Abstract
Background: We investigated the effect of corn-derived biodiesel glycerol on microRNAs (miRNAs) and mRNAs, which play a central role in regulating cell survival, apoptosis and carcinogenesis. Materials and Methods: Inbred Balb/c mice were treated with purified glycerol from biodiesel for 24 hours. After administration, we determined the expressions of miR-21, miR-27a, miR-34a, miR-93, miR-143, miR-146a, miR-148a, miR-155, miR-196a, miR-203, miR-205, miR-221 and nuclear factor kappa-light-chain enhancer of activated B-cells-1 (Nfκb1), mitogen-activated protein kinase-8 (Mapk8) and v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-ras) genes in the liver of mice. Results: We found a parallel altered expression of miRNAs and mRNAs in animals consuming biodiesel glycerol that compared to control mice; these alterations reached significant levels only in few cases. Conclusion: Biodiesel glycerol presents no higher risk for carcinogenicity or toxicity.
There are currently several companies which produce biodiesel from rapseed- or corn oil by trans-esterification. The by-product of trans-esterification is a fraction with a high percentage of glycerol. We have been examining whether the purified glycerol fraction from biodiesel is suitable for animal feeding, at the Institute of Public Health at the University of Pécs, since 2007 (1, 2).
In our previous studies, we examined the biological effects of biodiesel glycerol on two genes: nuclear factor kappa-light-chain enhancer of activated B-cells 1 (Nfκb1) and growth arrest and DNA damage-inducible protein 45 alpha (Gadd45a), which play a central role in cell survival and apoptosis. We also investigated the expression of genes for cytochrome P450, family 1, subfamily a polypeptide 1 (Cyp1a1) and cytochrome P450, family 2, subfamily e, polypeptide 1 (Cyp2e1), which encode metabolizing enzymes responsible for the oxidative transformation of trace lipid and methanol residues of biodiesel glycerol. In these ‘short-term’ animal studies we found that high purity biodiesel glycerol has no significant effect on the expression of Nfκb1 and Gadd45a, and has only a transient short-term effect on the gene expression levels of Cyp1a1 and Cyp2e1 (3, 4).
Szendi et al. also found no toxic effect of biodiesel glycerol in different ‘short-term’ and sub-chronic oral toxicity tests in Long-Evans rats (5, 6).
Recently miRNAs have become the focus of molecular genomic research. miRNAs are highly conserved, small, endogenous non-protein-coding RNAs; their genes are frequently located in cancer-associated genomic regions. They are partially complementary to one or more RNA transcripts and after binding they can inhibit protein translation. Several studies have shown, that miRNAs regulate cell proliferation and apoptosis, function as oncogenes or tumour suppressors, and also have a role in immune response. miRNA expression profiles can have predictive value for assessing chemical carcinogenesis (7-9).
In this investigation, we evaluated the expression of several miRNAs (miR-21, miR-27a, miR-34a, miR-93, miR-143, miR-146a, miR-148a, miR-155, miR-196a, miR-203, miR-205, miR-221) in liver of mice and compared it to the expression of the following mRNAs: Nfκb1, mitogen-activated protein kinase-8 (Mapk8) and v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-ras), in Balb/c mice after administration of corn-derived biodiesel glycerol.
In liver tissue miR-21, miR-27a, miR-34a, miR-93, miR-148a, miR-155 and miR-221 seem to be oncogenes, while miR-146a and miR-203 are tumour suppressor genes (7-15). K-ras is known as an oncogene, NFKB is an antiapoptotic transcription factor, which regulates genes via the mitogen activated protein kinase (MKK)/c-Jun N-terminal kinase (JNK) pathway that control cell proliferation and cell survival and mediate inflammatory response (16).
Materials and Methods
Five-week-old Balb/c inbred mice were fed with purified biodiesel glycerol (SZME3) in their diet for 24 hours. SZME3 was manufactured by KUKK R&D Ltd (Budapest, Hungary) and contains 85% glycerol, 5% vegetable oil, 2% phosphorus, 1% sodium and 2% potassium and less than 0.04% methanol.
The animal diet groups were maintained with twenty-two mice, twelve males and twelve females in each. Group one was given the SZME3 diet, namely 10% SZME3 by dry weight mixed in the standard chew pellet, while group two, representing the control group, consumed standard laboratory chew pellet. The standard rodent chew pellet was manufactured by Szinbád Ltd. (Gödöllő, Hungary; 86% dry matter, 20% crude protein, 18.20% enzyme protein, 0.97% lysine, 0.30% methionine, 0.64% cystein, 4% crude fat, 4.30% crude fibre, 1.08% Ca, 0.85% P, 0.20% Na, 18000 NE/kg vitamin A, 1000 NE/kg vitamin D, 75 mg/kg vitamin E).
Mice received humane care and the experiment was carried out under the approval of the Institutional Revision Board. During exposition, the behaviour of investigated mice was normal, they ate the same amount from SZME3-rich chew pellet as control animals from standard chew pellet, and their final weights were same. After 24 hours' administration, animals were autopsied by cervical dislocation and the livers of the mice were removed.
The livers were homogenized and pooled by group, and then total cellular RNA was isolated from the tissues with the High Pure miRNA Isolation Kit (Roche, Berlin, Germany), according to the manufacturer's instructions. Total RNA was reverse-transcribed to cDNA with the cDNA Synthesis System Kit (Roche) and AMV reverse transcriptase. The concentration of miRNA was determined by absorption photometry.
cDNA from total RNA was used for miRNA expression analysis in a LightCycler 480 Polymerase Chain Reaction (PCR) System using the LightCycler 480 SYBR Green I Master Kit. The PCR reactions were carried out in 96-well plates (Real-Time Ready Custom Panel 96; Roche). The PCR reaction mix contained 3 μl H2O, 10 μl LightCycler 480 SYBR Green I Master Mix, 2 μl of the equal mixture of the miRNA-specific forward and reverse primers and 5 μl of the sample cDNA. The PCR parameters were: pre-incubation of one cycle at 95°C for 10 min, amplification of 65 cycles at 95°C for 10 s, 42°C for 20 s and 72°C for 15 s. Melting curves were determined using one cycle: at 95°C for 5 s, 65°C for 30 s and melting at 97°C in a continuous detection mode. Detection was carried out in a LC480 PCR System with the Light Cycler 480 Software (Roche). Absolute quantification was calculated for all samples with the LightCycler 480 Software using the second derivate maximum. miRNA expressions were determined using 5 S RNA as internal control.
cDNA from total RNA was also used for mRNA expression analysis of K-ras, Nfκb1, Mapk8 and hypoxanthine guanine phosphoribosyl transferase (Hprt) in a LightCycler 2.0 carousel-based PCR system, using FAM labelled Universal Probe Library probes (Upl; Roche) and LightCycler 480 SYBR Green I (Roche). The PCR reactions were carried out in glass capillaries in 20 μl final volume. The reaction mix contained: 5 μl sample cDNA, 1 μl of the primer, 1 μl of the UPL probe, 4 μl of LightCycler 480 SYBR Green I Master Mix and 9 μl H2O. PCR parameters were: pre-incubation of one cycle at 95°C for 10 min, amplification of 45 cycles of denaturation at 95°C for 10 s, annealing at 55°C for 40 s, extension at 72°C for 1 s.
Primers and probes of investigated miRNAs and mRNAs.
Primers of miRNAs were synthesized by TIB Molbiol, ADR Logistics (Roche), and are shown in Table I. Probes and primers of mRNAs were designed and selected by the Roche database (www.applied-science.roche.com) and are also shown in Table I.
Gene expression of miRNAs, using 5S RNA as internal control, in the liver of mice after the addition of SZME3 to their diet.
All PCR reactions were run in triplicates, in separate runs. The concentrations of miRNAs and mRNAs for Nfκb1, Mapk8, K-ras and Hprt were determined in liver tissue and averaged. The gene expressions of Nfκb1, Mapk8, K-ras were calculated relative to the expression of Hprt. Statistical evaluation was carried out by paired t-test using the STATA Release 11 software for Windows (StataCorp LP, Texas, USA). Values of p<0.05 were considered to be statistically significant.
Results
Male mice exhibited more significant expression changes in the investigated miRNAs than did females.
In the group of female mice administered SZME3, the oncogene miR-27a was significantly de-regulated in parallel to the down-regulation of oncogene K-ras and antiapoptotic Nfkb1 and Mapk8, while two other oncogene miRNAs, miR-34a and miR-221, were found to be overexpressed.
In male mice, 24-hours' SZME3 administration resulted in the up-regulation of oncogene miR-93a and miR-221, while the oncogenes miR-155 with miR-196a were also observed to be down-regulated by the under-expression of K-ras. In male mice Nfkb1 expression was not altered, while Mapk8 was also down-regulated.
The investigated mRNA exhibited significant expression changes in all investigated animals and almost in all investigated genes, while the expression of oncogenes miR-21, miR-143, miR-148a and miR-205 and tumour suppressor miR-146a in the investigated animals, was similar to control. The results are shown in Tables II and III.
Discussion
Many studies have examined the expression profiles of miRNAs in different tumour types. The role in tumourigenesis, of the miRNAs examined in the present investigation, can be seen in Table IV.
Karakatsanis et al. examined patients with hepatocellular carcinoma, and found elevated expression of miR-21, miR-221, and up-regulation of miR-146a (10). Ji et al. investigated the oncogenic effect of miR-27a in rat liver (11). Welch et al. found that miR-34a functions as a potential tumour suppressor in neuroblastoma cells, as it directly targets the mRNA encoding the E2F3 protein, a potent transcriptional inducer of cell-cycle progression, while miR-34a is an oncogene in liver tissue (9, 17). Wang et al. found that the expression of miR-155 was significantly higher in the liver tissue of mice after a choline-rich diet. Choline presence indicates an inflammatory response and exposure to the agent via animal diet causes hepatocellular carcinoma (12). Tsai et al. analyzed gastric cancer samples, and their data suggest that miR-196a is an oncogene, since the expression level of miR-196a was significantly increased in primary gastric cancer tissues (18). Fang et al. found that miR-93 enhances cell survival, while promoting tumour growth (13). miR-203 was found to be a tumour suppressor miRNA in hepatocellular carcinoma, while it is up-regulated in several other types of tumours, for example breast, colorectal and ovarian cancer (14). miR-148a is also a tumour suppressor, it suppresses cell proliferation, cell progression and migration (15).
Gene expression of mRNAs relative to expression of hypoxanthine guanine phosphoribosyl transferase (Hprt) (%) in the liver of mice after addition of SZME3 to their diet.
miRNA regulation in cancer.
NFKB is a protein complex and has a central role in the regulation of cell proliferation, differentiation and apoptosis, as well as of many miRNAs. Ma et al. found a positive correlation between miR-34a, miR-155 and miR-221 up-regulation and Nfκb activation (19). The ret proto-oncogene (RET)/patched (PTC)/rat sarcoma virus oncogene (RAS)/v-raf murine sarcoma viral oncogene homolog B1 (BRAF) signalling pathway has a central role in papillary thyroid carcinoma, while miR-221, miR-222 and miR-146 were also strongly overexpressed in thyroid tumours compared with unaffected thyroid tissues (20). The MAPKs are a family of serine-threonine protein kinases that participate in major signalling systems, as they are also involved in the NFKB and RAS pathways. According to Paroo et al., the miRNA-generating complex is regulated by MAPK/ERK and this regulation is important in effecting mitogenic signalling; they demonstrated a direct connection between a cell signalling pathway and the core miRNAs (21).
In our investigation, the underexpression of antiapoptotic Nfκb1 and Mapk8, and oncogene K-ras coexisted with a lower concentration of oncogenes miR-27a, miR-155 and miR-196a, and with a higher concentration of tumour suppressor miR-203. There was no significant alteration in the expression of oncogenes miR-21, miR-143, miR-148a and miR-205 and tumor suppressor miR-146a. These data suggest that biodiesel glycerol may have a positive effect on the down-regulation of oncogene K-ras and by maintaining a low expression of Nfκb1 and Mapk8 it can facilitate the death of demaged cells. However the expression of oncogenes mir-34a, miR-93 in female mice and of miR-221 in both genders, did not follow the expression of examined mRNAs.
Summarizing our results, except for miR-34a, miR-93 and miR-221, the other gene expression alterations showed a positive correlation with cell death and tumour suppression. Presumably the biodiesel glycerol, with 85% glycerol and methanol concentration less than 0.04%, could be used as a food additive safely.
Acknowledgements
This work was funded by AGROÖK07, Project number: agrook07.
- Received September 11, 2012.
- Revision received October 18, 2012.
- Accepted October 22, 2012.
- Copyright © 2013 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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