HAND2-AS1 Promotes Ferroptosis to Reverse Lenvatinib Resistance in Hepatocellular Carcinoma by TLR4/NOX2/DUOX2 Axis.

INTRODUCTION: Lenvatinib resistance causes less than 40% of the objective response rate. Therefore, it is urgent to explore new therapeutic targets to reverse the lenvatinib resistance for HCC. HAND2-AS1 is a critical tumor suppressor gene in various cancers. METHODS: Here, we investigated the role of HAND2-AS1 in the molecular mechanism of lenvatinib resistance in HCC. It was found that HAND2-AS1 was lowly expressed in the HepG2 lenvatinib resistance (HepG2-LR) cells and HCC tissues and associated with progression-free intervals via TCGA. Overexpression of HAND2-AS1 (OE-HAND2-AS1) decreased the IC50 of lenvatinib in HepG2-LR cells to reverse lenvatinib resistance. Moreover, OE-HAND2-AS1 induced intracellular concentrations of malondialdehyde (MDA) and lipid ROS and decreased the ratio of glutathione to glutathione disulfide (GSH/GSSG) to promote ferroptosis. RESULTS: A xenograft model in which nude mice were injected with OE-HAND2-AS1 HepG2-LR cells confirmed that OE-HAND2-AS1 could reverse lenvatinib resistance and decrease tumor formation in vivo. HAND2-AS1 promoted the expression of ferroptosis-related genes (TLR4, NOX2, and DUOX2) and promoted ferroptosis to reverse lenvatinib resistance by increasing TLR4/ NOX2/DUOX2 via competing endogenous miR-219a-1-3p in HCC cells. Besides, patients with a low HAND2-AS1 level had early recurrence after resection. CONCLUSION: These findings suggested that HAND2-AS1 may be a potential therapeutic target and an indicator of early recurrence for HCC.

The utility of P-I-R classification in predicting the on-treatment histological and clinical outcomes of patients with hepatitis B and advanced liver fibrosis.

BACKGROUND AND AIMS: The predominantly progressive, indeterminate, and predominantly regressive (P-I-R) classification extends beyond staging and provides information on dynamic changes of liver fibrosis. However, the prognostic implication of P-I-R classification is not elucidated. Therefore, in the present research, we investigated the utility of P-I-R classification in predicting the on-treatment clinical outcomes. APPROACH AND RESULTS: In an extension study on a randomized controlled trial, we originally enrolled 1000 patients with chronic hepatitis B and biopsy-proven histological significant fibrosis, and treated them for more than 7 years with entecavir-based therapy. Among the 727 patients with a second biopsy at treatment week 72, we compared P-I-R classification and Ishak score changes in 646 patients with adequate liver sections for the histological evaluation. Progressive, indeterminate, and regressive cases were observed in 70%, 17%, and 13% of patients before treatments and 20%, 14%, and 64% after 72-week treatment, respectively, which could further differentiate the histological outcomes of patients with stable Ishak scores. The 7-year cumulative incidence of HCC was 1.5% for the regressive cases, 4.3% for the indeterminate cases, and 22.8% for the progressive cases ( p <0.001). After adjusting for age, treatment regimen, platelet counts, cirrhosis, Ishak fibrosis score changes, and Laennec staging, the posttreatment progressive had a HR of 17.77 (vs. posttreatment regressive; 95% CI: 5.55-56.88) for the incidence of liver-related events (decompensation, HCC, and death/liver transplantation). CONCLUSIONS: The P-I-R classification can be a meaningful complement to the Ishak fibrosis score not only in evaluating the histological changes but also in predicting the clinical outcomes.

Cancer-associated fibroblasts suppressed ferroptosis in glioblastoma via upregulating lncRNA DLEU1.

Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults. It is crucial to elucidate the mechanism underlying ferroptosis resistance in GBM. We used qRT-PCR to detect the level of DLEU1 and mRNAs of indicated genes, whereas protein levels were determined by Western blots. Fluorescence in situ hybridization assay (FISH) was applied to validate the sublocation of DLEU1 in GBM cells. Gene knockdown or overexpression was achieved by transient transfection. Ferroptosis markers were detected by indicated kits and transmission electron microscopy (TEM). RNA pull-down, RNA immunoprecipitation (RIP), chromatin immunoprecipitation (CHIP)-qPCR, and dual-luciferase assay were used to validate the direct interaction between indicated key molecules in the current study. We validated that the expression of DLEU1 was upregulated in GBM samples. DLEU1 knockdown exacerbated erastin-induced ferroptosis in LN229 and U251MG cells, as well as in the xenograft model. Mechanistically, we found that DLEU1 bound with ZFP36 and facilitated ZFP36 to degrade ATF3 mRNA, thus upregulating the expression of SLC7A11 to attenuate erastin-induced ferroptosis. Importantly, our results confirmed that cancer-associated fibroblasts (CAFs) conferred ferroptosis resistance in GBM. The stimulation of CAF-conditioned medium enhanced the activation of HSF1, and HSF1 transcriptionally increased the level of DLEU1 to regulate erastin-induced ferroptosis. This study identified DLEU1 as an oncogenic lncRNA that epigenetically downregulates ATF3 expression via binding with ZFP36 to facilitate ferroptosis resistance in GBM. The upregulation of DLEU1 in GBM might be attributed to CAF-induced HSF1 activation. Our study might provide a research basis for understanding CAF-induced ferroptosis resistance in GBM.

PGE2 pathway mediates oxidative stress-induced ferroptosis in renal tubular epithelial cells.

Prostaglandin E2 (PGE2) is one of the most abundant prostaglandins and has been implicated in various diseases. Here, we aimed to explore the role of the PGE2 pathway in mediating ferroptosis during acute kidney injury. When renal tubular epithelial cells stimulated by H2 O2 , the contents of glutathione (GSH) and glutathione peroxidase 4 (GPX4) decreased, whereas the level of lipid peroxide increased. Ferrostatin-1 can effectively attenuate these changes. In this process, the expression levels of cyclooxygenase (COX)-1 and COX-2 were up-regulated. Meanwhile, the expression of microsomal prostaglandin E synthase-2 was elevated, whereas the expression of microsomal prostaglandin E synthase-1 and cytosolic prostaglandin E synthase were down-regulated. Furthermore, the expression of 15-hydroxyprostaglandin dehydrogenase decreased. An excessive accumulation of PGE2 promoted ferroptosis, whereas the PGE2 inhibitor pranoprofen minimized the changes for COX-2, GSH, GPX4 and lipid peroxides. A decrease in the levels of the PGE2 receptor E-series of prostaglandin 1/3 partially restored the decline of GSH and GPX4 levels and inhibited the aggravation of lipid peroxide. Consistent with the in vitro results, increased PGE2 levels led to increased levels of 3,4-methylenedioxyamphetamine, Fe2+ accumulation and decreased GSH and GPX4 levels during renal ischaemia/reperfusion injury injury in mice. Our results indicate that the PGE2 pathway mediated oxidative stress-induced ferroptosis in renal tubular epithelial cells.

CircLRFN5 inhibits the progression of glioblastoma via PRRX2/GCH1 mediated ferroptosis.

BACKGROUND: Ferroptosis is a novel form of iron-dependent cell death and participates in the malignant progression of glioblastoma (GBM). Although circular RNAs (circRNAs) are found to play key roles in ferroptosis via several mechanisms, including regulating iron metabolism, glutathione metabolism, lipid peroxidation and mitochondrial-related proteins, there are many novel circRNAs regulating ferroptosis need to be found, and they may become a new molecular treatment target in GBM. METHODS: The expression levels of circLRFN5, PRRX2 and GCH1 were detected by qPCR, western blotting, and immunohistochemistry. Lentiviral-based infections were used to overexpress or knockdown these molecules in glioma stem cells (GSCs). The biological functions of these molecules on GSCs were detected by MTS (3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium), the 5-ethynyl-20-deoxyuridine (EdU) incorporation assay, transwell, neurosphere formation assays, Extreme Limiting Dilution Analysis (ELDA) and xenograft experiments. The content of ferroptosis levels in GSCs was detected by BODIPY 581/591 C11 assay, glutathione (GSH) assay and malondialdehyde (MDA) assay. The regulating mechanisms among these molecules were studied by RNA immunoprecipitation assay, RNA pull-down assay, ubiquitination assay, dual-luciferase reporter assay and chromatin immunoprecipitation assay. RESULTS: We found a novel circRNA circLRFN5 is downregulated in GBM and associated with GBM patients' poor prognosis. CircLRFN5 overexpression inhibits the cell viabilities, proliferation, neurospheres formation, stemness and tumorigenesis of GSCs via inducing ferroptosis. Mechanistically, circLRFN5 binds to PRRX2 protein and promotes its degradation via a ubiquitin-mediated proteasomal pathway. PRRX2 can transcriptionally upregulate GCH1 expression in GSCs, which is a ferroptosis suppressor via generating the antioxidant tetrahydrobiopterin (BH4). CONCLUSIONS: Our study found circLRFN5 as a tumor-suppressive circRNA and identified its role in the progression of ferroptosis and GBM. CircLRFN5 can be used as a potential GBM biomarker and become a target for molecular therapies or ferroptosis-dependent therapy in GBM.

COX-2/PGE2 Pathway Inhibits the Ferroptosis Induced by Cerebral Ischemia Reperfusion.

Cerebral ischemia reperfusion (I/R) injury easily develops in ischemic stroke, resulting in more serious injury. Ferroptosis is involved in cerebral I/R injury, but the mechanism remains unclear. Prostaglandin E2 (PGE2) is potential to regulate ferroptosis. This study mainly explored the regulation effects of PGE2 on ferroptosis induced by cerebral I/R. We first detected PGE2 levels and ferroptosis status in 11 human brain tissues. Then, we induced a cerebral I/R animal model to examine ferroptosis status in cerebral I/R. We further injected a ferroptosis inhibitor to define the response of the PGE2 pathway to ferroptosis. Finally, we injected PGE2 and pranoprofen to explore the regulation of the cyclooxygenases 2 (COX-2)/PGE2 pathway on ferroptosis in cerebral I/R. We found that PGE2 release was correlated with the levels of reactive oxygen species, malondialdehyde, glutathione peroxidase 4, COX-2, and Spermidine/spermine N1-acetyltransferase 1. Ferroptosis can be induced by cerebral I/R, while inhibition of ferroptosis induced by cerebral I/R can inactivate PGE2 synthases, degrade enzyme, and parts of PGE2 receptors, and reduce cerebral infarct volume. In turn, PGE2 inhibited ferroptosis through the reduction of Fe2+, glutathione oxidation, and lipid peroxidation, while pranoprofen, one of the COX inhibitors, played an opposite role. In conclusion, PGE2 was positively correlated with ferroptosis, inhibition of ferroptosis induced by cerebral I/R can inactivate COX-2/PGE2 pathway, and PGE2 inhibited ferroptosis induced by cerebral I/R, possibly via PGE2 receptor 3 and PGE2 receptor 4. Graphical abstract Inhibition of ferroptosis inactivates the COX-2/PGE2 pathway. Cerebral ischemia reperfusion injury induces the secretion of PGE2. After the inhibition of ferroptosis by Fer-1, the expression of cyclooxygenases (COX-1 and COX-2) decreased, and PGE2 synthases cPGES, mPGES-1, and mPGES-2 were also reduced. At the same time, the PGE2 degradation enzyme 15-PGDH was also reduced. Changes in these enzymes ultimately result in the declination of PGE2. Besides, the expression of PGE2 receptors EP3 and EP4 is also inhibited, indicating that the function they mediate is also impaired. In conclusion, after cerebral ischemia reperfusion injury, the inhibition of ferroptosis inactivates the COX-2/PGE2 pathway.

Regulation of PGE2 Pathway During Cerebral Ischemia Reperfusion Injury in Rat.

Stroke is an acute central nervous system disease with high morbidity and mortality rate. Cerebral ischemia reperfusion (I/R) injury is easily induced during the development or treatment of stroke and subsequently leads to more serious brain damage. Prostaglandin E2 (PGE2) is one of the most important inflammatory mediators in the brain and contributes to both physiological and pathophysiological functions. It may be upregulated and subsequently plays a key role in cerebral ischemia reperfusion injury. The synthesis and degradation of PGE2 is an extremely complex process, with multiple key stages and molecules. However, there are few comprehensive and systematic studies conducted to investigate the synthesis and degradation of PGE2 during cerebral I/R injury, which is what we want to demonstrate. In this study, qRT-PCR and immunoblotting demonstrated that the key enzymes in PGE2 synthesis, including COX-1, COX-2, mPGES-1 and mPGES-2, were upregulated during cerebral I/R injury, but 15-PGDH, the main PGE2 degradation enzyme, was downregulated. In addition, two of PGE2 receptors, EP3 and EP4, were also increased. Meanwhile, immunohistochemistry demonstrated the localization of these molecules in ischemic areas, including cortex, striatum and hippocampus, and reflected their expression patterns in different regions. Combining the results of PCR, Western blotting and immunohistochemistry, we can determine where the increase or decrease of these molecules occurs. Overall, these results further indicate a possible pathway that mediates enhanced production of PGE2, and thus that may impact production of inflammatory cytokines including IL-1ß and TNF-α during cerebral I/R injury.

Activation of the KDM5A/miRNA-495/YTHDF2/m6A-MOB3B axis facilitates prostate cancer progression.

BACKGROUND: Accumulating evidence supports that lysine-specific demethylase 5 (KDM5) family members act as oncogenic drivers. This study was performed to elucidate the potential effects of KDM5A on prostate cancer (PCa) progression via the miR-495/YTHDF2/m6A-MOB3B axis. METHODS: The expression of KDM5A, miR-495, YTHDF2 and MOB3B was validated in human PCa tissues and cell lines. Ectopic expression and knockdown experiments were developed in PCa cells to evaluate their effects on PCa cell proliferation, migration, invasion and apoptosis. Mechanistic insights into the interaction among KDM5A, miR-495, YTHDF2 and MOB3B were obtained after dual luciferase reporter, ChIP, and PAR-CLIP assays. Me-RIP assay was used to determine m6A modification level of MOB3B mRNA in PCa cells. Mouse xenograft models of PCa cells were also established to monitor the tumor growth. RESULTS: KDM5A was highly expressed in human PCa tissues and cell lines. Upregulated KDM5A stimulated PCa cell proliferation, migration and invasion, but reduced cell apoptosis. Mechanistically, KDM5A, as a H3K4me3 demethylase, bound to the miR-495 promoter, which led to inhibition of its transcription and expression. As a target of miR-495, YTHDF2 could inhibit MOB3B expression by recognizing m6A modification of MOB3B mRNA and inducing mRNA degradation. Furthermore, KDM5A was found to downregulate MOB3B expression, consequently augmenting PCa cell proliferation, migration and invasion in vitro and promoting tumor growth in vivo via the miR-495/YTHDF2 axis. CONCLUSION: In summary, our study highlights the potential of histone demethylase KDM5A activity in enhancing PCa progression, and suggests KDM5A as a promising target for PCa treatment.

Pharmacologic Blockade of 15-PGDH Protects Against Acute Renal Injury Induced by LPS in Mice.

Prostaglandin pathway plays multiple roles in various physiological and pathological conditions. The present study aimed to investigate the effect of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a key enzyme in the degradation of prostaglandins, on lipopolysaccharide (LPS)-induced acute kidney injury (AKI) in mice. In this study, male C57BL/6J mice were injected intraperitoneally with LPS (10 mg/kg). SW033291, a potent small-molecule inhibitor of 15-PGDH, was used to investigate the therapeutic potential of 15-PGDH inhibition on LPS-induced AKI. We discovered that the expression of 15-PGDH protein was upregulated in kidneys of LPS-stimulated mice, and it was mainly localized in the cytoplasm of renal tubular epithelial cells in renal cortex and outer medulla. SW033291 administration improved the survival rates of mice and attenuated renal injury of mice that were challenged by LPS. Additionally, inhibition of 15-PGDH also reversed LPS-induced apoptosis of renal cells, increased expression of anti-apoptotic protein Bcl-2, and downregulated expression of Fas, caspase-3, and caspase-8. Pretreatment of SW033291 enhanced autophagy in kidney cells after LPS stimulation. Our data also showed that inhibition of 15-PGDH relieved the level of lipid peroxidation and downregulated NADPH oxidase subunits induced by LPS in mice kidneys but had no significant effect on the release of inflammatory factors, such as IL-6, IL-1ß, TNF-α, and MCP-1. Our study demonstrated that inhibition of 15-PGDH could alleviate LPS-induced AKI by regulating the apoptosis, autophagy, and oxidative stress rather than inflammation in mice.

Exploring receptor selectivity of the chimeric relaxin family peptide R3/I5 by incorporating unnatural amino acids.

Relaxin family peptides perform a variety of biological functions by activating four G protein-coupled receptors, namely RXFP1-4. Our recent study demonstrated that selectivity of the chimeric relaxin family peptide R3/I5 towards the homologous RXFP3 and RXFP4 can be modulated by replacement of the highly conserved nonchiral B23Gly or B24Gly with some natural l-amino acids. To investigate the mechanism of this modulating effect, in the present study we incorporated unnatural amino acids into the B23 or B24 position of a semi-synthetic R3/I5 that was prepared by a novel sortase-catalysed ligation approach using synthetic relaxin-3 B-chain and recombinant INSL5 A-chain. R3/I5 was a weak agonist for RXFP3 after B23Gly was replaced by D-Ala or D-Ser, but a strong antagonist for this receptor after B23Gly was replaced by corresponding l-amino acids. However, these replacements always resulted in a weak agonist for RXFP4. Thus, configuration of the B23 residue of R3/I5 affected activation of RXFP3 but not RXFP4. For the B24 residue, both size and configuration affected receptor selectivity of R3/I5. l-amino acids with an appropriate size, such as L-Ser and L-Abu, had the greatest effect on increasing the selectivity of R3/I5 towards RXFP3 over the homologous RXFP4. Our present results provided new insights into receptor selectivity of R3/I5, and would facilitate design of novel agonists or antagonists for RXFP3 and RXFP4 in future studies.