The Transcription Factor SCL/TAL-1 Plays a Positive Role in the Erythropoietic Differentiation Via MEK/ERK Pathway in EPO-Induced K562 Cell Line
Authors: Gong Yuping , Zhou Ruiqing , Niu Ting 
Published: October 2019

Abstract
Abstract 4799

Objective
In the present study, EPO-induced K562 cell line was used to be the cell model of erythroid differentiation, the role and mechanism of transcript factor SCL/TAL-1 in the erythropoiesis was investigated.

Methods
Three plasmids, which included pTRIPdU3-RNAiTALh -EF1a-GFP (SCL/TAL1 shRNA to reduce the expression of SCL/TAL-1), pTRIP-EF1a-TAL1 (SCL/TAL-1 cDNA to enhance the expression of SCL/TAL-1) and control plasmid pTRIP-dU3-RNAiluc-EF1-GFP expressing EGFP gene, were transfected into K562 cell line via lentiviral vector system, and K562 SCL/TAL-1low, K562 SCL/TAL-1high and K562 LUC (control) were established and the effect of reducing or enhancing the expression of SCL/TAL-1 on the erythropoiesis of these three cell lines was investigated. After incubated with EPO-RPMI1640 medium in which EPO induced K562 cell line into erythropoiesis for 5 day, the mRNA levels of SCL/TAL-1 and erythroid related RhD, GPA, CD47 were detected by RT-PCR assay and erythroid antigen CD71, CD235a were examined by flow cytometry in the three cell lines. Effect of SCL/TAL-1 on key phosphorylated proteins, including p-PTEN, p-Akt, p-mTOR, p-P70 and p-4EBP-1 from PI3K/Akt/mTOR pathway and p-c-Raf, p-MEK and p-ERK1/2 from Raf/MEK/ERK pathway, in the downstream of EGFR signaling pathway were checked by Western Blot assay. Effect of MEK-ERK 1/2 inhibitor U0126 on the expression of SCL/TAL1 also examined.

Results
1. After 48h of transfect, more than 95% of K562 cells were GFP positive under the fluorescence microscope, indicating that infection rate of the plasmids in the K562 cells was more than 95%. 2. The results of RT-PCR showed SCL/TAL-1 mRNA expression in the K562 SCL/TAL-1low was significantly lower than that in the K562 LUC control (P <0.05). The mRNA levels of CD47 and RhD was also significantly lower and however, GPA just decreased slightly in comparison with the control. The mRNA levels of above erythroid antigens increased a little in K562-SCL/TAL-1high. 3. The FCM results showed the expression of CD71, CD235a obviously reduced in the K562 SCL/TAL-1low and positive rates were 10.4% and 76.5%, while the positive rates in the LUC control were 94.3% and 83.6%. The expression of CD71 and CD235a in K562-SCL/TAL-1high was similar to the control. 4. The level of p-MEK and p-ERK1/2 increased with transfect of SCL/TAL-1 cDNA and decreased after SCL/TAL-1 RNA interference. However, there were no obvious changes to be observed in PI3K-Akt-mTOR pathway, another important signal pathway. 5. There was no obvious alteration in SCL/TAL-1 level after treatment of MEK-ERK1/2 inhibitor, although MEK-ERK1/2 level reduced.

Conclusion
Our findings suggest that transcription factor SCL/TAL-1 plays a positive role in erythroid differentiation in EPO-induced K562 cell line. SCL/TAL-1 is located in the upstream of MEK-ERK1/2 and may regulate erythroid differentiation by affecting the phosphorylation levels of MEK-ERK1/2 pathway. Grant support: National Natural Science Foundation of China (No.30770912), Foundation of the Science & Technology Department of Sichuan Province (No.2008SZ0017).

Disclosures:
No relevant conflicts of interest to declare.