BACH1 silencing by siRNA inhibits migration of HT-29 colon cancer cells through reduction of metastasis-related genes
Introduction
Colorectal cancer (CRC), the most frequent cancers in humans, is the third most common cancers globally, with over one million new cases diagnosed worldwide every year [1], [2], [3]. Nearly 20% of patients are diagnosed at an advanced stage with metastatic disease. The survival rate of CRC detected at a localized stage is 90.3%, but after metastasis to adjacent organs or lymph nodes, the rate drops to 70.4% [4], [5], [6]. Approximately half of the CRC diagnosed patients eventually progress metastasis which is almost always fatal. The main common organ of metastases occurrence is the liver. It also gives metastasis to other organs like lung and bone [7], [8].
Early detection of cancer will reduce the number of cancer death and as development of a colorectal carcinoma may take several years, so there is a large window of opportunity for detection of tumors at a curable stage. More and more studies have been shown that ineffective therapies are due to lack of our understanding of precise molecular mechanism involved in carcinogenesis. So, alternative therapeutic approaches and the search for novel diagnostic and prognostic biomarkers is highly desired to prevent CRC-related deaths. Gene therapy is one such attempt [9], [10], [11], [12], [13]. Small interfering RNA (siRNA) has been broadly utilized to silence gene targets in many cancers and provides great therapeutic possibilities in the treatment of oncological patients [14], [15].
BACH1 is a protein member of cap ‘n’ collar (CNC) and basic region leucine zipper factor transcriptional superfamily. BACH1 is a transcriptional factor which forms a heterodimer with small Maf family, by linking to Maf recognition elements (MARE, Maf-related Antioxidant Response Elements) in gene promoter regions to mediate the gene transcription [16], [17], [18].
BACH1 plays a pivotal role in adjustment of oxidative stress and known as a repressor of its main target HOMX1 (hemoxygenas-1). Besides, BACH1 expression is greatly observed in human tissue that has been analyzed [19], [20]. Though BACH1 was mainly reported as a transcriptional repressor, it could also appear as an activator whether on different target genes or on the same genes in different cellular background [21]. Recently, growing evidence revealed BACH1 in the case of cancer progression. In 2011 Alvarez et al. predicted that BACH1 might be a regulator of the prostate cancer marker ACPP, although this was not experimentally verified [22]. Yajun Liang and colleagues revealed that ectopic expression of BACH1 is involved in enhancement malignancy of breast cancer cells while knockdown considerably suppresses these processes. BACH1 transcriptionally adjust several involved genes in the osteolytic metastasis of breast cancer, and more significantly, it promotes the invasiveness and metastasis of breast cancer cells [23]. Yun et al. demonstrated that RKIP prohibits let-7 target genes which are BACH1 and HMGA2 that in turn augment breast cancer metastasis genes MMP1, OPN, and CXCR4 and they reported BACH1 as a pro-metastatic gene and a direct target of the tumor suppressive microRNA Let-7 [24], [25], [26]. Moreover, in BoneKEy reports, transcription factor BACH1 is identified as a regulator of metastasis-related genes, including MMP1 and CXCR4, which are crucial in bone metastasis formation. Therefore, BACH1 may be a significant target for effective therapeutic intervention in tumor metastasis development. Although, decreased BACH1 expression in a mouse model of bone metastasis, remarkably reduced metastasis, and ectopic overexpression of the gene caused more malignant and aggressive cancer cells [27].
In the current study, we investigated the functional and molecular roles of BACH1 in colon cancer cells whether expression level is parallel with migration capacity and metastasis progression in vitro or not. We found that knockdown of BACH1 by small interference RNA (siRNA) prevented colon cancer cell growth and migration of HT-29 cells in vitro. Furthermore, CXCR4 and MMP1 expression were decreased after BACH1 siRNA-mediated knockdown in this cell line. This study provides evidence that BACH1 is associated with metastatic behavior of colon cancer cells.
Section snippets
Colon cancer samples
All tumor samples were provided from Emam Reza Hospital and all ethical approval were granted by ethic committee of Tabriz University of Medical Sciences. Forty pairs of colon cancer and adjacent non-cancerous colon tissues were obtained from patients who have diagnosed with stage II and III of colon cancer. Patients did not receive neoadjuvant therapy or chemotherapy before surgery. The tissue sections were evaluated and corroborated by a pathologist. The tumor samples were used for BACH1,
Correlation between BACH1 and metastasis-related genes expression of colon cancer samples
To investigate the role of BACH1 in CRC progression, we first examined the BACH1 expression in 40 matched pair samples of colon cancer tissue and marginal colon tissues in patients with CRC by qRT-PCR. Also, we investigated the MMP1 and CXCR4 expression in colon cancer tissues. The expression level of mentioned genes, BACH1, CXCR4 and MMP1 in all colon cancer tissues samples had an obvious elevated fold in contrary with normal samples (Fig. 1) suggesting that high expression of BACH1 might be
Discussion
Despite the fact that CRC mortality abated over last 30 years, the growing death statistic remains a matter of debate. Tumor cell migration and invasion through the basement membrane constitute the metastatic vital feature and invasion, which results in poor prognosis, is a feature of CRC [12], [28]. Through the past decades tremendously remarkable advances in the understanding of cancer biology especially the involved intricate molecular mechanisms in malignant progression of different cancers
Conflict of interest
The authors certify that there is no potential conflict of interest in relation to this article.
Acknowledgments
This study was supported by Immunology Research Center, Tabriz University of Medical Science, and Tabriz, Iran. I would like to acknowledge Miss.Leila Mohammadnez had for her cooperation and guidance through the experiments.
References (50)
- et al.
The molecular biology of colorectal carcinoma and its implications: a review
Surgeon
(2011) Therapeutic potentials of gene silencing by RNA interference: principles, challenges, and new strategies
Gene
(2014)- et al.
The small MAF transcription factors MAFF, MAFG and MAFK: current knowledge and perspectives
Biochim. Biophys. Acta (BBA)
(2012) Activity and expression of murine small Maf family protein MafK
J. Biol. Chem.
(1995)Small Maf proteins in mammalian gene control: mere dimerization partners or dynamic transcriptional regulators?
J. Mol. Biol.
(2008)Transcriptional network analysis identifies BACH1 as a master regulator of breast cancer bone metastasis
J. Biol. Chem.
(2012)A Drosophila protein that imparts directionality on a chromatin insulator is an enhancer of position-effect variegation
Cell
(1995)Molecularly targeted cancer therapy: some lessons from the past decade
Trends Pharmacol. Sci.
(2014)Expression of matrix metalloproteinase-1 in human colorectal carcinoma
Mod. Pathol.
(2000)Overexpression of collagenase 1 (MMP-1) is mediated by the ERK pathway in invasive melanoma cells role of braf mutation and fibroblast growth factor signaling
J. Biol. Chem.
(2004)