A novel zinc finger protein Zfp637 behaves as a repressive regulator in myogenic cellular differentiation.

Zinc finger proteins have been implicated as transcription factors in the differentiation and development of cells and tissues in higher organisms. The classical C2H2 zinc finger motif is one main type of motif of zinc finger proteins. Our previous studies have shown that Zfp637, which comprises six consecutively typical and one atypical C2H2 zinc finger motifs, is highly expressed in undifferentiated or poorly differentiated cell lines, but is moderately or slightly expressed in normal tissues. We have also demonstrated that Zfp637 can promote cell proliferation. However, its role in the regulation of cell differentiation remains unknown. We report here that endogenous Zfp637 as well as mTERT is expressed in proliferating C2C12 myoblasts and that their expression is downregulated during myogenic differentiation. Constitutive expression of Zfp637 in C2C12 myoblasts increased mTERT expression and telomerase activity, and promoted the progression of the cell cycle and cell proliferation. By contrast, endogenous repression of Zfp637 expression by RNA interference downregulated the mTERT gene and the activity of telomerase, and markedly reduced cell proliferation. Overexpression of Zfp637 also inhibited the expression of myogenic differentiation-specific genes such as MyoD and myogenin, and prevented C2C12 myoblast differentiation. Our results suggest that Zfp637 inhibits muscle differentiation through a defect in the cell cycle exit by potentially regulating mTERT expression in C2C12 myoblasts. This may provide a new research line for studying muscle differentiation.

[Construction of eukaryotic expression plasmid for a novel zinc finger protein gene Zfp637 and its effect on breast carcinoma cell EMT6].

OBJECTIVE: To construct the eukaryotic expression plasmid and establish stably transfected cell line, which contained the code gene of zinc finger protein637 (Zfp637), and to observe the effect of Zfp637 gene to the proliferation of tumor cells. METHODS: The Zfp637 DNA was amplified from the template of normal spleen tissue cDNA and cloned into the eukaryotic expression vector pEGFP-C3. The recombinant plasmid, named as pEGFP-Zfp637, was determined by restriction enzyme and sequencing analyses. Next the pEGFP-Zfp637 recombinant plasmid was transferred into mouse breast carcinoma EMT6 cells with lipofectamine 2000, and the stably transfected cells were selected by G418 (named Zfp637-EMT6). The growth condition of cells was observed, and subcellular localization of Zfp637 gene was located by fluorescence microscope at the same time. The Zfp637 mRNA expression in the transfected cells was detected by RT-PCR, and the proliferation of such cells was measured by MTT. RESULTS: The analysis confirmed that the recombinant pEGFP-Zfp637 contained the Zfp637 full-length cDNA. The Zfp637 mRNA was over-expressed stably in Zfp637-EMT6. The growth of Zfp637-EMT6 was increased obviously when compared with the negative control group and blank group. CONCLUSION: The recombinant pEGFP-Zfp637 has been constructed successfully, and the expression of the Zfp637 gene can promote the proliferation of cells. This recombinant eukaryotic expression plasmid can be used in advanced studies in the biological effects of Zfp637 and anti-tumor gene therapy.

[Synergic effect of Na(+)-K(+) ATPaseB1 and adriamycin on inhibition of cell proliferation and reversal of drug resistance in breast cancer MCF-7 cells].

BACKGROUND AND OBJECTIVE: Na(+)-K(+)ATPase (Na(+)-K(+) pump) is an important cell energy conversion system which is probably associated with tumor metastasis. Expression of its B1 subunit gene-ATP1B1 is high in well differentiated and low in poorly differentiated tumor cells. This study was to investigate the synergic effect of Na(+)-K(+) ATPaseB1 and adriamycin (ADM) on inhibition of cell proliferation and reversal of drug resistance in MCF-7 and MCF-7/ADM cells. METHODS: Growth of MCF-7 and MCF-7/ADM cells transfected with PEGFP-ATP1B1 and shATP1B1 were measured by MTT. Intracellular fluorescence intensity of ADM was analyzed by inverted fluorescence microscopy and flow cytometry. ATP enzyme activity was measured by ultramicro-ATP enzyme, and mRNA expression of multi-drug resistance gene MDR1 was detected by RT-PCR and real-time PCR. The expression of P-glycoprotein (P-gp) was analyzed by western blot. RESULTS: The sensitivity of MCF-7 and MCF-7/ADM cells transfected with pEGFP-ATP1B1 to ADM was higher in comparing to the negative control ADM-C3 (transfected with vector pEGFP-C3) and the control ADM-RPMI-1640 (cultured with RPMI-1640), and the differences between ADM-ATP1B1 and ADM-RPMI-1640 groups were statistically significant at different concentrations of adriamycin (P<0.05). After the B1 subunit was silenced, the sensitivity of cells to ADM in the ADM-shNC group was higher than that in the shATP and ADM-RPMI-1640 groups. The mean fluorescence intensity of ADM in the ADM-ATP1B1 group was higher than that in the ADM-C3 and ADM-RPMI-1640 groups (P<0.05). ATP enzyme activity was significantly higher in ADM-ATP1B1 group comparing to the ADM-RPMI-1640 group (P<0.05). mRNA expression of MDR1 gene and protein expression of P-gp were not significantly different among the ADM-ATP1B1 group and two control groups (P>0.05). CONCLUSION: Na(+)-K(+) ATPase B1 can synergize with ADM and reverse drug resistance to ADM in the MCF-7/ADM cell line. This may be related to ATP enzyme activity, but not to influencing the expression of MDR1 gene.