Expression Of Zinc Finger Protein521on Mouse Breast Cancer Cells And Regulation Of MiR-9-1

Background&ObjectiveBreast cancer is one of the high incidence malignant tumors in female, and its metastatic mechanisms are very complex. Zinc finger protein521(Zfp521), which belongs to the superfamily of C2H2-type zinc finger proteins, comprises1311amino acids and contains30Kruppel-like zinc. It is wildly expressed in cerebellum, dorsal root ganglion, spleen, adrenal gland, lymph nodes, thymus and fetal tissue, etc al, but low expression in normal breast tissue. The expression of Zfp521in breast cancer cells remains no report. This study will investigate the expression of Zfp521and its possible role in mouse breast cancer cells and the effect of microRNA-9-1(miR-9-1) which is the most important microRNA in promoting tumor metastasis, in the regulation of Zfp521.Materials and MethodsMouse breast cancer cell line4T1is selected to be the experimental cells. RNA interference and miR-9-1lentivirus vector are applied to construct Zfp521siRNA4T1cells and miR-9-1overexpressing4T1cells. The cells were divided into:4T1(untransfected group),4T1SiRNA (transfected with Zfp521siRNA),4T1siRNA control (transfected with negative control siRNA),4Tlmir (infected with miR-9-l-LV),4Tlmir control (infected with FU-RNAi-NC-LV) groups. The ability of invasion and migration was detected by scratch test, Transwell chamber test and invasion assay. The viability of4T1was detected by MTT. The expression of Zfp521and E-cadherin were detected by immunocytochemistry. The expression of miR-9-1was detected by real-time PCR. The role of miR-9-1in the regulation of Zfp521was evaluated by double-fluorescence staining method.Results1. Zfp521and E-cadherin are expressed in4T1cells.2. After transfection of Zfp521siRNA, part of the cell morphology were transformed from round epithelial cells to spindle interstitial cells, and cells arranged loosely, separated each other, tending to epithelial-mesenchymal transition. The highest transfection efficiency was on4th and the efficiency of transfecion was up to81.1±3.96%under an invert fluorescence microscope. MTT assay showed that the viability of4T1sjRNA cells was significantly lower than4T1and4T1siRNAcontrol groups, and there was statistical significance(P<0.01). There was a lower expression of Zfp521and E-Cadherin in4T1siRNA group than4T1and4T1siRNA control groups(P<0.01). Transwell chamber test and scratch test showed that the migration and invasion ability of4T1siRNA group was stronger than4T1and4TlsiRNA control groups(P＜O.Ol).3. The best infection efficiency was on4th and up to90.5±5.16%in4Tlmir group, and GFP could be continuously expressed after passaged. The results of real-time PCR showed that the expression of miR-9-1in4Tlmir group was more than6times as much as the one in4T1and4Tlmir control groups. The expression of Zfp521and E-Cadherin in4Tlmir group were significantly lower than in4Tland4Tlmir control groups(P<0.01). Transwell chamber test and scratch test showed that the migration and invasion ability of4Tlmir group was stronger than4T1 group and4Tlmir control group (P<0.01)4. Double-fluorescence staining method showed that miR-9-1could inhibite the expression of Zfp521by binding it, and the inhibition rate was about25.26%.Conclusion1. Zfp521is expressed in4T1cells.2. Zfp521can promote the metastasis of4T1cells.3. MiR-9-1may promote the metastasis of4T1breast cancer cells by downregulating the expression of Zfp521.