3.2 AREG and EREG are regulated epigenetically
3.2.2 AREG and EREG expression increases after treatment with his-
Figure 6:
120 h: 144 h:
Figure 6: AREG andEREG mRNA expression after Valproat treatment: Five cell lines were treated with 1 mM or 2 mM Valproat (Valproat 1 or 2) for 120 and 144 h. The data were sorted according to theAREG mRNA expression of the untreated cells (see figure 3). In each plot the relative quantification of the expression is shown compared to the untreated control.
Figure 7:
120 h: 144 h:
Figure 7: AREG protein expression after Valproat treatment: Five cell lines were treated with 1 mM or 2 mM Valproat (Valproat 1 or 2) for 120 and 144 h. The data were sorted according to the AREG mRNA expression of the untreated cells (see figure 3). In both plots the relative AREG level is shown compared to the untreated control.
To test if AREG and EREG expression can also be changed by HDACis, the cells
were treated for 120 and 144 h with 1 and 2 mM Valproat as described in section 2.3.4. As shown in figure 6 and 7, there is no correlation between theAREG andEREG expression in untreated cells and the change of AREG and EREG expression by Valproat, as seen for DAC treated cells (see figure 4). Treatment with 2 mM Valproat increased theAREG mRNA expression only in LIM1215 by a factor of three to four at both timepoints. For the other cells, Valproat seemed to affect the expression in a different way. For SW480 cells, an increased AREG mRNA expression was also seen. Compared to the untreated control, an increase by a factor of 2.5 was seen after 120 h treatment. However, at 144 h AREG mRNA expression declined again to 1.5 fold. Valproat affected theEREG mRNA expression to a larger extent. For the very low expressing cells LIM1215 and SW480 treatment with 1 and 2 mM Valproat increased the EREG mRNA level between 5 and 60 fold at both timepoints. In contrast, RKO only showed an increased EREG mRNA level after 120 h, when treated with 1 mM Valproat. The EREG protein expression after Valproat treatment was tested by Stephan Bartels (Charit´e Berlin, Institute for Pathology) in his Master thesis.241 But similar to the results obtained for untreated cell lines in this work (see figure 3) EREG protein was not detected in any of the tested samples, irrespective of Valproat treatment.
Valproat treatment influenced the AREG protein levels (figure 7) similar to the AREG mRNA expression. LIM1215 increased levels up to 5 times. However, unlike AREG mRNA expression, 2 mM of Valproat led to an increase of AREG protein in RKO and SW480 after 144 h of treatment. Thus, mRNA expression and protein levels are not consistent following inhibition of histone deacetylation.
Figure 8:
120 h: 144 h:
Figure 8: AREG and EREG mRNA expression after TSA treatment: Five cell lines were treated with 25 ng/ml TSA for 120 and 144 h. The data were sorted according to the AREG mRNA expression of the untreated cells (see figure 3). In each plot the relative quantification of the expression is shown compared to the untreated control.
Figure 9:
120 h: 144 h:
Figure 9: AREG protein expression after TSA treatment: Five cell lines were treated with 25 ng/ml TSA for 120 and 144 h. The data were sorted according to theAREG mRNA expression of the untreated cells (see figure 3). In both plots the relative AREG level is shown compared to the untreated control.
To confirm the influence of histone deacetylation on AREG or EREG expression, the effect of other HDACis was tested. Trichostatin A (TSA) led to an up-regulation of the AREG mRNA level in all tested cells after treatment for 120 h and in all cells except SW480 cells after 144 h (see figure 8). However, in that cell line EREG mRNA
expression increased by about 90 fold compared to the untreated control. For the other cell lines except HCT116 cells theEREG mRNA level was also increased upon treatment with TSA. AREG protein levels increased also for all cells but in contrast to the mRNA result, the strongest effect was seen in SW480 cells (see figure 9).
Figure 10:
LIM1215:
SW480:
Figure 10: AREG mRNA expression after treatment with different HDACis: LIM1215 and SW480 were treated with different HDACis for 144 h (TSA: 25 ng/ml, Cambinol 20µM, SAHA 1µM, Valproat 1 mM, DMSO: solvent control for TSA and Cambinol, Methanol: solvent control for SAHA and Valproat). In both plots the relative quantification of the expression is shown compared to the untreated control.
To investigate, if inhibition of histone deacetylases generally leads to an up-regulation of AREG, and to address, if the abrupt changes in the AREG expression are common in the cells, LIM1215 and SW480 cells were tested for their AREG mRNA expression after treatment with the previously tested TSA and Valproat, but also with two additional HDACis, SAHA and Cambinol. AREG mRNA was analyzed for 6 time-points between 24 h and 144 h. In figure 10 is shown that all four HDACis in LIM1215 and all HDACis except Cambinol in SW480 led to an up-regulation of AREG mRNA.
In contrast to LIM1215, the HDACis led to a higher up-regulation of AREG mRNA expression in SW480 at early timepoints, but the effect diminished towards the late timepoints, although the cells were treated daily with the HDACis. In LIM1215 the AREG mRNA expression remained almost stable at all timepoints.
To conclude, AREG and EREG expression were mainly increased by the DNA methyltransferase inhibitor DAC in different cells. The effect depended on the initial AREG orEREG expression. Expression was also increased by HDACi treatment but in this case it did not depend on initialAREG orEREG expression. In contrast to expres-sion changes by DAC treatment, expresexpres-sion changes by treatment with HDACis strongly varied between the timepoints of the experiments and between different experiments, too.
Also discrepancies betweenAREG and EREG mRNA and protein expression were seen after treatment. Nevertheless, the experiments show that AREG and EREG might be regulated by epigenetic mechanisms. In the next step, the potential epigenetic regulatory control regions of both genes were identified.