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BACKGROUND: Colorectal cancer (CRC) is a major cause of cancer-related deaths worldwide, and chemoresistance is a major obstacle in its treatment. Despite advances in therapy, the molecular mechanism underlying chemoresistance in CRC is not fully understood. Recent studies have implicated the key roles of long noncoding RNAs (lncRNAs) in the regulation of CRC chemoresistance. METHODS: In this study, we investigated the role of the lncRNA LINC01852 in CRC chemoresistance. LINC01852 expression was evaluated in multiple CRC cohorts using quantitative reverse transcription PCR. We conducted in vitro and in vivo functional experiments using cell culture and mouse models. RNA pull-down, RNA immunoprecipitation, chromatin immunoprecipitation, and dual luciferase assays were used to investigate the molecular mechanism of LINC01852 in CRC. RESULTS: Our findings revealed that a lncRNA with tumor-inhibiting properties, LINC01852, was downregulated in CRC and inhibited cell proliferation and chemoresistance both in vitro and in vivo. Further mechanistic investigations revealed that LINC01852 increases TRIM72-mediated ubiquitination and degradation of SRSF5, inhibiting SRSF5-mediated alternative splicing of PKM and thereby decreasing the production of PKM2. Overexpression of LINC01852 induces a metabolic switch from aerobic glycolysis to oxidative phosphorylation, which attenuates the chemoresistance of CRC cells by inhibiting PKM2-mediated glycolysis. CONCLUSIONS: Our results demonstrate that LINC01852 plays an important role in repressing CRC malignancy and chemoresistance by regulating SRSF5-mediated alternative splicing of PKM, and that targeting the LINC01852/TRIM72/SRSF5/PKM2 signaling axis may represent a potential therapeutic strategy for CRC.
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Neoplasias Colorrectales , ARN Largo no Codificante , Animales , Ratones , Humanos , Empalme Alternativo , Resistencia a Antineoplásicos , Carcinogénesis , Transformación Celular Neoplásica , Inmunoprecipitación de CromatinaRESUMEN
Many studies have shown that tumor cells that survive radiotherapy are more likely to metastasize, but the underlying mechanism remains unclear. Here we aimed to identify epithelial-mesenchymal transition (EMT)-related key genes, which associated with prognosis and radiosensitivity in rectal cancer. First, we obtained differentially expressed genes by analyzing the RNA expression profiles of rectal cancer retrieved from The Cancer Genome Atlas database, EMT-related genes, and radiotherapy-related databases, respectively. Then, Lasso and Cox regression analyses were used to establish an EMT-related prognosis model (EMTPM) based on the identified independent protective factor Fibulin5 (FBLN5) and independent risk gene EHMT2. The high-EMTPM group exhibited significantly poorer prognosis. Then, we evaluated the signature in an external clinical validation cohort. Through in vivo experiments, we further demonstrated that EMTPM effectively distinguishes radioresistant from radiosensitive patients with rectal cancer. Moreover, individuals in the high-EMTPM group showed increased expression of immune checkpoints compared to their counterparts. Finally, pan-cancer analysis of the EMTPM model also indicated its potential for predicting the prognosis of lung squamous cell carcinoma and breast cancer patients undergoing radiotherapy. In summary, we established a novel predictive model for rectal cancer prognosis and radioresistance based on FBLN5 and EHMT2 expressions, and suggested that immune microenvironment may be involved in the process of radioresistance. This predictive model could be used to select management strategies for rectal cancer.
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Nitrogen mustard (NM) is an alkylating vesicant that causes severe pulmonary injury. Currently, there are no effective means to counteract vesicant-induced lung injury. MG53 is a vital component of cell membrane repair and lung protection. Here, we show that mice with ablation of MG53 are more susceptible to NM-induced lung injury than the wild-type mice. Treatment of wild-type mice with exogenous recombinant human MG53 (rhMG53) protein ameliorates NM-induced lung injury by restoring arterial blood oxygen level, by improving dynamic lung compliance and by reducing airway resistance. Exposure of lung epithelial and endothelial cells to NM leads to intracellular oxidative stress that compromises the intrinsic cell membrane repair function of MG53. Exogenous rhMG53 protein applied to the culture medium protects lung epithelial and endothelial cells from NM-induced membrane injury and oxidative stress, and enhances survival of the cells. Additionally, we show that loss of MG53 leads to increased vulnerability of macrophages to vesicant-induced cell death. Overall, these findings support the therapeutic potential of rhMG53 to counteract vesicant-induced lung injury.
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Lesión Pulmonar Aguda , Mecloretamina , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/tratamiento farmacológico , Lesión Pulmonar Aguda/genética , Animales , Células Endoteliales/metabolismo , Pulmón/metabolismo , Mecloretamina/uso terapéutico , Mecloretamina/toxicidad , Proteínas de la Membrana/metabolismo , Ratones , Proteínas Recombinantes/metabolismoRESUMEN
BACKGROUND: Long noncoding RNAs (lncRNAs) have driven research focused on their effects as oncogenes or tumor suppressors involved in carcinogenesis. However, the functions and mechanisms of most lncRNAs in colorectal cancer (CRC) remain unclear. METHODS: The expression of DLGAP1-AS2 was assessed by quantitative RT-PCR in multiple CRC cohorts. The impacts of DLGAP1-AS2 on CRC growth and metastasis were evaluated by a series of in vitro and in vivo assays. Furthermore, the underlying mechanism of DLGAP1-AS2 in CRC was revealed by RNA pull down, RNA immunoprecipitation, RNA sequencing, luciferase assays, chromatin immunoprecipitation, and rescue experiments. RESULTS: We discovered that DLGAP1-AS2 promoted CRC tumorigenesis and metastasis by physically interacting with Elongin A (ELOA) and inhibiting its protein stability by promoting tripartite motif containing 21 (Trim21)-mediated ubiquitination modification and degradation of ELOA. In particular, we revealed that DLGAP1-AS2 decreases phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP) expression by inhibiting ELOA-mediated transcriptional activating of LHPP and thus blocking LHPP-dependent suppression of the AKT signaling pathway. In addition, we also demonstrated that DLGAP1-AS2 was bound and stabilized by cleavage and polyadenylation specificity factor (CPSF2) and cleavage stimulation factor (CSTF3). CONCLUSIONS: The discovery of DLGAP1-AS2, a promising prognostic biomarker, reveals a new dimension into the molecular pathogenesis of CRC and provides a prospective treatment target for this disease.
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Neoplasias Colorrectales , MicroARNs , ARN Largo no Codificante , Humanos , Carcinogénesis/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular , Transformación Celular Neoplásica/genética , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Neoplasias Colorrectales/patología , Elonguina/genética , Elonguina/metabolismo , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismoRESUMEN
BACKGROUND: SLCO4A1-AS1 was found to be upregulated in several cancer types, including colorectal cancer (CRC). However, the detailed roles of SLCO4A1-AS1 in CRC remain to be elucidated. Therefore, we investigated the functions, mechanism, and clinical significance of SLCO4A1-AS1 in colorectal tumourigenesis. METHODS: We measured the expression of SLCO4A1-AS1 in CRC tissues using qRT-PCR and determined its correlation with patient prognosis. Promoter methylation analyses were used to assess the methylation status of SLCO4A1-AS1. Gain- and loss-of-function assays were used to evaluate the effects of SLCO4A1-AS1 on CRC growth in vitro and in vivo. RNA pull-down, RNA immunoprecipitation, RNA-seq, luciferase reporter and immunohistochemistry assays were performed to identify the molecular mechanism of SLCO4A1-AS1 in CRC. RESULTS: SLCO4A1-AS1 was frequently upregulated in CRC tissues based on multiple CRC cohorts and was associated with poor prognoses. Aberrant overexpression of SLCO4A1-AS1 in CRC is partly attributed to the DNA hypomethylation of its promoter. Ectopic SLCO4A1-AS1 expression promoted CRC cell growth, whereas SLCO4A1-AS1 knockdown repressed CRC proliferation both in vitro and in vivo. Mechanistic investigations revealed that SLCO4A1-AS1 functions as a molecular scaffold to strengthen the interaction between Hsp90 and Cdk2, promoting the protein stability of Cdk2. The SLCO4A1-AS1-induced increase in Cdk2 levels activates the c-Myc signalling pathway by promoting the phosphorylation of c-Myc at Ser62, resulting in increased tumour growth. CONCLUSIONS: Our data demonstrate that SLCO4A1-AS1 acts as an oncogene in CRC by regulating the Hsp90/Cdk2/c-Myc axis, supporting SLCO4A1-AS1 as a potential therapeutic target and prognostic factor for CRC.
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Neoplasias Colorrectales , MicroARNs , ARN Largo no Codificante , Carcinogénesis/genética , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular/genética , Neoplasias Colorrectales/genética , Quinasa 2 Dependiente de la Ciclina , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas Proto-Oncogénicas c-myc , ARN sin Sentido , Transducción de Señal/genéticaAsunto(s)
Antiinflamatorios/uso terapéutico , Subtipo H1N1 del Virus de la Influenza A , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Proteínas de Motivos Tripartitos/uso terapéutico , Animales , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Infecciones por Orthomyxoviridae/mortalidad , Proteínas Recombinantes/uso terapéutico , Resultado del TratamientoRESUMEN
As a potential tumor suppressor, the detailed clinical application value of sorting nexin 1 (SNX1) has not been elucidated in colorectal cancer (CRC). The aim of the present study was to evaluate the expression of SNX1 in CRC tissues and to determine its correlation with clinicopathologic characteristics and its impact on patient's prognosis. We detected the expression of SNX1 mRNA in 72 CRC patients and SNX1 protein in 237 CRC patients by real-time polymerase chain reaction (RT-PCR) and immunohistochemical staining, respectively. Relationship between the expression of SNX1 and various clinicopathological features in these patients was evaluated. Both the mRNA and protein expression of SNX1 were remarkably decreased in CRC tissues compared with paired non-cancerous tissues, and the down-regulation of SNX1 protein was strongly associated with poor differentiation and poor overall survival (OS) rate of CRC patients. Ectopic SNX1 expression repressed CRC cell growth and promoted tumor sensitivity to most commonly used chemotherapeutic drugs (oxaliplatin and 5-Fluorouracil). In conclusion, overexpression of SNX1 may serve as a new therapeutic strategy for CRC.
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Neoplasias Colorrectales/genética , Neoplasias Colorrectales/mortalidad , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica , Nexinas de Clasificación/genética , Adulto , Anciano , Antineoplásicos/farmacología , Biomarcadores de Tumor , Línea Celular Tumoral , Proliferación Celular , Neoplasias Colorrectales/patología , Regulación hacia Abajo , Femenino , Fluorouracilo/farmacología , Humanos , Estimación de Kaplan-Meier , Metástasis Linfática , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Estadificación de Neoplasias , Pronóstico , Carga TumoralRESUMEN
Originally discovered in neuronal guidance, the Slit-Robo pathway is emerging as an important player in human cancers. However, its involvement and mechanism in colorectal cancer (CRC) remains to be elucidated. Here, we report that Slit2 expression is reduced in CRC tissues compared with adjacent noncancerous tissues. Extensive promoter hypermethylation of the Slit2 gene has been observed in CRC cells, which provides a mechanistic explanation for the Slit2 downregulation in CRC. Functional studies showed that Slit2 inhibits CRC cell migration in a Robo-dependent manner. Robo-interacting ubiquitin-specific protease 33 (USP33) is required for the inhibitory function of Slit2 on CRC cell migration by deubiquitinating and stabilizing Robo1. USP33 expression is downregulated in CRC samples, and reduced USP33 mRNA levels are correlated with increased tumor grade, lymph node metastasis and poor patient survival. Taken together, our data reveal USP33 as a previously unknown tumor-suppressing gene for CRC by mediating the inhibitory function of Slit-Robo signaling on CRC cell migration. Our work suggests the potential value of USP33 as an independent prognostic marker of CRC.
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Movimiento Celular/genética , Neoplasias Colorrectales/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Proteínas del Tejido Nervioso/genética , Receptores Inmunológicos/genética , Transducción de Señal/genética , Ubiquitina Tiolesterasa/genética , Células CACO-2 , Línea Celular , Línea Celular Tumoral , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Metilación de ADN/genética , Regulación hacia Abajo/genética , Genes Supresores de Tumor/fisiología , Células HCT116 , Células HEK293 , Células HT29 , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Metástasis Linfática/genética , Metástasis Linfática/patología , Proteínas del Tejido Nervioso/metabolismo , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , Receptores Inmunológicos/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Proteínas RoundaboutRESUMEN
It has been well established that a starvation-induced decrease in insulin/IGF-I and serum amino acids effectively suppresses the mammalian target of rapamycin (mTor) signaling to induce autophagy, which is a major degradative cellular pathway in skeletal muscles. In this study, we investigated the systematic effects of exercise on the mTor signaling of skeletal muscles. Wild type C57BL/6J mice were starved for 24h under synchronous autophagy induction conditions. Under these conditions, endogenous LC3-II increased, while both S6-kinse and S6 ribosomal protein were dephosphorylated in the skeletal muscles, which indicated mTor inactivation. Using GFP-LC3 transgenic mice, it was also confirmed that fluorescent GFP-LC3 dots in the skeletal muscles increased, including soleus, plantaris, and gastrocnemius, which clearly showed autophagosomal induction. These starved mice were then subjected to a single bout of running on a treadmill (12m/min, 2h, with a lean of 10 degrees). Surprisingly, biochemical analyses revealed that the exercise elicited a decrease in the LC3-II/LC3-I ratio as well as an inversion from the dephosphorylated state to the rephosphorylated state of S6-kinase and ribosomal S6 in these skeletal muscles. Consistently, the GFP-LC3 dots of the skeletal muscles were diminished immediately after the exercise. These results indicated that exercise suppressed starvation-induced autophagy through a reactivation of mTor signaling in the skeletal muscles of these starved mice.
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Condicionamiento Físico Animal , Serina-Treonina Quinasas TOR/metabolismo , Animales , Autofagia , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/metabolismo , Músculo Esquelético/metabolismo , Fosforilación , Proteínas Quinasas S6 Ribosómicas/metabolismo , Carrera , Transducción de Señal , InaniciónRESUMEN
Double minute chromosomes (DMs) are a hallmark of gene amplification. The relationship between the formation of DMs and the amplification of DM-carried genes remains to be clarified. The human colorectal cancer cell line NCI-H716 and human malignant primitive neuroectodermal tumor cell line SK-PN-DW are known to contain many DMs. To examine the amplification of DM-carried genes in tumor cells, we performed Affymetrix SNP Array 6.0 analyses and verified the regions of amplification in NCI-H716 and SK-PN-DW tumor cells. We identified the amplification regions and the DM-carried genes that were amplified and overexpressed in tumor cells. Using RNA interference, we downregulated seven DM-carried genes, (NDUFB9, MTSS1, NSMCE2, TRIB1, FAM84B, MYC and FGFR2) individually and then investigated the formation of DMs, the amplification of the DM-carried genes, DNA damage and the physiological function of these genes. We found that suppressing the expression of DM-carried genes led to a decrease in the number of DMs and reduced the amplification of the DM-carried genes through the micronuclei expulsion of DMs from the tumor cells. We further detected an increase in the number of γH2AX foci in the knockdown cells, which provides a strong link between DNA damage and the loss of DMs. In addition, the loss of DMs and the reduced amplification and expression of the DM-carried genes resulted in a decrease in cell proliferation and invasion ability.
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Biomarcadores de Tumor/genética , Cromosomas Humanos/genética , Neoplasias Colorrectales/patología , Amplificación de Genes , Micronúcleos con Defecto Cromosómico , Tumores Neuroectodérmicos Primitivos/patología , Polimorfismo de Nucleótido Simple/genética , Biomarcadores de Tumor/metabolismo , Western Blotting , Ciclo Celular , Movimiento Celular , Núcleo Celular/genética , Proliferación Celular , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Daño del ADN/genética , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Histonas/genética , Histonas/metabolismo , Humanos , Tumores Neuroectodérmicos Primitivos/genética , Tumores Neuroectodérmicos Primitivos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
Gastric cancer (GC) is a rising global health issue, with increasing incidence and mortality rates. The pathogenesis of GC is highly complex and involves a combination of genetic and environmental factors. Therefore, identifying new genes and pathways that contribute to the development and progression of GC is essential for improving diagnosis and treatment outcomes. Long noncoding RNAs (lncRNAs) have recently emerged as a promising area of research in understanding the molecular mechanisms underlying various cancers, including GC. These RNA molecules are longer than 200 nucleotides and do not code proteins. Although initially considered "junk DNA", lncRNAs have been demonstrated to play significant roles in various biological processes, including cell proliferation, differentiation, and apoptosis, as well as in the pathogenesis of various cancers. In this study, we screened clinical specimens for a novel lncRNA, LINC00853, which showed high expression in GC tissues and promoted the proliferation, migration, and invasion of GC cells. Furthermore, in vivo experiments confirmed its ability to facilitate the growth and metastasis of GC. These results suggest that LINC00853 plays a crucial role in the development and progression of GC.
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ARN Largo no Codificante , Neoplasias Gástricas , Humanos , Neoplasias Gástricas/patología , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Transformación Celular Neoplásica/genética , Proliferación Celular/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/genética , Movimiento Celular/genéticaRESUMEN
Ferulic acid (FA), a naturally occurring polyphenol abundant in vegetables and rice bran, is known to possess a potent antioxidant activity, thereby protecting cells from oxidative stress. In the present study, we show that in addition to its known anti-oxidant activity, ferulic acid exerts substantial inhibitory activity on cellular mammalian target of rapamycin (mTor)-signaling pathways. In HeLa cells and mouse primary hepatocytes cultured with conventional nutrient-rich media, ferulic acid (1 mM) elicited dephosphorylation of S6 kinase and its substrate ribosomal S6. The dephosphorylating activity of ferulic acid was almost comparable to that of rapamycin, an established mTor inhibitor (TORC1). We next investigated the effect of ferulic acid on autophagy, a major cellular degradative process, which significantly contributes to the maintenance of cell homeostasis. Using a conventional green fluorescent protein-microtubule-associated protein IA/IB light chain 3 (GFP-LC3) dot assay to evaluate autophagy flux, we showed that ferulic acid caused a significant increase in GFP-LC3 dots under serum-rich conditions in HeLa cells. The enhancement of autophagic flux by ferulic acid was almost equivalent to that of rapamycin. Furthermore, ferulic acid significantly enhanced autophagic degradation of (14)C-leucine-labeled long-lived proteins of cultured mouse hepatocytes under nutrient-rich conditions, but not nutrient-deprived conditions. These results indicate that ferulic acid is almost the equivalent of rapamycin in the ability to inhibit mTor (TORC1), which makes it a potent activator of basal autophagy.
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Ácidos Cumáricos/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Animales , Autofagia/efectos de los fármacos , Células Cultivadas , Células HeLa , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Proteínas Quinasas S6 Ribosómicas , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
MicroRNAs (miRNAs) are a class of non-coding single-stranded RNA molecules with a length of approximately 18-25 nt nucleotides that regulate gene expression post-transcriptionally. MiR-204-5p originates from the sixth intron of the transient receptor potential cation channel subfamily M member 3 (TRPM3) gene. MiR-204-5p is frequently downregulated in various cancer types and is related to the clinicopathological characteristics and prognosis of cancer patients. So far, many studies have determined that miR-204-5p functions as a tumor suppressor for its extensive and powerful capacity to inhibit tumor proliferation, metastasis, autophagy, and chemoresistance in multiple cancer types. MiR-204-5p appears to be a promising prognostic biomarker and a therapeutic target for human cancers. This review summarized the latest advances on the role of miR-204-5p in human cancers.
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MicroARNs , Humanos , Línea Celular Tumoral , MicroARNs/genética , Genes Supresores de Tumor , Pronóstico , Proliferación Celular/genética , Regulación Neoplásica de la Expresión GénicaRESUMEN
Signaling receptor and transporter of retinol STRA6 (STRA6) plays a primary role in transporting retinol. Several studies have reported that STRA6 is involved in several pathways related to tumorigenesis and progression. However, the exact functions and mechanisms of STRA6 in colorectal cancer (CRC) remain unclear. In our work, STRA6 was highly up-regulated in CRC and promoted the proliferation of CRC cells. Additionally, we discovered that STRA6 suppresses apoptosis partly by controlling BCL2 expression, which in turn causes CRC to become resistant to LOHP treatment. Our study demonstrates that STRA6 is a potential prognostic factor and oncogene in CRC by promoting CRC growth and chemoresistance.
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Neoplasias Colorrectales , Vitamina A , Humanos , Oxaliplatino/farmacología , Oxaliplatino/uso terapéutico , Regulación hacia Arriba , Vitamina A/metabolismo , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Pronóstico , Resistencia a Antineoplásicos , Línea Celular Tumoral , Proliferación Celular , Proteínas de la Membrana/metabolismoRESUMEN
Long noncoding RNAs (lncRNAs) play an important role in tumor progression. Small nucleolar RNA host gene 15 (SNHG15) is a lncRNA that has been confirmed to play an oncogenic role in multiple cancer types. However, its role in glycolysis and chemoresistance in colorectal cancer (CRC) is unclear. The expression of SNHG15 in CRC was analyzed using data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases by bioinformatics methods. Cell Counting Kit-8 (CCK-8) and colony formation assays were used to evaluate cell viability. Cell sensitivity to 5-fluorouracil (5-FU) was detected by CCK-8. Glucose absorption and lactate production were used to evaluate the impact of SNHG15 on glycolysis. RNA-seq, real-time fluorescence quantitative reverse transcription PCR (RT-qPCR) and Western blotting (WB) were used to reveal the potential molecular mechanism of SNHG15 in CRC. SNHG15 was upregulated in CRC tissues compared with paired noncancerous tissues. Ectopic SNHG15 expression increased proliferation, 5-FU chemoresistance, and glycolysis in CRC cells. In contrast, SNHG15 knockdown inhibited CRC proliferation, 5-FU chemoresistance and glycolysis. Multiple pathways, including apoptosis and glycolysis, were potentially regulated by SNHG15 based on RNA-seq and pathway enrichment analyses. RT-qPCR and WB experiments confirmed that SNHG15 promoted the expression of TYMS, BCL2, GLUT1 and PKM2 in CRC cells. In conclusion, SNHG15 promotes 5-FU chemoresistance and glycolysis in CRC by potentially regulating the expression of TYMS, BCL2, GLUT1 and PKM2 and appears to be a new target for cancer therapy.
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Neoplasias Colorrectales , MicroARNs , ARN Largo no Codificante , Humanos , Transportador de Glucosa de Tipo 1/metabolismo , Resistencia a Antineoplásicos/genética , Línea Celular Tumoral , Proliferación Celular , Fluorouracilo/farmacología , Neoplasias Colorrectales/patología , Glucólisis , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Largo no Codificante/genética , MicroARNs/genética , Regulación Neoplásica de la Expresión GénicaRESUMEN
BACKGROUND: Mantle cell lymphoma (MCL) is a rare B-cell non-Hodgkin lymphoma subtype which remains incurable despite multimodal approach including chemoimmunotherapy followed by stem cell transplant, targeted approaches such as the BTK inhibitor ibrutinib, and CD19 chimeric antigen receptor (CAR) T cells. CD74 is a nonpolymorphic type II integral membrane glycoprotein identified as an MHC class II chaperone and a receptor for macrophage migration inhibitory factor. Our group previously reported on CD74's abundant expression in MCL and its ability to increase via pharmacological inhibition of autophagosomal degradation. Milatuzumab, a fully humanized anti-CD74 monoclonal antibody, demonstrated significant activity in preclinical lymphoma models but failed to provide meaningful benefits in clinical trials mainly due to its short half-life. We hypothesized that targeting CD74 using a CAR-T cell would provide potent and durable anti-MCL activity. METHODS: We engineered a second generation anti-CD74 CAR with 4-1BB and CD3ζ signaling domains (74bbz). Through in silico and rational mutagenesis on the scFV domain, the 74bbz CAR was functionally optimized for superior antigen binding affinity, proliferative signaling, and cytotoxic activity against MCL cells in vitro and in vivo. RESULTS: Functionally optimized 74bbz CAR-T cells (clone 42105) induced significant killing of MCL cell lines, and primary MCL patient samples including one relapse after commercial CD19 CAR-T cell therapy with direct correlation between antigen density and cytotoxicity. It significantly prolonged the survival of an animal model established in NOD-SCIDγc-/- (NSG) mice engrafted with a human MCL cell line Mino subcutaneously compared to controls. Finally, while CD74 is also expressed on normal immune cell subsets, treatment with 74bbz CAR-T cells resulted in minimal cytotoxicity against these cells both in vitro and in vivo. CONCLUSIONS: Targeting CD74 with 74bbz CAR-T cells represents a new cell therapy to provide a potent and durable and anti-MCL activity.
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Ischemic injury to the heart induces mitochondrial dysfunction due to increasing oxidative stress. MG53, also known as TRIM72, is highly expressed in striated muscle, is secreted as a myokine after exercise, and is essential for repairing damaged plasma membrane of many tissues by interacting with the membrane lipid phosphatidylserine (PS). We hypothesized MG53 could preserve mitochondrial integrity after an ischemic event by binding to the mitochondrial-specific lipid, cardiolipin (CL), for mitochondria protection to prevent mitophagy. Fluorescent imaging and Western blotting experiments showed recombinant human MG53 (rhMG53) translocated to the mitochondria after ischemic injury in vivo and in vitro. Fluorescent imaging indicated rhMG53 treatment reduced superoxide generation in ex vivo and in vitro models. Lipid-binding assay indicated MG53 binds to CL. Transfecting cardiomyocytes with the mitochondria-targeted mt-mKeima showed inhibition of mitophagy after MG53 treatment. Overall, we show that rhMG53 treatment may preserve cardiac function by preserving mitochondria in cardiomyocytes. These findings suggest MG53's interactions with mitochondria could be an attractive avenue for developing MG53 as a targeted protein therapy for cardioprotection.
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Proteínas Portadoras , Miocitos Cardíacos , Proteínas Portadoras/metabolismo , Humanos , Isquemia/metabolismo , Lípidos , Mitocondrias/metabolismo , Miocitos Cardíacos/metabolismo , Estrés Oxidativo , ReperfusiónRESUMEN
Tumor-associated macrophages (TAMs) are one of the most abundant cell types in colorectal cancer (CRC) tumor microenvironment (TME). Recent studies observed complicated "cross-talks" between cancer cells and macrophages in TME. However, the underlying mechanisms are still poorly elucidated. Here, PD-L1 levels are very low in CRC cells but highly abundant in TAMs, and a specific PD-L1+CD206+ macrophage subpopulation are identified, which is induced by tumor cells and associated with a poor prognosis. Mechanistic investigations reveal that CRC cells can secrete small extracellular vesicles (sEVs) taken up by macrophages that induce M2 like polarization and PD-L1 expression, resulting in increased PD-L1+CD206+ macrophage abundance and decreased T cell activity in CRC TME. sEV-derived miR-21-5p and miR-200a are identified as key signaling molecules mediating the regulatory effects of CRC on macrophages. Further studies reveal that CRC-derived miR-21-5p and miR-200a synergistically induces macrophage M2 like polarization and PD-L1 expression by regulating the PTEN/AKT and SCOS1/STAT1 pathways, resulting in decreased CD8+ T cell activity and increased tumor growth. This study suggests that inhibiting the secretion of specific sEV-miRNAs from CRC and targeting PD-L1 in TAMs may serve as novel methods for CRC treatment as well as a sensitization method for anti-PD-L1 therapy in CRC.
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Antígeno B7-H1/metabolismo , Neoplasias Colorrectales , Vesículas Extracelulares , Línea Celular Tumoral , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Vesículas Extracelulares/metabolismo , Humanos , Escape del Tumor , Microambiente Tumoral , Macrófagos Asociados a TumoresRESUMEN
Rationale: While reactive oxygen species (ROS) has been recognized as one of the main causes of cardiac injury following myocardial infarction, the clinical application of antioxidants has shown limited effects on protecting hearts against ischemia-reperfusion (I/R) injury. Thus, the precise role of ROS following cardiac injury remains to be fully elucidated. Objective: We investigated the role of mitsugumin 53 (MG53) in regulating necroptosis following I/R injury to the hearts and the involvement of ROS in MG53-mediated cardioprotection. Methods and Results: Antioxidants were used to test the role of ROS in MG53-mediated cardioprotection in the mouse model of I/R injury and induced human pluripotent stem cells (hiPSCs)-derived cardiomyocytes subjected to hypoxia or re-oxygenation (H/R) injury. Western blotting and co-immunoprecipitation were used to identify potential cell death pathways that MG53 was involved in. CRISPR/Cas 9-mediated genome editing and mutagenesis assays were performed to further identify specific interaction amino acids between MG53 and its ubiquitin E3 ligase substrate. We found that MG53 could protect myocardial injury via inhibiting the necroptosis pathway. Upon injury, the generation of ROS in the infarct zone of the hearts promoted interaction between MG53 and receptor-interacting protein kinase 1 (RIPK1). As an E3 ubiquitin ligase, MG53 added multiple ubiquitin chains to RIPK1 at the sites of K316, K604, and K627 for proteasome-mediated RIPK1 degradation and inhibited necroptosis. The application of N-acetyl cysteine (NAC) disrupted the interaction between MG53 and RIPK1 and abolished MG53-mediated cardioprotective effects. Conclusions: Taken together, this study provided a molecular mechanism of a potential beneficial role of ROS following acute myocardial infarction. Thus, fine-tuning ROS levels might be critical for cardioprotection.
RESUMEN
Single-domain antibodies, VHHs or nanobodies, represent a promising set of alternatives to conventional therapeutic antibodies, gaining substantial attention in the field of cancer immunotherapy. However, inherent drawbacks of nanobodies such as fast clearance from blood circulation and lack of immune effector functions often led to unsatisfactory therapeutic efficacy. We previously reported that dinitrophenyl modification of an anti-EGFR VHH conferred Fc-dependent immune effector functions and elongated serum half-life on it through recruiting of hapten antibodies, resulting in improved immunotherapy efficacy in vivo. In the present work, we further tested the versatility of this approach in the case of an anti-PD-L1 blockade VHH (KN035). Site-specific dinitrophenyl conjugation did not impair the binding capacity of KN035 portion to PD-L1, but indirectly restored its immune effector functions, manifested by the observed antibody dependent cell-mediated cytotoxicity, antibody-dependent cellular phagocytosis and complement-dependent cytotoxicity against PD-L1 positive tumor cells. Significant delay of blood clearance of dinitrophenylated KN035 was evidenced by the prolonged half-life of ca. 22 h. This approach, using small hapten molecule conjugation, loaded additional antibody-mediated tumor killing mechanisms to PD-L1 blockade VHH and therefore improved efficacy is anticipated in the future in vivo therapeutic studies. Thus, our results underscore the power of this versatile approach for achieving desirable properties of VHH-based or similar therapeutics.