miR-515-3p, miR-623, miR-1272 and Notch3 protein as new biomarkers of Hepatocellular carcinoma

Backgrounds Hepatocellular carcinoma (HCC) is a diversity of hepatocellular neoplasms and is more prevalence in people with chronic liver disease and cirrhosis. It has been revealed that modification in miRNA regulation possibly will be elaborated in HCC pathogenesis. Materials and methods In this research 40 samples of HCC subjects and 40 samples of healthy liver were considered. Total RNA was obtained from paraffin-embedded tissue blocks and miR-515, miR-623 and miR-1272 gene expression levels were quantified by Real-Time Quantitative Reverse Transcription PCR. Likewise, the Notch protein quantity was assayed in ffpe materials by immunohistochemistry. Results Our study disclosed that Notch protein deals was ominously elevated in cancer cells than healthy cells (p<0.05). Data analysis also displayed that miR-515, miR-623 and miR-1272 expression levels were 3.8, 4.7, and 2.9 fold in normal tissues, respectively (p<0.05). Furthermore, it was found that expression levels of these genes are not dependent by hepatitis B and hepatic cirrhosis and it could be used as a marker of high specificity and sensitivity for the diagnosis of HCC. Discussions Our study demonstrated main role of miR-515, miR-623 and miR-1272 in HCC pathogenesis and similarly disclosed that these genes expression could be utilized in HCC prognosis.

Different concentrations of Chinese medicine Xiaoyingdaotan Decoction on Hashimoto’s thyroiditis mouse model

Background Through the detection of Notch/Treg/Th17 pathway related factors, to explore the effect of different concentrations of Chinese medicine Xiaoyingdaotan Decoction on Hashimoto's thyroiditis mouse model. Methods To make the mouse model of Hashimoto's thyroiditis, and to administer the Chinese medicine Xiaoyingdaotan Decoction of different concentrations by gavage. After the treatment, the expression of serum Notch protein and Treg/Th17 cytokine levels in mice were detected. The quantitative data conformed to the normal distribution with t test, and did not conform to the normal distribution with Wilcoxon rank sum test. Results The serum Notch protein expression of mice in the model group was significantly higher than that of the other groups, and the difference was statistically significant (P<0.001). Comparing the high-dose Chinese medicine group with the low-dose Chinese medicine group and the model group, the level of the key activation protein FOX-P3 in serum of Treg cells in mice was significantly increased, and the difference was statistically significant (P<0.05). Compared with the model group, the serum TGF-β levels of each group of traditional Chinese medicine were significantly higher than that of the model group, and the difference was statistically significant (P<0.05). The levels of key activating proteins STAT3, RORγt and IL-22 in serum of Th17 cells in each group of traditional Chinese medicine mice were significantly lower than those in the model group, with statistical differences (P<0.05). Conclusion Different concentrations of Chinese medicine Xiaoyingdaotan Decoction can down-regulate the expression of Notch protein in HT mouse model, and can effectively regulate Treg/Th17 cytokines.

The Ubiquitin Conjugating Enzyme UbcD1 is Required for Notch Signaling Activation During Drosophila Wing Development

Notch signaling pathway plays crucial roles in animal development. Protein ubiquitination contributes to Notch signaling regulation by governing the stability and activity of major signaling components. Studies in Drosophila have identified multiple ubiquitin ligases and deubiquitinating enzymes that modify Notch ligand and receptor proteins. The fate of ubiquitinated substrates depend on topologies of the attached ubiquitin chains, which are determined by the ubiquitin conjugating enzymes (E2 enzymes). However, which E2 enzymes participate in Notch signal transduction remain elusive. Here, we report that the E2 enzyme UbcD1 is required for Notch signaling activation during Drosophila wing development. Mutations of UbcD1 lead to marginal nicks in the adult wing and reduction of Notch signaling targets expression in the wing imaginal disc. Genetic analysis reveal that UbcD1 functions in the signaling receiving cells prior to cleavage of the Notch protein. We provide further evidence suggesting that UbcD1 is likely involved in endocytic trafficking of Notch protein. Our results demonstrate that UbcD1 positively regulates Notch signaling and thus reveal a novel role of UbcD1 in development.

miR-27-3p Enhances the Sensitivity of Triple-Negative Breast Cancer Cells to the Antitumor Agent Olaparib by Targeting PSEN-1, the Catalytic Subunit of Γ-Secretase

Olaparib has been used in the treatment of triple-negative breast cancer (TNBC) with BRCA mutations. In the present study, we demonstrated the effect of miR-27-3p on the γ-secretase pathway by regulating the sensitivity of TNBC cells to olaparib. miR-27-3p, a microRNA with the potential to target PSEN-1, the catalytic subunit of γ-secretase mediating the second step of the cleavage of the Notch protein, was identified by the online tool miRDB and found to inhibit the expression of PSEN-1 by directly targeting the 3’-untranslated region (3’-UTR) of PSEN-1. The overexpression of miR-27-3p inhibited the activation of the Notch pathway via the inhibition of the cleavage of the Notch protein, mediated by γ-secretase, and, in turn, enhanced the sensitivity of TNBC cells to the antitumor agent olaparib. Transfection with PSEN-1 containing mutated targeting sites for miR-27-3p or the expression vector of the Notch protein intracellular domain (NICD) almost completely blocked the effect of miR-27-3p on the Notch pathway or the sensitivity of TNBC cells to olaparib, respectively. Therefore, our results suggest that the miR-27-3p/γ-secretase axis participates in the regulation of TNBC and that the overexpression of miR-27-3p represents a potential approach to enhancing the sensitivity of TNBC to olaparib.

Pharmacogenetics of quetiapine

(1) Introduction: Quetiapine (QTP) is a dibenzothiazepine derivative, a second generation antipsychotic (AP), which is structurally similar to clozapine. The main indications for use are schizophrenia and depressive disorder. Under manic episodes in bipolar disorder can be used alone or in combination with lithium. The frequency of prescribing QTP is on average 11,987 per 100,000 population, with a positive trend in dynamics: a growth rate of more than 800% within the period 2002 to 2017.(2) Purpose: The review of studies of pharmacogenetic pharmacokinetic and pharmacogenetic pharmacodynamic markers of QTP efficacy and safety.(3) Materials and Methods: A search was carried out for publications of the Science Index, PubMed, Web of Science, Springer databases by keywords and their combinations over the last 10 years. In addition, the review includes earlier publications of historical interest. Despite extensive searches of these commonly used databases and search terms, it cannot be ruled out that some publications may have been missed.(4) Results: The review considers the following pharmacokinetic markers of QTP efficacy and safety: genes are coding isoforms of cytochrome P450 (CYP2D6, CYP2C19, CYP3A4, CYP3A5), P-glycoprotein (ABCB1); pharmacogenetic pharmacodynamic markers of the efficacy and safety of QTP : genes of dopamine receptor isoform (DRD3), dopamine transporter (SCL1A1) and catecholO-methyltransferase (COMT), serotonin receptor isoforms (HTR2C), melanocortin receptor (MC4R), NOTCH protein (NOTCH4), phosphodiesterase 4D (PDE4D), SPoPL protein (SPoPL), multiple EGFlike domain (MEGF10), protocadherin-7 (PCDH7), contactin-associated protein 5 (CNTNAP5) , TRAF2 and NCK-interacting protein kinase (TNIK), spermatogenesis-associated protein 6 (SPATA6L), neurobihin (NBEA), synaptic vesicle protein-2C (SVC2) .(5) Conclusion: Disclosure of pharmacogenetic markers of pharmacokinetics and pharmacodynamics of QTP efficacy and safety in the treatment of patients with schizophrenia and other psychiatric disorders, may provide a key to developing a strategy for its personalized prevention of adverse grug reactions (ADRs) and therapy strategy in real clinical practice.

Structural and Functional Dissection of the 5′ Region of the Notch Gene in Drosophila melanogaster

Notch is a key factor of a signaling cascade which regulates cell differentiation in all multicellular organisms. Numerous investigations have been directed mainly at studying the mechanism of Notch protein action; however, very little is known about the regulation of activity of the gene itself. Here, we provide the results of targeted 5′-end editing of the Drosophila Notch gene in its native environment and genetic and cytological effects of these changes. Using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9 (CRISPR/Cas9) system in combination with homologous recombination, we obtained a founder fly stock in which a 4-kb fragment, including the 5′ nontranscribed region, the first exon, and a part of the first intron of Notch, was replaced by an attachment Phage (attP) site. Then, fly lines carrying a set of six deletions within the 5′untranscribed region of the gene were obtained by ΦC31-mediated integration of transgenic constructs. Part of these deletions does not affect gene activity, but their combinations with transgenic construct in the first intron of the gene cause defects in the Notch target tissues. At the polytene chromosome level we defined a DNA segment (~250 bp) in the Notch5′-nontranscribed region which when deleted leads to disappearance of the 3C6/C7 interband and elimination of CTC-Factor (CTCF) and Chromator (CHRIZ) insulator proteins in this region.

Evolving Notch polyQ tracts reveal possible solenoid interference elements

ABSTRACTPolyglutamine (polyQ) tracts in regulatory proteins are extremely polymorphic. As functional elements under selection for length, triplet repeats are prone to DNA replication slippage and indel mutations. Many polyQ tracts are also embedded within intrinsically disordered domains, which are less constrained, fast evolving, and difficult to characterize. To identify structural principles underlying polyQ tracts in disordered regulatory domains, here I analyze deep evolution of metazoan Notch polyQ tracts, which can generate alleles causing developmental and neurogenic defects. I show that Notch features polyQ tract turnover that is restricted to a discrete number of conserved “polyQ insertion slots”. Notch polyQ insertion slots are: (i) identifiable by an amphipathic “slot leader” motif; (ii) conserved as an intact C-terminal array in a 1-to-1 relationship with the N-terminal solenoid-forming ankyrin repeats (ARs); and (iii) enriched in carboxamide residues (Q/N), whose sidechains feature dual hydrogen bond donor and acceptor atoms. Correspondingly, the terminal loop and β-strand of each AR feature conserved carboxamide residues, which would be susceptible to folding interference by hydrogen bonding with residues outside the ARs. I thus suggest that Notch polyQ insertion slots constitute an array of AR interference elements (ARIEs). Notch ARIEs would dynamically compete with the delicate serial folding induced by adjacent ARs. Huntingtin, which harbors solenoid-forming HEAT repeats, also possesses a similar number of polyQ insertion slots. These results strongly suggest that intrinsically disordered interference arrays featuring carboxamide and polyQ enrichment are coupled proteodynamic modulators of solenoids.SIGNIFICANCENeurodegenerative disorders are often caused by expanded polyglutamine (polyQ) tracts embedded in the disordered regions of regulatory proteins, which are difficult to characterize structurally. To identify functional principles underlying polyQ tracts in disordered regulatory domains, I analyze evolution of the Notch protein, which can generate polyQ-related alleles causing neurodevelopmental defects. I show that Notch evolves polyQ tracts that come and go in a few conserved “polyQ insertion slots”. Several features suggest these slots are ankyrin repeat (AR) interference elements, which dynamically compete with the delicate solenoid formed by Notch. Huntingtin, whose polyQ expansions causes Huntington’s Disease in humans, also has solenoid-forming modules and polyQ insertion slots, suggesting a common architectural principle underlies solenoid-forming polyQ-rich proteins.