Truncated forms of RUNX3 Unlike Full Length Protein Alter Cell Proliferation in a TGF-β Context Dependent Manner

Abstract

The Runt related transcription factors (RUNX) are recognized as key players in suppressing or promoting tumor growth. RUNX3, a member of this family, is known as a tumor suppressor in many types of cancers, although such a paradigm was challenged by some researchers. The TGF-β pathway governs major upstream signals to activate RUNX3. RUNX3 protein consists of several regions and domains. The Runt domain is a conserved DNA binding domain and is considered as the main part of RUNX proteins. Herein, we compared the effects of Runt domains and full-Runx3 in cell viability by designing two constructs of Runx3, including N-terminal region and Runt domain. We investigated the effect of full-Runx3, N-t, and RD on growth inhibition in AGS, MCF-7, A549, and HEK293 cell lines which are different in TGF-β sensitivity, in the absence and presence of TGF-β. The full length RUNX3 did not notably inhibit growth of these cell lines while, the N-t and RD truncates showed different trends in these cell lines. Cell proliferation in the TGF-β impaired context cell lines (AGS and MCF-7) significantly decrease while in the A549 significantly increase. On the other hand, transfection of N-t and RD did not considerably affect the cell proliferation in the HEK293.Our results show that full-lenght RUNX3 did not affect the cell viability. Conversely, the N-t and RD constructs significantly changed cell proliferation. Therefore, therapeutic potentials for these truncated proteins are suggested in tumors with RUNX proteins dysfunction, even in the TGF-β impair context.

Keywords: Apoptosis; Cancer; Domain Analysis; Runt; TGF-β.

Figure 1

Schematic illustration of the structure of full-length RUNX3(4) and its deletion derivatives. The numbers represent the positions of amino acids. AD: transcription activation domain, ID: transcription inhibition domain, NLS: nuclear localization signal

Figure 2

AGS cells were transfected by Polyfect reagent using different number of cellS and different amount of EGFP-plasmid and Polyfect reagent. 24 h. after transfection cells were examined for transfection efficiency by fluorescence microscopy

Figure 3

Effect of full-RUNX3 on Cell Viability in the Absence and Presence of TGF-β in AGS, MCF-7, A549, and HEK cell lines. Cells were transfected with RUNX3. 24 h. after transfection, the medium of the transfected cells was exchanged in the absence or presence of 10ng/mL TGF-β. After 48 h. cell proliferation was evaluated by MTT assay. Empty vector (pcDNA3) was used as a control. Data presented as Mean ± SE of three independent experiments

Figure 4

Effect of RUNX3, N-t, and RD on Cell Viability in the Absence and Presence of TGF-β. Cells were transfected with RUNX3, N-t, and RD. 24 h. after transfection, the medium of the transfected cells was exchanged in the absence or presence of 10ng/mL TGF-β. After 48 h. cell proliferation was evaluated by MTT assay. a) AGS b) MCF-7 c) A549 d) HEK293cell line. Empty vector (pcDNA3) was used as a control. Data presented as Mean ± SE of three independent experiments (n=3, * and # p<0.05, ** and ## p<0.01, ***and ### p<0.001). The sign of * correspond with construct vs pcDNA3 and the sign of # correspond with comparison of the same construct with and without TGF-β