MiR-320 and miR-494 affect cell cycles of primary murine bronchial epithelial cells exposed to benzo[a]pyrene

Abstract

MicroRNAs (miRNAs) are a class of small noncoding RNA molecules with profound impact on various biological processes. Some miRNAs are involved in tumorigenesis by regulation of cell cycle progression. Here, we cultured primary murine bronchial epithelial cells and then examined the expression of miR-320 and miR-494 in cells exposed to benzo[a]pyrene (B[a]P). To better characterize roles of miR-320 and miR-494 in cell cycle progression, we used miRNA inhibitors to downregulate expression of miRNAs and determined cell cycle distribution and expression of cyclin-dependent kinases 6 (CDK6) by flow cytometric analysis. Treating cells with 1 μM B[a]P for 24 h resulted in time-dependent increases in miR-320 and miR-494 expression. Moreover, G1 arrest and downregulated expression of CDK6 were shown in the treated cells. Flow cytometric analysis indicated a relief of G1 arrest and an elevated expression of CDK6 after inhibition of the expressions of miR-320 and miR-494 in cells exposed to B[a]P. These results suggest that expression levels of miRNA-320 and miR-494, which regulate B[a]P-exposed cell cycle progression, may impact G1/S transition through CDK6, and provide further insights into functions of miRNAs in cell cycle of primary murine bronchial epithelial cells exposed to B[a]P.

Introduction

MicroRNAs (miRNAs) are a novel class of 19–23 nucleotide (nt) noncoding RNA molecules that regulate gene expression by targeting the 3′-untranslated region (3′UTR) of mRNAs with consequent inhibition of protein translation or degradation of target mRNA (Bartel, 2004). Since their discovery in 2001, there has been impressive progress in miRNA research. Many miRNAs are highly conserved between a variety of evolutionary distinguished species. MiRNA-mediated regulation of gene expression is a wide-spread phenomenon in eukaryotic organisms. MiRNAs play evolutionarily conserved roles in cellular development and function (O’Connell et al., 2008). Moreover, miRNA expression profiles are very informative because they reflect the developmental lineage and differentiation state of the tumors, and differ between normal tissues and derived tumors and between tumor types (Lujambio et al., 2008). Altered expression of specific miRNAs has been found in the pathogenesis of diverse human cancers, including lung cancer (Yanaihara et al., 2006), breast cancer (Lehmann et al., 2008), hepatocellular carcinoma (Budhu et al., 2008), and gastric cancer (Zhang et al., 2008). However, down-regulation or up-regulation of any particular miRNAs in cancer does not sufficiently address the role of these changes in tumorigenesis. Furthermore, the expression of miRNAs, like many genes important in toxicology, can be regulated by xenobiotics such as carcinogens. But data are scarce on the role of miRNAs in carcinogenesis arising from exposure to environmental factors. In our earlier study, miR-320 and miR-494 were highly expressed in human bronchial epithelial cells exposed to anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (anti-BPDE) (Shen et al., 2009). Some other studies also have reported overexpression of miR-320 or miR-494 in the human Burkitt lymphoma-derived cell line (Sander et al., 2008) and a leukemia cell line (Schaar et al., 2009). But their roles in carcinogenesis have not been explored.

Benzo[a]pyrene (B[a]P), a model polycyclic aromatic hydrocarbon (PAH), is well-known from experimental and epidemiological studies to be carcinogenic in both humans and animals. PAHs, products of the incomplete combustion of organic materials, are abundantly present in coal tar, cigarette smoke, charcoal-broiled and smoked foods (Curfs et al., 2004). Thus, there is a possibility for either environmental or occupational exposure to B[a]P. B[a]P is metabolized to the ultimate reactive form, anti-BPDE, by microsomal cytochrome P450 enzymes (Curfs et al., 2004). Anti-BPDE binds to DNA, most commonly to guanines, forming bulky adducts that induce GC → TA transversions, which may be responsible for tumor initiation (Denissenko et al., 1996, Rojas et al., 2004). B[a]P is known to be present in cigarette smoke and implicated as a causative substance in the development of smoking-related lung cancer (Sanchez-Cespedes et al., 2001, Alexandrov et al., 2002). But the exact underlying mechanism has still not been elucidated.

Carcinogenesis is considered to be the result of dysregulation of the cell cycle machinery (Sun et al., 2008). In mammalian cells, cellular proliferation requires progression through four different phases of the cell cycle: G1 → S → G2 → M. It is now known that progression through different phases of cell cycle is controlled by several cell cycle checkpoints, including G1/S, G2/M and the spindle checkpoint. Transitions between cell-cycle phases are mediated by cyclin-dependent kinases (CDKs) and their modulators (Kim et al., 2009). CDK6 functions early in G1 phase of the cell cycle to link growth regulatory signals to the control of cell development. Cell tumorigenesis can result from deregulated activity of CDK6 with subsequent, inappropriate inactivation of pRb in several tissue types. The G1 restriction point has been found to be lost in many human cancers (Whitehurst et al., 2008, Li et al., 2009, Hirt et al., 2009). B[a]P exposure led to p53-independent G1 arrest in 3T3 fibroblasts (Vaziri and Faller, 1997). Emerging evidence suggests that several miRNAs target genes that are involved in cell cycle progression and cellular proliferation (Wu et al., 2007). The miR-106b family is overexpressed in multiple tumor types and their presence has been correlated with the expression of genes that regulate the cell cycle (Ivanovska et al., 2008). MiR-106b and miR-93 interfere with TGFb-induced cell-cycle arrest, mainly by inhibiting the expression of p21 at the posttranscriptional level (Petrocca et al., 2008). However, little is known about the functions of miRNAs in the processes of B[a]P-exposed cell cycle alteration.

In our previous study, miR-320 and miR-494 were the most highly expressed in human bronchial epithelial cells transformed by anti-BPDE, compared with vehicle-treated control cells (Shen et al., 2009). In the present work, for the first time, we investigated the roles of miR-320 and miR-494 in cell cycle of primary murine bronchial epithelial cells exposed to B[a]P.