Differential methylation of the two CpG dinucleotides within the gives rise to obligatory of preference transcription
Some of our recent data contradict such a claim, showing that the absence of the minor Gai3 isoform cannot be compensated by the remaining Gai2 isoform. Gprotein signaling pathways come in at least two different shapes: a canonical and a non-canonical pathway which may mechanistically establish non-redundant distinct functions. Future works have to concentrate on solving this question. The current study displays intriguing and highly significant differences between the two Gai-isoforms albeit it employed a relatively small number of animals. One obvious limitation is the fact that global knockout animals, which lack the respective Gaiisoform in every tissue or organ, were studied. For future directions of research, in particular additional tools are required to decipher the specific functions of the two Gai isoforms in cardiac and non-cardiac cells, e.g. cardiomyocytes, endothelial or immune cells. Ideally, experimental approaches may include detailed analyses of tissue-specific mouse models where the Gai gene of interest is deleted in a constitutive or inducible manner. This allows elucidating the individual contribution of the Gaiisoforms to the ischemic reperfusion injury in the heart. Furthermore with this approach an up regulation of the remaining isoform may be prevented. Whereas an appropriate Gai2-model is available the corresponding Gai3-mouse model has not been created so far. In conclusion, we provide strong evidence that both the deficiency for Gai2 and for Gai3 has profound and opposite effects on IR injury in mice. This may open the rationale to develop biased GiPCR drugs which may allow a different regulation of Gai2 and Gai3 by the same receptor. Targeting transcription factors therapeutically remains a challenge, as they are not conventional ‘‘druggable’’ molecules, such as proteins with enzymatic activity that can be inhibited by small molecules or receptor proteins that can be targeted by antibodies. The discovery of RNA interference has revolutionized this field as, theoretically, any target can be hit with this strategy. RNA interference consists of a doublestranded small interfering RNA with a length of about 20-30 nucleotides that leads to a sequence specific enzymatic cleavage of a target mRNA through complementary base pairing. Although promising, the clinical application of siRNAs continues to face problems related to their effective cellular delivery. Therefore, the development of delivery systems that can protect and transport siRNA is a field of active research. Chitosan is a polymer of b-1-4 N-acetylglucosamine and D-glucosamine residues derived by partial deacetylation of chitin. Since this is a natural, biocompatible, biodegradable, mucoadhesive and non-toxic polymer with a relative low-cost production, it has been broadly studied for the delivery of both plasmid DNA and siRNA due to its capacity, when positively charged, to protect nucleic acids from degradation by endonucleases. Primary amine residues of CH are protonated at pH values below its pKa giving it the capacity to complex anionic compounds, such as the phosphate groups of nucleic acids, enabling the formation of nanoparticles by electrostatic interactions between both functional groups. A number of CH modifications have been proposed to enhance the efficacy of CH as a nucleic acid vector, namely the introduction of imidazole moieties into the CH backbone which has proven effective in promoting the escape of the nanoparticles from the endocytic pathway. The partial quaternization of CH gives origin to trimethylchitosan, which has fixed positive charges, being soluble at a wider pH range and exhibiting enhanced mucoadhesive potential. CDX2, a transcription factor belonging to the caudal-related homeobox gene family, is a master regulator of intestinal cell survival and differentiation. Besides its involvement in the normal development of the intestine, it is also present in every foci of aberrant intestinal differentiation, such as intestinal metaplasia of the stomach, which is a precursor lesion of gastric cancer. It was shown that CDX2 regulates its own expression and is bound to its own promoter in mouse intestine and in human gastric IM, suggesting that a positive autoregulatory mechanism could be critical for the maintenance of the intestinal phenotype. In colorectal cancer, there are multiple evidences that CDX2 has a tumor suppressor function. However, it was also recently described as a lineage-survival oncogene in this context, which might extend to other cancer types associated with intestinal differentiation. Thus, CDX2 appears as an obvious therapeutic target of premalignant lesions with aberrant intestinal differentiation, for which specific treatments are lacking, and might also constitute an adjuvant therapy in cancer. In our study we used a nanoparticle delivering system of siRNA directed to CDX2, using CHimi and TMC as vectors, and showed that this system is able to downregulate CDX2 expression in gastric cell lines, and reaches the gastric mucosa in mouse gastric explants. We next examined the cellular uptake of the nanoparticle formulations by AGS and IPA220 cells. FITC-labelled siRNA was used to assess the percentage of internalization by flow cytometry, 24 hours post-transfection. Our data shows that TMC nanoparticles were taken up more efficiently compared to the CHimi nanoparticles, which might be explained by the fact that transfection is performed under physiological conditions in which CHimi nanoparticles decrease their complexation capacity and tend to aggregate.