ABSTRACT
Objective:Enriching and isolating breast cancer stem cells from breast cancer transplantation tumors in nude mice.Methods:Human breast cancer MDA-MB-231 cells were injected into the right axilla subcutaneous of 20 nude mice, and the tumor growth was observed .After 30 days, tumors were isolated and stained with hematoxylin and eosin, and then tumor cells from tissues were isolated. DMEM medium containing serum was used to cultivate isolated transplantation tumor cells, cell morphology and growth were also observed. Flow cytometry was used to detect the proportion of stem cells (CD44 +/CD24 -/low cells) in transplantation tumor cells. Serum-free DMEM medium containing epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and B27 cell supplement were used to cultivate transplantation tumor cells and to obtain cell microspheres. The proportion of stem cells on the 10th day in cell microspheres was detected by using flow cell sorter and stem cells were isolated according to the markers of cell surface. Results:After subcutaneously injecting MDA-MB-231 cells into 20 nude mice for 9 days, 17 nude mice had subcutaneous tumors with more parenchymal cells, little interstitial cells, arranged cords tumor cells, large volume of the cell and abundant cytoplasm, the nuclei in different sizes and hyperchromatic state, mitotic more common, the nucleoli clear and obvious pleomorphy. After cultivating transplantation tumor cells with DMEM medium containing serum, the cells began to grow adherent after 24 h, and the adherent proportion rose to 60% after 3 days; after 7 days, the cell proliferation was accelerated; and the cell morphology was more consistent, most of which were spindle shaped and were not significantly different from MDA-MB-231 cells; the proportion of stem cells in transplantation tumor cells was (0.10±0.02)%. After cultivating transplantation tumor cells with serum-free DMEM medium containing cell cultured supplement, the cells grow in spherical patterns, the proportion of stem cells in cell microspheres got up to (70.47±2.03)% on the 10th day.Conclusions:Subcutaneously injecting MDA-MB-231 cells in nude mice can build breast cancer nude mice ectopic transplantation tumor model. Breast cancer stem cells in the transplantation tumors can be enriched from isolated transplantation tumor cells through serum-free medium, and more stem cells can be isolated to provide the research basis for the biological characteristics of breast cancer stem cells.
ABSTRACT
BACKGROUND: The transcription factor Runx2 is the key factor that regulates osteogenic differention and bone development. It has been reported that the C2C12 mesenchymal cells can be induced to differentiate into osteoblasts by Runx2 overexpression, but the molecular mechanism of induction is stil largely unclear. OBJECTIVE: To investigate the role of the members of the miR-376 family during Runx2-induced osteogenic differentiation in C2C12 cells. METHODS: The expression of the members of the miR-376 family was detected by real-time quantitative PCR at different time points using C2C12/Runx2Dox sub-line with conditional Runx2 expression. In miR-376b-3p-transfected C2C12/Runx2Dox cells, the expression of osteoblast markers, such as alkaline phosphatase and osteocalcin, was detected by real-time quantitative PCR, and the alkaline phosphatase activity was also examined by alkaline phosphatase staining. The putative miR-376b-3p targets were commonly predicted by online tools (miRanda, miRWalk and TargetScan). The functional classification of these putative targets was performed by DAVID Bioinformatics Resources database. RESULTS AND CONCLUSION: The expression of miR-376b-3p was significantly increased during Runx2-induced osteogenic differentiation of C2C12 cells, but the expression of other members was not changed. Transfection of miR-376b-3p mimic upregulated alkaline phosphatase expression, but had no effect on osteocalcin expression. The alkaline phosphatase activity was also increased by transfection of miR-376b-3p. The functional classification of miR-376b-3p putative targets showed that miR-376b-3p is involved in the skeleton development, indicating the role of miR-376b-3p in osteoblast differentiation. Taken together, these results suggest that Runx2 promotes early osteogenic differentiation in C2C12 cells by regulating the expression of genes related to osteogenic differentiation through upregulation of miR-376b-3p.