Tuberculosis is one of the most important infectious diseases worldwide and macrophage apoptosis is the major host defense mechanism against TB. We attempted to characterize the role of miRNA ...(miR)-125b-5p on mycobacterium tuberculosis (Mtb) infection and macrophages behaviors in vitro. According to fluorescence-activated cell separation (FACS), primary monocytes (CD14
+
) in TB patients were accumulated, and apoptotic monocytes were decreased. Peripheral blood mononuclear cells (PBMCs)-derived macrophages (MDMs) and monocytic cells THP-1-derived macrophage-like cells (TDMs) in vitro were used to be infected with H37Rv. After infection, colony-forming units assay revealed the increase of bacterial activity, FACS demonstrated the decrease of apoptosis rate of MDMs and TDMs, as well as promoted levels of IL-6, TNF-α, Bax, and Bim and suppressed levels of IL-10 and Bcl-2, examined by enzyme-linked immunosorbent assay (ELISA) and western blot assay. Expression of miR-125b-5p and DNA damage-regulated autophagy modulator 2 (DRAM2) was examined, and real-time PCR and western blot assay showed that miR-125b-5p was upregulated, whereas DRAM2 was downregulated in primary monocytes and H37Rv-infected macrophages (MDMs and TDMs). Moreover, blocking miR-125b-5p could attenuated H37Rv-induced bacterial activity and inflammatory response of MDMs and TDMs, accompanied with apoptosis inhibition. Whereas these effects of miR-125b-5p knockdown were abolished by downregulating DRAM2. In mechanism, DRAM2 was a downstream target of miR-125b-5p, as evidenced by dual-luciferase reporter assay. Collectively, silencing miR-125b-5p could protect human macrophages against Mtb infection through promoting apoptosis and inhibiting inflammatory response via targeting DRAM2, suggesting a novel target for Mtb eliminating.
Abbreviations: TB: tuberculosis; PBMCs: peripheral blood mononuclear cells; Mtb: mycobacterium tuberculosis; AFB: acid fast bacilli; FITC: fluorescein isothiocyanate; MDMs: monocytes-derived macrophages; TDMs: THP-1-derived macrophage-like cells; ERFP: Mtb-enhanced red fluorescent protein; CFU: colony-forming units; ELISA: enzyme-linked immunosorbent assay; FACS: fluorescence-activated cell separation; PI: propidium iodide; DRAM2: DNA damage-regulated autophagy modulator 2; Real-time PCR: real-time polymerase chain reaction; in-miR-125b-5p: miR-125b-5p inhibitor; si-DRAM2: siRNA against DRAM2
Autophagy is an important antimicrobial effector process that defends against Mycobacterium tuberculosis (Mtb), the human pathogen causing tuberculosis (TB). MicroRNAs (miRNAs), endogenous noncoding ...RNAs, are involved in various biological functions and act as post-transcriptional regulators to target mRNAs. The process by which miRNAs affect antibacterial autophagy and host defense mechanisms against Mtb infections in human monocytes and macrophages is largely uncharacterized. In this study, we show that Mtb significantly induces the expression of MIR144*/hsa-miR-144-5p, which targets the 3′-untranslated region of DRAM2 (DNA damage regulated autophagy modulator 2) in human monocytes and macrophages. Mtb infection downregulated, whereas the autophagy activators upregulated, DRAM2 expression in human monocytes and macrophages by activating AMP-activated protein kinase. In addition, overexpression of MIR144* decreased DRAM2 expression and formation of autophagosomes in human monocytes, whereas inhibition of MIR144* had the opposite effect. Moreover, the levels of MIR144* were elevated, whereas DRAM2 levels were reduced, in human peripheral blood cells and tissues in TB patients, indicating the clinical significance of MIR144* and DRAM2 in human TB. Notably, DRAM2 interacted with BECN1 and UVRAG, essential components of the autophagic machinery, leading to displacement of RUBCN from the BECN1 complex and enhancement of Ptdlns3K activity. Furthermore, MIR144* and DRAM2 were critically involved in phagosomal maturation and enhanced antimicrobial effects against Mtb. Our findings identify a previously unrecognized role of human MIR144* in the inhibition of antibacterial autophagy and the innate host immune response to Mtb. Additionally, these data reveal that DRAM2 is a key coordinator of autophagy activation that enhances antimicrobial activity against Mtb.
Ginsenoside Rb2, a major active component of Panax ginseng, has various physiological activities, including anticancer and anti-inflammatory effects. However, the mechanisms underlying the ...rejuvenation effect of Rb2 in human skin cells have not been elucidated.
We performed a senescence-associated β-galactosidase staining assay to confirm cellular senescence in human dermal fibroblasts (HDFs). The regulatory effects of Rb2 on autophagy were evaluated by analyzing the expression of autophagy marker proteins, such as microtubule-associated protein 1A/1B-light chain (LC) 3 and p62, using immunoblotting. Autophagosome and autolysosome formation was monitored using transmission electron microscopy. Autophagic flux was analyzed using tandem-labeled GFP-RFP-LC3, and lysosomal function was assessed with Lysotracker. We performed RNA sequencing to identify potential target genes related to HDF rejuvenation mediated by Rb2. To verify the functions of the target genes, we silenced them using shRNAs.
Rb2 decreased β-galactosidase activity and altered the expression of cell cycle regulatory proteins in senescent HDFs. Rb2 markedly induced the conversion of LC3-Ⅰ to LC3-Ⅱ and LC3 puncta. Moreover, Rb2 increased lysosomal function and red puncta in tandem-labeled GFP-RFP-LC3, which indicate that Rb2 promoted autophagic flux. RNA sequencing data showed that the expression of DNA damage-regulated autophagy modulator 2 (DRAM2) was induced by Rb2. In autophagy signaling, Rb2 activated the AMPK-ULK1 pathway and inactivated mTOR. DRAM2 knockdown inhibited autophagy and Rb2-restored cellular senescence.
Rb2 reverses cellular senescence by activating autophagy via the AMPK-mTOR pathway and induction of DRAM2, suggesting that Rb2 might have potential value as an antiaging agent.
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Autophagy is a critical cellular homeostatic process that controls the turnover of damaged organelles and proteins. Impaired autophagic activity is involved in a number of diseases, including ...idiopathic pulmonary fibrosis suggesting that altered autophagy may contribute to fibrogenesis. However, the specific role of autophagy in lung fibrosis is still undefined. In this study, we show for the first time, how autophagy disruption contributes to bleomycin-induced lung fibrosis in vivo using an Atg4b-deficient mouse as a model. Atg4b-deficient mice displayed a significantly higher inflammatory response at 7 d after bleomycin treatment associated with increased neutrophilic infiltration and significant alterations in proinflammatory cytokines. Likewise, we found that Atg4b disruption resulted in augmented apoptosis affecting predominantly alveolar and bronchiolar epithelial cells. At 28 d post-bleomycin instillation Atg4b-deficient mice exhibited more extensive and severe fibrosis with increased collagen accumulation and deregulated extracellular matrix-related gene expression. Together, our findings indicate that the ATG4B protease and autophagy play a crucial role protecting epithelial cells against bleomycin-induced stress and apoptosis, and in the regulation of the inflammatory and fibrotic responses.
Pathogenic variants in DNA-damage regulated autophagy modulator 2 gene (DRAM2) cause a rare autosomal recessive retinal dystrophy and its disease course is not well understood. We present two ...Slovenian patients harboring a novel DRAM2 variant and a detailed review of all 23 other patients described to date. Whole exome and whole genome sequencing were performed in the two patients, and both underwent ophthalmological examination with a 2-year follow-up. PubMed was searched for papers with clinical descriptions of DRAM2 retinopathy. Patient 1 was homozygous for a novel variant, p.Met1?, and presented with the acute onset of photopsia and retina-wide retinopathy at the age of 35 years. The patient was first thought to have an autoimmune retinopathy and was treated with mycophenolate mofetil, which provided some symptomatic relief. Patient 2 was compound heterozygous for p.Met1? and p.Leu246Pro and presented with late-onset maculopathy at the age of 59 years. On review, patients with DRAM2 retinopathy usually present in the third decade with central visual loss, outer retinal layer loss on optical coherence tomography and a hyperautofluorescent ring on fundus autofluorescence. Either cone–rod or rod–cone dystrophy phenotype is observed on electroretinography, reflecting the importance of DRAM2 in both photoreceptor types. Non-null variants can result in milder disease.
-associated retinopathy is a rare inherited retinal dystrophy, and its outcome has not been determined. A single retinal involvement by a mutation of the
gene is unexplained. We found three unrelated ...patients with a disease-causing
variant in a biallelic state from 1555 Japanese individuals of 1314 families with inherited retinal dystrophy. We reviewed their medical records and examined their peripheral lymphocytes by transmission electron microscopy (TEM).
was a 38-year-old woman who complained of night blindness and reduced vision. She developed macular degeneration at age 43 years.
and
were a man and a woman both of whom noticed night blindness in their 30s. Both had a degeneration in the macula and midperiphery in their 40s, which progressed to a diffuse retinal degeneration in their 60s when their vision was reduced to hand motions. Three novel
variants were identified. TEM of the lymphocytes of
and
showed abnormal structures in 40.6% and 0.3% of the peripheral lymphocytes, respectively. We concluded that the
-associated retinopathy of our patients was a progressive rod-cone dystrophy, and the visual outcome was poor. The systemic effect of
mutations may be compensable and have variations.
Damage-regulated autophagy modulator 2(DRAM2) is associated with autophagy processes. However, the role of DRAM2 in the progression of human neoplasms is still unknown. Here, we show that DRAM2 may ...act as an oncogenic regulator in non-small cell lung cancer (NSCLC).
Tumor specimens from 259 NSCLC patients were collected and analyzed. Transwell migration, cell cycle analysis, MTT and colony formation assays were performed to determine the effect of DRAM2 overexpression and knockdown on NSCLC-cell migration and proliferation. Western blotting confirmed the expression of DRAM2, p53, and the other involved proteins.
DRAM2 was preferentially upregulated in NSCLC tissues and higher expression of DRAM2 in NSCLC correlated with tumor node metastases stage and lymph node metastasis. Additionally, DRAM2 overexpression promoted cell metastasis and proliferation in vitro, while knockdown of DRAM2 expression yielded opposite result. Furthermore, DRAM2 overexpression increased the expression of proteins RAC1, RHOA, RHOC, ROCK1, and decreased RHOB expression, all of which are cell migration factors. DRAM2 overexpression also increased proteins CDK4, CyclinD3, and decreased p27 expression, all of which are cell cycle-related factors. Consistently knocked down DRAM2 had the opposite effect. We also found that DRAM2 expression was negatively correlated to p53 expression. Knockdown of DRAM2 caused an increase of p53 and p21 expression, and overexpression of p53 caused a decrease of DRAM2 expression. Finally, absence of p53 did not influence the function of DRAM2 in NSCLC, but overexpression of p53 repressed its function.
DRAM2 plays an oncogenic role in NSCLC via regulating p53 expression. Therefore, DRAM2 may act as an oncogene in NSCLC and could serve as a prognostic factor and potential target for NSCLC treatment.
Bladder transitional cell carcinoma (BTCC) is one of the most prevalent human malignant diseases. Gemcitabine is commonly applied in the treatment of BTCC while acquired gemcitabine resistance has ...caused a severe impediment to recovery. This study aimed to investigate the function of
in regulating gemcitabine resistance of BTCC.
GSE77883 was introduced to screen out the differentially expressed autophagy-related genes in T24 cells and gemcitabine-resistant T24-GEM cells. After establishing T24-GEM cells ourselves, aberrant expression of
was detected by qRT-PCR and Western blot. After stably manipulating the expression of
in T24 and T24-GEM cells, the changes of cell biological functions under gemcitabine treatment were compared, including cell viability, apoptosis and autophagy, using colony formation, flow cytometry and electron microscopy respectively.
was up-regulated in gemcitabine-resistant T24-GEM cells. Silencing of
in T24-GEM cells inhibited the cell autophagy induced by treatment with gemcitabine and contributed to attenuated gemcitabine resistance. Also, overexpression of
in T24 cells enhanced the autophagy, strengthened the chemoresistance and decreased the cell apoptosis rate under the treatment with gemcitabine.
Our data suggested that downregulation of
rescued the sensitivity of T24-GEM cells to gemcitabine, providing an appropriate therapeutic target for BTCC treatment.
Rare
DRAM2
coding variants cause retinal dystrophy with early macular involvement via unknown mechanisms. We found that
DRAM2
is ubiquitously expressed in the human eye and expression changes were ...observed in eyes with more common maculopathy such as Age-related Macular Degeneration (AMD). To gain insights into pathogenicity of DRAM2-related retinopathy, we used a combination of
in vitro
and
in vivo
models. We found that
DRAM2
loss in human pluripotent stem cell (hPSC)-derived retinal organoids caused the presence of additional mesenchymal cells. Interestingly,
Dram2
loss in mice also caused increased proliferation of cells from the choroid
in vitro
and exacerbated choroidal neovascular lesions
in vivo
. Furthermore, we observed that
DRAM2
loss in human retinal pigment epithelial (RPE) cells resulted in increased susceptibility to stress-induced cell death
in vitro
and that
Dram2
loss in mice caused age-related photoreceptor degeneration. This highlights the complexity of DRAM2 function, as its loss in choroidal cells provided a proliferative advantage, whereas its loss in post-mitotic cells, such as photoreceptor and RPE cells, increased degeneration susceptibility. Different models such as human pluripotent stem cell-derived systems and mice can be leveraged to study and model human retinal dystrophies; however, cell type and species-specific expression must be taken into account when selecting relevant systems.
Although Hodgkin lymphoma (HL) is curable with current therapy, at least 20% of patients relapse or fail to make complete remission. In addition, patients who achieve long-term disease-free survival ...frequently undergo infertility, secondary malignancies, and cardiac failure, which are related to chemotherapeutic agents and radiation therapies. Hence, new therapeutic strategies able to counteract the HL disease in this important patient population are still a matter of study. Estrogens, in particular 17β-estradiol (E2), have been suggested to play a role in lymphoma cell homeostasis by estrogen receptors (ER) β activation. On these bases, we investigated whether the ligation of ERβ by a selective agonist, the 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN), could impact HL tumor growth. We found that DPN-mediated ERβ activation led to a reduction of in vitro cell proliferation and cell cycle progression by inducing autophagy. In nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice engrafted with HL cells, ERβ activation by DPN was able to reduce lymphoma growth up to 60% and this associated with the induction of tumor cell autophagy. Molecular characterization of ERβ-induced autophagy revealed an overexpression of damage-regulated autophagy modulator 2 (DRAM2) molecule, whose role in autophagy modulation is still debated. After ERβ activation, both DRAM2 and protein 1 light chain 3 (LC3), a key actor in the autophagosome formation, strictly interacted each other and localized at mitochondrial level.Altogether these results suggest that targeting ERβ with selective agonists might affect HL cell proliferation and tumor growth via a mechanism that brings into play DRAM2-dependent autophagic cascade.
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