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Calorie restriction (CR) is suggested preventing development of mammary tumors (MT) however the mechanism remains to be clarified. We aimed to determine the miRNA profile in mice applied to two different CR protocols; chronic (CCR) and intermittent (ICR) and follow the MT development. In addition, the roles of miRNAs involved in adiponectin and/or leptin signaling pathways were investigated. Mice were enrolled into ad-libitum (AL), CCR, or ICR which was three-weeks AL feeding followed by one-week of 60% CR in cyclic manner. Blood and tissue collection were performed at weeks 10, 17/18, 49/50 and 81/82. Long-term CCR provided better protection compared to ICR for MT development with a delay in the MT occurrence. Adiponectin expression in mammary fat pad were significantly higher in CCR group compared to AL. Using GeneChip™ Array, 250 of 3,195 miRNAs were differentially expressed among the dietary groups. 13 of 250 miRNAs were related to adiponectin and/or leptin signaling genes. Results were verified by RT-PCR. Specifically, miR-326-3p, miR-500-3p and miR-129-5p which are adiponectin and/or leptin signaling related may play important roles in the preventive effects of CR in MT development and in ageing. Thus, these miRNAs might be putative biomarkers to target for diagnostic and treatment purposes. Novelty Bullets • Type of Calorie restriction and micro RNA interaction is related to ageing. • miR-326-3p, miR-500-3p and miR-129-5p expression levels were differentially expressed in MT development and in ageing. • The adiponectin and/or leptin signaling pathways related genes are regulated by certain miRNAs in the protective effects of CR

Aim: Leptin activates multiple intracellular signaling pathways, including JAK/STAT, by binding to its receptor. Leptin is also an important regulator of glucose homeostasis. Although both glucose and leptin increase breast cancer cell proliferation in vitro, whether the enhancing effect of glucose on the proliferation of breast cancer cells is mediated by the leptin signaling pathway is not known. The aim of this study was to investigate the effect of different glucose concentrations on the leptin signaling pathway in MCF-7 and T47D breast cancer cells. Material and Methods: MCF-7 and T47D cell proliferation in different glucose concentrations (2.5 mM, 5 mM, 25 mM, or 50 mM) were assayed using CCK-8 assay. Leptin, leptin receptors (ObR, ObRb) as well as STAT3 mRNA and protein levels in both cell lines in different glucose concentrations were examined by RT-PCR and western blot, respectively. Results: Incubation in 2.5 mM, 5 mM, 25 mM, or 50 mM glucose for 72h significantly increased the proliferation of both MCF-7 and T47D cells compared to 0 mM glucose incubated cells (P<0.001). mRNA levels of leptin, ObR, ObRb or STAT3 in 2.5 mM, 5 mM, 25 mM, or 50 mM glucose incubated cells were not significantly different in both cell lines compared to 0 mM (p>0.05). However, ObR protein levels in MCF-7 cells incubated in 25 mM glucose was significantly lower compared to 0 mM glucose by western blot (p<0.05). Conclusion: Our data suggest that the enhancing effect of glucose on breast cancer cell proliferation is not mediated by the JAK/STAT pathway.

Calorie restriction (CR) has been shown to be one of the most effective methods in alleviating the effects of ageing and age-related diseases such as increasing longevity and preventing dementia, and Alzheimer’s Disease. Although the protective effects of CR have been reported, the exact molecular mechanism still needs to be clarified. This study aims to determine differentially expressed (DE) miRNAs and altered gene pathways due to long-term chronic (CCR) and intermittent (ICR) CR in the brain of mice to understand the preventive roles of miRNAs in long-term CR. Ten weeks old mice were enrolled into three different dietary groups; ad-libitum, CCR or ICR, and fed until 82 weeks old mice. miRNAs were analyzed using GeneChip 4.1 microarray and the target of DE miRNAs was determined using miRNA target databases. Out of a total 3,163 analyzed miRNAs, 55 of them were differentially expressed either by different CR protocols or by ageing. Brain samples from the CCR group had increased expression levels of mmu-miR-713 while decreasing expression levels of mmu-miR-184-3p and mmu-miR-351-5p compared to the other dietary groups. Also, current results indicated that CCR showed better preventive effects than that of ICR. Thus, CCR may perform its protective effects by modulating these specific miRNAs since they are shown to play roles in neurogenesis, chromatin, and histone regulation. In conclusion, these three miRNAs could be potential targets for neurodegenerative and ageing-related diseases and may play important roles in the protective effects of CR in the brain.

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