HIF-3α-Induced miR-630 Expression Promotes Cancer Hallmarks in Cervical Cancer Cells by Forming a Positive Feedback Loop

Background Hypoxia has crucial functions in cervical cancer development and metastasis by inducing the level of numerous genes, including microRNA genes. But we know little about how the hypoxia factors and microRNAs orchestrate to regulate hallmarks of cervical cancer cells. Methods We used RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing experiments (ChIP-seq) to investigate the targets of HIF-3α or miR-630. ChIP-qPCR and RT-qPCR were used to validate the sequencing results of ChIP-seq and RNA-seq. Cellular, molecular and radiation experiments were used to explore the functions of miR-630.Results Here, we show that hypoxia-induced overexpression HIF-3α increased the expression of dozens of miRNAs, including miR-630. Further experiment showed the activation of miR-630 was induced by hypoxia treatment. We showed that hypoxia induces expression of HIF-3α to activate miR-630 expression by directly binding to its promoter. Meanwhile, miR-630 positively regulates HIF-1α expression, but represses HIF-1α. Stable overexpression of miR-630 in HeLa cells promotes cancer hallmarks, including radioresistance, inhibition of apoptosis, increased migration and invasion, and EMT-mediated metastasis. Stable inhibition of miR-630 showed opposite features. Conclusion Taken together, our findings implicate a novel hypoxia-induced HIF3a-miR-630 regulatory feedback loop contributing to metastasis and progression of cervical cancer cells, and suggest HIF3a and miR630 might be applied as a potential biomarker and therapeutic target for cervical cancer.

The role of USP7 in the Shoc2 - ERK1/2 signaling axis and Noonan-like syndrome with loose anagen hair (NSLAH)

The ERK1/2 signaling pathway is critical in organismal development and tissue morphogenesis. Deregulation of this pathway leads to congenital abnormalities with severe developmental dysmorphisms. The core ERK1/2 cascade relies on scaffold proteins such as Shoc2 to guide and fine-tune its signals. Mutations in shoc2 lead to the development of the pathology termed Noonan-like Syndrome with Loose Anagen Hair (NSLAH). However, the mechanisms underlying the functions of Shoc2 and its contributions to disease progression remain unclear. Here we show that ERK1/2 pathway activation triggers the interaction of Shoc2 with the ubiquitin-specific protease USP7. We identify that in the Shoc2 module USP7 functions as a molecular “switch” that controls the E3 ligase HUWE1 and the HUWE1-induced regulatory feedback loop. We also demonstrate that disruption of Shoc2-USP7 binding leads to aberrant activation of the Shoc2-ERK1/2 axis. Importantly, our studies reveal a possible role for USP7 in the pathogenic mechanisms underlying NSLAH extending our understanding of how ubiquitin-specific proteases regulate intracellular signaling.

Long non-coding RNA SLC2A1-AS1 induced by GLI3 promotes aerobic glycolysis and progression in esophageal squamous cell carcinoma by sponging miR-378a-3p to enhance Glut1 expression

Background Emerging evidence demonstrates that lncRNAs play pivotal roles in tumor energy metabolism; however, the detailed mechanisms of lncRNAs in the regulation of tumor glycolysis remain largely unknown. Methods The expression of SLC2A1-AS1 was investigated by TCGA, GEO dataset and qRT-PCR. The binding of GLI3 to SLC2A1-AS1 promoter was detected by Luciferase Reporter Assay System and Ago2-RIP assay. FISH was performed to determine the localization of SLC2A1-AS1 in ESCC cells. Double Luciferase Report assay was used to investigate the interaction of miR-378a-3p with SLC2A1-AS1 and Glut1. Gain-of-function and Loss-of-function assay were performed to dissect the function of SLC2A1-AS1/miR-378a-3p/Glut1 axis in ESCC progression in vitro and in vivo. Results We identified a novel lncRNA SLC2A1-AS1 in ESCC. SLC2A1-AS1 was frequently overexpressed in ESCC tissues and cells, and its overexpression was associated with TNM stage, lymph node metastasis and poor prognosis of ESCC patients. Importantly, GLI3 and SLC2A1-AS1 formed a regulatory feedback loop in ESCC cells. SLC2A1-AS1 promoted cell growth in vitro and in vivo, migration and invasion, and suppressed apoptosis, leading to EMT progression and increased glycolysis in ESCC cells. SLC2A1-AS1 functioned as ceRNA for sponging miR-378a-3p, resulting in Glut1 overexpression in ESCC cells. MiR-378a-3p inhibited cell proliferation and invasion as well as induced apoptosis, resulting in reduced glycolysis, which was partly reversed by SLC2A1-AS1 or Glut1 overexpression in ESCC cells. Conclusion SLC2A1-AS1 plays important roles in ESCC development and progression by regulating glycolysis, and SLC2A1-AS1/miR-378a-3p/Glut1 regulatory axis may be a novel therapeutic target in terms of metabolic remodeling of ESCC patients.

A Positive Regulatory Feedback Loop between EKLF/KLF1 and TAL1/SCL Sustaining the Erythropoiesis

The erythroid Krüppel-like factor EKLF/KLF1 is a hematopoietic transcription factor binding to the CACCC DNA motif and participating in the regulation of erythroid differentiation. With combined use of microarray-based gene expression profiling and the promoter-based ChIP-chip assay of E14.5 fetal liver cells from wild type (WT) and EKLF-knockout (Eklf−/−) mouse embryos, we identified the pathways and direct target genes activated or repressed by EKLF. This genome-wide study together with the molecular/cellular analysis of the mouse erythroleukemic cells (MEL) indicate that among the downstream direct target genes of EKLF is Tal1/Scl. Tal1/Scl encodes another DNA-binding hematopoietic transcription factor TAL1/SCL, known to be an Eklf activator and essential for definitive erythroid differentiation. Further identification of the authentic Tal gene promoter in combination with the in vivo genomic footprinting approach and DNA reporter assay demonstrate that EKLF activates the Tal gene through binding to a specific CACCC motif located in its promoter. These data establish the existence of a previously unknow positive regulatory feedback loop between two DNA-binding hematopoietic transcription factors, which sustains mammalian erythropoiesis.

Drosophila primary microRNA-8 encodes a microRNA-encoded peptide acting in parallel of miR-8

Background Recent genome-wide studies of many species reveal the existence of a myriad of RNAs differing in size, coding potential and function. Among these are the long non-coding RNAs, some of them producing functional small peptides via the translation of short ORFs. It now appears that any kind of RNA presumably has a potential to encode small peptides. Accordingly, our team recently discovered that plant primary transcripts of microRNAs (pri-miRs) produce small regulatory peptides (miPEPs) involved in auto-regulatory feedback loops enhancing their cognate microRNA expression which in turn controls plant development. Here we investigate whether this regulatory feedback loop is present in Drosophila melanogaster. Results We perform a survey of ribosome profiling data and reveal that many pri-miRNAs exhibit ribosome translation marks. Focusing on miR-8, we show that pri-miR-8 can produce a miPEP-8. Functional assays performed in Drosophila reveal that miPEP-8 affects development when overexpressed or knocked down. Combining genetic and molecular approaches as well as genome-wide transcriptomic analyses, we show that miR-8 expression is independent of miPEP-8 activity and that miPEP-8 acts in parallel to miR-8 to regulate the expression of hundreds of genes. Conclusion Taken together, these results reveal that several Drosophila pri-miRs exhibit translation potential. Contrasting with the mechanism described in plants, these data shed light on the function of yet undescribed primary-microRNA-encoded peptides in Drosophila and their regulatory potential on genome expression.

A Positive Regulatory Feedback Loop Between 1 EKLF/ KLF1 and TAL1/SCL2 Sustaining the Erythropoiesis

The erythroid Krppel-like factor EKLF/KLF1 is a hematopoietic transcription factor binding to CACCC DNA motif and participating in the regulation of erythroid differentiation. With combined use of microarray-based gene expression profiling and promoter-based ChIP-chip assay of E14.5 fetal liver cells from wild type (WT) and EKLF-knockout (Eklf-/-) mouse embryos, we have identified the pathways and direct target genes activated or repressed by EKLF. This genome-wide study together with molecular/ cellular analysis of mouse erythroleukemic cells (MEL) indicate that among the downstream direct target genes of EKLF is Tal1/Scl. Tal1/Scl encodes another DNA-binding hematopoietic transcription factor TAL1/SCL known to be an Eklf activator and essential for definitive erythroid differentiation. Further identification of the authentic Tall gene promoter in combination with in vivo genomic footprinting approach and DNA reporter assay demonstrate that EKLF activates Tall gene through binding to a specific CACCC motif located in its promoter. These data establish the existence of a previously unknow positive regulatory feedback loop between two DNA-binding hematopoietic transcription factors that sustains the mammalian erythropoiesis.

Drosophila primary microRNA-8 encodes a microRNA encoded peptide acting in parallel of miR-8

SummaryBackgroundRecent genome-wide studies of many species reveal the existence of a myriad of RNAs differing in size, coding potential and function. Among these are the long non-coding RNAs, some of them producing functional small peptides via the translation of short ORFs. It now appears that any kind of RNA presumably has a potential to encode small peptides. Accordingly, our team recently discovered that plant primary transcripts of microRNAs (pri-miRNAs) produce small regulatory peptides (miPEPs) involved in auto-regulatory feedback loops enhancing their cognate microRNA expression which in turn controls plant development. Here we investigate whether this regulatory feedback loop is present in Drosophila melanogaster.ResultsWe perform a survey of ribosome profiling data and reveal that many pri-miRNAs exhibit ribosome translation marks. Focusing on miR-8, we show that pri-miR-8 can produce a miPEP-8. Functional assays performed in Drosophila reveal that miPEP-8 affects development when overexpressed or knocked down. Combining genetic and molecular approaches as well as genome-wide transcriptomic analyses, we show that miR-8 expression is independent of miPEP-8 activity and that miPEP-8 acts in parallel to miR-8 to regulate the expression of hundreds of genes.ConclusionTaken together, these results reveal that several Drosophila pri-miRNAs exhibit translation potential. Contrasting with the mechanism described in plants, these data shed light on the function of yet un-described pri-microRNA encoded peptides in Drosophila and their regulatory potential on genome expression.

Disrupted circadian core-clock oscillations in Type 2 Diabetes are linked to altered rhythmic mitochondrial metabolism

SummaryCircadian rhythms are generated by an auto-regulatory feedback loop composed of transcriptional activators and repressors. Disruption of circadian rhythms contributes to Type 2 diabetes (T2D) pathogenesis. We elucidated whether altered circadian rhythmicity of clock genes is associated with metabolic dysfunction in T2D. Transcriptional cycling of core clock genes ARNTL, CLOCK, CRY1 and NR1D1 was altered in skeletal muscle from individuals with T2D and this was coupled with reduced number and amplitude of cycling genes and disturbed circadian oxygen consumption. Mitochondrial associated genes were enriched for differential circadian amplitudes in T2D, and positively correlated with insulin sensitivity. ChIP- sequencing identified CLOCK and BMAL1 binding to circadian mitochondrial genes associated with insulin sensitivity, implicating regulation by the core clock. Mitochondria disruption altered core-clock gene expression and free-radical production, phenomena that were restored by resveratrol treatment. We identify bi-directional communication between mitochondrial function and rhythmic gene expression, processes which are disturbed in diabetes.

Insulin Receptors and Intracellular Ca2+ 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 Aβ oligomer-evoked Ca2+ influx by activating N-methyl-D-aspartate receptors (NMDARs) with Aβ oligomers directly, or indirectly through Aβ-induced release of glutamate, an endogenous NMDAR ligand. In the present opinion article, we highlight evidence that IR activity and free intracellular Ca2+ concentration [Ca2+]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 [Ca2+]i inhibits activation of IR and, vice versa, insulin-induced activation of IR inhibits the glutamate-evoked rise in [Ca2+]i. In theory, such a double-negative regulatory feedback loop predicts that any condition leading to an increase of [Ca2+]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. This model also predicts that any intervention aiming to maintain low [Ca2+]i may be useful for treating central insulin resistance.

Post-Translational Modifications of Nitrate Reductases Autoregulates Nitric Oxide Biosynthesis in Arabidopsis

Nitric oxide (NO) is a regulator of growth, development, and stress responses in living organisms. Plant nitrate reductases (NR) catalyze the reduction of nitrate to nitrite or, alternatively, to NO. In plants, NO action and its targets remain incompletely understood, and the way NO regulates its own homeostasis remains to be elucidated. A significant transcriptome overlapping between NO-deficient mutant and NO-treated wild type plants suggests that NO could negatively regulate its biosynthesis. A significant increase in NO content was detected in transgenic plants overexpressing NR1 and NR2 proteins. In turn, NR protein and activity as well as NO content, decreased in wild-type plants exposed to a pulse of NO gas. Tag-aided immunopurification procedures followed by tandem mass spectrometry allowed identifying NO-triggered post-translational modifications (PTMs) and ubiquitylation sites in NRs. Nitration of tyrosine residues and S-nitrosation of cysteine residues affected key amino acids involved in binding the essential FAD and molybdenum cofactors. NO-related PTMs were accompanied by ubiquitylation of lysine residues flanking the nitration and S-nitrosation sites. NO-induced PTMs of NRs potentially inhibit their activities and promote their proteasome-mediated degradation. This auto-regulatory feedback loop may control nitrate assimilation to ammonium and nitrite-derived production of NO under complex environmental conditions.