| 【Background】Metastasis is a major cause of death in patients with hepatocellular carcinoma(HCC), although survival has improved due to advances in surgical techniques. Cancer stem cells(CSCs) are characterized by their capacity for indefinite self-renew. In several types of cancer, side populations(SPs) have been shown to be enriched for cells with CSC-like activity and a CSC phenotype. Recent studies have suggested that SP cells may serve as an ideal model for stem cell for research. Micro RNAs(mi RNAs) are frequently dysregulated in cancer where they may behave as tumor suppressor genesor oncogenes. Recently, it was reported that mi R-200 a plays a crucial role in the development of cancer through its regulation of epithelial to mesenchymal transition, cell migration, proliferation and metastasis. Previously, it was shown that mesenchymal to epithelial transition(MET) is promoted by repression of the zinc finger E-box-binding 1(ZEB1) and ZEB2 expression. This study aims to the cancer stem cell research, and ultimately leads to development of new ideas for prevention and therapy for HCC. 【Objective】In present studies, micro RNAs are mainly taken in conventional tumor cells. In this study, the pathway of mi R-200a/ZEB was taken in side populations of HCC, which were more related to tumor invasion and metastasis. Thus, further studies of SP cells from HCC and the components of this pathway may provide new insights into the treatment of this deadly disease. 【Methods】1. The sort of side population: HCC cells were analyzed by dual wavelength FACS after incubation with Hoechst 33342. The expression of ALBU(a marker of mature hepatocytes) and KRT14(a marker of liver stem cells) in side population cells and in non-side population cells that were isolated from HCC cell lines were detected.2. Functional analysis of mi R-200 a in vitro: The detection of the expression of mi R-200 a and downstream molecules ZEB. The technology of chemistry, PCR and western-blot detect EMT markers of E-cadherin, vimentin, N-cadherin.3. Mi R-200 a induces the metastasis of SP cells through the transactivation of ZEB2 expression: To detect ZEB whether antagonizing mi R-200a’s affection in SP malignant phenotype.4. In vivo metastasis assays:(1) tumor size;(2) time of form;(3) metastasis;(4) expression of E-cadherin、vimentin and N-cadherin. 【Results】1. In this study we found that mi R-200 a expression was significantly downregulated in SP cells of human HCC cell lines and HCC tissues. As in the case with HCC cell lines, mi R-200 a expression was much lower in HCC tissues from patients who developed metastasis than in patients who did not develop metastasis. In addition, these clinical data strongly indicate that low expression of mi R-200 a contributes to the metastasis of HCC;2. We sorted SP cells by flow cytometry successfully;3. Mi R-200 a was low-expression in SP cells;4. The down-regulation of mi R-200 a may promote the metastasis of SP cells, whereas the up-regulation of mi R-200 a inhibited the metastasis of SP cells;5. We found that mi R-200 a inhibited ZEB2 expression by binding to the ZEB2 promoter, which inhibits epithelial to mesenchymal transition. Mi R-200 a decreased N-cadherin expression and increased E-cadherin expression. The up-regulation of ZEB2 expression inhibited the increase in E-cadherin expression and the loss of N-cadherin expression. Thus, the mi R-200a-mediated ZEB2/EMT signaling pathway is essential for SP cells in HCC cell lines to metastasize. 【Conclusion】In this study, we show that low expression of mi R-200 a promotes human HCC SP cells to metastasize through the transactivation of ZEB2 expression, which results in the induction of EMT. |
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