The effect of VISTA and CTLA-4 genes suppression on growth and migration inhibition in breast cancer cell line

Abstract

Breast carcinoma remains a complex heterogeneous disorder, being the most frequent malignant disease in women worldwide. Regulating immune checkpoints provide a breakthrough in cancer treatment, such as breast cancer therapy. VISTA, a novel checkpoint regulator, is a type I transmembrane protein that negatively regulates the immune system. Blocking this novel immune checkpoint can enhance the efficacy of cancer treatment. CTLA-4 is an inhibitory immune checkpoint that expresses in immune cells and tumor cells. Among all new therapies using small interfering RNA (siRNA) for silencing genes is a promising approach. In this study, we examine the effect of VISTA and CTLA-4 genes suppression on growth, apoptosis, and migration inhibition in MCF-7 breast cancer cells. Materials and methods: In the current study, we evaluate the effect of suppression CTLA-4 and VISTA on the MCF-7 cells proliferation and survival by MTT assay. Wound healing assay used for determining the invasion and migration ability of breast cancer cells after silencing CTLA-4 and VISTA genes. Flow cytometry was used to study the induction of apoptosis and the cell cycle arrest in CTLA-4/VISTA downregulated MCF-7 cells. For evaluating the expression level of apoptosis-related genes, qRT-PCR was performed. Moreover, to assess the effect of CTLA-VISTA knockdown cancer cells on T lymphocytes, we designed a co-culture system to examine the change of T cells cytokine genes profile. Results: The expression of CTLA-4 and VISTA genes decreased after transfecting specific siRNAs in MCF-4 cells. Inhibition of both CTLA-4 and VISTA led to the relative reduction of MCF-7 cells proliferation. Further, flow cytometry results showed arrest in the sub-G1 phase and relative induction in apoptosis after silencing CTLA-4 and VISTA compared to downregulating each of them individually. Assessing the expression level of apoptosis-related genes showed no significant change after CTLA-4/VISTA knockdown.