pLVX-IRES-Hyg

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with high proliferative activity. TNBC tumors exhibit elevated MYC expression and altered expression of MYC-regulated genes, which are associated with tumor progression and poor prognosis. However, the underlying mechanisms by which MYC maintains high expression and mediates TNBC tumorigenesis require further exploration. ACTL6A binds to MYC and inhibits glycogen synthase kinase 3 beta (GSK3β)-induced MYC T58 phosphorylation, thereby inhibiting and stabilizing MYC ubiquitination. Furthermore, ACTL6A promotes the recruitment of MYC and the histone acetyltransferase KAT5 to the CDK2 promoter, leading to overactivation of CDK2 transcription. In TNBC cells in vitro and in vivo, ACTL6A overexpression promotes cell proliferation and tumor growth, whereas silencing ACTL6A inhibits cell proliferation and tumor growth, which is dependent on MYC signaling. Furthermore, combined treatment with paclitaxel and CDK2 inhibitors showed synergistic effects in tumor suppression.

In this study, pLVX-IRES-Hyg vector was used to construct stable cell lines. Human ACTL6A cDNA was PCR amplified and cloned into the pLVX-IRES-Hyg vector. Two shRNA against ACTL6A and one shRNA against MYC were cloned into the pSuper-retro-neo vector. Use transfection reagent for plasmid transfection. Cells (2 × 105) were seeded and infected with retroviruses generated from pLVX-IRES-Hyg-ACTL6A and pSuper-retro-neo-ACTL6A-shRNA/-MYC-shRNA for 3 days. Stable cell lines expressing ACTL6A, ACTL6A-shRNA and MYC-shRNA were selected with 200μg/mL hygromycin and 400μg/mLG418 respectively for 7 days.

Figure 1. Western blot analysis of ACTL6A and MYC proteins in control, ACTL6A overexpressing, and knockdown SUM159PT cells. (Jian Y, et al. 2021)