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Squamate reptiles are central for studying phenotypic correlates of evolutionary transitions from oviparity to viviparity because these transitions are numerous, with many of them being recent. Several models of life-history theory predict that viviparity is associated with increased female size, and thus more female-biased sexual size dimorphism (SSD). Yet, the corresponding empirical evidence is overall weak and inconsistent. The lizard Zootoca vivipara, which occupies a major part of Northern Eurasia and includes four viviparous and two non-sister oviparous lineages, represents an excellent model for testing these predictions. We analysed how sex-specific body size and SSD is associated with parity mode, using body length data for nearly 14,000 adult individuals from 97 geographically distinct populations, which cover almost the entire species' range and represent all six lineages. Our analyses controlled for lineage identity, climatic seasonality (the strongest predictor of geographic body size variation in previous studies of this species) and several aspects of data heterogeneity. Parity mode, lineage and seasonality are significantly associated with female size and SSD; the first two predictors accounted for 14%-26% of the total variation each, while seasonality explained 5%-7%. Viviparous populations exhibited a larger female size than oviparous populations, with no concomitant differences in male size. The variation of male size was overall low and poorly explained by our predictors. Albeit fully expected from theory, the strong female bias of the body size differences between oviparous and viviparous populations found in Z. vivipara is not evident from available data on three other lizard systems of closely related lineages differing in parity mode. We confront this pattern with the data on female reproductive traits in the considered systems and the frequencies of evolutionary changes of parity mode in the corresponding lizard families and speculate why the life-history correlates of live-bearing in Z. vivipara are distinct. Comparing conspecific populations, our study provides the most direct evidence for the predicted effect of parity mode on adult body size but also demonstrates that the revealed pattern may not be general. This might explain why across squamates, viviparity is only weakly associated with larger size.
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BACKGROUND: During pseudoglandular stage of the human lung development the primitive bronchial buds are initially conformed by simple tubules lined by endoderm-derived epithelium surrounded by mesenchyme, which will progressively branch into airways and start to form distal epithelial saculles. For first time alveolar type II (AT2) pneumocytes appears. This study aims to characterize the genes and microRNAs involved in this differentiation process and decipher its role in the starting alveolar differentiation. METHODS: Gene and microRNA profiling was performed in human embryonic lungs from 7 to 12 post conception weeks (pcw). Protein expression location of candidate genes were analyzed by immunofluorescense in embryonic lung tissue sections. mRNA/miRNA target pairs were identified using computational approaches and their expression was studied in purified epithelial/mesenchymal cell populations and in isolated tips and stalks from the bronchial tree. Additionally, silencing experiments in human embryonic lung mesenchymal cells and in human embryonic tip-derived lung organoids were performed, as well as organoid differentiation studies. AT2 cell markers were studied by qRT-PCR and by immunofluorescence. The TGFB-ß phosphorylated pathways was analyzed with membrane protein arrays. Lung explants were cultured in air/liquid interface with/without peptides. RESULTS: We identified 88 differentially expressed genes, including IGFBP3. Although IGFBP3 mRNA was detected in both epithelial and mesenchymal populations, the protein was restricted to the epithelium, indicating post-transcriptional regulation preventing IGFBP3 protein expression in the mesenchyme. MicroRNA profiling identified miR-34a as an IGFBP3 regulator. miR-34a was up-regulated in mesenchymal cells, and its silencing in human embryonic lung mesenchymal cells increased IGFBP3 levels. Additionally, IGFBP3 expression showed a marked downregulation from 7 to 12 pcw, suggesting its involvement in the differentiation process. The differentiation of human tip-derived lung embryonic organoids showed a drastic reduction in IGFBP3, supported by the scRNAseq data. IGFBP3 silencing in organoids activated an alveolar-like differentiation process characterized by stem cell markers downregulation and upregulation of AT2 markers. This process was mediated by TGFß signalling inhibition and BMP pathway activation. CONCLUSIONS: The IGFBP3/miR-34a axis restricts IGFBP3 expression in the embryonic undifferentiated lung epithelium, and the progressive downregulation of IGFBP3 during the pseudoglandular stage is required for alveolar differentiation.
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Diferenciación Celular , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina , Pulmón , MicroARNs , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Pulmón/metabolismo , Pulmón/embriología , Pulmón/citología , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/citología , Alveolos Pulmonares/metabolismo , Alveolos Pulmonares/citología , Regulación del Desarrollo de la Expresión Génica , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/citologíaRESUMEN
PURPOSE: TIGIT blockade in our ex vivo model of bone marrow (BM) reduced the number of malignant plasma cells (PC) in only half of patients with multiple myeloma. Here, we wanted to investigate whether increased expression of TIGIT ligands may inhibit T-cell immune response promoting resistance to TIGIT blockade. EXPERIMENTAL DESIGN: We first characterized the number and phenotype of BM macrophages in different stages of the disease by multiparameter flow cytometry. We assessed the effect of TIGIT ligands on PC survival by performing experiments in the ex vivo BM model and analyzed changes in gene expression by using NanoString technology and real-time PCR. RESULTS: The frequency of BM macrophages was significantly decreased in multiple myeloma, which was accompanied by changes in their immunophenotype. Moreover, we found a higher number of malignant PC in ex vivo BM cells cultured onto the poliovirus receptor (PVR) and nectin-2 compared with control, suggesting that both ligands may support PC survival. In addition, the presence of PVR, but not nectin-2, overcame the therapeutic effect of TIGIT blockade or exogenous IL2. Furthermore, exogenous IL2 increased TIGIT expression on both CD4+ and CD8+ T cells and, indirectly, PVR on BM macrophages. Consistently, PVR reduced the number of cytotoxic T cells and promoted a gene signature with reduced effector molecules. CONCLUSIONS: IL2 induced TIGIT on T cells in the BM, in which increased PVR expression resulted in cytotoxic T-cell inhibition, promoting PC survival and resistance to TIGIT blockade.
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Macrófagos , Mieloma Múltiple , Receptores Inmunológicos , Receptores Virales , Mieloma Múltiple/patología , Mieloma Múltiple/genética , Mieloma Múltiple/metabolismo , Mieloma Múltiple/tratamiento farmacológico , Receptores Inmunológicos/antagonistas & inhibidores , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Humanos , Macrófagos/metabolismo , Macrófagos/inmunología , Receptores Virales/genética , Receptores Virales/metabolismo , Resistencia a Antineoplásicos/genética , Nectinas/genética , Médula Ósea/patología , Médula Ósea/metabolismo , Línea Celular TumoralRESUMEN
The early mechanisms of spontaneous tumor initiation that precede malignancy are largely unknown. We show that reduced aPKC levels correlate with stem cell loss and the induction of revival and metaplastic programs in serrated- and conventional-initiated premalignant lesions, which is perpetuated in colorectal cancers (CRCs). Acute inactivation of PKCλ/ι in vivo and in mouse organoids is sufficient to stimulate JNK in non-transformed intestinal epithelial cells (IECs), which promotes cell death and the rapid loss of the intestinal stem cells (ISCs), including those that are LGR5+. This is followed by the accumulation of revival stem cells (RSCs) at the bottom of the crypt and fetal-metaplastic cells (FMCs) at the top, creating two spatiotemporally distinct cell populations that depend on JNK-induced AP-1 and YAP. These cell lineage changes are maintained during cancer initiation and progression and determine the aggressive phenotype of human CRC, irrespective of their serrated or conventional origin.
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Neoplasias Colorrectales , Células Epiteliales , Metaplasia , Proteína Quinasa C , Animales , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/genética , Proteína Quinasa C/metabolismo , Proteína Quinasa C/genética , Metaplasia/patología , Metaplasia/metabolismo , Ratones , Humanos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Células Madre/metabolismo , Células Madre/patología , Factor de Transcripción AP-1/metabolismo , Factor de Transcripción AP-1/genética , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Proteínas Señalizadoras YAP/metabolismo , Transformación Celular Neoplásica/patología , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Organoides/metabolismo , Organoides/patología , Linaje de la Célula , Isoenzimas/metabolismo , Isoenzimas/genética , Isoenzimas/deficiencia , Células Madre Neoplásicas/patología , Células Madre Neoplásicas/metabolismoRESUMEN
The metabolic and signaling pathways regulating aggressive mesenchymal colorectal cancer (CRC) initiation and progression through the serrated route are largely unknown. Although relatively well characterized as BRAF mutant cancers, their poor response to current targeted therapy, difficult preneoplastic detection, and challenging endoscopic resection make the identification of their metabolic requirements a priority. Here, we demonstrate that the phosphorylation of SCAP by the atypical PKC (aPKC), PKCλ/ι promotes its degradation and inhibits the processing and activation of SREBP2, the master regulator of cholesterol biosynthesis. We show that the upregulation of SREBP2 and cholesterol by reduced aPKC levels is essential for controlling metaplasia and generating the most aggressive cell subpopulation in serrated tumors in mice and humans. Since these alterations are also detected prior to neoplastic transformation, together with the sensitivity of these tumors to cholesterol metabolism inhibitors, our data indicate that targeting cholesterol biosynthesis is a potential mechanism for serrated chemoprevention.
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Proteína Quinasa C , Transducción de Señal , Animales , Humanos , Ratones , Transformación Celular Neoplásica/genética , Colesterol , Células Epiteliales/metabolismo , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismoRESUMEN
BACKGROUND: Posterior tibial tendon (PTT) tendoscopy and medializing calcaneal osteotomy (MCO) are among the available techniques for patients presenting with symptomatic flexible hindfoot valgus (stage IA) progressive collapsing foot deformity (PCFD). The aim of this study was to determine clinical and radiographic outcomes of combined PTT tendoscopy and MCO for patients presenting with symptomatic stage IA PCFD. METHODS: A retrospective cohort study was performed in order to determine clinical and radiographic outcomes of 30 combined PTT tendoscopies and MCO on 27 patients presenting with symptomatic stage IA PCFD, with a minimum follow-up of 24 months. Patient satisfaction was assessed at last available follow-up as very satisfied, satisfied, and unsatisfied. Clinical assessment was performed evaluating preoperative and last available follow-up visual analog scale for pain (VAS-P), Foot and Ankle Outcome Score (FAOS), and the 36-Item Short Form Health Survey (SF-36). Magnetic resonance imaging (MRI) was performed preoperatively on all patients. Standard weightbearing anteroposterior, lateral, and long axial view radiographs of the foot and ankle were taken preoperatively, immediate postoperatively, at 6 weeks, 3 months, 6 months, 1 year postoperatively, and last follow-up evaluation available for each patient. RESULTS: The mean follow-up was 38.6 (range, 26-62) months. We registered 27 very satisfied, 1 satisfied, and 2 unsatisfied patients. There was statistically significant improvement on all clinical scores (VAS-P, FAOS and SF-36), as well as on lateral talo-first metatarsal and hindfoot alignment angles. We found low-grade PTT tears in 5 patients (16.67%) in whom preoperative MRI documented PTT tenosynovitis alone. CONCLUSION: We found that combined PTT tendoscopy and MCO provide significant clinical and radiographic improvement for patients presenting with symptomatic stage IAB PCFD. PTT tendoscopy should be considered in the treatment of all surgically addressed flexible valgus feet as it detects tendon tears which are frequently missed on an MRI. LEVEL OF EVIDENCE: Level IV, retrospective case series.
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Pie Plano , Deformidades del Pie , Humanos , Estudios Retrospectivos , Tendones/cirugía , Osteotomía/métodos , Articulación del Tobillo , Pie Plano/diagnóstico por imagen , Pie Plano/cirugíaRESUMEN
Understanding of the ankle and subtalar joint ligaments is essential to recognize and manage foot and ankle disorders. The stability of both joints relies on the integrity of its ligaments. The ankle joint is stabilized by the lateral and medial ligamentous complexes while the subtalar joint is stabilized by its extrinsic and intrinsic ligaments. Most injuries to these ligaments are linked with ankle sprains. Inversion or eversion mechanics affect the ligamentous complexes. A profound knowledge of the ligament's anatomy allows orthopedic surgeons to further understand anatomic or nonanatomic reconstructions.
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Traumatismos del Tobillo , Articulación del Tobillo , Inestabilidad de la Articulación , Humanos , Tobillo , Articulación del Tobillo/anatomía & histología , Inestabilidad de la Articulación/diagnóstico , Inestabilidad de la Articulación/etiología , Ligamentos Articulares , Articulación TalocalcáneaRESUMEN
Imaging organoid culture provides an excellent tool for studying complex diseases such as cancer. However, retaining the morphology of intact organoids for immunolabeling has been challenging. Here, we describe a protocol for immunofluorescence staining in intact colorectal cancer organoids derived from mice. We also describe additional steps for co-culture with mouse fibroblasts to enable the study of interactions with other cellular components of the tissue microenvironment. For complete details on the use and execution of this protocol, please refer to Martinez-Ordoñez et al. (2023).1.
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The monitoring of patients with dementia who receive comprehensive care in day centers allows formal caregivers to make better decisions and provide better care to patients. For instance, cognitive and physical therapies can be tailored based on the current stage of disease progression. In the context of day centers of the Mexican Federation of Alzheimer, this work aims to design and evaluate Alzaid, a technological platform for assisting formal caregivers in monitoring patients with dementia. Alzaid was devised using a participatory design methodology that consisted in eliciting and validating requirements from 22 and 9 participants, respectively, which were unified to guide the construction of a high-fidelity prototype evaluated by 14 participants. The participants were formal caregivers, medical staff, and management. This work contributes a high-fidelity prototype of a technological platform for assisting formal caregivers in monitoring patients with dementia considering restrictions and requirements of four Mexican day centers. In general, the participants perceived the prototype as quite likely to be useful, usable, and relevant in the job of monitoring patients with dementia (p-value < 0.05). By evaluating and designing Alzaid that unifies requirements for monitoring patients of four day centers, this work is the first effort towards a standard monitoring process of patients with dementia in the context of the Mexican Federation of Alzheimer.
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Enfermedad de Alzheimer , Demencia , Humanos , Cuidadores/psicología , Monitoreo FisiológicoRESUMEN
Mesenchymal colorectal cancer (mCRC) is microsatellite stable (MSS), highly desmoplastic, with CD8+ T cells excluded to the stromal periphery, resistant to immunotherapy, and driven by low levels of the atypical protein kinase Cs (aPKCs) in the intestinal epithelium. We show here that a salient feature of these tumors is the accumulation of hyaluronan (HA) which, along with reduced aPKC levels, predicts poor survival. HA promotes epithelial heterogeneity and the emergence of a tumor fetal metaplastic cell (TFMC) population endowed with invasive cancer features through a network of interactions with activated fibroblasts. TFMCs are sensitive to HA deposition, and their metaplastic markers have prognostic value. We demonstrate that in vivo HA degradation with a clinical dose of hyaluronidase impairs mCRC tumorigenesis and liver metastasis and enables immune checkpoint blockade therapy by promoting the recruitment of B and CD8+ T cells, including a proportion with resident memory features, and by blocking immunosuppression.
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Neoplasias Colorrectales , Ácido Hialurónico , Microambiente Tumoral , Humanos , Linfocitos T CD8-positivos/patología , Neoplasias Colorrectales/patología , Ácido Hialurónico/metabolismo , Inmunoterapia , Sarcoma/patología , Microambiente Tumoral/fisiologíaRESUMEN
Air-liquid organotypic culture models enable the study of the cellular crosstalk in the tumor microenvironment. This 3D assay recapitulates the tumor niche more faithfully than 2D culture systems and represents a versatile platform that can be easily adapted to different types of cancer cells, stromal components, or ECM composition. Here, we detail the steps to build an organotypic culture including the preparation of the organotypic structure, organotypic gels, cell seeding, gel casting, membrane processing, and image and data analysis. For complete details on the use and execution of this protocol, please refer to Linares et al. (2022).
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Neoplasias , Microambiente Tumoral , Línea Celular TumoralRESUMEN
In resected non-small cell lung cancer (NSCLC), post-surgical recurrence occurs in around 40% of patients, highlighting the necessity to identify relapse biomarkers. An analysis of the extracellular vesicle (EV) cargo from a pulmonary tumor-draining vein (TDV) can grant biomarker identification. We studied the pulmonary TDV EV-miRNAome to identify relapse biomarkers in a two-phase study (screening and validation). In the screening phase, a 17-miRNA relapse signature was identified in 18 selected patients by small RNAseq. The most expressed miRNA from the signature (EV-miR-203a-3p) was chosen for further validation. Pulmonary TDV EV-miR-203a-3p was studied by qRT-PCR in a validation cohort of 70 patients, where it was found to be upregulated in relapsed patients (p = 0.0194) and in patients with cancer spread to nearby lymph nodes (N+ patients) (p = 0.0396). The ROC curve analysis showed that TDV EV-miR-203a-3p was able to predict relapses with a sensitivity of 88% (AUC: 0.67; p = 0.022). Moreover, patients with high TDV EV-miR-203a-3p had a shorter time to relapse than patients with low levels (43.6 vs. 97.6 months; p = 0.00703). The multivariate analysis showed that EV-miR-203a-3p was an independent, predictive and prognostic post-surgical relapse biomarker. In conclusion, pulmonary TDV EV-miR-203a-3p is a promising new relapse biomarker for resected NSCLC patients.
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Carcinoma de Pulmón de Células no Pequeñas , Vesículas Extracelulares , Neoplasias Pulmonares , MicroARNs/genética , Biomarcadores , Biomarcadores de Tumor/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/cirugía , Vesículas Extracelulares/genética , Vesículas Extracelulares/patología , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/cirugía , Recurrencia Local de Neoplasia/genéticaRESUMEN
Reduced p62 levels are associated with the induction of the cancer-associated fibroblast (CAF) phenotype, which promotes tumorigenesis in vitro and in vivo through inflammation and metabolic reprogramming. However, how p62 is downregulated in the stroma fibroblasts by tumor cells to drive CAF activation is an unresolved central issue in the field. Here we show that tumor-secreted lactate downregulates p62 transcriptionally through a mechanism involving reduction of the NAD+/NADH ratio, which impairs poly(ADP-ribose)-polymerase 1 (PARP-1) activity. PARP-1 inhibition blocks the poly(ADP-ribosyl)ation of the AP-1 transcription factors, c-FOS and c-JUN, which is an obligate step for p62 downregulation. Importantly, restoring p62 levels in CAFs by NAD+ renders CAFs less active. PARP inhibitors, such as olaparib, mimick lactate in the reduction of stromal p62 levels, as well as the subsequent stromal activation both in vitro and in vivo, which suggests that therapies using olaparib would benefit from strategies aimed at inhibiting CAF activity.
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Fibroblastos Asociados al Cáncer , Neoplasias , Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos/metabolismo , Ácido Láctico/metabolismo , NAD/metabolismo , Neoplasias/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismoRESUMEN
BACKGROUND: When genes responsible for normal embryonic development are abnormally expressed in adults, it can lead to tumor development. This can suggest that the same mechanism that controls embryonic differentiation can also control tumor differentiation. We hypothesize that the malignant phenotype of lung cancer cells could acquire benign characteristics when in contact with an embryonic lung microenvironment. We cultured two lung cancer cell lines in embryonic lung mesenchyme-conditioned medium and evaluated morphological, functional and molecular changes. METHODS: The human embryonic mesenchymal lung-conditioned medium (hEML-CM) was obtained by culturing lung cells from embryos in the pseudoglandular stage of development. The NSCLC cell lines A549 and H1299 we cultured in the hEML-CM and in a tumor-conditioned medium. Morphological changes were analyzed with optical and transmission electron microscopy. To evaluate the functional effect of conditioned medium in tumor cells, we analyzed cell proliferation, migration, colony formation capacity in 2D and 3D and in vivo tumor growth capacity. The expression of the pluripotency genes OSKM, the adenocarcinoma marker NKX2-1, the lung surfactant proteins SFTP, the myofibroblast marker MYH and DNMT3A/3B was analyzed with qRT-PCR and the presence of the myofibroblast markers vimentin and α-SMA with immunofluorescence. Transcriptomic analysis was performed using Affymetrix arrays. RESULTS: The A549 and H1299 cells cultured in hEML-CM lost their epithelial morphology, acquired mesodermal characteristics, and decreased proliferation, migration, and colony formation capacity in 2D and 3D, as well as reduced its capacity to growth in vivo. The expression of OSKM, NKX2-1 and SFTP decreased, while that of DNMT3A/3B, vimentin, α-SMA and MYH increased. Distant matrix analysis based on transcriptomic profile showed that conditioned cells were closer to myoblast and human lung fibroblast than to normal epithelial immortalized lung cells. A total of 1631 for A549 and 866 for H1299 differentially expressed genes between control and conditioned cells were identified. CONCLUSIONS: To the best of our knowledge, this is the first study to report that stimuli from the embryonic lung can modulate the malignant phenotype of lung cancer cells, control their growth capacity and activate their differentiation into myofibroblasts. These findings could lead to new strategies for lung cancer management.
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Adenocarcinoma del Pulmón/genética , Células Madre Embrionarias Humanas/metabolismo , Neoplasias Pulmonares/genética , Miofibroblastos/metabolismo , Adenocarcinoma del Pulmón/fisiopatología , Animales , Diferenciación Celular , Línea Celular Tumoral , Proliferación Celular , Humanos , Neoplasias Pulmonares/fisiopatología , Masculino , Ratones , Ratones Desnudos , FenotipoRESUMEN
In resected non-small cell lung cancer (NSCLC), postsurgical recurrence is the major factor affecting long-term survival. The identification of biomarkers in extracellular vesicles (EV) obtained from serial blood samples after surgery could enhance early detection of relapse and improve NSCLC outcome. Since EV cargo contains long non-coding RNAs (lncRNAs), we aimed to analyze whether the oncogenic lncRNA HOTTIP, which higher expression in tumor tissue was related to worse outcome in NSCLC, could be detected in EV from NSCLC patients and serve as recurrence biomarker. After purification of EVs by ultracentrifugation in 52 serial samples from 18 NSCLC patients, RNA was isolated and HOTTIP was quantified by Real time PCR. We observed that patients that relapsed after surgery displayed increased postsurgical EV HOTTIP levels in comparison with presurgical levels. In the relapsed patients with several samples available between surgery and relapse, we observed an increment in the EV HOTTIP levels when approaching to relapse, which indicated its potential utility for monitoring disease recurrence. When we focused in EV HOTTIP levels in the first post-surgical sample, we observed that the detection of an increment of the expression levels in comparison to presurgical sample, predicted recurrence with high sensitivity (85.7%) and specificity (90.9%) and that patients had shorter time to relapse and shorter overall survival. In conclusion, our pilot study showed that EV HOTTIP is a potential biomarker for monitoring disease recurrence after surgery in NSCLC.
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Preoperative chemoradiotherapy (CRT) is a standard treatment for locally advanced rectal cancer (RC) patients, but its use in non-responders can be associated with increased toxicities and resection delay. LincRNA-p21 is a long non-coding RNA involved in the p53 pathway and angiogenesis regulation. We aimed to study whether lincRNA-p21 expression levels can act as a predictive biomarker for neoadjuvant CRT response. We analyzed RNAs from pretreatment biopsies from 70 RC patients treated with preoperative CRT. Pathological response was classified according to the tumor regression grade (TRG) Dworak classification. LincRNA-p21 expression was determined by RTqPCR. The results showed that lincRNA-p21 was upregulated in stage III tumors (p = 0.007) and in tumors with the worst response regarding TRG (p = 0.027) and downstaging (p = 0.016). ROC curve analysis showed that lincRNA-p21 expression had the capacity to distinguish a complete response from others (AUC:0.696; p = 0.014). LincRNA-p21 was shown as an independent marker of preoperative CRT response (p = 0.047) and for time to relapse (TTR) (p = 0.048). In conclusion, lincRNA-p21 is a marker of advanced disease, worse response to neoadjuvant CRT, and shorter TTR in locally advanced RC patients. The study of lincRNA-p21 may be of value in the individualization of pre-operative CRT in RC.
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During the last years, the study of extracellular vesicles (EVs) and its cargo has gained interest in the scientific media. EVs have been found in all biofluids and it is postulated that all cells are capable to secrete a wide variety of these vesicles, which play a key role in different cell-to-cell communication processes as well as in the microenvironment modulation. In the EV cargo, DNA, protein, and RNA molecules can be found, including long noncoding RNAs (lncRNAs). Several authors consider the study of EV lncRNAs an ideal source of biomarkers due to the easy sampling of EVs in different biofluids and the high specificity of the lncRNA expression pattern.In the present chapter, a detailed explanation of the EV isolation workflow followed by RNA isolation and lncRNA gene expression study is provided for two sample sources: blood plasma and cell culture conditioned media. EVs from both plasma samples and cell cultured media are isolated using sequential ultracentrifugation method (UC), which has been reported as one of the best methods available to date in terms of purity. UC is followed by RNA extraction based on the combination of phenol/guanidine-based lysis of samples with silica-membrane-based purification of total RNA. LncRNA quantification is performed by qRT-PCR. This chapter includes detailed discussion on lncRNA quantification using hydrolysis probes, recommended housekeeping genes and evaluation of methods for comparing lncRNA levels between EVs and its parental cells. In summary, we describe here the main steps for a successful isolation of the EVs-lncRNA cargo, paying attention to how overcome the different challenges found in the experimental procedure and in the data analysis of lncRNA expression from this source.
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Ácidos Nucleicos Libres de Células , Medios de Cultivo Condicionados/metabolismo , Vesículas Extracelulares/metabolismo , ARN Largo no Codificante/genética , Biomarcadores , Fraccionamiento Químico/métodos , Exosomas/metabolismo , Humanos , Biopsia Líquida/métodos , Reacción en Cadena de la Polimerasa , ARN Largo no Codificante/sangre , ARN Largo no Codificante/aislamiento & purificación , ARN Largo no Codificante/metabolismo , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: Circular RNAs (circRNAs) are non-coding RNAs with a circular structure that have recently emerged as important regulators of tumorogenesis. Recently, several circRNAS, including circ-10720 have been related to epithelial-mesenchymal transition (EMT) process. In the present study, we have analyzed the role of circ-10720 in non-small-cell lung cancer (NSCLC) and studied its prognostic relevance in resected stage I-IIIa NSCLC patients. METHODS: Circ-10720 expression was analyzed using a custom TaqMan assay in four NSCLC cell lines (HCC44, A549, H23 and H1299) and in the normal immortalized lung cell line BEAS2B. Silencing of circ-10720 was performed using two custom siRNAs which were transfected using lipofectamine 2000. Protein levels were evaluated by Western blot and immunofluorescence. Wound healing and invasion assays were performed to evaluate the impact the circRNA on cell motility. Apoptosis was analyzed by evaluation of Caspase 3-7 activity and proliferation by MTS assay. Moreover, the expression levels of the circRNA were studied in 119 resected NSCLC patients. The expression in tumor tissue was correlated with the main clinicopathological characteristics and with time to relapse (TTR). RESULTS: Circ-10720 was overexpressed in HCC44 and A549 and underexpressed in H23 and H1299 NSCLC cell lines in comparison to BEAS2B normal immortalized lung cell line. CircRNA knockdown in the two circ-10720 overexpressing cell lines was associated with a decrease of Vimentin (VIM) and an increase of E-cadherin (CDH1) protein levels, loss of mesenchymal phenotype, and a significant reduction of migration and invasion capacity. After silencing circ-10720, the apoptosis rate increased and the proliferation was significantly reduced. Furthermore, circ-10720 was upregulated in tumor vs. normal tissue from 119 resected NSCLC patients. In the group of patients not receiving adjuvant treatment, those with high levels of circ-10720 had a shorter TTR than those with low levels and emerged as an independent prognostic value in the multivariate analysis. In tumor tissue, circ-10720 levels positively correlated with the EMT gene Twist1 levels. CONCLUSIONS: Circ-10720 regulates EMT, apoptosis and proliferation and acts as a biomarker of relapse in NSCLC.
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BACKGROUND: A therapeutic approach for the treatment of glucocorticoid-induced skeletal muscle atrophy should be based on the knowledge of the molecular mechanisms determining the unbalance between anabolic and catabolic processes and how to re-establish this balance. Here, we investigated whether the obestatin/GPR39 system, an autocrine signalling system acting on myogenesis and with anabolic effects on the skeletal muscle, could protect against chronic glucocorticoid-induced muscle atrophy. METHODS: In this study, we used an in vivo model of muscle atrophy induced by the synthetic glucocorticoid dexamethasone to examine the liaison molecules that define the interaction between the glucocorticoid receptor and the obestatin/GPR39 systems. The findings were extended to in vitro effects on human atrophy using human KM155C25 myotubes. RESULTS: KLF15 and FoxO transcription factors were identified as direct targets of obestatin signalling in the control of proteostasis in skeletal muscle. The KLF15-triggered gene expression program, including atrogenes and FoxOs, was regulated via KLF15 ubiquitination by the E3 ubiquitin ligase NEDD4. Additionally, a specific pattern of FoxO post-translational modification, including FoxO4 phosphorylation by Akt pathway, was critical in the regulation of the ubiquitin-proteasome system. The functional cooperativity between Akt and NEDD4 in the regulation of FoxO and KLF15 provides integrated cues to counteract muscle proteostasis and re-establish protein synthesis. CONCLUSIONS: The effective control of FoxO activity in response to glucocorticoid is critical to counteract muscle-related pathologies. These results highlight the potential of the obestatin/GPR39 system to fine-tune the effects of glucocorticoids on skeletal muscle wasting.
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Transducción de Señal , Ghrelina , Glucocorticoides , Humanos , Factores de Transcripción de Tipo Kruppel , Músculo Esquelético , Atrofia Muscular/inducido químicamente , Atrofia Muscular/tratamiento farmacológico , Ubiquitina-Proteína Ligasas Nedd4 , Receptores Acoplados a Proteínas G/genéticaRESUMEN
In vivo interrogation of the functional role of genes implicated in colorectal cancer (CRC) is limited by the need for physiological models that mimic the disease. Here, we describe a protocol that provides the steps required for the orthotopic co-implantation of tumoral and stromal cells into the cecum and rectum to investigate the crosstalk between the tumor and its microenvironment. This protocol recapitulates metastases to the lymph nodes, liver, and lungs observed in human CRC. For complete details on the use and execution of this protocol, please refer to Kasashima et al. (2020).