Regulatory Mechanisms Of CD44v6 And CD44 Cross-linking In The Malignant Progression Of Breast Cancer

Objectives To study the difference of CD44v6 expression and CD44 cross-linking on the surface of breast cancer cells with different metastasis potential,and to explore its mechanism in the migration and invasion of breast cancer cells,so as to provide a new strategy for the search for biomarkers and therapeutic targets for breast cancer.Methods 1.From the perspective of miRNA specifically targeting CD44v6,to explore its effect on the migration and invasion ability of breast cancer cells(1)MCF-7,T-47 D,BT-549 and Hs-578 t breast cancer cells with different metastasis capacity were selected;(2)The expression of CD44v6 in different breast cancer cells was detected by Western blot assay;(3)The migration and invasion of breast cancer cells were analyzed by Wound healing and Transwell experiments;(4)Using bioinformatics methods,miRNAs targeting CD44 exon v6 were analyzed.By combining the prediction results of miRwalk3.0 and RNAhybrid software,11 miRNAs with high matching degree were selected for verification and finally determined as miR-193b-5p;(5)The binding target predicted by bioinformatics was verified by dual-luciferase reporter gene assay;(6)After overexpression of miR-193b-5p,the effect of miR-193b-5p on the expression of CD44v6 was verified by Western blot and qRT-PCR experiments;(7)Based on Wound healing and Transwell experiments,the effects of down-regulation of CD44v6 expression by miR-193b-5p on the migration and invasion of breast cancer cells were analyzed;(8)The effect of miR-193b-5p-CD44v6 on the proliferation ability of breast cancer cells was analyzed by CCK-8 proliferation detection experiment and plate cloning experiment;(9)qRT-PCR experiment was used to analyze the expression level of miR-193b-5p in the serum of normal control,benign breast tumor and breast cancer patients.The possibility of using miR-193b-5p as a potential biological diagnostic marker was analyzed by ROC curve.2.CD44 cross-linking on the migration and invasion of breast cancer cells.(1)Four types of breast cancer cells with different metastatic capacities,MCF-7,T-47 D,BT-549 and MDA-MB-231,were selected.Differences in CD44 protein expression and cross-linking level of the four breast cancer cells were analyzed by Western blot assay,and differences in migration and invasion ability of the four breast cancer cells were analyzed by Transwell assay;(2)To analyze the difference of CD44 de-crosslinking ability on the surface of breast cancer cells by different concentrations of hyaluronidase;(3)Transwell experiment was conducted to explore the effect of CD44 cross-linking on the migration and invasion of breast cancer cells;(4)The effects of CD44 cross-linking on ERM complex and Merlin protein expression and phosphorylation were investigated by Western blot assay;(5)The correlation between p-Moesin and malignant progression of breast cancer was further verified by TCGA data and immunohistochemical staining of breast cancer tissue chips.Results1.The expression level of CD44v6 was positively correlated with the migration and invasion of breast cancer cells;2.miR-193b-5p can down-regulate the expression of CD44v6 by specifically targeting CD44 exon v6,thus inhibiting the migration and invasion of breast cancer cells;3.Compared with the serum samples of normal and benign tumors,miR-193b-5p had low expression in the serum of breast cancer patients,and ROC curve analysis suggested that miR-193b-5p had potential as a diagnostic marker;4.The degree of CD44 cross-linking was positively correlated with the migration and invasion of breast cancer cells;5.CD44 cross-linking regulates the migration and invasion of breast cancer cells by enhancing the phosphorylation level of downstream Moesin protein;6.p-Moesin is highly expressed in both ductal carcinoma in situ and metastatic breast cancer metastasis,suggesting that p-moesin is related to breast cancer metastasis.Conclusions1.miR-193b-5p specifically targets CD44v6 and inhibits the migration and invasion of breast cancer cells by reducing the expression of CD44v6;2.miR-193b-5p expression in serum samples of breast cancer patients has the potential to become a diagnostic marker of breast cancer;3.CD44 cross-linking promoted the migration and invasion of breast cancer cells by phosphorylating the downstream Moesin protein.Combined with the immunohistochemical results of clinical samples,it was suggested that the expression of p-Moesin was related to the malignant progression of breast cancer.