Abstract
Background: microRNA expression profile analysis provides evaluation of the early stages of carcinogenesis. This study focuses on early alteration of miRNA expression after treatment with different carcinogens. Materials and Methods: Mice were intraperitoneally injected with one dose of 7,12-dimethylbenz(α)anthracene (DMBA) and N-methyl-N-nitrosourea (MNU). The expression of miRNAs were analyzed 3 and 6 hours after the treatment, using quantitative real-time-polymerase chain reaction. Results: Underexpression of miR-34a and miR-155 were detected in the liver, spleen and kidneys at 3 and 6 hours after MNU treatment. In the lungs and kidneys, the expression of miR-21 was significantly elevated 6 hours after DMBA treatment, while in the liver, MNU induced higher expression levels of miR-21 at 3 and 6 hours compared to treatment with DMBA. Conclusion: The different response of miRNAs to carcinogens emphasizes their possible role as potential epidemiological biomarkers in early phases of environmental tumorigenesis.
7,12-Dimethylbenz(α)anthracene (DMBA) and N-methyl-N-nitrosourea (MNU) are two important chemical carcinogens. There is evidence that a single dose of these agents is able to induce various types of cancer in animal models (1, 2). One of the major differences between the two agents is in their activation. DMBA is a polycyclic aromatic hydrocarbon which must be metabolized by P450 enzymes to become mutagenic, while MNU requires no metabolic activation (3). MNU serves as a tumor initiator by transferring methyl groups in DNA, whereas DMBA is involved in initiation, as well as in promotion, of tumorigenesis (4, 5). In previous studies, we reported that DMBA and MNU caused overexpression of H-Ras, c-Myc and p53 genes 24-48 hours after the treatment (6, 7). Additionally, we compared the short-term effects (within 24 hours) of DMBA and MNU on the level of oncosupressor genes (8). We observed that overexpression of c-Myc, H-Ras and p53, induced by carcinogens, was strongly linked to the activation and metabolism of DMBA and MNU.
MicroRNAs (miRNAs) represent an newly emerging field of cancer research. By regulating gene expression, miRNAs play an important role in the control of cellular processes. To date, more than 17,000 miRNAs have been identified, out of which approximately 1,400 miRNAs can be found in humans (9). There is evidence that miRNA expression can respond very sensitively to carcinogen exposure, which provides a good model for investigating the acute effect of carcinogenic agents (10). Based on toxicological research focusing on miRNA expression, Chen demonstrated that the miRNA expression pattern can be specific to different types of exposure to carcinogens and their target tissues (11).
We previously investigated the acute effect (24 hours) of DMBA and MNU on miRNA expression in healthy tissues. We analyzed the alteration of miRNA expression induced by intraperitoneal injections of these carcinogens (12, 13). According to our results, there was a marked difference in miRNA expression detected at 24 hours with that at one week after the exposure to DMBA, but there was no significant difference between the two groups after MNU treatment.
The aim of this short-term experiment was to reveal the differences between DMBA-, and MNU-induced tumorigenesis at the level of miRNA expression. We selected miRNAs including let-7a, miR-21, miR-34a, miR-146a and miR-155 to determine the modification in their expression after carcinogenic exposure in vivo.
Materials and Methods
Six groups of 6-week-old CBA/Ca H2k inbred mice constituted the experiment (Table I). Each group consisted of 6 male and 6 female animals, weighing 20-25 g. Two groups were treated intraperitoneally with a single dose of DMBA at the onset of the experiment. Another two groups of animals were given intraperitoneal injection of MNU at the start of the investigation. Mice belonging to control groups received standard laboratory chew pellet and tap water ad libitum. At the 3- and 6-hour timepoints of the experiment, mice were euthanized and autopsied (Table I). The animal experimental procedures were carried out in accordance with the Institutional Revision Board.
Preparation of groups for this study.
During the autopsies, the liver, spleen, lungs and kidneys were removed and the tissue samples from the dissected organs were homogenized. Total RNA was extracted using TRIZOL reagent according to the manufacturer's protocol (Molecular Research Center Inc., Cincinnati, OH, USA). The quality of the isolated RNA was checked by absorption photometry at 260/280 nm. Total RNA was exposed to RNAase-free DNAase and 2 μl were reverse transcribed into cDNA using Transcriptor First Strand cDNA Synthesis Kit (Roche, Berlin, Germany).
The expression of the investigated miRNAs was determined by quantitative real-time polymerase chain reaction (PCR). PCR primers were designed using the primer finder database (www.applied-science.roche.com) and were synthesized by TIB Molbiol, ADR Logistics, (Roche Warehouse, Budapest, Hungary). The sequences of the primers are listed in Table II. The PCR was performed on a LightCycler 2.0 carousel-based PCR system (Roche, Berlin, Germany). The 20-μl PCR reaction mixture included: 7.2 μl LightCycler RNA master SYBR Green I fluorescent labeled dye, 1.3 μl Mn(OAc)2 stock solution, 2 μl specific primer at 0.5 μM final concentration, 1 μl template miRNA and 8.2 μl H2O. The reaction mixtures were incubated for 30 s at 95°C, followed by 45 three-step amplification cycles (95°C for 5 s, 50°C for 15 s, 72°C for 5 s). The statistical calculation of differences in expression was performed using the Statistical Program for Social Science 19.0 (SPSS) software (IBM, Armonk, NY, USA). Student's t-test was performed between control and treated groups, and p-values less than 0.05 were considered statistically significant.
Results
In the liver, MNU induced higher expression of miR-21 at the 3- and 6-hour timepoints compared to the values detected in control and DMBA-treated groups (Table III). DMBA exposure significantly elevated the expression of miR-21 in lungs and kidneys 6 hours after the treatment compared to MNU (Tables V and VI).
Except for the kidneys, the expression level of miR-155 was elevated in DMBA-treated groups at the 3- and 6-hour timepoints in comparison with groups which received MNU treatment (Tables III, IV and V). In the kidneys, there was no difference between DMBA and MNU-treated groups 3 hours after the exposure, while at the 6 hour timepoint, DMBA treatment resulted in a significantly higher expression level than did MNU (Table VI).
A comparison of miR-34a expression between DMBA- and MNU-treated groups revealed that it was significantly higher in liver and kidney tissues 3 and 6 hours after DMBA treatment and at 6 hours in spleen, while MNU induced a higher expression level of miR-34a in lungs compared to DMBA (Tables III, IV, V and VI).
The expression of let-7a did not markedly differ between DMBA- and MNU-treated groups (Tables III, IV and VI). There was a relevant difference observed in the expression of miR-146a in the kidneys, where the expression levels detected in DMBA- and MNU-treated groups were found to decrease below those of the control groups (Table VI).
Discussion
With the recognition of the crucial role of miRNAs in carcinogenesis, there is increasing interest in the relationship between the miRNA expression profile and exposure to carcinogenic agents. Recently, several studies reported on the role of miRNAs in genotoxicogical research such as vinyl-carbamate, 2- acetylaminofluore, N-ethyl-N-nitrosurea (ENU), DMBA, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butatone, 1,3,5-trinitro-1,3,5-triazine, tamoxifen and cigarette smoke (14-20). In the recent study we paralelly investigated the impact of DMBA and MNU treatment on miRNA expression in mice.
According to our results, the exposure of mice to MNU resulted in lower expression of miR-34a compared to those of controls and DMBA-treated groups in all investigated organs except for the lungs. miR-34a is recognized as a tumor supressor whose activation is strongly linked to the p53 pathway (21). miR-34a targeting B-cell lymphoma-2 (BCL2), cyclin D1, MYCN, cyclin-dependent kinase-4 (CDK4), sirtuin-1 (SIRT1) genes can influence all steps of carcinogenesis (22, 23). Chen et al. investigated the acute effect of N-ethyl-N-nitrosurea on miR-34a expression in mice (24). ENU, similarly to MNU, acts as an alkylating agent. In contrast to our results, they showed that exposure of mice to ENU induced a markedly elevated level of miR-34a after 24 hours of treatment in spleen. When Le et al. exposed rats to ENU for 1, 3, 7, 15, 30 and 120 days, they detected an elevated level of miR-34 family in mouse liver 7 and 15 days after the treatment (25).
PCR primer sequences.
Results of statistical analysis. Presented fold change values are the gene expression ratios of 7,12-dimethylbenz(α)anthracene (DMBA)- and N-methyl-N-nitrosourea (MNU)-treated mice, relative to the controls in liver.
Results of statistical analysis. Presented fold change values are the gene expression ratios of 7,12-dimethylbenz(α)anthracene (DMBA)- and N-methyl-N-nitrosourea (MNU)-treated mice, relative to the controls in spleen.
Results of statistical analysis. Presented fold change values are the gene expression ratios of 7,12-dimethylbenz(α)anthracene (DMBA)- and N-methyl-N-nitrosourea (MNU)-treated mice, relative to the controls in lungs.
Results of statistical analysis. Presented fold change values are the gene expression ratios of 7,12-dimethylbenz(α)anthracene (DMBA)- and N-methyl-N-nitrosourea (MNU)-treated mice, relative to the controls in kidneys.
Based on the differences in miRNA expression between controls and treated groups, the effect of MNU appeared more dominant on miR-21, miR-34a and miR-155 expression compared to DMBA. These very early responses of miRNAs in the MNU-treated groups can be explained by the fact that MNU is a direct-acting carcinogen, while DMBA is an indirect carcinogenic agent. Metabolic activation of DMBA by cytochrome P450 enzymes, which are mainly synthesized in liver, may explain the higher expression levels of miR-34a and miR-155 in liver of DMBA-treated mice (26).
In the investigated organs, exposure to DMBA induced significantly higher expression levels of miR-155 than MNU. miR-155 has been identified as an oncomiR contributing to the developement of hematopoietic malignancies (27). Increased expression of miR-155 was shown in different types of human cancer by targeting tumor supressor genes including tumor protein p53 inducible nuclear protein-1 (TP53INP1) and RhoA (28). Similarly to miR-155, miR-21 functions as oncogenic miRNA. Negatively regulating the expression of p53, phosphatase and tensin homolog (PTEN), programmed cell death-4 (PDCD4) and tropomyosin-1 (alpha) (TPM1) genes miR-21 can contribute to the process of carcinogenesis (29, 30). miR-21 was expressed at a higher level in mouse liver after MNU treatment compared to DMBA, while DMBA induced the overexpression of miR-21 6 hours after the treatment in lung and kidney tissues compared to MNU.
There was no considerable difference between the effect of the two agents on let-7a expression except for the lungs. While up-regulation of let-7a was detected at the 6-hour timepoint in lungs after DMBA treatment, an opposite tendency was observed at the 3-hour timepoint. Repression of c-Myc and RAS genes by let-7a can contribute to pathogenesis of different types of human cancer such as tumors of lung, colon, stomach and breast (31, 32).
In conclusion, in the current study we provided evidence that DMBA and MNU differentially altered the expession of miR-21, miR-34a and miR-155, and the changes in their expression is strongly linked to the activation of carcinogens. The early alteration in the expression levels of the investigated miRNAs induced by carcinogenic agents (DMBA, MNU) support their role in initial processes of chemical carcinogenesis. Our results suggest that further analysis of miRNA expression shows promise for explanation of the early stage of chemically-induced tumorgenesis and primary tumor prevention based on miRNAs as early biomarkers.
- Received September 25, 2012.
- Revision received October 26, 2012.
- Accepted October 29, 2012.
- Copyright © 2013 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
