Rosa canina extract relieves methylation alterations of pancreatic genes in STZ-induced diabetic rats : Gene methylation in diabetic rats treated with an extract.

BACKGROUND: Diabetes is a chronic metabolic disease that affects many parts of the body. Considering diabetes as a beta cells' defect and loss, the focus is on finding mechanisms and compounds involved in stimulating the function and regeneration of pancreatic ß-cells. DNA methylation as an epigenetic mechanism plays a pivotal role in the ß-cells' function and development. Considering the regenerative and anti-diabetic effects of Rosa canina extract, this study aimed to assess the methylation levels of Pdx-1, Pax-4, and Ins-1 genes in diabetic rats treated with Rosa Canina extract. METHODS AND RESULTS: Streptozotocin-induced diabetic rats were used to evaluate the frequency of Pdx-1, Pax-4, and Ins-1 gene methylation. Treatment groups were exposed to Rosa canina as spray-dried and decoction extracts. Following blood glucose measurement, pancreatic DNA was extracted and bisulfited. Genes' methylation was measured using MSP-PCR and qRT-PCR techniques. Oral administration of Rosa canina extracts significantly reduced blood sugar levels in diabetic rats compared to the control group. The methylation levels of the Pdx-1, Pax-4, and Ins-1 genes promoter in streptozotocin-induced diabetic rats increased compared to the control rats while, the treatment of diabetic rats with Rosa canina extracts, spray-dried samples especially, led to a decreased methylation in these genes. CONCLUSION: The results of this study showed that Rosa canina extract as a spray-dried sample could be effective in treating diabetes by regulating the methylation of genes including Pdx-1, Pax-4, and Ins-1 involved in the activity and regeneration of pancreatic islet cells.

DUX4 is a common driver of immune evasion and immunotherapy failure in metastatic cancers.

Cancer immune evasion contributes to checkpoint immunotherapy failure in many patients with metastatic cancers. The embryonic transcription factor DUX4 was recently characterized as a suppressor of interferon-γ signaling and antigen presentation that is aberrantly expressed in a small subset of primary tumors. Here, we report that DUX4 expression is a common feature of metastatic tumors, with ~10-50% of advanced bladder, breast, kidney, prostate, and skin cancers expressing DUX4. DUX4 expression is significantly associated with immune cell exclusion and decreased objective response to PD-L1 blockade in a large cohort of urothelial carcinoma patients. DUX4 expression is a significant predictor of survival even after accounting for tumor mutational burden and other molecular and clinical features in this cohort, with DUX4 expression associated with a median reduction in survival of over 1 year. Our data motivate future attempts to develop DUX4 as a biomarker and therapeutic target for checkpoint immunotherapy resistance.

Over time cancer patients can become resistant to traditional treatments such as chemotherapy and radiotherapy. In some cases, this can be counteracted by administering a new type of treatment called immune checkpoint inhibition which harnesses a patient's own immune system to eradicate the tumor. However, a significant proportion of cancers remain resistant, even when these immunotherapy drugs are used. This is potentially caused by tumors reactivating a gene called DUX4, which is briefly turned on in the early embryo shortly after fertilization, but suppressed in healthy adults. Activation of DUX4 during the early stages of cancer has been shown to remove the cell surface proteins the immune system uses to recognize tumors. However, it remained unclear whether DUX4 changes the response to immunotherapy in more advanced cancers which have begun to spread and metastasize to other parts of the body. To investigate, Pineda and Bradley analyzed publicly available sequencing data which revealed the genes turned on and off in patients with different types of cancer. The analysis showed that DUX4 is reactivated in approximately 10­50% of advanced bladder, breast, kidney, prostate and skin cancers. Next, Pineda and Bradley studied a cohort of patients with advanced bladder cancer who had been treated with immune checkpoint inhibitors. They found that patients with tumors in which DUX4 had been turned back on had shorter survival times than patients who had not reactivated the gene. These results suggest that the activity of DUX4 could be used to predict which patients with advanced bladder cancer may benefit from immune checkpoint inhibitors. In the future, this work could be extended to see if DUX4 could be used as a prognostic tool for other types of cancer. Future studies could also investigate if the DUX4 gene could be a therapeutic target for mitigating resistance to immunotherapy in metastatic cancers.

CUX1 attenuates the apoptosis of renal tubular epithelial cells induced by contrast media through activating the PI3K/AKT signaling pathway.

OBJECTIVE: Contrast media (CM) is a commonly applied drug in medical examination and surgery. However, contrast-induced acute kidney injury (CIAKI) poses a severe threat to human life and health. Notably, the CUT-like homeobox 1 (CUX1) gene shows protective effects in a variety of cells. Therefore, the objective of this study was to provide a new target for the treatment of CIAKI through exploring the role and possible molecular mechanism of CUX1 in CIAKI. METHOD: Blood samples were collected from 20 patients with CIAKI and healthy volunteers. Human kidney 2 (HK-2) cells were incubated with 200 mg/mL iohexol for 6 h to establish a contrast-induced injury model of HK-2 cells. Subsequently, qRT-PCR was used to detect the relative mRNA expression of CUX1; CCK-8 and flow cytometry to assess the proliferation and apoptosis of HK-2 cells; the levels of IL(interleukin)-1ß, tumor necrosis factor alpha (TNF-α) and malondialdehyde (MDA) in cells and lactate dehydrogenase (LDH) activity in cell culture supernatant were detect; and western blot to observe the expression levels of CUX1 and the PI3K/AKT signaling pathway related proteins [phosphorylated phosphoinositide 3-kinase (p-PI3K), PI3K, phosphorylated Akt (p-AKT), AKT]. RESULTS: CUX1 expression was significantly downregulated in blood samples of patients with CIAKI and contrast-induced HK-2 cells. Contrast media (CM; iohexol) treatment significantly reduced the proliferation of HK-2 cells, promoted apoptosis, stimulated inflammation and oxidative stress that caused cell damage. CUX1 overexpression alleviated cell damage by significantly improving the proliferation level of HK-2 cells induced by CM, inhibiting cell apoptosis, and reducing the level of LDH in culture supernatant and the expression of IL-1ß, TNF-α and MDA in cells. CM treatment significantly inhibited the activity of PI3K/AKT signaling pathway activity. Nevertheless, up-regulating CUX1 could activate the PI3K/AKT signaling pathway activity in HK-2 cells induced by CM. CONCLUSION: CUX1 promotes cell proliferation, inhibits apoptosis, and reduces inflammation and oxidative stress in CM-induced HK-2 cells to alleviate CM-induced damage. The mechanism of CUX1 may be correlated with activation of the PI3K/AKT signaling pathway.

TBX3 is essential for establishment of the posterior boundary of anterior genes and upregulation of posterior genes together with HAND2 during the onset of limb bud development.

During limb bud formation, axis polarities are established as evidenced by the spatially restricted expression of key regulator genes. In particular, the mutually antagonistic interaction between the GLI3 repressor and HAND2 results in distinct and non-overlapping anterior-distal Gli3 and posterior Hand2 expression domains. This is a hallmark of the establishment of antero-posterior limb axis polarity, together with spatially restricted expression of homeodomain and other transcriptional regulators. Here, we show that TBX3 is required for establishment of the posterior expression boundary of anterior genes in mouse limb buds. ChIP-seq and differential gene expression analysis of wild-type and mutant limb buds identifies TBX3-specific and shared TBX3-HAND2 target genes. High sensitivity fluorescent whole-mount in situ hybridisation shows that the posterior expression boundaries of anterior genes are positioned by TBX3-mediated repression, which excludes anterior genes such as Gli3, Alx4, Hand1 and Irx3/5 from the posterior limb bud mesenchyme. This exclusion delineates the posterior mesenchymal territory competent to establish the Shh-expressing limb bud organiser. In turn, HAND2 is required for Shh activation and cooperates with TBX3 to upregulate shared posterior identity target genes in early limb buds.

Joint-specific regulation of homeobox D10 expression in rheumatoid arthritis fibroblast-like synoviocytes.

Rheumatoid arthritis (RA) is a systemic immune-mediated disease characterized by joint inflammation and destruction. The disease typically affects small joints in the hands and feet, later progressing to involve larger joints such as the knees, shoulders, and hips. While the reasons for these joint-specific differences are unclear, distinct epigenetic patterns associated with joint location have been reported. In this study, we evaluated the unique epigenetic landscapes of fibroblast-like synoviocytes (FLS) from hip and knee synovium in RA patients, focusing on the expression and regulation of Homeobox (HOX) transcription factors. These highly conserved genes play a critical role in embryonic development and are known to maintain distinct expression patterns in various adult tissues. We found that several HOX genes, especially HOXD10, were differentially expressed in knee FLS compared with hip FLS. Epigenetic differences in chromatin accessibility and histone marks were observed in HOXD10 promoter between knee and hip FLS. Histone modification, particularly histone acetylation, was identified as an important regulator of HOXD10 expression. To understand the mechanism of differential HOXD10 expression, we inhibited histone deacetylases (HDACs) with small molecules and siRNA. We found that HDAC1 blockade or deficiency normalized the joint-specific HOXD10 expression patterns. These observations suggest that epigenetic differences, specifically histone acetylation related to increased HDAC1 expression, play a crucial role in joint-specific HOXD10 expression. Understanding these mechanisms could provide insights into the regional aspects of RA and potentially lead to therapeutic strategies targeting specific patterns of joint involvement during the course of disease.

Gene regulatory landscape of cerebral cortex folding.

Folding of the cerebral cortex is a key aspect of mammalian brain development and evolution, and defects are linked to severe neurological disorders. Primary folding occurs in highly stereotyped patterns that are predefined in the cortical germinal zones by a transcriptomic protomap. The gene regulatory landscape governing the emergence of this folding protomap remains unknown. We characterized the spatiotemporal dynamics of gene expression and active epigenetic landscape (H3K27ac) across prospective folds and fissures in ferret. Our results show that the transcriptomic protomap begins to emerge at early embryonic stages, and it involves cell-fate signaling pathways. The H3K27ac landscape reveals developmental cell-fate restriction and engages known developmental regulators, including the transcription factor Cux2. Manipulating Cux2 expression in cortical progenitors changed their proliferation and the folding pattern in ferret, caused by selective transcriptional changes as revealed by single-cell RNA sequencing analyses. Our findings highlight the key relevance of epigenetic mechanisms in defining the patterns of cerebral cortex folding.

An Efficient Transgenesis Approach for Gene Delivery in the Mouse Embryonic Heart.

The mammalian heart is a complex organ formed during development via highly diverse populations of progenitor cells. The origin, timing of recruitment, and fate of these progenitors are vital for the proper development of this organ. The molecular mechanisms that govern the morphogenesis of the heart are essential for understanding the pathogenesis of congenital heart diseases and embryonic cardiac regeneration. Classical approaches to investigate these mechanisms employed the generation of transgenic mice to assess the function of specific genes during cardiac development. However, mouse transgenesis is a complex, time-consuming process that often cannot be performed to assess the role of specific genes during heart development. To address this, we have developed a protocol for efficient electroporation and culture of mouse embryonic hearts, enabling transient transgenesis to rapidly assess the effect of gain- or loss-of-function of genes involved in cardiac development. Using this methodology, we successfully overexpressed Meis1 in the embryonic heart, with a preference for epicardial cell transfection, demonstrating the capabilities of the technique.

Association of the SHOX2 and RASSF1A methylation levels with the pathological evolution of early-stage lung adenocarcinoma.

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.

Bacteroides uniformis CECT 7771 requires adaptive immunity to improve glucose tolerance but not to prevent body weight gain in diet-induced obese mice.

BACKGROUND: The metabolic disturbances of obesity can be mitigated by strategies modulating the gut microbiota. In this study, we sought to identify whether innate or adaptive immunity mediates the beneficial metabolic effects of the human intestinal bacterium Bacteroides uniformis CECT 7771 in obesity. METHODS: We evaluated the effects of orally administered B. uniformis on energy homeostasis, intestinal immunity, hormone levels, and gut microbiota in wild-type and Rag1-deficient mice with diet-induced obesity. We also assessed whether B. uniformis needed to be viable to exert its beneficial effects in obesity and to directly induce immunoregulatory effects. RESULTS: The administration of B. uniformis to obese mice improved glucose tolerance and insulin secretion, restored the caloric intake suppression after an oral glucose challenge, and reduced hyperglycemia. The pre- and post-prandial glucose-related benefits were associated with restoration of the anti-inflammatory tone mediated by type 2 macrophages and regulatory T cells (Tregs) in the lamina propria of the small intestine. Contrastingly, B. uniformis administration failed to improve glucose tolerance in obese Rag1-/- mice, but prevented the increased body weight gain and adiposity. Overall, the beneficial effects seemed to be independent of enteroendocrine effects and of major changes in gut microbiota composition. B. uniformis directly induced Tregs generation from naïve CD4+ T cells in vitro and was not required to be viable to improve glucose homeostasis but its viability was necessary to prevent body weight gain in diet-induced obese wild-type mice. CONCLUSIONS: Here we demonstrate that B. uniformis modulates the energy homeostasis in diet-induced obese mice through different mechanisms. The bacterium improves oral glucose tolerance by adaptive immunity-dependent mechanisms that do not require cell viability and prevents body weight gain by adaptive immunity-independent mechanisms which require cell viability. Video Abstract.

HOXB2 promotes cisplatin resistance by upregulating lncRNA DANCR in ovarian cancer.

Ovarian cancer (OV) is a highly fatal malignant disease that commonly manifests at an advanced stage. Drug resistance, particularly platinum resistance, is a leading cause of treatment failure because first-line systemic chemotherapy primarily relies on platinum-based regimens. By analyzing the gene expression levels in the Cancer Genome Atlas database, Genotype-Tissue Expression database, and Gene Expression Omnibus datasets, we discerned that HOXB2 was highly expressed in OV and was associated with poor prognosis and cisplatin resistance. Immunohistochemistry and loss-of-function experiments on HOXB2 were conducted to explore its role in OV. We observed that suppressing HOXB2 could impair the growth and cisplatin resistance of OV in vivo and in vitro. Mechanical investigation and experimental validation based on RNA-Seq revealed that HOXB2 regulated ATP-binding cassette transporter members and the ERK signaling pathway. We further demonstrated that HOXB2 modulated the expression of long non-coding RNA DANCR, a differentiation antagonizing non-protein coding RNA, and thus influenced its downstream effectors ABCA1, ABCG1, and ERK signaling to boost drug resistance and cancer proliferation. These results verified that high expression of HOXB2 correlated with platinum resistance and poor prognosis of OV. Therefore, targeting HOXB2 may be a promising strategy for OV therapy.

Ki67 deficiency impedes chromatin accessibility and BCR gene rearrangement.

The proliferation marker Ki67 has been attributed critical functions in maintaining mitotic chromosome morphology and heterochromatin organization during the cell cycle, indicating a potential role in developmental processes requiring rigid cell-cycle control. Here, we discovered that despite normal fecundity and organogenesis, germline deficiency in Ki67 resulted in substantial defects specifically in peripheral B and T lymphocytes. This was not due to impaired cell proliferation but rather to early lymphopoiesis at specific stages where antigen-receptor gene rearrangements occurred. We identified that Ki67 was required for normal global chromatin accessibility involving regulatory regions of genes critical for checkpoint stages in B cell lymphopoiesis. In line with this, mRNA expression of Rag1 was diminished and gene rearrangement was less efficient in the absence of Ki67. Transgenes encoding productively rearranged immunoglobulin heavy and light chains complemented Ki67 deficiency, completely rescuing early B cell development. Collectively, these results identify a unique contribution from Ki67 to somatic antigen-receptor gene rearrangement during lymphopoiesis.

HOXD10 attenuates renal fibrosis by inhibiting NOX4-induced ferroptosis.

In chronic kidney disease (CKD), renal fibrosis is an unavoidable result of various manifestations. However, its pathogenesis is not yet fully understood. Here, we revealed the novel role of Homeobox D10 (HOXD10) in CKD-related fibrosis. HOXD10 expression was downregulated in CKD-related in vitro and in vivo fibrosis models. UUO model mice were administered adeno-associated virus (AAV) containing HOXD10, and HOXD10 overexpression plasmids were introduced into human proximal tubular epithelial cells induced by TGF-ß1. The levels of iron, reactive oxygen species (ROS), lipid ROS, the oxidized glutathione/total glutathione (GSSG/GSH) ratio, malonaldehyde (MDA), and superoxide dismutase (SOD) were determined using respective assay kits. Treatment with AAV-HOXD10 significantly attenuated fibrosis and renal dysfunction in UUO model mice by inhibiting NOX4 transcription, ferroptosis pathway activation, and oxidative stress. High levels of NOX4 transcription, ferroptosis pathway activation and profibrotic gene expression induced by TGF-ß1/erastin (a ferroptosis agonist) were abrogated by HOXD10 overexpression in HK-2 cells. Moreover, bisulfite sequencing PCR result determined that HOXD10 showed a hypermethylated level in TGF-ß1-treated HK-2 cells. The binding of HOXD10 to the NOX4 promoter was confirmed by chromatin immunoprecipitation (ChIP) analysis and dual-luciferase reporter assays. Targeting HOXD10 may represent an innovative therapeutic strategy for fibrosis treatment in CKD.

Genome-wide identification and expression analysis of the KNOX family and its diverse roles in response to growth and abiotic tolerance in sweet potato and its two diploid relatives.

KNOXs, a type of homeobox genes that encode atypical homeobox proteins, play an essential role in the regulation of growth and development, hormonal response, and abiotic stress in plants. However, the KNOX gene family has not been explored in sweet potato. In this study, through sequence alignment, genomic structure analysis, and phylogenetic characterization, 17, 12 and 11 KNOXs in sweet potato (I. batatas, 2n = 6x = 90) and its two diploid relatives I. trifida (2n = 2x = 30) and I. triloba (2n = 2x = 30) were identified. The protein physicochemical properties, chromosome localization, phylogenetic relationships, gene structure, protein interaction network, cis-elements of promoters, tissue-specific expression and expression patterns under hormone treatment and abiotic stresses of these 40 KNOX genes were systematically studied. IbKNOX4, -5, and - 6 were highly expressed in the leaves of the high-yield varieties Longshu9 and Xushu18. IbKNOX3 and IbKNOX8 in Class I were upregulated in initial storage roots compared to fibrous roots. IbKNOXs in Class M were specifically expressed in the stem tip and hardly expressed in other tissues. Moreover, IbKNOX2 and - 6, and their homologous genes were induced by PEG/mannitol and NaCl treatments. The results showed that KNOXs were involved in regulating growth and development, hormone crosstalk and abiotic stress responses between sweet potato and its two diploid relatives. This study provides a comparison of these KNOX genes in sweet potato and its two diploid relatives and a theoretical basis for functional studies.

Analysis of CDO1, PITX2, and CDH13 Gene Methylation in Early Endometrial Cancer for Prediction of Medical Treatment Outcomes.

An observational cohort study of patients diagnosed with endometrial cancer (EC) stage IA G1, or atypical endometrial hyperplasia (AEH), undergoing organ-preserving treatment, was conducted. OBJECTIVE OF THE STUDY: To determine CDO1, PITX2, and CDH13 gene methylation levels in early endometrial cancer and atypical hyperplasia specimens obtained before organ-preserving treatment in the patients with adequate response and with insufficient response to hormonal treatment. MATERIALS AND METHODS: A total of 41 endometrial specimens obtained during diagnostic uterine curettage in women with EC (n = 28) and AEH (n = 13), willing to preserve reproductive function, were studied; 18 specimens of uterine cancer IA stage G1 from peri- and early postmenopausal women (comparison group) were included in the study. The control group included 18 endometrial specimens from healthy women obtained by diagnostic curettage for missed abortion and/or intrauterine adhesions. Methylation levels were analyzed using the modified MS-HRM method. RESULTS: All 13 women with AEH had a complete response (CR) to medical treatment. In the group undergoing organ-preserving treatment for uterine cancer IA stage G1 (n = 28), 14 patients had a complete response (EC CR group) and 14 did not (EC non-CR group). It was found that all groups had statistically significant differences in CDO1 gene methylation levels compared to the control group (p < 0.001) except for the EC CR group (p = 0.21). The p-value for the difference between EC CR and EC non-CR groups was <0.001. The differences in PITX2 gene methylation levels between the control and study groups were also significantly different (p < 0.001), except for the AEH group (p = 0.21). For the difference between EC CR and EC non-CR groups, the p-value was 0.43. For CDH13 gene methylation levels, statistically significant differences were found between the control and EC non-CR groups (p < 0.001), and the control and EC comparison groups (p = 0.005). When comparing the EC CR group with EC non-CR group, the p-value for this gene was <0.001. The simultaneous assessment of CDO1 and CDH13 genes methylation allowed for an accurate distinction between EC CR and EC non-CR groups (AUC = 0.96). CONCLUSION: The assessment of CDO1 and CDH13 gene methylation in endometrial specimens from patients with endometrial cancer (IA stage G1), scheduled for medical treatment, can predict the treatment outcome.

Genome-wide identification of the plant homeodomain-finger family in rye and ScPHD5 functions in cold tolerance and flowering time.

KEY MESSAGE: 111 PHD genes were newly identified in rye genome and ScPHD5's role in regulating cold tolerance and flowering time was suggested. Plant homeodomain (PHD)-finger proteins regulate the physical properties of chromatin and control plant development and stress tolerance. Although rye (Secale cereale L.) is a major winter crop, PHD-finger proteins in rye have not been studied. Here, we identified 111 PHD genes in the rye genome that exhibited diverse gene and protein sequence structures. Phylogenetic tree analysis revealed that PHDs were genetically close in monocots and diverged from those in dicots. Duplication and synteny analyses demonstrated that ScPHDs have undergone several duplications during evolution and that high synteny is conserved among the Triticeae species. Tissue-specific and abiotic stress-responsive gene expression analyses indicated that ScPHDs were highly expressed in spikelets and developing seeds and were responsive to cold and drought stress. One of these genes, ScPHD5, was selected for further functional characterization. ScPHD5 was highly expressed in the spike tissues and was localized in the nuclei of rye protoplasts and tobacco leaves. ScPHD5-overexpressing Brachypodium was more tolerant to freezing stress than wild-type (WT), with increased CBF and COR gene expression. Additionally, these transgenic plants displayed an extremely early flowering phenotype that flowered more than two weeks earlier than the WT, and vernalization genes, rather than photoperiod genes, were increased in the WT. RNA-seq analysis revealed that diverse stress response genes, including HSPs, HSFs, LEAs, and MADS-box genes, were also upregulated in transgenic plants. Our study will help elucidate the roles of PHD genes in plant development and abiotic stress tolerance in rye.

Comprehensive genetic analysis of facioscapulohumeral muscular dystrophy by Nanopore long-read whole-genome sequencing.

BACKGROUND: Facioscapulohumeral muscular dystrophy (FSHD) is a high-prevalence autosomal dominant neuromuscular disease characterized by significant clinical and genetic heterogeneity. Genetic diagnosis of FSHD remains a challenge because it cannot be detected by standard sequencing methods and requires a complex diagnosis workflow. METHODS: We developed a comprehensive genetic FSHD detection method based on Oxford Nanopore Technologies (ONT) whole-genome sequencing. Using a case-control design, we applied this procedure to 29 samples and compared the results with those from optical genome mapping (OGM), bisulfite sequencing (BSS), and whole-exome sequencing (WES). RESULTS: Using our ONT-based method, we identified 59 haplotypes (35 4qA and 24 4qB) among the 29 samples (including a mosaic sample), as well as the number of D4Z4 repeat units (RUs). The pathogenetic D4Z4 RU contraction identified by our ONT-based method showed 100% concordance with OGM results. The methylation levels of the most distal D4Z4 RU and the double homeobox 4 gene (DUX4) detected by ONT sequencing are highly consistent with the BSS results and showed excellent diagnostic efficiency. Additionally, our ONT-based method provided an independent methylation profile analysis of two permissive 4qA alleles, reflecting a more accurate scenario than traditional BSS. The ONT-based method detected 17 variations in three FSHD2-related genes from nine samples, showing 100% concordance with WES. CONCLUSIONS: Our ONT-based FSHD detection method is a comprehensive method for identifying pathogenetic D4Z4 RU contractions, methylation level alterations, allele-specific methylation of two 4qA haplotypes, and variations in FSHD2-related genes, which will all greatly improve genetic testing for FSHD.

Long non-coding RNA RP11-197K6.1 as ceRNA promotes colorectal cancer progression via miR-135a-5p/DLX5 axis.

BACKGROUND: Colorectal cancer (CRC) remains a major global health challenge, with high incidence and mortality rates. The role of long noncoding RNAs (lncRNAs) in cancer progression has received considerable attention. The present study aimed to investigate the function and mechanisms underlying the role of lncRNA RP11-197K6.1, microRNA-135a-5p (hsa-miR-135a-5p), and DLX5 in CRC development. METHODS: We analyzed RNA sequencing data from The Cancer Genome Atlas Colorectal Cancer dataset to identify the association between lncRNA RP11-197K6.1 and CRC progression. The expression levels of lncRNA RP11-197K6.1 and DLX5 in CRC samples and cell lines were determined by real-time quantitative PCR and western blotting assays. Fluorescence in situ hybridization was used to confirm the cellular localization of lncRNA RP11-197K6.1. Cell migration capabilities were assessed by Transwell and wound healing assays, and flow cytometry was performed to analyze apoptosis. The interaction between lncRNA RP11-197K6.1 and miR-135a-5p and its effect on DLX5 expression were investigated by the dual-luciferase reporter assay. Additionally, a xenograft mouse model was used to study the in vivo effects of lncRNA RP11-197K6.1 on tumor growth, and an immunohistochemical assay was performed to assess DLX5 expression in tumor tissues. RESULTS: lncRNA RP11-197K6.1 was significantly upregulated in CRC tissues and cell lines as compared to that in normal tissues, and its expression was inversely correlated with patient survival. It promoted the migration and metastasis of CRC cells by interacting with miR-135a-5p, alleviated suppression of DLX5 expression, and facilitated tumor growth. CONCLUSION: This study demonstrated the regulatory network and mechanism of action of the lncRNA RP11-197K6.1/miR-135a-5p/DLX5 axis in CRC development. These findings provided insights into the molecular pathology of CRC and suggested potential therapeutic targets for more effective treatment of patients with CRC.

Knockdown of PHOX2B in the retrotrapezoid nucleus reduces the central CO2 chemoreflex in rats.

PHOX2B is a transcription factor essential for the development of different classes of neurons in the central and peripheral nervous system. Heterozygous mutations in the PHOX2B coding region are responsible for the occurrence of Congenital Central Hypoventilation Syndrome (CCHS), a rare neurological disorder characterised by inadequate chemosensitivity and life-threatening sleep-related hypoventilation. Animal studies suggest that chemoreflex defects are caused in part by the improper development or function of PHOX2B expressing neurons in the retrotrapezoid nucleus (RTN), a central hub for CO2 chemosensitivity. Although the function of PHOX2B in rodents during development is well established, its role in the adult respiratory network remains unknown. In this study, we investigated whether reduction in PHOX2B expression in chemosensitive neuromedin-B (NMB) expressing neurons in the RTN altered respiratory function. Four weeks following local RTN injection of a lentiviral vector expressing the short hairpin RNA (shRNA) targeting Phox2b mRNA, a reduction of PHOX2B expression was observed in Nmb neurons compared to both naive rats and rats injected with the non-target shRNA. PHOX2B knockdown did not affect breathing in room air or under hypoxia, but ventilation was significantly impaired during hypercapnia. PHOX2B knockdown did not alter Nmb expression but it was associated with reduced expression of both Task2 and Gpr4, two CO2/pH sensors in the RTN. We conclude that PHOX2B in the adult brain has an important role in CO2 chemoreception and reduced PHOX2B expression in CCHS beyond the developmental period may contribute to the impaired central chemoreflex function.

miR-133a and miR-135a Regulate All-Trans Retinoic Acid-Mediated Differentiation in Pediatric Acute Myeloid Leukemia by Inhibiting CDX2 Translation and Serve as Prognostic Biomarkers.

Background: Acute myeloid leukemia (AML) is a type of blood cancer characterized by excessive growth of immature myeloid cells. Unfortunately, the prognosis of pediatric AML remains unfavorable. It is imperative to further our understanding of the mechanisms underlying leukemogenesis and explore innovative therapeutic approaches to enhance overall disease outcomes for patients with this condition. Methods: Quantitative reverse-transcription PCR was used to quantify the expression levels of microRNA (miR)-133a and miR-135a in 68 samples from 59 pediatric patients with AML. Dual-luciferase reporter transfection assay, Cell Counting Kit-8 assay, and western blot analysis were used to investigate the functions of miR-133a and miR-135a. Results: Our study found that all-trans-retinoic acid (ATRA) promoted the expression of miR-133a and miR-135a in AML cells, inhibited caudal type homeobox 2 (CDX2) expression, and subsequently inhibited the proliferation of AML cells. Additionally, miR-133a and miR-135a were highly expressed in patients with complete remission and those with better survival. Conclusions: miR-133a and miR-135a may play an antioncogenic role in pediatric AML through the ATRA-miRNA133a/135a-CDX2 pathway. They hold promise as potentially favorable prognostic indicators and novel therapeutic targets for pediatric AML.

USP11 regulates proliferation and apoptosis of human spermatogonial stem cells via HOXC5-mediated canonical WNT/ß-catenin signaling pathway.

Spermatogonial stem cells (SSCs) are capable of transmitting genetic information to the next generations and they are the initial cells for spermatogenesis. Nevertheless, it remains largely unknown about key genes and signaling pathways that regulate fate determinations of human SSCs and male infertility. In this study, we explored the expression, function, and mechanism of USP11 in controlling the proliferation and apoptosis of human SSCs as well as the association between its abnormality and azoospermia. We found that USP11 was predominantly expressed in human SSCs as shown by database analysis and immunohistochemistry. USP11 silencing led to decreases in proliferation and DNA synthesis and an enhancement in apoptosis of human SSCs. RNA-sequencing identified HOXC5 as a target of USP11 in human SSCs. Double immunofluorescence, Co-immunoprecipitation (Co-IP), and molecular docking demonstrated an interaction between USP11 and HOXC5 in human SSCs. HOXC5 knockdown suppressed the growth of human SSCs and increased apoptosis via the classical WNT/ß-catenin pathway. In contrast, HOXC5 overexpression reversed the effect of proliferation and apoptosis induced by USP11 silencing. Significantly, lower levels of USP11 expression were observed in the testicular tissues of patients with spermatogenic disorders. Collectively, these results implicate that USP11 regulates the fate decisions of human SSCs through the HOXC5/WNT/ß-catenin pathway. This study thus provides novel insights into understanding molecular mechanisms underlying human spermatogenesis and the etiology of azoospermia and it offers new targets for gene therapy of male infertility.