miR-376b controls starvation and mTOR inhibition-related autophagy by targeting ATG4C and BECN1

Macroautophagy (autophagy) is the major intracellular degradation pathway for long-lived proteins and organelles. It helps the cell to survive a spectrum of stressful conditions including starvation, growth factor deprivation and misfolded protein accumulation. Moreover, abnormalities of autophagy play a role in major health problems including cancer and neurodegenerative diseases. Yet, mechanisms controlling autophagic activity are not fully understood. Here, we describe hsa-miR-376b (miR-376b) as a new microRNA (miRNA) regulating autophagy. We showed that miR-376b expression attenuated starvation- and rapamycin-induced autophagy in MCF-7 and Huh-7 cells. We discovered autophagy proteins ATG4C and BECN1 (Beclin 1) as cellular targets of miR-376b. Indeed, upon miRNA overexpression, both mRNA and protein levels of ATG4C and BECN1 were decreased. miR-376b target sequences were present in the 3' UTR of ATG4C and BECN1 mRNAs and introduction of mutations abolished their miR-376b responsiveness. Antagomir-mediated inactivation of the endogenous miR-376b led to an increase in ATG4C and BECN1 levels. Therefore, miR-376b controls autophagy by directly regulating intracellular levels of two key autophagy proteins, ATG4C and BECN1.     

Granular cell tumor presenting as a tongue nodule: two case reports

Introduction

Granular cell tumor is an uncommon neoplasm that can occur in any part of the body, including the orofacial region. The tumor is usually benign, but there are reports of cases in which the tumor shows a locally aggressive behavior, malignancy, and distant metastases. The most widely accepted hypothesis is that granular cell tumor arises from the altered metabolism of Schwann cells. The tumor is typically asymptomatic and appears as a nodule that does not exceed 3 cm.

Case presentation

In case 1, a 26-year-old Caucasian man was seen at the Oral Medicine out-patient clinic of the São José dos Campos Dental School, Universidade Estadual Paulista, with a 'small blister on the tongue', which he had noted approximately three years ago. The nodule was located on the dorsum of the tongue, measured about 1.5 cm in diameter, and was not tender to palpation. Treatment consisted of an excisional biopsy performed on the basis of the diagnostic hypothesis of granular cell tumor, which was confirmed by microscopic analysis. In case 2, a 31-year-old Caucasian woman attended the out-patient clinic of the São José dos Campos Dental School, Universidade Estadual Paulista, with a five-year history of a 'painful lump on the tongue'. Intra-oral examination revealed the presence of a nodular lesion measuring approximately 0.8 cm in diameter, which was located deep in the submucosa of the right lateral margin of the tongue. Treatment consisted of an excisional biopsy performed on the basis of the differential diagnosis of neurofibroma and granular cell tumor. Microscopic analysis defined the final diagnosis of granular cell tumor.

Conclusions

Granular cell tumor is an uncommon tumor that must be carefully diagnosed and treated correctly.

     

Prokaryotic diversity in Tuz Lake, a hypersaline environment in Inland Turkey

Tuz Lake is an inland thalassohaline water body located in central Anatolia that contributes to 60% of the total salt production in Turkey per year. The microbiota inhabiting this lake has been studied by FISH, denaturing gradient gel electrophoresis of PCR-amplified fragments of 16S rRNA genes, and 16S rRNA gene clone library analysis. Total cell counts per milliliter (1.38 × 10⁷) were in the range of the values normally found for hypersaline environments. The proportion of Bacteria to Archaea in the community detectable by FISH was one to three. 16S rRNA gene clone libraries indicated that the archaeal assemblage was dominated by members of the Square Haloarchaea of the Walsby group, although some other groups were also found. Bacteria were dominated by members of the Bacteroidetes , including Salinibacter ruber -related phylotypes. Because members of Bacteroidetes are widely present in different hypersaline environments, a phylogenetic analysis of 16S rRNA gene sequences from Bacteroidetes retrieved from these environments was carried out in order to ascertain whether they formed a unique cluster. Sequences retrieved from Tuz Lake and a group of sequences from other hypersaline environments clustered together in a branch that could be considered as the 'halophilic branch' within the Bacteroidetes phylum.     

The genotoxic risk of underground coal miners from Turkey

A cytogenetic monitoring study was carried out on a group of workers from a bituminous coal mine in Zonguldak province of Turkey, to investigate the genotoxic risk of occupational exposure to coal mine dust. Cytogenetic analysis, namely sister chromatid exchanges (SCEs), chromosomal aberrations (CAs) and micronucleus (MN) tests were performed on a strictly selected group of 39 workers and compared to 34 controls matched for gender, age, and habit. Smoking and age were considered as modulating factors. Both SCE and CA frequencies in coal miners appeared significantly higher than in controls. Similarly, there was a significant increase in the frequency of total micronuclei in exposed group as compared to control group. The effect of smoking on the level of SCE and MN was significant in the control group. A positive correlation between the age and the level of SCE was also found in controls. The frequencies of both SCE and CA were significantly enhanced with the years of exposure. The results of this study demonstrated that occupational exposure to coal mine dust leads to a significant induction of cytogenetic damage in peripheral lymphocytes of workers engaged in underground coal mining.     

Inhibition of Cell Proliferation and Migration by miR-509-3p That Targets CDK2, Rac1, and PIK3C2A

CDK2 is a key regulator of cell cycle progression. In this study, we screened for miRNAs targeting CDK2 using a luciferase-3′-untranslated region reporter assay. Among 11 hit miRNAs, miR-509-3p reduced CDK2 protein levels and significantly inhibited cancer cell growth. Microarray, Western blotting, and luciferase reporter analyses revealed additional targets of miR-509-3p, including Rac1 and PIK3C2A. Overexpression of miR-509-3p induced G1 cell-cycle arrest and inhibited colony formation and migration. RNAi experiments indicated that the growth-inhibitory effects of miR-509-3p may occur through down-regulation of CDK2, Rac1, and PIK3C2A. Targeting of multiple growth regulatory genes by miR-509-3p may contribute to effective anti-cancer therapy.     

Immunohistochemistry of matrix metalloproteinases (MMP), their substrates, and their inhibitors (TIMP) during trophoblast invasion in the human placenta

The invasion of extravillous trophoblast cells into the maternal endometrium is one of the key events in human placentation. The ability of these cells to infiltrate the uterine wall and to anchor the placenta to it as well as their ability to infiltrate and to adjust utero-placental vessels to pregnancy depends, among other things, on their ability to secrete enzymes that degrade the extracellular matrix. Most of the latter enzymes belong to the family of matrix metalloproteinases. Their activity is regulated by the tissue inhibitors of matrix metalloproteinases. We have studied the distribution patterns of matrix metalloproteinases-1, -2, -3, and -9 and their inhibitors TIMP-1 and TIMP-2 as compared to the distribution of their substrates along the invasive pathway of extravillous trophoblast of 1st, 2nd, and 3rd trimester placentas by means of light microscopy on paraffin and cryostat sections as well as at the ultrastructural level (only 3rd trimester placenta). The comparison of different methods proved to be necessary, since the immunohistochemical distribution patterns of these soluble enzymes are considerably influenced by the pretreatment of tissues. All three methods revealed immunoreactivities of both, proteinases and their inhibitors, not only intracellularly in the extravillous trophoblast but also extracellularly in its surrounding matrix, the distribution patterns depending on the stage of pregnancy and on the degree of differentiation of trophoblast cells along their invasive pathway. Within the extracellular matrix, immunolocalization of matrix metalloproteinases as well as their inhibitors showed a specific relation to certain extracellular matrix molecules.     

Circadian Disorganization Alters Intestinal Microbiota

Intestinal dysbiosis and circadian rhythm disruption are associated with similar diseases including obesity, metabolic syndrome, and inflammatory bowel disease. Despite the overlap, the potential relationship between circadian disorganization and dysbiosis is unknown; thus, in the present study, a model of chronic circadian disruption was used to determine the impact on the intestinal microbiome. Male C57BL/6J mice underwent once weekly phase reversals of the light:dark cycle (i.e., circadian rhythm disrupted mice) to determine the impact of circadian rhythm disruption on the intestinal microbiome and were fed either standard chow or a high-fat, high-sugar diet to determine how diet influences circadian disruption-induced effects on the microbiome. Weekly phase reversals of the light:dark (LD) cycle did not alter the microbiome in mice fed standard chow; however, mice fed a high-fat, high-sugar diet in conjunction with phase shifts in the light:dark cycle had significantly altered microbiota. While it is yet to be established if some of the adverse effects associated with circadian disorganization in humans (e.g., shift workers, travelers moving across time zones, and in individuals with social jet lag) are mediated by dysbiosis, the current study demonstrates that circadian disorganization can impact the intestinal microbiota which may have implications for inflammatory diseases.