RESUMO
INTRODUCTION: The study explored the effects of SHOX2 and RASSF1A DNA methylation in lung cancer (LC). METHOD: Bronchoalveolar lavage fluid (BALF) samples as well as LC and normal adjacent tissues were collected from 72 LC patients and 35 patients with benign pulmonary nodules. Quantitative analysis of SHOX2 and RASSF1A DNA methylation was performed in benign pulmonary nodules and different stages of LC. The diagnostic value of SHOX2 and RASSF1A DNA methylation in LC and benign pulmonary nodules was determined by receiver operating characteristics analysis. Gain/loss-of-function experiments were constructed in LC cells and mouse models of xenograft and pulmonary nodule metastasis. The levels of SHOX2 and transfer-associated genes were tested through quantitative reverse transcription polymerase chain reaction and Western blot. Malignant phenotype of LC cells was assessed by functional experiment. The tumor volume and weight of mice in xenograft models were measured. Pulmonary nodule metastasis was determined through HE staining assay. 5-azacytidine appeared as a positive control drug. RESULT: SHOX2 DNA methylation or RASSF1A DNA methylation had diagnostic efficiency in pulmonary nodules and early LC, with the two combined having better diagnostic value. SHOX2 expression was upregulated in LC. Similar to 5-azacytidine, SHOX2 knockdown inhibited LC cell viability, migration, and invasion in vitro as well as restrained LC tumorigenesis and pulmonary nodule metastasis in vivo, whereas overexpressed SHOX2 had the opposite effects. CONCLUSION: The combination of SHOX2 and RASSF1A DNA methylation had a diagnostic value in pulmonary nodules and early LC. SHOX2 positively modulated the tumorigenesis and metastasis of LC by regulating DNA methylation processes.
Assuntos
Metilação de DNA , Proteínas de Homeodomínio , Neoplasias Pulmonares , Proteínas Supressoras de Tumor , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/diagnóstico , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Animais , Camundongos , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Masculino , Feminino , Pessoa de Meia-Idade , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Idoso , Proliferação de Células , Biomarcadores Tumorais/genética , Movimento CelularRESUMO
Background The methylation of SHOX2 and RASSF1A shows promise as a potential biomarker for the early screening of lung cancer, offering a solution to remedy the limitations of morphological diagnosis. The aim of this study is to diagnose lung adenocarcinoma by measuring the methylation levels of SHOX2 and RASSF1A, and provide an accurate pathological diagnosis to predict the invasiveness of lung cancer prior to surgery.Material and methods The methylation levels of SHOX2 and RASSF1A were quantified using a LungMe® test kit through methylation-specific PCR (MS-PCR). The diagnostic efficacy of SHOX2 and RASSF1A and the cutoff values were validated using ROC curve analysis. The hazardous factors influencing the invasiveness of lung adenocarcinoma were calculated using multiple regression.Results: The cutoff values of SHOX2 and RASSF1A were 8.3 and 12.0, respectively. The sensitivities of LungMe® in IA, MIA and AIS patients were 71.3% (122/171), 41.7% (15/36), and 16.1% (5/31) under the specificity of 94.1% (32/34) for benign lesions. Additionally, the methylation level of SHOX2, RASSF1A and LungMe® correlated with the high invasiveness of clinicopathological features, such as age, gender, tumor size, TNM stage, pathological type, pleural invasion and STAS. The tumor size, age, CTR values and LungMe® methylation levels were identified as independent hazardous factors influencing the invasiveness of lung adenocarcinoma.Conclusion: SHOX2 and RASSF1A combined methylation can be used as an early detection indicator of lung adenocarcinoma. SHOX2 and RASSF1A combined (LungMe®) methylation is significantly correlated to age, gender, tumor size, TNM stage, pathological type, pleural invasion and STAS. The SHOX2 and RASSF1A methylation levels, tumor size and CTR values could predict the invasiveness of the tumor prior to surgery, thereby providing guidance for the surgical procedure.
Assuntos
Adenocarcinoma de Pulmão , Biomarcadores Tumorais , Metilação de DNA , Proteínas de Homeodomínio , Neoplasias Pulmonares , Estadiamento de Neoplasias , Proteínas Supressoras de Tumor , Humanos , Proteínas Supressoras de Tumor/genética , Masculino , Feminino , Pessoa de Meia-Idade , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Idoso , Proteínas de Homeodomínio/genética , Biomarcadores Tumorais/genética , Adulto , Curva ROCRESUMO
Methylation of the promoters of SHOX2 and RASSF1A (LungMe®) exhibits promise as a potential molecular biomarker for diagnosing lung cancer. This study sought to assess the aberrant methylation of SHOX2 and RASSF1A in broncho-exfoliated cells (BEC) and compare it with conventional cytology, histology examination, immunohistochemistry, and serum tumor markers to evaluate the overall diagnostic efficiency for lung cancer. This study recruited 240 patients, including 185 malignant cases and 55 benign cases. In our observation, we noted a slight reduction in the detection sensitivity, however, the ΔCt method exhibited a significant enhancement in specificity when compared to Ct judgment. Consequently, the ΔCt method proves to be a more appropriate approach for interpreting methylation results. The diagnostic sensitivity of cytology and histology was in ranged from 20.0%-35.1% and 42.9%-80%, respectively, while the positive detection rate of LungMe® methylation ranged from 70.0% to 100%. Additionally, our findings indicate a higher prevalence of SHOX2( +) among patients exhibiting medium and high expression of Ki67 (P < 0.01), as opposed to those with low expression of Ki67, but RASSF1A methylation did not show this phenomenon (P = 0.35). Furthermore, CEA, SCCA, and CYFRA21-1 showed positive detection rates of 48.8%, 26.2%, and 55.8%, respectively. Finally, we present a comprehensive lung cancer diagnostic work-up, including LumgMe® methylation. The combined analysis of SHOX2 and RASSF1A methylation serves as a powerful complement and extension to conventional methods, enhancing the accuracy of a lung cancer diagnosis with satisfactory sensitivity and specificity.
Assuntos
Antígenos de Neoplasias , Queratina-19 , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Metilação de DNA , Antígeno Ki-67/metabolismo , Proteínas de Homeodomínio/genéticaRESUMO
AIM: To investigate the diagnostic value of combined detection of SHOX2 and RASSF1A gene methylation with carcinoembryonic antigen (CEA) level in diagnosing malignant pleural effusion. METHODS: Between March 2020 and December 2021, we enrolled 68 patients with pleural effusion admitted to the Department of Respiratory and critical care medicine of Foshan Second People's Hospital. The study group included 35 cases of malignant pleural effusion and 33 cases of benign pleural effusion. Methylation of the short homeobox 2 genes (SHOX2) and RAS-related region family 1A gene (RASSF1A) in pleural effusion samples were detected by real-time fluorescence quantitative PCR, and the level of carcinoembryonic antigen (CEA) in pleural effusion samples was detected by immune flow cytometry fluorescence quantitative chemiluminescence. RESULTS: SHOX2 or RASSF1A gene methylation was detected in 5 cases in the benign pleural effusion group and 25 patients in the malignant pleural effusion group. The positive rate of SHOX2 or RASSF1A gene methylation in the malignant pleural effusion group was significantly higher than in the benign pleural effusion group (71.4% vs. 15.2%, P < 0.01). Positive CEA (CEA > 5 ng/m) was detected in 1 case in the benign pleural effusion group and 26 patients in the malignant pleural effusion group. The CEA-positive rate in the malignant pleural effusion group was significantly higher than in the benign pleural effusion group (74.3% vs. 3%, P < 0.01). When SHOX2 and RASSF1A gene methylation was combined with CEA detection, 6 cases were positive in the benign pleural effusion group, and 31 patients were positive in the malignant pleural effusion group. The positive rate of combined detection in the malignant pleural effusion group was significantly higher than in the benign pleural effusion group (88.6% vs. 18.2%, P < 0.01). The sensitivity, specificity, accuracy, positive predictive value, negative predictive value, and Youden's index of SHOX2, RASSF1A gene methylation combined with CEA in diagnosing malignant pleural effusion were 88.6%, 81.8%, 85.3%, 83.8%, 87.1% and 0.7 respectively. CONCLUSION: The combined detection of SHOX2 and RASSF1A gene methylation with CEA level in pleural effusion has a high diagnostic value for malignant pleural effusion.
Assuntos
Derrame Pleural Maligno , Derrame Pleural , Humanos , Derrame Pleural Maligno/diagnóstico , Derrame Pleural Maligno/genética , Antígeno Carcinoembrionário , Metilação , Derrame Pleural/genética , Exsudatos e Transudatos , Proteínas de Homeodomínio/genéticaRESUMO
BACKGROUND: Short stature homeobox 2 (SHOX2) is significant gene in the development and progression of multiple types of tumors. Nonetheless, the biological role of SHOX2 within pan-cancer datasets has not been investigated. Thus, comprehensive bioinformatics analyses of pan-cancer datasets were conducted to explore how SHOX2 regulates tumorigenesis. METHODS: A variety of tumor datasets and online analytical tools, including SangerBox, TIMER2, LinkedOmic, GEPIA2 and cBioPortal, were applied to explore SHOX2 expression in various tumors. To ascertain the connections between SHOX2 expression and genetic alterations, SHOX2-related genes and tumor immunity, the pan-cancer datasets were examined. In vitro assays were applied to verify the biological functions of SHOX2 in glioma cells via CCK-8, wound healing, Transwell and colony formation assays. RESULTS: Analyses found that SHOX2 was overexpressed in multiple cancer types. SHOX2 expression level was significantly correlated with isocitrate dehydrogenase (IDH), 1p/19q, O6-methylguanine DNA methyltransferase (MGMT) status and new types of glioma patients. High mRNA expression levels of SHOX2 were associated with a poor prognosis in multiple tumor patients. KEGG enrichment analysis showed that SHOX2-related genes were associated with cell cycle and DNA damage repair. Genetic alterations of SHOX2 were identified in multiple types of cancers, including duplications and deep mutations. Immune analysis showed that SHOX2 was closely correlated with the tumor mutation burden (TMB), microsatellite instability (MSI), neoantigen and neoantigens and immune checkpoint (ICP) in a variety of tumors and could influence the immunotherapy sensitivity of cancers. CCK-8, wound healing, Transwell and colony formation experiments showed that SHOX2 knockdown inhibited glioma cell proliferation, migration, invasion and colony formation abilities. CONCLUSION: SHOX2 was overexpressed in multiple cancer types in TCGA cohort. SHOX2 knockdown inhibited glioma cell proliferation, migration and colony formation ability. Our study showed that SHOX2 may be an immunotherapeutic and promising prognostic biomarker in certain types of tumors.
Assuntos
Glioma , Sincalida , Humanos , Prognóstico , Glioma/genética , Glioma/terapia , Divisão Celular , Biomarcadores , Proteínas de Homeodomínio/genéticaRESUMO
MiRNAs are essential epigenetic modulators that can regulate protein expression. According to the principle of base complementary pairing, miRNA is partially or completely complementary to the 3'-UTR region of its target gene, by which it inhibits the translation of the targeted gene. This study investigated the role of miR-24-1-5p in clear cell renal cell carcinoma (ccRCC). Data in TCGA-KIRC denoted that miR-24-1-5p was under-expressed in ccRCC. Bioinformatics analysis predicted that its target gene was SHOX2, which was significantly expressed in cancer tissues. Dual luciferase assay verified the targeting relationship between miR-24-1-5p and SHOX2. Cell function experiments demonstrated that overexpression of miR-24-1-5p significantly inhibited SHOX2 level and the malignant phenotypes of ccRCC cells. The above results illustrated that miR-24-1-5p/SHOX2 axis was critical for the oncogenesis and development of ccRCC, which might be helpful for us to understand the mechanism and novel therapeutic methods of ccRCC.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , MicroRNAs , Humanos , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Neoplasias Renais/genética , Neoplasias Renais/patologia , Linhagem Celular Tumoral , MicroRNAs/genética , MicroRNAs/metabolismo , Proliferação de Células/genética , Movimento Celular/genética , Regiões 3' não Traduzidas , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genéticaRESUMO
The sinoatrial node (SAN), the primary cardiac pacemaker, consists of a head domain and a junction/tail domain that exhibit different functional properties. However, the underlying molecular mechanism defining these two pacemaker domains remains elusive. Nkx2-5 is a key transcription factor essential for the formation of the working myocardium, but it was generally thought to be detrimental to SAN development. However, Nkx2-5 is expressed in the developing SAN junction, suggesting a role for Nkx2-5 in SAN junction development and function. In this study, we present unambiguous evidence that SAN junction cells exhibit unique action potential configurations intermediate to those manifested by the SAN head and the surrounding atrial cells, suggesting a specific role for the junction cells in impulse generation and in SAN-atrial exit conduction. Single-cell RNA-seq analyses support this concept. Although Nkx2-5 inactivation in the SAN junction did not cause a malformed SAN at birth, the mutant mice manifested sinus node dysfunction. Thus, Nkx2-5 defines a population of pacemaker cells in the transitional zone. Despite Nkx2-5 being dispensable for SAN morphogenesis during embryogenesis, its deletion hampers atrial activation by the pacemaker.
Assuntos
Relógios Biológicos/genética , Linhagem da Célula/genética , Proteína Homeobox Nkx-2.5/fisiologia , Miócitos Cardíacos/citologia , Nó Sinoatrial/citologia , Nó Sinoatrial/fisiologia , Animais , Separação Celular , Embrião de Mamíferos , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Átrios do Coração/citologia , Átrios do Coração/embriologia , Camundongos , Camundongos Transgênicos , Morfogênese/genética , Contração Miocárdica/genética , Miócitos Cardíacos/fisiologia , GravidezRESUMO
BACKGROUND: DNA methylation on cytosine in the CpG dinucleotides is one of the most common epigenetic perturbations taking place during cancer initiation, progression, occurrence and resistance therapy. DNA methylation seems to be sufficiently stable epigenetic modification to be utilized as a cancer biomarker in in vitro diagnostic (IVD) settings. Nowadays, the SHOX2 methylation (mSHOX2) is one of the most valuable DNA methylation biomarkers of lung cancer that is the leading cause of cancer death. It is being continuously validated across ethnicities, lifestyles and lifespan. This study focused on characteristics of mSHOX2 in Vietnamese patients with lung cancer since a lack of investigation and evidence of its utility in this country. METHODS: The probe and primer sets were designed according to the MethyLight method for quantitative assessment of the mSHOX2 in 214 formalin-fixed paraffin-embedded (FFPE) lung tissues and 57 plasma samples. RESULTS: mSHOX2 in FFPE tissues allowed discriminating benign and malignant lung diseases with 60% (95% CI 50.7-68.8%) sensitivity and 90.4% (95% CI 82.6-95.5%) specificity. Importantly, based on mSHOX2 in plasma, lung cancer could be detected with 83.3% (95% CI 65.3-94.4%) sensitivity and 92.6% (95% CI 75.7-99.1%) specificity, respectively. There were insignificant associations between mSHOX2 with age, cancer stage, EGFR mutation and serum CEA, CYFRA21-1 concentrations except for that gender. CONCLUSION: Our study indicated that mSHOX2 was satisfactory for distinguishing malignant from benign lung tissue and noninvasively detecting lung cancer.
Assuntos
Metilação de DNA , Proteínas de Homeodomínio , Neoplasias Pulmonares , Antígenos de Neoplasias , Povo Asiático , Biomarcadores Tumorais/genética , Proteínas de Homeodomínio/genética , Humanos , Queratina-19 , Neoplasias Pulmonares/patologiaRESUMO
The sinoatrial node (SAN) is the primary pacemaker in the heart. During cardiogenesis, Shox2 and Nkx2-5 are co-expressed in the junction domain of the SAN and regulate pacemaker cell fate through a Shox2-Nkx2-5 antagonism. Cx40 is a marker of working myocardium and an Nkx2-5 transcriptional output antagonized by Shox2, but the underlying regulatory mechanisms remain elusive. Here we characterized a bona fide myocardial-specific Gja5 (coding gene of Cx40) distal enhancer consisting of a pair of Nkx2-5 and Shox2 co-bound elements in the regulatory region of Gja5. Transgenic reporter assays revealed that neither element alone, but the conjugation of both elements together, drives myocardial-specific transcription. Genetic analyses confirmed that the activation of this enhancer depends on Nkx2-5 but is inhibited by Shox2 in vivo, and its presence is essential for Gja5 expression in the myocardium but not the endothelial cells of the heart. Furthermore, chromatin conformation analysis showed an Nkx2-5-dependent loop formation between these two elements and the Gja5 promoter in vivo, indicating that Nkx2-5 bridges the conjugated activation of this enhancer by pairing the two elements to the Gja5 promoter.
Assuntos
Conexinas/biossíntese , Proteína Homeobox Nkx-2.5/metabolismo , Proteínas de Homeodomínio/metabolismo , Miocárdio/metabolismo , Regiões Promotoras Genéticas , Nó Sinoatrial/embriologia , Transcrição Gênica , Animais , Conexinas/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteína Homeobox Nkx-2.5/genética , Proteínas de Homeodomínio/genética , Camundongos , Camundongos TransgênicosRESUMO
Haploinsufficiency of Meis homeobox 2 (MEIS2), encoding a transcriptional regulator, is associated with human cleft palate, and Meis2 inactivation leads to abnormal palate development in mice, implicating MEIS2 functions in palate development. However, its functional mechanisms remain unknown. Here we observed widespread MEIS2 expression in the developing palate in mice. Wnt1Cre -mediated Meis2 inactivation in cranial neural crest cells led to a secondary palate cleft. Importantly, about half of the Wnt1Cre ;Meis2f/f mice exhibited a submucous cleft, providing a model for studying palatal bone formation and patterning. Consistent with complete absence of palatal bones, the results from integrative analyses of MEIS2 by ChIP sequencing, RNA-Seq, and an assay for transposase-accessible chromatin sequencing identified key osteogenic genes regulated directly by MEIS2, indicating that it plays a fundamental role in palatal osteogenesis. De novo motif analysis uncovered that the MEIS2-bound regions are highly enriched in binding motifs for several key osteogenic transcription factors, particularly short stature homeobox 2 (SHOX2). Comparative ChIP sequencing analyses revealed genome-wide co-occupancy of MEIS2 and SHOX2 in addition to their colocalization in the developing palate and physical interaction, suggesting that SHOX2 and MEIS2 functionally interact. However, although SHOX2 was required for proper palatal bone formation and was a direct downstream target of MEIS2, Shox2 overexpression failed to rescue the palatal bone defects in a Meis2-mutant background. These results, together with the fact that Meis2 expression is associated with high osteogenic potential and required for chromatin accessibility of osteogenic genes, support a vital function of MEIS2 in setting up a ground state for palatal osteogenesis.
Assuntos
Proteínas de Homeodomínio/metabolismo , Osteogênese , Palato/metabolismo , Animais , Sítios de Ligação , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Endogâmicos C57BL , Crista Neural/citologia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Palato/embriologia , Ligação ProteicaRESUMO
Touch and mechanical sensations require the development of several different kinds of sensory neurons dedicated to respond to certain types of mechanical stimuli. The transcription factor Shox2 (short stature homeobox 2) is involved in the generation of TRKB+ low-threshold mechanoreceptors (LTMRs), but mechanisms terminating this program and allowing alternative fates are unknown. Here, we show that the conditional loss of the miR-183-96-182 cluster in mouse leads to a failure of extinction of Shox2 during development and an increase in the proportion of Aδ LTMRs (TRKB+/NECAB2+) neurons at the expense of Aß slowly adapting (SA)-LTMRs (TRKC+/Runx3-) neurons. Conversely, overexpression of miR-183 cluster that represses Shox2 expression, or loss of Shox2, both increase the Aß SA-LTMRs population at the expense of Aδ LTMRs. Our results suggest that the miR-183 cluster determines the timing of Shox2 expression by direct targeting during development, and through this determines the population sizes of Aδ LTMRs and Aß SA-LTMRs.
Assuntos
Proteínas de Homeodomínio/metabolismo , Mecanorreceptores/metabolismo , MicroRNAs/genética , Células Receptoras Sensoriais/citologia , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Diferenciação Celular/genética , Proteínas do Olho/metabolismo , Feminino , Humanos , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Gravidez , Proteínas Tirosina Quinases/metabolismoRESUMO
BACKGROUND: Urodele amphibians are capable of regenerating their organs after severe damage. During such regeneration, participating cells are given differentiation instructions by the surrounding cells. Limb regeneration has been investigated as a representative phenomenon of organ regeneration. Cells known as blastema cells are induced after limb amputation. In this process, dermal fibroblasts are dedifferentiated and become undifferentiated similar to limb bud cells. Just like limb bud cells, the induced blastema cells are positioned along the three limb developmental axes: the dorsoventral, the anteroposterior, and the proximodistal. The accurate developmental axes are essential for reforming the structures correctly. Despite the importance of the developmental axes, the relationship between the newly establishing developmental axes and existing limb axes was not well described with molecular markers. RESULTS: In this study, we grafted skin from GFP-transgenic axolotls and traced the cell lineage with position-specific gene expressions in order to investigate the correlation of the newly established axes and cellular origin. Shh- and Lmx1b-expressing cells emerged from the posterior skin and dorsal skin, respectively, even though the skin was transplanted to an inconsistent position. Shox2, a posterior marker gene, could be activated in cells derived from distal skin. CONCLUSIONS: Our results suggest that the location memories on anteroposterior and dorsoventral axes are relatively stable in a regenerating blastema though cellular differentiation is reprogrammed.
Assuntos
Ambystoma mexicanum/embriologia , Ambystoma mexicanum/fisiologia , Animais , Diferenciação Celular/fisiologia , Extremidades/embriologia , Extremidades/fisiologia , Imunofluorescência , Hibridização In Situ , Regeneração/fisiologiaRESUMO
During mammalian palatogenesis, cranial neural crest-derived mesenchymal cells undergo osteogenic differentiation and form the hard palate, which is divided into palatine process of the maxilla and the palatine. However, it remains unknown whether these bony structures originate from the same cell lineage and how the hard palate is patterned at the molecular level. Using mice, here we report that deficiency in Shox2 (short stature homeobox 2), a transcriptional regulator whose expression is restricted to the anterior palatal mesenchyme, leads to a defective palatine process of the maxilla but does not affect the palatine. Shox2 overexpression in palatal mesenchyme resulted in a hyperplastic palatine process of the maxilla and a hypoplastic palatine. RNA sequencing and assay for transposase-accessible chromatin-sequencing analyses revealed that Shox2 controls the expression of pattern specification and skeletogenic genes associated with accessible chromatin in the anterior palate. This highlighted a lineage-autonomous function of Shox2 in patterning and osteogenesis of the hard palate. H3K27ac ChIP-Seq and transient transgenic enhancer assays revealed that Shox2 binds distal-acting cis-regulatory elements in an anterior palate-specific manner. Our results suggest that the palatine process of the maxilla and palatine arise from different cell lineages and differ in ossification mechanisms. Shox2 evidently controls osteogenesis of a cell lineage and contributes to the palatine process of the maxilla by interacting with distal cis-regulatory elements to regulate skeletogenic gene expression and to pattern the hard palate. Genome-wide Shox2 occupancy in the developing palate may provide a marker for identifying active anterior palate-specific gene enhancers.
Assuntos
Diferenciação Celular/genética , Proteínas de Homeodomínio/genética , Osteogênese/genética , Palato Duro/metabolismo , Animais , Padronização Corporal/genética , Linhagem da Célula/genética , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Humanos , Maxila/citologia , Maxila/embriologia , Maxila/metabolismo , Camundongos Knockout , Camundongos Transgênicos , Palato Duro/citologia , Palato Duro/embriologia , Transdução de Sinais/genéticaRESUMO
Disorder of long non-coding RNAs (LncRNAs) is found in various types of cancers and demonstrated to be associated with tumor occurrence and development. Our study found that lncRNA insulin growth factor 2 antisense (IGF2-AS) is up-regulated in gastric adenocarcinoma (GAC) tissues and correlated with poor prognosis in patients with GAC. Cell counting kit-8 (CCK8), colony formation, wound healing and transwell assays revealed that knockdown of IGF2-AS in BGC823 and SGC7901 cells significantly suppressed cell proliferation, migration and invasion. While, overexpression of IGF2-AS in AGS and MGC803 cells exhibited the opposite effects. RNA-FISH and subcellular fractionation assay found that most IGF2-AS was distributed in the cytoplasm, suggesting that IGF2-AS functioned as a potential ceRNA. RNA binding protein immunoprecipitation (RIP) assays further confirmed this assumption. By informatics prediction and luciferase reporter assay, we found that IGF2-AS functioned as an efficient miR-503 sponge and the level of miR-503 showed an inverse correlation with IGF2-AS. Short stature homeobox 2 (SHOX2) is predicted and verified as a target of miR-503. Moreover, IGF2-AS expression exhibited a negative correlation with miR-503 and a positive correlation with IGF2-AS. Subsequent rescue assay revealed that down-regulation of miR-503 or restoration of SHOX2 canceled IGF2-AS depletion-induced depression in proliferation and motility of BGC823 and SGC7901 cells. Meanwhile, up-regulation of miR-503 or down-regulation of SHOX2 decreased IGF2-AS overexpression induced promotion in proliferation and motility of AGS and MGC803 cells. In vivo tumorigenicity assay showed that knockdown of IGF2-AS significantly reduced tumor volume. Taken together, our results demonstrated that IGF2-AS takes important regulatory parts in GAC development by functioning as a ceRNA to regulate SHOX2 via sponging miR-503.
Assuntos
Adenocarcinoma/genética , Proteínas de Homeodomínio/genética , MicroRNAs/genética , RNA Longo não Codificante/genética , Neoplasias Gástricas/genética , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Idoso , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Masculino , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Prognóstico , Proteínas , Neoplasias Gástricas/mortalidade , Neoplasias Gástricas/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Vertebrate appendage patterning is programmed by Hox-TALE factor-bound regulatory elements. However, it remains unclear which cell lineages are commissioned by Hox-TALE factors to generate regional specific patterns and whether other Hox-TALE co-factors exist. In this study, we investigated the transcriptional mechanisms controlled by the Shox2 transcriptional regulator in limb patterning. Harnessing an osteogenic lineage-specific Shox2 inactivation approach we show that despite widespread Shox2 expression in multiple cell lineages, lack of the stylopod observed upon Shox2 deficiency is a specific result of Shox2 loss of function in the osteogenic lineage. ChIP-Seq revealed robust interaction of Shox2 with cis-regulatory enhancers clustering around skeletogenic genes that are also bound by Hox-TALE factors, supporting a lineage autonomous function of Shox2 in osteogenic lineage fate determination and skeleton patterning. Pbx ChIP-Seq further allowed the genome-wide identification of cis-regulatory modules exhibiting co-occupancy of Pbx, Meis and Shox2 transcriptional regulators. Integrative analysis of ChIP-Seq and RNA-Seq data and transgenic enhancer assays indicate that Shox2 patterns the stylopod as a repressor via interaction with enhancers active in the proximal limb mesenchyme and antagonizes the repressive function of TALE factors in osteogenesis.
Assuntos
Padronização Corporal , Extremidades/embriologia , Proteínas de Homeodomínio/metabolismo , Osteogênese , Animais , Sequência de Bases , Sítios de Ligação/genética , Padronização Corporal/genética , Linhagem da Célula , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Elementos Facilitadores Genéticos , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Proteínas de Homeodomínio/genética , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Modelos Biológicos , Motivos de Nucleotídeos/genética , Osteogênese/genética , Ligação ProteicaRESUMO
In humans, atrial fibrillation is often triggered by ectopic pacemaking activity in the myocardium sleeves of the pulmonary vein (PV) and systemic venous return. The genetic programs that abnormally reinforce pacemaker properties at these sites and how this relates to normal sinoatrial node (SAN) development remain uncharacterized. It was noted previously that Nkx2-5, which is expressed in the PV myocardium and reinforces a chamber-like myocardial identity in the PV, is lacking in the SAN. Here we present evidence that in mice Shox2 antagonizes the transcriptional output of Nkx2-5 in the PV myocardium and in a functional Nkx2-5(+) domain within the SAN to determine cell fate. Shox2 deletion in the Nkx2-5(+) domain of the SAN caused sick sinus syndrome, associated with the loss of the pacemaker program. Explanted Shox2(+) cells from the embryonic PV myocardium exhibited pacemaker characteristics including node-like electrophysiological properties and the capability to pace surrounding Shox2(-) cells. Shox2 deletion led to Hcn4 ablation in the developing PV myocardium. Nkx2-5 hypomorphism rescued the requirement for Shox2 for the expression of genes essential for SAN development in Shox2 mutants. Similarly, the pacemaker-like phenotype induced in the PV myocardium in Nkx2-5 hypomorphs reverted back to a working myocardial phenotype when Shox2 was simultaneously deleted. A similar mechanism is also adopted in differentiated embryoid bodies. We found that Shox2 interacts with Nkx2-5 directly, and discovered a substantial genome-wide co-occupancy of Shox2, Nkx2-5 and Tbx5, further supporting a pivotal role for Shox2 in the core myogenic program orchestrating venous pole and pacemaker development.
Assuntos
Proteínas de Homeodomínio/fisiologia , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Veias Pulmonares/metabolismo , Nó Sinoatrial/metabolismo , Fatores de Transcrição/fisiologia , Animais , Relógios Biológicos , Diferenciação Celular , Linhagem da Célula , Separação Celular , Eletrocardiografia , Corpos Embrioides/metabolismo , Citometria de Fluxo , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Coração/embriologia , Proteína Homeobox Nkx-2.5 , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Fenótipo , Estrutura Terciária de Proteína , Proteínas com Domínio T/metabolismoRESUMO
Atrial fibrillation (AF), as the most common sustained cardiac arrhythmia, is associated with substantially increased morbidity and mortality. Aggregating evidence demonstrates that genetic defects play a crucial role in the pathogenesis of AF, especially in familial AF. Nevertheless, AF is of pronounced genetic heterogeneity, and in an overwhelming majority of cases the genetic determinants underlying AF remain elusive. In the current study, 162 unrelated patients with familial AF and 238 unrelated healthy individuals served as controls were recruited. The coding exons and splicing junction sites of the SHOX2 gene, which encodes a homeobox-containing transcription factor essential for proper development and function of the cardiac conduction system, were sequenced in all study participants. The functional effect of the mutant SHOX2 protein was characterized with a dual-luciferase reporter assay system. As a result, a novel heterozygous SHOX2 mutation, c.580C>T or p.R194X, was identified in an index patient, which was absent from the 476 control chromosomes. Genetic analysis of the proband's pedigree revealed that the nonsense mutation co-segregated with AF in the family with complete penetrance. Functional assays demonstrated that the mutant SHOX2 protein had no transcriptional activity compared with its wild-type counterpart. In conclusion, this is the first report on the association of SHOX2 loss-of-function mutation with enhanced susceptibility to familial AF, which provides novel insight into the molecular mechanism underpinning AF, suggesting potential implications for genetic counseling and individualized management of AF patients.
Assuntos
Fibrilação Atrial/metabolismo , Proteínas de Homeodomínio/metabolismo , Fibrilação Atrial/genética , Códon sem Sentido/genética , Feminino , Células HEK293 , Proteínas de Homeodomínio/genética , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Linhagem , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The formation and conduction of electrocardiosignals and the synchronous contraction of atria and ventricles with rhythmicity are both triggered and regulated by the cardiac conduction system (CCS). Defect of this system will lead to various types of cardiac arrhythmias. In recent years, the research progress of molecular genetics and developmental biology revealed a clearer understanding of differentiation and development of the cardiac conduction system and their regulatory mechanisms. Short stature homeobox 2 (Shox2) transcription factor, encoded by Shox2 gene in the mouse, is crucial in the formation and differentiation of the sinoatrial node (SAN). Shox2 drives embryonic development processes and is widely expressed in the appendicular skeleton, palate, temporomandibular joints, and heart. Mutations of Shox2 can lead to dysembryoplasia and abnormal phenotypes, including bradycardiac arrhythmia. In this review, we provide a summary of the latest research progress on the regulatory effects of the Shox2 gene in differentiation and development processes of the cardiac conduction system, hoping to deepen the knowledge and understanding of this systematic process based on the cardiac conduction system. Overall, the Shox2 gene is intimately involved in the differentiation and development of cardiac conduction system, especially sinoatrial node. We also summarize the current information about human SHOX2. This review article provides a new direction in biological pacemaker therapies.
Assuntos
Sistema de Condução Cardíaco/metabolismo , Proteínas de Homeodomínio/metabolismo , Nó Sinoatrial/citologia , Nó Sinoatrial/embriologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Relógios Biológicos , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Sistema de Condução Cardíaco/embriologia , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Humanos , CamundongosRESUMO
Esophageal squamous cell carcinoma (ESCC) is the most common histological type in China. MicroRNAs are endogenously expressed in mammals and play a significant role in tumor invasion and metastasis by targeting potential downstream genes. In the present study, microarray analysis showed that miR-375 expression was distinctly downregulated in ESCC compared with that in normal esophageal epithelium tissues. Then, luciferase reporter assay showed that SHOX2 was the direct downstream target of miR-375 and this interaction was confirmed by the rescue experiments. Quantitative polymerase chain reaction results also showed that SHOX2 expression was upregulated in ESCC cells and tissues. Further analysis showed that SHOX2 induced proliferation, invasion, and metastasis of ESCC both in vivo and in vitro. Moreover, the interaction between miR-375 and SHOX2 affected the epithelial-to-mesenchymal transition. We conclude that miR-375 may suppress invasion and metastasis of ESCC by directly targeting SHOX2. The miR-375/SHOX2 axis may be a novel therapeutic target for ESCC.
Assuntos
Carcinoma de Células Escamosas/genética , Movimento Celular/genética , Neoplasias Esofágicas/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , MicroRNAs/genética , Regiões 3' não Traduzidas/genética , Animais , Western Blotting , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Feminino , Perfilação da Expressão Gênica/métodos , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Metástase Neoplásica , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante HeterólogoRESUMO
Purkinje cells of the developing cerebellum secrete the morphogen sonic hedgehog (SHH), which is required to maintain the proliferative state of granule cell precursors (GCPs) prior to their differentiation and migration to form the internal granule layer (IGL). Despite a wealth of knowledge regarding the function of SHH during cerebellar development, the upstream regulators of Shh expression during this process remain largely unknown. Here we report that the murine short stature homeobox 2 (Shox2) gene is required for normal Shh expression in dorsal-residing Purkinje cells. Using two different Cre drivers, we show that elimination of Shox2 in the brain results in developmental defects in the inferior colliculus and cerebellum. Specifically, loss of Shox2 in the cerebellum results in precocious differentiation and migration of GCPs from the external granule layer (EGL) to the IGL. This correlates with premature bone morphogenetic protein 4 (Bmp4) expression in granule cells of the dorsal cerebellum. The size of the neonatal cerebellum is reduced in Shox2-mutant animals, which is consistent with a reduction in the number of GCPs present in the EGL, and could account for the smaller vermis and thinner IGL present in adult Shox2mutants. Shox2-mutant mice also display reduced exploratory activity, altered gait and impaired motor coordination. Our findings are the first to show a role for Shox2 in brain development. We provide evidence that Shox2 plays an important role during cerebellar development, perhaps to maintain the proper balance of Shh and Bmp expression levels in the dorsal vermis, and demonstrate that in the absence of Shox2, mice display both cerebellar impairments and deficits in motor coordination, ultimately highlighting the importance of Shox2 in the cerebellum.