4 C in PR cells; The attachment and detachment assay, the wound-healing assay and the transwell assay were applied in detecting biological activities of PR cells transfected with SEMA4 C si RNA. ⑻ q RT-PCR and the western blotting were used to detect the expression of markers of EMT in PR cells transfected with SEMA4 C si RNA. ⑼ We conducted SRB assay in PR cells treated with mi R-125 b mimic or SEMA4 C si RNA to detect the changes of PR cells to paclitaxel sensitivity.Results: ⑴ q RT-PCR revealed that mi R-125 b level were lower in breast cancer and PR cells compared with adjacent normal tissue and parental cells. ⑵ The capability of PR cells in attachment and detachment, migration and invasion have decreased(P<0.05)after mi R-125 mimics treatment compared with the control group; We observed that mi R-125 b mimic treatment led to PR cells changed from elongated, fibroblastoid morphology to a rounded shape. ⑶ We observed that E-cadherin significantly elevated in PR cells after mi R-125 mimic treatment. Otherwise, Vimentin, Snail and Slug decreased compared with control group(P<0.05). ⑷ Bioinformatics technology showd that SEMA4 C is one of the potential target genes of mi R-125 b and Luciferase test, q RT-PCR, the western blotting further verify it. ⑸ Immunohistochemical experiment confirmed high expression of SEMA4 C in breast cancer which is negatively related with the expression of mi R-125 b, and clinical pathology data confirmd that both are negatively related with the expression of cerb B-2 as well as lymphatic metastasis of breast cancer. ⑹ RNAi confirmed that SEMA4 C si RNA can greatly decrease the expression of SEMA4 C in PR cells. SEMA4 C si RNA decreased the abilities of attachment, detachment, migration and invasion of PR cells.(P <0.05). ⑺ After transfecting with SEMA4 C si RNA, We found the expression of E-cadherin increased(P<0.05);otherwise Snail,Slug andVimentin reduced(P<0.05). ⑻ SRB assay confirm up-regulation of mi R-125 b or depletion of SEMA4 C enhances PR cells to paclitaxel sensitivity.Conclusion: These findings suggest that mi R-125 b decreased in epithelial-mesenchymal transition(EMT) change of PR cells, and may play a critical role in EMT via SEMA4 C which is one of its target genes. Up-regulation of mi R-125 b or targeting SEMA4 C could reverse EMT and can also be helpful to clinical breast cancer adjuvant chemotherapy plan formulation. |
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