Potential chemopreventive effect of “ Procont ” on miRNA expression in CBA / CA mice

Background: There is emerging molecular evidence concerning themodification of miR expression pattern after the treatment with chemopreventive agents. We investigated whether a potential chemopreventive agent called “Procont” deriving from a biological systemhas a positive effect on the DMBA induced dysregulation of let-7a, miR-21 and miR-146amiRNAs. Materials and Methods: CBA/CA H2 k inbred micewere fed by Procont for 7 days before the intraperitonealinjection of DMBA. After one weekof the beginning of Procont diet weanalized the let7a, miR-21, miR-146agene expression in vital organs of mice. Results: The Procont feeding significantly decreased all of the investigated miRNA expression compared with either the mice on a normal diet in the control group or mice were exposed to DMBA alone. Conclusion: The results indicate that“Procont” diet has a potential chemopreventiveeffect in mice exposed to DMBA.


Introduction
MicroRNAs represent a small group of small noncoding RNAs, whose mature form are 18-25 nucleotides in length (1).Over the past several years it has become clear that alterations in the expression of miRNA genes contribute to the pathogenesis of most human malignances (2).Among many miRNA, miR-21 was found overexpressed in different type of cancers (3).Inhibiting the expression of several pro -apoptotic gene and tumor suppressor gene such as p53, PTEN, PDCD4, TPM1 miR-21 contribute to genesis and progression of many type of cancer (4).Let-7a is a member of the let-7 miRNA family and required for timing of cell fate determination.Partial upregulation of this miRNA in the stem cells is required for their terminal differentiation at the adult stage (5).Let-7a can induce apoptosis and inhibits tumorgenesis by regulating oncogens such as RAS and c-myc (6).It has been suggested that RAS and c-myc expression is negatively regulated by let-7a (7).MiR-146a is mainly involved in the activation of the NF-κB protein regulating innate and adaptive immune responses and inflammation (8).With the recognition that inflammatory conditions are often associated with carcinogenesis, it was natural to suspect that there is a link between NF-kB and carcinogenesis (9) Recently several studies have attempted to reveal the alteration in miRNA expression profile as the early response to environmental exposures.According Izotti et al. the cigarette smoke induces the alterations of miRNA expression in rats after 28 days of exposure, they demonstrated considerably downregulation in the case of 24 miRNAs involving in cellular stress, cell proliferation and differentiation (10).In an another study Progribny et al. exposed rats to tamoxifen a potential hepatocarcinogen agent mainly showed upregulation of micro-RNAs, functioning oncogens in hepatocellular carcinogenesis (11).Zhang and Pan tested the effect of hexahydro-1, 3, 5trinitro -1,3, 5triazine (RDX) on the micro-RNA expression in mice's liver, they found a significant upregulation of oncogenic miRNAs and a significant down-regulation of tumor-suppressing miRNAs (12).
In our previous study we investigated the effect of DMBA on the micro-RNA expression in CBA/CA H2 k inbred mice after 24 hours and 1 week of the exposure (13).The expression levels of miR-21, miR-146a and let-7a were significantly higher in the vital organs of the mice after 24 hours of DMBA exposure compared to the control group.While a significant down-regulation of these miRNAs was seen 7 days after the DMBA administration.This early response to DMBA on miRNA level provide us a new in vivo model for testing chemopreventing agents and monitoring their potential chemopreventive effects via molecular epidemiological biomarkers such as onco/supressor genes and miRNAs.
Based on data, confirmed the early modification of miR expression after carcinogenic exposure, our research group elaborated an animal model in order to reveal the potential chemopreventive effect of "Procont" consumption on miRNA expression in mice exposed to DMBA."Procont" is a protein complex isolated from colostrum of cows with leucosis.The Hungarian patent of Procont (P0200172) was registered under international patent number: PCT/HU0300004 in 2003.Bioactive compounds of Procont include 40 and 60 kDA proteins.
These proteins is degraded enzymatically in stomach and is accompanied by the release of 9-12 kDa biologicaly active fragments.

Materials and methods
6-week-old CBA/CA H2 k haplotype mice of both sex were used in this experiment.
The average animal weight were 20 g.Mice were divided into three groups (6-6 in each group, male and female).The preparation of groups is shown in Table 1.
The first group was the control group where animals consumed the standard laboratory chew pellet and tap water ad libitum.The second group received intraperitoneal injection of 7, 12 -dimethylbenz(α )anthracene in a single 20 mg/kg animal weight dose (0,4 mg DMBA dissolved in 0,1 ml corn oil) at the start of the examination.Intraperitoneal gavage decreased the first pass elimination of DMBA in the liver.The animals belonging to the third group consumed "Procont" for 7 day before the exposure of DMBA.The feed were provided ad libitum and prepared according to the instructions of the manufacturers (30 g of the agent was added to 90 g vehicle and 1500 g powder feed, thoroughly mixed, reshaped into food pellets).
Student's t test was performed between the groups and 95 % confidence interval of the differendce was calculated in the case of each organ.Values were expressed as the mean ± 2 SD.The calculation was performed using Statistical Program for Social Science 19.0 (SPSS) software.

Results
In this study we compared the effect of Procont consumption on mice exposed to DMBA with the mice were treated with DMBA alone as shown Figure 1-3.The results of the statistical analysis are shown in Table 2.
The DMBA exposition caused increased expression of the let-7a and miR-21 genes by 1,1 -3,02 fold change in all examined organs compared to the controls (Figure 1, 2).On the other hand, there was no significant effect of DMBA on the expression of miR-146 a gene except for lungs (Figure 3).Dietary administration of Procont before DMBA treatment caused a strongly decrease in the level of miRNAs compared to the animals were treated with the carcinogen agent alone (Figure 1-3).The miR-146a level in kidneys was detected to be more than six times higher in DMBA treated group than in mice consumed Procont before DMBA gavage.We observed the lowest expression of let-7a in spleen and lung tissues at the DMBA exposed mice treated with Procont.The mir-21 and miR-146a gene expression also showed markedly reduction in mice lung relative to the other organ (Figure 2, 3).Especially the expression of miR-21, where we found nine times lower expression (Figure 2).

Discussion
It has been demonstrated that administration of chemopreventive agents can target multiple cellular signaling patways.Several animal model was developed by our research group to evaluate the potential chemopreventive effect of different compounds by analysing the changes in oncogene and tumor supressor gene expression.The results of one of these studies supported, that the coadministration of the chemopreventive agent "Afobazole" and carcinogenic agents DMBA resulted in decrease of DMBA induced overexpression of Ha-ras and p53 gene (14).In an other study we examined the effect of "CoD TM tea" on the expression pattern of c-myc, Ha-ras, Bcl-2 and K-ras proto-oncogene and p53 tumor supressor gene.According to the results the experimental agent was able to decrease the DMBA-induced overexpression of onco-and tumor supressor genes (15).for assessing the capability of a chemopreventive agent to modulate the altered miRNA expression profile induced by environmental carcinogens.
Based on our data, Procont seems to induce a very characteristic decrease in the expression level of let-7a, miR-146a and onco-miR-21 after just 7 days consumption in mice were exposure to a potential carcinogenic agent.According the results of the present study the potential chemopreventive effect of "Procont" could be confirmed.We have several undergoing experiments that clarifying the chemoprentive effects of "Procont" and could give deeper insight into the biochemical action of Procont.

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photometry at 260/280nm.Optical density of the RNA was between 1.9 and 2.1.

Figure 1 .
Figure 1.Let-7a gene expression pattern in liver, spleen, lung and kidney of mice.

Figure 2 .
Figure 2. miR-21 gene expression pattern in liver, spleen, lung and kidney of mice.

Table 1 .
Preparation of groups

Table 2 .
Results of statistical analysis.* 95 % confidence interval of the difference