Skip to Content
MilliporeSigma
  • Long non-coding RNA ARHGAP5-AS1 inhibits migration of breast cancer cell via stabilizing SMAD7 protein.

Long non-coding RNA ARHGAP5-AS1 inhibits migration of breast cancer cell via stabilizing SMAD7 protein.

Breast cancer research and treatment (2021-08-10)
Chen-Long Wang, Jing-Chi Li, Ci-Xiang Zhou, Cheng-Ning Ma, Di-Fei Wang, Lu-Lu Wo, Ming He, Qianqian Yin, Jian-Rong He, Qian Zhao
ABSTRACT

Tumor metastasis is the main cause of death from breast cancer patients and cell migration plays a critical role in cancer metastasis. Recent studies have shown long non-coding RNAs (lncRNAs) play an essential role in the initiation and progression of cancer. In the present study, the role of an LncRNA, Rho GTPase Activating Protein 5- Antisense 1 (ARHGAP5-AS1) in breast cancer was investigated. RNA sequencing was performed to find out dysregulated LncRNAs in MDA-MB-231-LM2 cells. Transwell migration assays and F-actin staining were utilized to estimate cell migration ability. RNA pulldown assays and RNA immunoprecipitation were used to prove the interaction between ARHGAP5-AS1 and SMAD7. Western blot and immunofluorescence imaging were used to examine the protein levels. Dual luciferase reporter assays were performed to evaluate the activation of TGF-β signaling. We analyzed the RNA-seq data of MDA-MB-231 and its highly metastatic derivative MDA-MB-231-LM2 cell lines (referred to as LM2) and identified a novel lncRNA (NR_027263) named as ARHGAP5-AS1, which expression was significantly downregulated in LM2 cells. Further functional investigation showed ARHGAP5-AS1 could inhibit cell migration via suppression of stress fibers in breast cancer cell lines. Afterwards, SMAD7 was further identified to interact with ARHGAP5-AS1 by its PY motif and thus its ubiquitination and degradation was blocked due to reduced interaction with E3 ligase SMURF1 and SMURF2. Moreover, ARHGAP5-AS1 could inhibit TGF-β signaling pathway due to its inhibitory role on SMAD7. ARHGAP5-AS1 inhibits breast cancer cell migration via stabilization of SMAD7 protein and could serve as a novel biomarker and a potential target for breast cancer in the future.

MATERIALS
Product Number
Brand
Product Description

Roche
Biotin RNA Labeling Mix, sufficient for 20 reactions (transcription), pkg of 40 μL, solution
Millipore
Protease Inhibitor Cocktail Set III, EDTA-Free, Protease inhibitor cocktail III, EDTA-free for inhibiting aspartic, cysteine, and serine proteases as well as aminopeptidases in mammalian cells and tissues.
Sigma-Aldrich
Monoclonal ANTI-FLAG® M2 antibody produced in mouse, 1 mg/mL, clone M2, affinity isolated antibody, buffered aqueous solution (50% glycerol, 10 mM sodium phosphate, and 150 mM NaCl, pH 7.4)
Sigma-Aldrich
Magna RIP® RNA-Binding Protein Immunoprecipitation Kit, RNA Immunoprecipitation (RIP) Kit containing all necessary reagents to perform 12 individual RNA-binding protein immunoprecipitation (RIP) reactions using protein A/G magnetic beads.