[Effects of receptor-interacting protein 140 gene knockdown on the proliferation of microglioma cells: experiment with rat microglioma cells].

OBJECTIVE: To investigate the effects of receptor-interacting protein (RIP)140 gene knockdown on the proliferation of microglioma cells. METHODS: Mouse microglioma cells of the line BV-2 were cultured and transfected with 2 kinds of recombinant RIP140-shRNA plasmids (V2MM-71674 and V2MM-71080) or blank plasmid MSCV-EGFP. Real-time PCR and Western blotting were used to detect the mRNA and protein expression of RIP140; and the cell proliferation was detected by MTT assay. RESULTS: There were not significant differences in the RIP140 mRNA and protein expression between the BV-2 and BV-2-MGCV-EGFP groups. Compared to those of the BV-2 group, the RIP140 mRNA expression levels of the BV-2-71674 and BV-2-71080 groups were lower by 73% and 75% respectively. The protein expression levels of the BV-2-71674 and BV-2-71080 groups were remarkably lower than those of the BV-2 and BV-MSGV-EGFP groups. MTT assay showed that there were not significant differences in the proliferation rates at different time points between the BV-2 and BV-2-MSCV-EGFP groups, however, the proliferation rates at the time points of 24, 48, 72, and 96 h of the BV-2-71674 and BV-2-71080 groups were significantly lower than those of the BV-2 group (all P<0.01). CONCLUSION: RIP140 gene knockdown effectively inhibits the proliferation of microglioma cells.

Overexpression or knock-down of runt-related transcription factor 1 affects BCR-ABL-induced proliferation and migration in vitro and leukemogenesis in vivo in mice.

BACKGROUND: Runt-related transcription factor 1 (Runx1) plays a crucial role in hematogenesis and its dysfunction may contribute to leukemogenesis. However, it is not clear whether or not abnormal expression of Runx1 will induce leukemia and how the change of Runx1 expression level could affect BCR-ABL-induced leukemogenesis. In the present study, we aimed to analyze if abnormal expression of Runx1 in BaF3 cells alone would induce leukemogenesis. And we also wanted to know if abnormal expression of Runx1 in leukemic cells would affect leukemogenesis. Furthermore, we investigated whether overexpression or knock-down of Runx1 in BaF3 cells would induce leukemogenesis. METHODS: Plasmids containing full-length Runx1 cDNA were transduced into BaF3 cells and BaF3-P185wt cells (BCR-ABL transformed BaF3 cells) by electroporation. Plasmids containing a short hairpin RNA of Runx1 were transduced into BaF3 cells and BaF3-P185wt cells by electroporation. Runx1 expression level was quantified by Western blotting and quantitative real-time PCR. The effects of overexpression or knock-down of Runx1 on proliferation, apoptosis and migration of cells were detected in vitro. Then, using MSCV-P185wt-EGFP as a control, we transplanted MSCV-P185wt-Runx1 cells or MSCV-P185wt-shRNA cells into Balb/c mice through tail vein and observed tumorgenesis of the different phenotypes. RESULTS: In vitro analysis revealed that overexpression of Runx1 in P185wt cells could inhibit cell proliferation and slow down cell migration; while knock-down of Runx1 could promote cell proliferation and speed up cell migration. In vivo analysis indicated that mice transplanted with MSCV-P185wt-Runx1 survived longer than controls. In contrast, mice transplanted with MSCV-P185wt-shRNA survived shorter than the control group. Gross pathological analysis revealed that the MSCV-P185wt-Runx1 group had less severe splenomegaly and hepatomegaly compared to the control group, and the MSCV-P185wt-shRNA group had more severe splenomegaly and hepatomegaly. No splenomegaly or hepatomegaly was detected in mice transplanted with MSCV-BaF3-Runx1 cells or MSCV-BaF3-shRNA cells. Both the mice of MSCV-BaF3-Runx1 group and MSCV-BaF3-shRNA group were healthy with no sign of leukemia for up to three months. CONCLUSIONS: Overexpression or knock-down of Runx1 gene in BaF3 cells alone could not induce leukemogenesis. However, in BaF3-P185wt cells, alteration of Runx1 expression could affect BCR-ABL-induced proliferation and migration in vitro and leukemogenesis in vivo.