A double-negative feedback loop mediated by non-coding RNAs contributes to tooth morphogenesis.

Tooth morphogenesis is a critically ordered process manipulated by a range of signaling factors. Particularly, the involvement of fine-tuned signaling mediated by non-coding RNAs has been of longstanding interest. Here, we revealed a double-negative feedback loop acted by a long non-coding RNA (LOC102159588) and a microRNA (miR-133b) that modulated tooth morphogenesis of miniature swine. Mechanistically, miR-133b repressed the transcription of LOC102159588 through downstream target Sp1. Conversely, LOC102159588 not only inhibited the transport of pre-miR-133b from the nucleus to the cytoplasm by regulating exportin-5 but also served as a sponge in the cytoplasm, suppressing functional miR-133b. Together, the double-negative feedback loop maintained normal tooth morphogenesis by modulating endogenous apoptosis. Related disruptions would lead to an arrest of tooth development and may result in tooth malformations.

A unique circ_0067716/EIF4A3 double-negative feedback loop impacts malignant transformation of human bronchial epithelial cells induced by benzo(a)pyrene.

Benzo(a)pyrene as a pervasive environmental contaminant is characterized by its substantial genotoxicity, and epidemiological investigations have established a correlation between benzo(a)pyrene exposure and the susceptibility to human lung cancer. Notably, much research has focused on the link between epigenetic alterations and lung cancer induced by chemicals, although circRNAs are also emerging as relevant contributors to the carcinogenic process of benzo(a)pyrene. In this study, we identified circ_0067716 as being significantly upregulated in response to stress injury and downregulated during malignant transformation induced by benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE) in human bronchial epithelial cells. The observed differential expression of circ_0067716 in cells treated with BPDE for varying durations suggests a strong correlation between this circRNA and BPDE exposure. The tissue samples of lung cancer patients also suggest that a lower circ_0067716 expression is associated with BPDE-DNA adduct levels. Remarkably, we demonstrate that EIF4A3, located in the nucleus, interacts with the flanking sequences of circ_0067716 and inhibits its biogenesis. Conversely, circ_0067716 is capable of sequestering EIF4A3 in the cytoplasm, thereby preventing its translocation into the nucleus. EIF4A3 and circ_0067716 can form a double-negative feedback loop that could be affected by BPDE. During the initial phase of BPDE exposure, the expression of circ_0067716 was increased in response to stress injury, resulting in cell apoptosis through the involvement of miR-324-5p/DRAM1/BAX axis. Subsequently, as cellular adaptation progressed, long-term induction due to BPDE exposure led to an elevated EIF4A3 and a reduced circ_0067716 expression, which facilitated the proliferation of cells by stabilizing the PI3K/AKT pathway. Thus, our current study describes the effects of circ_0067716 on the genotoxicity and carcinogenesis induced by benzo(a)pyrene and puts forwards to the possible regulatory mechanism on the occurrence of smoking-related lung cancer, providing a unique insight based on epigenetics.

The miR-200-Zeb1 axis regulates key aspects of ß-cell function and survival in vivo.

OBJECTIVE: The miR-200-Zeb1 axis regulates the epithelial-to-mesenchymal transition (EMT), differentiation, and resistance to apoptosis. A better understanding of these processes in diabetes is highly relevant, as ß-cell dedifferentiation and apoptosis contribute to the loss of functional ß-cell mass and diabetes progression. Furthermore, EMT promotes the loss of ß-cell identity in the in vitro expansion of human islets. Though the miR-200 family has previously been identified as a regulator of ß-cell apoptosis in vivo, studies focusing on Zeb1 are lacking. The aim of this study was thus to investigate the role of Zeb1 in ß-cell function and survival in vivo. METHODS: miR-200 and Zeb1 are involved in a double-negative feedback loop. We characterized a mouse model in which miR-200 binding sites in the Zeb1 3'UTR are mutated (Zeb1200), leading to a physiologically relevant upregulation of Zeb1 mRNA expression. The role of Zeb1 was investigated in this model via metabolic tests and analysis of isolated islets. Further insights into the distinct contributions of the miR-200 and Zeb1 branches of the feedback loop were obtained by crossing the Zeb1200 allele into a background of miR-141-200c overexpression. RESULTS: Mild Zeb1 derepression in vivo led to broad transcriptional changes in islets affecting ß-cell identity, EMT, insulin secretion, cell-cell junctions, the unfolded protein response (UPR), and the response to ER stress. The aggregation and insulin secretion of dissociated islets of mice homozygous for the Zeb1200 mutation (Zeb1200M) were impaired, and Zeb1200M islets were resistant to thapsigargin-induced ER stress ex vivo. Zeb1200M mice had increased circulating proinsulin levels but no overt metabolic phenotype, reflecting the strong compensatory ability of islets to maintain glucose homeostasis. CONCLUSIONS: This study signifies the importance of the miR-200-Zeb1 axis in regulating key aspects of ß-cell function and survival. A better understanding of this axis is highly relevant in developing therapeutic strategies for inducing ß-cell redifferentiation and maintaining ß-cell identity in in vitro islet expansion.

Multi-Stability and Consequent Phenotypic Plasticity in AMPK-Akt Double Negative Feedback Loop in Cancer Cells.

Adaptation and survival of cancer cells to various stress and growth factor conditions is crucial for successful metastasis. A double-negative feedback loop between two serine/threonine kinases AMPK (AMP-activated protein kinase) and Akt can regulate the adaptation of breast cancer cells to matrix-deprivation stress. This feedback loop can significantly generate two phenotypes or cell states: matrix detachment-triggered pAMPKhigh/ pAktlow state, and matrix (re)attachment-triggered pAkthigh/ pAMPKlow state. However, whether these two cell states can exhibit phenotypic plasticity and heterogeneity in a given cell population, i.e., whether they can co-exist and undergo spontaneous switching to generate the other subpopulation, remains unclear. Here, we develop a mechanism-based mathematical model that captures the set of experimentally reported interactions among AMPK and Akt. Our simulations suggest that the AMPK-Akt feedback loop can give rise to two co-existing phenotypes (pAkthigh/ pAMPKlow and pAMPKhigh/pAktlow) in specific parameter regimes. Next, to test the model predictions, we segregated these two subpopulations in MDA-MB-231 cells and observed that each of them was capable of switching to another in adherent conditions. Finally, the predicted trends are supported by clinical data analysis of The Cancer Genome Atlas (TCGA) breast cancer and pan-cancer cohorts that revealed negatively correlated pAMPK and pAkt protein levels. Overall, our integrated computational-experimental approach unravels that AMPK-Akt feedback loop can generate multi-stability and drive phenotypic switching and heterogeneity in a cancer cell population.

Insulin Receptors and Intracellular Ca 2+ Form a Double-Negative Regulatory Feedback Loop Controlling Insulin Sensitivity.

Since the discovery of insulin and insulin receptors (IR) in the brain in 1978, numerous studies have revealed a fundamental role of IR in the central nervous system and its implication in regulating synaptic plasticity, long-term potentiation and depression, neuroprotection, learning and memory, and energy balance. Central insulin resistance has been found in diverse brain disorders including Alzheimer's disease (AD). Impaired insulin signaling in AD is evident in the activation states of IR and downstream signaling molecules. This is mediated by Aß oligomer-evoked Ca 2+ influx by activating N-methyl-D-aspartate receptors (NMDARs) with Aß oligomers directly, or indirectly through Aß-induced release of glutamate, an endogenous NMDAR ligand. In the present opinion article, we highlight evidence that IR and free intracellular Ca 2+ concentration [Ca 2+] i form a double-negative regulatory feedback loop controlling insulin sensitivity, in which mitochondria play a key role, being involved in adenosine triphosphate (ATP) synthesis and IR activation. We found recently that the glutamate-evoked rise in [Ca 2+] i inhibits activation of IR and, vice versa, insulin-induced activation of IR inhibits the glutamate-evoked rise in [Ca 2+] i . In theory, such a double-negative feedback loop generates bistability. Thus, a stable steady state could exist with high [Ca 2+] i and nonactive IR, or with active IR and low [Ca 2+] i, but no stable steady state is possible with both high [Ca 2+] i and active IR. Such a circuit could toggle between a high [Ca 2+] i state and an active IR state in response to glutamate and insulin, respectively. This model predicts that any condition leading to an increase of [Ca 2+] i may trigger central insulin resistance and explains why central insulin resistance is implicated in the pathogenesis of AD, with which glutamate excitotoxicity is a comorbid condition. The model also predicts that any intervention aiming to maintain low [Ca 2+] i may be useful for treating central insulin resistance.

A Double Negative Feedback Loop between mTORC1 and AMPK Kinases Guarantees Precise Autophagy Induction upon Cellular Stress.

Cellular homeostasis is controlled by an evolutionary conserved cellular digestive process called autophagy. This mechanism is tightly regulated by the two sensor elements called mTORC1 and AMPK. mTORC1 is one of the master regulators of proteostasis, while AMPK maintains cellular energy homeostasis. AMPK is able to promote autophagy by phosphorylating ULK1, the key inducer of autophagosome formation, while mTORC1 downregulates the self-eating process via ULK1 under nutrient rich conditions. We claim that the feedback loops of the AMPK-mTORC1-ULK1 regulatory triangle guarantee the appropriate response mechanism when nutrient and/or energy supply changes. In our opinion, there is an essential double negative feedback loop between mTORC1 and AMPK. Namely, not only does AMPK downregulate mTORC1, but mTORC1 also inhibits AMPK and this inhibition is required to keep AMPK inactive at physiological conditions. The aim of the present study was to explore the dynamical characteristic of AMPK regulation upon various cellular stress events. We approached our scientific analysis from a systems biology perspective by incorporating both theoretical and molecular biological techniques. In this study, we confirmed that AMPK is essential to promote autophagy, but is not sufficient to maintain it. AMPK activation is followed by ULK1 induction, where protein has a key role in keeping autophagy active. ULK1-controlled autophagy is always preceded by AMPK activation. With both ULK1 depletion and mTORC1 hyper-activation (i.e., TSC1/2 downregulation), we demonstrate that a double negative feedback loop between AMPK and mTORC1 is crucial for the proper dynamic features of the control network. Our computer simulations have further proved the dynamical characteristic of AMPK-mTORC1-ULK1 controlled cellular nutrient sensing.

Copy number gain of ZEB1 mediates a double-negative feedback loop with miR-33a-5p that regulates EMT and bone metastasis of prostate cancer dependent on TGF-ß signaling.

Background: The reciprocal repressive loop between ZEB1 and miRNAs has been extensively reported to play an important role in tumor progression and metastasis of various human tumor types. The aim of this study was to elucidate the role and the underlying mechanism of the double-negative feedback loop between ZEB1and miR-33a-5p in bone metastasis of prostate cancer (PCa). Methods: miR-33a-5p expression was examined in 40 bone metastatic and 165 non-bone metastatic PCa tissues by real-time PCR. Statistical analysis was performed to evaluate the clinical correlation between miR-33a-5p expression and clinicopathological characteristics, and overall and bone metastasis-free survival in PCa patients. The biological roles of miR-33a-5p in bone metastasis of PCa were investigated both by EMT and the Transwell assay in vitro, and by a mouse model of left cardiac ventricle inoculation in vivo. siRNA library, real-time PCR and chromatin immunoprecipitation (ChIP) were used to identify the underlying mechanism responsible for the decreased expression of miR-33a-5p in PCa. Bioinformatics analysis, Western blotting and luciferase reporter analysis were employed to examine the relationship between miR-33a-5p and its potential targets. Clinical correlation of miR-33a-5p with its targets was examined in human PCa tissues and primary PCa cells. Results: miR-33a-5p expression was downregulated in PCa tissues with bone metastasis and bone-derived cells, and low expression of miR-33a-5p strongly and positively correlated with advanced clinicopathological characteristics, and shorter overall and bone metastasis-free survival in PCa patients. Upregulating miR-33a-5p inhibited, while silencing miR-33a-5p promoted EMT, invasion and migration of PCa cells. Importantly, upregulating miR-33a-5p significantly repressed bone metastasis of PC-3 cells in vivo. Our results further revealed that recurrent ZEB1 upregulation induced by copy number gains transcriptionally inhibited miR-33a-5p expression, contributing to the reduced expression of miR-33a-5p in bone metastatic PCa tissues. In turn, miR-33a-5p formed a double negative feedback loop with ZEB1 in target-independent manner, which was dependent on TGF-ß signaling. Finally, the clinical negative correlations of miR-33a-5p with ZEB1 expression and TGF-ß signaling activity were demonstrated in PCa tissues and primary PCa cells. Conclusion: Our findings elucidated that copy number gains of ZEB1-triggered a TGF-ß signaling-dependent miR-33a-5p-mediated negative feedback loop was highly relevant to the bone metastasis of PCa.

Dysregulation of the miR-194-CUL4B negative feedback loop drives tumorigenesis in non-small-cell lung carcinoma.

Cullin 4B (CUL4B), a scaffold protein that assembles CRL4B ubiquitin ligase complexes, is overexpressed in many types of cancers and represses many tumor suppressors through epigenetic mechanisms. However, the mechanisms by which CUL4B is upregulated remain to be elucidated. Here, we show that CUL4B is upregulated in non-small-cell lung carcinoma (NSCLC) tissues and is critically required for cell proliferation and migration in vitro and for xenograft tumor formation in vivo. We found that microRNA-194 (miR-194) and CUL4B protein were inversely correlated in cancer specimens and demonstrated that miR-194 could downregulate CUL4B by directly targeting its 3'-UTR. We also showed that CUL4B could be negatively regulated by p53 in a miR-194-dependent manner. miR-194 was further shown to attenuate the malignant phenotype of lung cancer cells by downregulating CUL4B. Interestingly, CRL4B also epigenetically represses miR-194 by catalyzing monoubiquitination at H2AK119 and by coordinating with PRC2 to promote trimethylation at H3K27 at the gene clusters encoding miR-194. RBX1, another component in CRL4B complex, is also targeted by miR-194 in NSCLC cells. Our results thus establish a double-negative feedback loop between miR-194 and CRL4B, dysregulation of which contributes to tumorigenesis. The function of miR-194 as a negative regulator of CUL4B has therapeutic implications in lung cancer.