Endoplasmic reticulum stress: molecular mechanism and therapeutic targets.

The endoplasmic reticulum (ER) functions as a quality-control organelle for protein homeostasis, or "proteostasis". The protein quality control systems involve ER-associated degradation, protein chaperons, and autophagy. ER stress is activated when proteostasis is broken with an accumulation of misfolded and unfolded proteins in the ER. ER stress activates an adaptive unfolded protein response to restore proteostasis by initiating protein kinase R-like ER kinase, activating transcription factor 6, and inositol requiring enzyme 1. ER stress is multifaceted, and acts on aspects at the epigenetic level, including transcription and protein processing. Accumulated data indicates its key role in protein homeostasis and other diverse functions involved in various ocular diseases, such as glaucoma, diabetic retinopathy, age-related macular degeneration, retinitis pigmentosa, achromatopsia, cataracts, ocular tumors, ocular surface diseases, and myopia. This review summarizes the molecular mechanisms underlying the aforementioned ocular diseases from an ER stress perspective. Drugs (chemicals, neurotrophic factors, and nanoparticles), gene therapy, and stem cell therapy are used to treat ocular diseases by alleviating ER stress. We delineate the advancement of therapy targeting ER stress to provide new treatment strategies for ocular diseases.

Endothelial progenitor cell-derived exosomes promote anti-inflammatory macrophages via SOCS3/JAK2/STAT3 axis and improve the outcome of spinal cord injury.

BACKGROUND: Macrophage in the spinal cord injury (SCI) area imparts a chronic pro-inflammation effect that challenges the recovery of SCI. Previously, endothelial progenitor cell-produced exosomes (EPC-EXOs) have been noticed to facilitate revascularization and inflammation control after SCI. However, their effects on macrophage polarization remained unclear. This study aimed to investigate the EPC-EXOs' role in macrophage polarization and reveal its underlying mechanism. METHODS: We extracted the macrophages and EPC from the bone marrow suspension of C57BL/L mice by centrifugation. After cell identification, the EPC-EXOs were collected by ultra-high-speed centrifugation and exosome extraction kits and identified by transmission electron microscopy and nanoparticle tracking analysis. Then, macrophages were cultured with EPC-EXOs in different concentrations. We labeled the exosome to confirm its internalization by macrophage and detected the macrophage polarization marker level both in vitro and in vivo. We further estimated EPC-EXOs' protective effects on SCI by mice spinal cord tissue H&E staining and motor behavior evaluation. Finally, we performed RT-qPCR to identify the upregulated miRNA in EPC-EXOs and manipulate its expression to estimate its role in macrophage polarization, SOCS3/JAK2/STAT3 pathway activation, and motor behavior improvement. RESULTS: We found that EPC-EXOs decreased the macrophages' pro-inflammatory marker expression and increased their anti-inflammatory marker expression on the 7 and 14 days after SCI. The spinal cord H&E staining results showed that EPC-EXOs raised the tissue-sparing area rate significantly after 28 days of SCI and the motor behavior evaluation indicated an increased BMS score and motor-evoked potential by EPC-EXOs treatment after SCI. The RT-qPCR assay identified that miR-222-3P upregulated in EPC-EXOs and its miRNA-mimic also decreased the pro-inflammatory macrophages and increased the anti-inflammatory macrophages. Additionally, miR-222-3P mimic activated the SOCS3/JAK2/STAT3 pathway, and SOCS3/JAK2/STAT3 pathway inhibition blocked miR-2223P's effects on macrophage polarization and mouse motor behavior. CONCLUSION: Comprehensively, we discovered that EPC-EXOs-derived miR-222-3p affected macrophage polarization via SOCS3/JAK2/STAT3 pathway and promoted mouse functional repair after SCI, which reveals EPC-EXOs' role in modulation of macrophage phenotype and will provide a novel interventional strategy to induce post-SCI recovery.

Identification of Cathepsin B as a Therapeutic Target for Ferroptosis of Macrophage after Spinal Cord Injury.

Hemorrhage and immune cell infiltration are the main pathological features of spinal cord injury (SCI). Excessive iron deposition is caused by leaking hemosiderin which may over-activate ferroptosis pathways, resulting in lipid peroxidation and mitochondrial dysfunction in cells. Inhibiting ferroptosis after SCI has been shown to aid functional recovery. However, the essential genes involved in cellular ferroptosis following SCI are still unknown. Here we show that Ctsb is a statistical significance gene by collecting multiple transcriptomic profiles and identifying differentially expressed ferroptosis-related genes, which are abundantly expressed in myeloid cells after SCI and widely distributed at the epicenter of the injury. The expression score of ferroptosis, calculated by ferroptosis driver/suppressor genes, was high in macrophages. Furthermore, we discovered that inhibiting cathepsin B (CTSB), specifically with a small-molecule drug, CA-074-methyl ester (CA-074-me), reduced lipid peroxidation and mitochondrial dysfunction in macrophages. We also found that alternatively activated M2-polarized macrophages are more susceptible to hemin-induced ferroptosis. Consequently, CA-074-me could reduce ferroptosis, induce M2 macrophage polarization, and promote the neurological function recovery of mice after SCI. Our study comprehensively analyzed the ferroptosis after SCI from the perspective of multiple transcriptomes and provided a novel molecular target for SCI treatment.

MAEL Augments Cancer Stemness Properties and Resistance to Sorafenib in Hepatocellular Carcinoma through the PTGS2/AKT/STAT3 Axis.

Cancer stem cells (CSCs) are responsible for tumorigenesis, therapeutic resistance, and metastasis in hepatocellular cancer (HCC). Cancer/testis antigen Maelstrom (MAEL) is implicated in the formation of CSC phenotypes, while the exact role and underlying mechanism remain unclear. Here, we found the upregulation of MAEL in HCC, with its expression negatively correlated with survival outcome. Functionally, MAEL promoted tumor cell aggressiveness, tumor stem-like potentials, and resistance to sorafenib in HCC cell lines. Transcriptional profiling indicated the dysregulation of stemness in MAEL knockout cells and identified PTGS2 as a critical downstream target transactivated by MAEL. The suppression effect of MAEL knockout in tumor aggressiveness was rescued in PTGS2 overexpression HCC cells. A molecular mechanism study revealed that the upregulation of PTGS2 by MAEL subsequently resulted in IL-8 secretion and the activation of AKT/NF-κB/STAT3 signaling. Collectively, our work identifies MAEL as an important stemness regulation gene in HCC. Targeting MAEL or its downstream molecules may provide a novel possibility for the elimination of CSC to enhance therapeutic efficacy for HCC patients in the future.

PITX2C increases the stemness features of hepatocellular carcinoma cells by up-regulating key developmental factors in liver progenitor.

BACKGROUND: Tumor cells exhibited phenotypic and molecular characteristics similar to their lineage progenitor cells. Liver developmental signaling pathways are showed to be associated with HCC development and oncogenesis. The similarities of expression profiling between liver progenitors (LPs) and HCC suggest that understanding the molecular mechanism during liver development could provide insights into HCC. METHODS: To profile the dynamic gene expression during liver development, cells from an in vitro liver differentiation model and two paired hepatocellular carcinoma (HCC) samples were analyzed using deep RNA sequencing. The expression levels of selected genes were analyzed by qRT-PCR. Moreover, the role of a key transcription factor, pituitary homeobox 2 (PITX2), was characterized via in vitro and vivo functional assays. Furthermore, molecular mechanism studies were performed to unveil how PITX2C regulate the key developmental factors in LPs, thereby increasing the stemness of HCC. RESULTS: PITX2 was found to exhibit a similar expression pattern to specific markers of LPs. PITX2 consists of three isoforms (PITX2A/B/C). The expression of PITX2 is associated with tumor size and overall survival rate, whereas only PITX2C expression is associated with AFP and differentiation in clinical patients. PITX2A/B/C has distinct functions in HCC tumorigenicity. PITX2C promotes HCC metastasis, self-renewal and chemoresistance. Molecular mechanism studies showed that PITX2C could up-regulate RALYL which could enhance HCC stemness via the TGF-ß pathway. Furthermore, ChIP assays confirmed the role of PITX2C in regulating key developmental factors in LP. CONCLUSION: PITX2C is a newly discovered transcription factor involved in hepatic differentiation and could increase HCC stemness by upregulating key transcriptional factors related to liver development.

Elevated expression of RIT1 hyperactivates RAS/MAPK signal and sensitizes hepatocellular carcinoma to combined treatment with sorafenib and AKT inhibitor.

Hyperactivation of RAS/MAPK signaling is commonly observed in hepatocellular carcinoma (HCC). Gain-of-function mutations of canonical RAS genes, however, are rarely detected and it remains unclear how the activity of this pathway is turned on during hepatocarcinogenesis. We performed a comprehensive analysis of RAS superfamily genetic alterations across ten subfamilies, 152 members in 377 HCC patients from the Cancer Genome Atlas database. RIT1 (Ras-like without CAAX 1) was the most frequently altered RAS member amplified in 13% of the HCC cohort. Both genomic amplification and CREB-mediated transcriptional activation contributed to the elevated RIT1 expression, and its overexpression correlated with RAS/MAPK activation and poor prognosis. Then, we found that RIT1-induced angiogenesis via the MEK/ERK/EIF4E/HIF1-α/VEGFA axis. MAP3K11 and MAP3K12, in addition to CRAF, could mediate this process by binding to RIT1. Moreover, RIT1 increased the phosphorylation of p38 MAPK and AKT to promote cell survival under reactive oxygen species stress. Based on this mechanistic understanding, we treated RIT1-overexpressing HCC with combined regimen sorafenib plus AKT inhibitor, and achieved enhanced antitumor effects in vivo. Our study reveals RAS "orphan" member RIT1 as the most common genetic alteration of RAS family in HCC and combination of sorafenib with AKT inhibitor might be a promising treatment strategy for RIT1-overexpressing HCC.

RALYL increases hepatocellular carcinoma stemness by sustaining the mRNA stability of TGF-ß2.

Growing evidences suggest that cancer stem cells exhibit many molecular characteristics and phenotypes similar to their ancestral progenitor cells. In the present study, human embryonic stem cells are induced to differentiate into hepatocytes along hepatic lineages to mimic liver development in vitro. A liver progenitor specific gene, RALY RNA binding protein like (RALYL), is identified. RALYL expression is associated with poor prognosis, poor differentiation, and metastasis in clinical HCC patients. Functional studies reveal that RALYL could promote HCC tumorigenicity, self-renewal, chemoresistance, and metastasis. Moreover, molecular mechanism studies show that RALYL could upregulate TGF-ß2 mRNA stability by decreasing N6-methyladenosine (m6A) modification. TGF-ß signaling and the subsequent PI3K/AKT and STAT3 pathways, upregulated by RALYL, contribute to the enhancement of HCC stemness. Collectively, RALYL is a liver progenitor specific gene and regulates HCC stemness by sustaining TGF-ß2 mRNA stability. These findings may inspire precise therapeutic strategies for HCC.

MicroRNA-9 facilitates hypoxia-induced injury and apoptosis in H9c2 cells via targeting CDK8.

Hypoxia plays an important role in many heart diseases. MicroRNA-9 (miR-9) has been reported to be involved in hypoxia-induced cell proliferation, injury and apoptosis in cardiomyocytes. However, the underlying mechanism still remains poorly understood. The expression levels of miR-9 and cyclin-dependent kinase 8 (CDK8) were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The relative protein expression was measured by Western blot. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), lactate dehydrogenase (LDH) measurement, flow cytometry assays were conducted to detect cell proliferation, the release of LDH and cell apoptosis, respectively. The potential relationship between miR-9 and CDK8 was predicted by online database, and confirmed by dual-luciferase reporter assay. We found that miR-9 was increased, while CDK8 was decreased in hypoxia-treated H9c2 cells. miR-9 down-regulation or CDK8 up-regulation promoted cell proliferation, while repressed cell damage and apoptosis in hypoxia-induced H9c2 cells. Moreover, CDK8 was identified to be target of miR-9, and CDK8 knockdown could reverse the effects of miR-9 inhibitor on cell proliferation, damage and apoptosis in hypoxia-treated H9c2 cells. Besides, miR-9 could regulate the Wnt/b-catenin pathway by targeting CDK8 in hypoxic-induced H9c2 cells. In conclusion, miR-9 repressed cell proliferation and promoted cell damage and apoptosis by binding to CDK8 through the Wnt/ ß-catenin pathway in hypoxic-induced H9c2 cells, which provided a new direction for further studying the treatment of hypoxia-aroused heart diseases.