Proteomics Analysis Identified Hub Genes Associated With RNAi-Induced Silencing of G1 Cyclins in MDA-MB-231 Cells

Abstract

Background: The G1 cyclins are the most potent candidates in the pathogenesis of breast cancer. This study was designed to analyze the synergistic effect of G1 cyclins silencing on the proliferation of breast cancer cells and to identify G1 cyclins-molecular targets by proteomics approach.Methods: The MDA-MB-231 cells were transfected by a dual shRNA vector targeting G1 cyclins through a bidirectional survivin promoter. Silencing efficacy and cell proliferation were evaluated by real-time PCR, Western blot, and MTS assays, respectively. The protein expression profile was evaluated by 2D gel electrophoresis and mass spectroscopy. Further, bioinformatics tools were applied to identify the molecular targets by G1 cyclins and their possible biological consequences.Results: In response to G1 cyclins silencing, the proliferation of cells exposed to the dual shRNA vector decreased significantly at 72 h post-transfection. The reduction of G1 cyclins proteins was following their mRNA expression level as well. Protein signature of cells was altered in response to the silencing of G1 cyclins, 13 up-regulated, and seven down-regulated. Network analysis of G1 cyclins-regulated proteins identified ACTB, HSP90AA1, ALB, and HSPA5 as the hub genes according to the degree method.The regulated proteins by G1 cyclins participate in cancer-related pathways such as PI3K-Akt signaling pathway and pathways in cancer (HSP90AA1; HSP90AB1; HSP9B1), HIF-1 signaling pathway (LDHA; ALDOA; PGK1), apoptosis and proteoglycans in cancer (ACTB).Conclusions: We identified the G1 cyclins-regulated proteins and their mode of action in the pathogenesis of breast cancer. Our findings suggested that G1 cyclins participate in various biological functions. Meanwhile, it offers a new perception of the interactions among G1 cyclins-regulated proteins to identify targeted treatment. It follows-up research in the field of breast cancer.

In vivo assessment of simultaneous G1 cyclins silencing by a tumor-specific bidirectional promoter on the mammary tumor in nude mice

“…By silencing of this mitotic marker, the proliferation of cells is reduced and apoptosis is induced ( 16 ). Our earlier experiment also revealed the simultaneously silencing of G1 cyclins by the dual shRNA vector (bipSUR) in MDA-MB-231cells ( 17 ). Thus, this human triple-negative breast cancer cell line was selected as the cell model and nude mice were selected as the animal model for this in vivo study.…”

“…Our research team has also previously indicated the activity of this bidirectional promoter in breast cancer in vitro and in vivo ( 18 ). The anti-proliferative effect of bipSUR in both triple positive and negative breast cancer cells, MCF-7 and MDA-MB-231, has been demonstrated ( 17 ). These genes are over-expressed in all breast cancer cell lines.…”

“…The bipSUR targets the G1 cyclins through a bidirectional survivin promoter that has been made in the Golestan University of Medical Sciences and Health Services ( 17 , 18 ). This plasmid harbors the sequences of shRNAs against cyclin D1 (NM_053056.2) and cyclin E (NM_001238.2) genes; their transcription is controlled by bidirectional survivin promoter.…”