The Proliferation and Differentiation of Erythroid Cell Lines Affected by TGIF1 through Regulating the Expression of Erythroid Specific Factors, Iron Metabolism and Apoptosis-related Genes

Haimin Tan^{1, #}, Hongyu Zhu^{2, #}, Huimin Chen¹, Yu Duan¹, Xiaoqiao Zhou¹, Mengjiao Lou¹, Xiaoping Xia^{1, *}

¹Department of Laboratory, The Fourth Affiliated Hospital Zhejiang University School of Medicine, Yiwu 322000, PR China - ²Department of Hemodialysis Room, The Fourth Affiliated Hospital Zhejiang University School of Medicine, Yiwu 322000, PR China

Objective: To analyze the effect of transforming growth influencing factor 1 (TGIF1) on the proliferation and differentiation of erythroid cell lines by regulating the expression of erythroid-specific factors, iron metabolism and apoptosis-related genes. 

Method: TF-1 cells were put into a cell incubator for cultivation, appropriate amount of cells were taken for electrotransformation, the TGIF1 interference plasmids were cloned and cultured and introduced into TF-1 cells, and after screening by G418 drug, stable cell lines with TGIF1 knockdown were established in erythroid TF-1 cells. Next, the TGIF1 interference group was cultured, and a negative control group was set. The expression of TGIF1 of cells in each group was observed and measured using an inverted fluorescence microscope, real-time fluorescence quantitative PCR, and Western blotting. The γ globin, ε globin, erythroid-specific factor DNaseⅠ hypersensitive point (GATA1), Kruppel-like factor 1 (KLF1), FOG1, nuclear transcription factor erythrocyte 2 (NF-E2), iron metabolism-related factors IRP2, hepcidin, apoptosis-related genes Bax, and Bcl-2 expression levels were measured using a real-time fluorescence quantitative PCR method. The expressions of the CD235a and CD71 of cells in each group were then measured using flow cytometry. Finally, the changes of cell proliferation in each group were measured using a CCK-8 method.

Results: Compared with the control group, the expression levels of TGIF1, γ globin, ε globin, CD235a, CD71, GATA1, KLF1, FOG1, NF-E2, IRP2, and Bcl-2 of the cells in the TGIF1 interference group were significantly lower, and the expression levels of hepcidin and Bax were significantly higher (P<0.01). There was no significant difference in the cell proliferation level between the two groups at 24h (P>0.05); from 48h, the cell proliferation ability of the TGIF1 interference group was significantly lower than that of the control group (P<0.01). 

Conclusion: Down-regulation of TGIF1 expression can significantly inhibit the proliferation and differentiation of erythroid cell lines, which may be achieved by affecting the expression levels of erythroid-specific factors, iron metabolism, and apoptosis-related genes.