Elsevier

Biomedicine & Pharmacotherapy

Volume 84, December 2016, Pages 191-198
Biomedicine & Pharmacotherapy

BACH1 silencing by siRNA inhibits migration of HT-29 colon cancer cells through reduction of metastasis-related genes

https://doi.org/10.1016/j.biopha.2016.09.021Get rights and content

Abstract

Background

Metastasis to distant organs is a hallmark of many tumor cells. BACH1 (BTB and CNC homology 1) is a transcriptional factor which promotes the migration and invasion of breast cancer cells. BACH1 expression and its target genes are intimately associated with the metastasis possibility of clinical samples, and BACH1 reduction leads to meaningful depletion in metastasis. The evaluation of BACH1 role in colon cancer remains elusive. This study seeks to further investigate the role of BACH1 in colon cancer cells.

Methods

Quantitative RT-PCR (qRT-PCR) was used to detect BACH1 expression and other related metastatic genes following siRNA knockdown in colon cancer HT-29 cells. And the protein level assessed by Western blot. MTT assay was to measure the changed cell viability after BACH1 siRNA transfection. Scratch-wound motility assays measured capacity of tumor cell migration of HT-29 cells after BACH1 silencing.

Results

The inhibitory effect of BACH1 was performed by siRNA knockdown using highly metastatic HT-29 colon cell lines. Quantitative RT-PCR and Western blot analysis revealed that the expression levels of BACH1 mRNA and protein in HT29 cells were significantly suppressed after transfection. Conversely, the BACH1 expression increased migration. Also the CXCR4 and MMP1 expression levels decreased following BACH1 knockdown in HT-29 cells.

Conclusion

Our results indicated that BACH1 down-regulation in HT29 CRC cells had no effect on cell growth but did inhibit cell migration by decreasing metastasis-related genes expression. Collectively, these results suggest that BACH1 may function as an oncogenic driver in colon cancer and may represent as a potential target of gene therapy for CRC treatment.

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.

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