Widespread microRNA degradation elements in target mRNAs can assist the encoded proteins

Binding of microRNAs (miRNAs) to mRNAs normally results in post-transcriptional repression of gene expression. However, extensive base-pairing between miRNAs and target RNAs can trigger miRNA degradation, a phenomenon called target RNA-directed miRNA degradation (TDMD). Here, we systematically analyzed Argonaute-CLASH (cross-linking, ligation, and sequencing of miRNA–target RNA hybrids) data and identified numerous candidate TDMD triggers, focusing on their ability to induce nontemplated nucleotide addition at the miRNA 3′ end. When exogenously expressed in various cell lines, eight triggers induce degradation of corresponding miRNAs. Both the TDMD base-pairing and surrounding sequences are essential for TDMD. CRISPR knockout of endogenous trigger or ZSWIM8, a ubiquitin ligase essential for TDMD, reduced miRNA degradation. Furthermore, degradation of miR-221 and miR-222 by a trigger in BCL2L11, which encodes a proapoptotic protein, enhances apoptosis. Therefore, we uncovered widespread TDMD triggers in target RNAs and demonstrated an example that could functionally cooperate with the encoded protein.

Mst2 Overexpression Inhibits Thyroid Carcinoma Growth and Metastasis by Disrupting Mitochondrial Fitness and Endoplasmic Reticulum Homeostasis

Although the incidence of thyroid carcinoma has increased over the past several decades, it has an excellent prognosis and overall 5-year survival, with a stable mortality rate, except in cases with advanced stages or rare malignant tumor types. Biomarkers have emerged as effective targets of molecular therapy against thyroid carcinoma due to their rapid and convenient detection; however, there has been little clinical application. Macrophage stimulating 2 (Mst2) is a proapoptotic protein with implications in carcinogenesis and metastasis. We found that Mst2 overexpression-induced endoplasmic reticulum (ER) stress in MDA-T32 thyroid carcinoma cells, accompanied by elevated caspase-12 activity, increased apoptotic rate, and reduced cell viability. In addition, Mst2 overexpression contributed to mitochondrial damage, as evidenced by increased mitochondrial oxidative stress and activated the mitochondrial apoptotic pathway. Inhibition of the JNK pathway abolished these effects. These results show Mst2 to be a novel tumor suppressor that induces mitochondrial dysfunction and ER stress via the JNK pathway. Thus, Mst2 could potentially serve as a biomarker for developing targeted therapy against thyroid carcinoma.

Downregulation of ATXN3 Enhances the Sensitivity to AKT Inhibitors (Perifosine or MK-2206), but Decreases the Sensitivity to Chemotherapeutic Drugs (Etoposide or Cisplatin) in Neuroblastoma Cells

BackgroundChemotherapy resistance is the major cause of failure in neuroblastoma (NB) treatment. ATXN3 has been linked to various types of cancer and neurodegenerative diseases; however, its roles in NB have not been established. The aim of our study was to explore the role of ATXN3 in the cell death induced by AKT inhibitor (perifosine or MK-2206) or chemotherapy drugs (etoposide or cisplatin) in NB cells.MethodsThe expressions of ATXN3 and BCL-2 family members were detected by Western blot. Cell survival was evaluated by CCK8, cell confluence was measured by IncuCyte, and apoptosis was detected by flow cytometry. AS and BE2 were treated with AKT inhibitors or chemotherapeutics, respectively.ResultsDownregulation of ATXN3 did not block, but significantly increased the perifosine/MK-2206-induced cell death. Among the BCL-2 family members, the expression of pro-apoptotic protein BIM and anti-proapoptotic protein Bcl-xl expression increased significantly when ATXN3 was down-regulated. Downregulation of BIM protected NB cells from the combination of perifosine/MK-2206 and ATXN3 downregulation. Downregulation of ATXN3 did not increase, but decrease the sensitivity of NB cells to etoposide/cisplatin, and knockdown of Bcl-xl attenuated this decrease in sensitivity.ConclusionDownregulation of ATXN3 enhanced AKT inhibitors (perifosine or MK-2206) induced cell death by BIM, but decreased the cell death induced by chemotherapeutic drugs (etoposide or cisplatin) via Bcl-xl. The expression of ATXN3 may be an indicator in selecting different treatment regimen.

Modern aspects of the etiology and pathogenesis of hyperplastic endometrial processes

The article presents modern approaches to the etiology, classification and pathogenesis of endometrial hyperplastic processes. Hyperplastic processes of the endometrium, representing an urgent medical and social problem, which is due to the peculiarities of the clinical manifestation of the disease (uterine bleeding), reduced reproductive potential, high recurrence rate, limited conservative treatment of this pathology in pre- and postmenopausal women, due to concomitant extragenital diseases and a high risk of malignancy.On the basis of new criteria of pathomorphological diagnostics, drawing parallels with the accumulated experience and clinical data, the immunohistochemical profile for each histotype of hyperplastic process of endometrium is created, certain diagnostic determinants are defined. Namely, simple and complex endometrial hyperplasia without atypia are characterized by imbalance between proliferation and apoptosis, manifested in moderate activation of the intratissue APUD system, lack of activation of proapoptotic protein p53, increased levels of antiapoptotic protein 2. Atypical forms of endometrial hyperplasia (simple and complex) are characterized by expression in the epithelial component of the proapoptotic protein p53, an increase in the level of antiapoptotic protein Bcl-2 in 2-2.5 times, expressed by activation of the intratissue APUD system.

Pannexin-1 Contributes to the Apoptosis of Spinal Neurocytes in Spinal Cord Injury

Currently, the role of Pannexin-1, a homomeric membrane hemichannel on the neuron cell membrane, in the development of spinal cord injury (SCI) is largely unknown. Herein, we assessed the contribution of Panx1 in the development of SCI. The SCI in vitro model was established using rat primary spinal neurocytes treated with hydrogen peroxide (H2O2). Effects of Panx1 overexpression or depletion in spinal neurocytes were analyzed by lentivirus-mediated transfection of Panx1 and interference sh-Panx1. Decreased cell viability was seen in SCI cells, which was further enhanced under Panx1 overexpression and mitigated by Panx1 deficiency. H2O2 induced an increase of intracellular Ca2+ signal and upregulated level of the proapoptotic protein Bax, and apoptosis pathway proteins including cleaved Caspase-3 and PARP1, which was enhanced by Panx1 overexpression or attenuated by Panx1 depletion. On the other hand, H2O2 treatment suppressed the level of antiapoptotic protein Bcl-2, which was further decreased by Panx1 overexpression or mitigated by Panx1 depletion. The results indicate that Panx1 was involved in the intracellular Ca2+ overload of SCI cells by accelerating extracellular Ca2+ influx, which promoted the apoptosis of spinal neurocytes through Ca2+ dependent pathways, thus aggravating the secondary injury of SCI.

PDCD4–MAPK–NF-κB Positive Loop Simultaneously Promotes Microglia Activation and Neuron Apoptosis During Neuroinflammation

Neuroinflammation and neuron injury are common features of the central nervous system (CNS) diseases. It is of great significance to identify their shared regulatory mechanisms and explore the potential therapeutic targets. Programmed cell death factor 4 (PDCD4), an apoptosis-related molecule, extensively participates in tumorigenesis and inflammatory diseases, but its expression and biological function during CNS neuroinflammation remain unclear. In the present study, utilizing the lipopolysaccharide (LPS)-induced neuroinflammation model in mice, we reported an elevated expression of PDCD4 both in injured neurons and activated microglia of the inflamed brain. A similar change in PDCD4 expression was observed in vitro in the microglial activation model. Silencing PDCD4 by shRNA significantly inhibited the phosphorylation of MAPKs (p38, ERK, and JNK), prevented the phosphorylation and nuclear translocation of NF-κB p65, and thus attenuated the LPS-induced microglial inflammatory activation. Interestingly, LPS also required the MAPK/NF-κB signaling activation to boost PDCD4 expression in microglia, indicating the presence of a positive loop. Moreover, a persistent elevation of PDCD4 expression was detected in the H2O2-induced neuronal oxidative damage model. Knocking down PDCD4 significantly inhibited the expression of proapoptotic protein BAX, suggesting the proapoptotic activity of PDCD4 in neurons. Taken together, our data indicated that PDCD4 may serve as a hub regulatory molecule that simultaneously promotes the microglial inflammatory activation and the oxidative stress-induced neuronal apoptosis within CNS. The microglial PDCD4–MAPK–NF-κB positive feedback loop may exaggerate the vicious cycle of neuroinflammation and neuronal injury and thus may become a potential therapeutic target for neuroinflammatory diseases.

TPEN Selectively Eliminates Lymphoblastic B Cells from Pediatric Acute Lymphoblastic Lleukemia Patients

B-acute lymphoblastic leukemia (B-ALL) is a hematologic disorder characterized by abnormal proliferation and accumulation of immature B-lymphoblast arrested at various differentiation stages. Despite some advances in treatment, there is still an important percentage of pediatric patients with precursor-B ALL who relapsed. Therefore, alternative therapies are needed to improve cure rates for pediatric patients. TPEN is a pro-oxidant agent capable of selectively inducing apoptosis in leukemia cells. Consequently, TPEN has been suggested as a potential agent for oxidative therapy. However, it is not yet known whether TPEN can selectively destroy leukemia cells in a more disease-like milieu e.g., bloodstream and bone marrow (BM) in vivo. In this investigation, we report for the first time that TPEN significantly induces apoptosis in CD34+/CD19+ cells from whole bone marrow de novo B-ALL (n=5) and refractory B-ALL (n=6) patients by oxidative stress (OS, n=8). We found that TPEN significantly increased not only positive cell counts for the oxidation of the stress sensor protein DJ-1 as a sign of the formation of H2O2, but also significantly increased positive cell counts for the proapoptotic protein TP53, PUMA, and CASPASE-3 as indicative of apoptosis in B-ALL cells irrespective of diagnostic status (de novo or refractory) and sex. Understanding the TPEN-induced cell death in leukemia cells provides insight into more effective therapeutic prooxidant-inducing anticancer agents.

Cullin-5 neddylation-mediated NOXA degradation is enhanced by PRDX1 oligomers in colorectal cancer

NOXA, a BH3-only proapoptotic protein involved in regulating cell death decisions, is highly expressed but short-lived in colorectal cancer (CRC). Neddylated cullin-5 (CUL5)-mediated ubiquitination and degradation of NOXA is crucial to prevent its overaccumulation and maintain an appropriate action time. However, how this process is manipulated by CRC cells commonly exposed to oxidative stress remain unknown. The peroxiredoxin PRDX1, a conceivable antioxidant overexpressed in CRC tissues, has been shown to inhibit apoptosis and TRAF6 ubiquitin-ligase activity. In this study, we found that PRDX1 inhibits CRC cell apoptosis by downregulating NOXA. Mechanistically, PRDX1 promotes NOXA ubiquitination and degradation, which completely depend on CUL5 neddylation. Further studies have demonstrated that PRDX1 oligomers bind with both the Nedd8-conjugating enzyme UBE2F and CUL5 and that this tricomplex is critical for CUL5 neddylation, since silencing PRDX1 or inhibiting PRDX1 oligomerization greatly dampens CUL5 neddylation and NOXA degradation. An increase in reactive oxygen species (ROS) is not only a hallmark of cancer cells but also the leading driving force for PRDX1 oligomerization. As shown in our study, although ROS play a role in upregulating NOXA mRNA transcription, ROS scavenging in CRC cells by N-acetyl-L-cysteine (NAC) can significantly reduce CUL5 neddylation and extend the NOXA protein half-life. Therefore, in CRC, PRDX1 plays a key role in maintaining intracellular homeostasis under conditions of high metabolic activity by reinforcing UBE2F-CUL5-mediated degradation of NOXA, which is also evidenced in the resistance of CRC cells to etoposide treatment. Based on these findings, targeting PRDX1 could be an effective strategy to overcome the resistance of CRC to DNA damage-inducing chemotherapeutics.

Early Lipid Raft-Related Changes: Interplay between Unilateral Denervation and Hindlimb Suspension

Muscle disuse and denervation leads to muscle atrophy, but underlying mechanisms can be different. Previously, we have found ceramide (Cer) accumulation and lipid raft disruption after acute hindlimb suspension (HS), a model of muscle disuse. Herein, using biochemical and fluorescent approaches the influence of unilateral denervation itself and in combination with short-term HS on membrane-related parameters of rat soleus muscle was studied. Denervation increased immunoexpression of sphingomyelinase and Cer in plasmalemmal regions, but decreased Cer content in the raft fraction and enhanced lipid raft integrity. Preliminary denervation suppressed (1) HS-induced Cer accumulation in plasmalemmal regions, shown for both nonraft and raft-fractions; (2) HS-mediated decrease in lipid raft integrity. Similar to denervation, inhibition of the sciatic nerve afferents with capsaicin itself increased Cer plasmalemmal immunoexpression, but attenuated the membrane-related effects of HS. Finally, both denervation and capsaicin treatment increased immunoexpression of proapoptotic protein Bax and inhibited HS-driven increase in antiapoptotic protein Bcl-2. Thus, denervation can increase lipid raft formation and attenuate HS-induced alterations probably due to decrease of Cer levels in the raft fraction. The effects of denervation could be at least partially caused by the loss of afferentation. The study points to the importance of motor and afferent inputs in control of Cer distribution and thereby stability of lipid rafts in the junctional and extrajunctional membranes of the muscle.