Utility of SHOX2 and RASSF1A gene methylation detection on the residual cytology material from endobronchial ultrasound-guided transbronchial needle aspiration.

Objective: This study aims to assess the effectiveness of Short Stature Homeobox 2 (SHOX2) and RAS Association Domain Family 1 Isoform A (RASSF1A) gene methylation detection in residual liquid-based cytology (LBC) materials from Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration (EBUS-TBNA) and investigate the diagnostic accuracy of a comprehensive diagnostic approach. Material and Methods: Between June 2022 and May 2023, a total of 110 cases that underwent EBUS-TBNA were enrolled in the study. SHOX2 and RASSF1A genes methylation detection using the residual cytological material, LBC, and cell block (CB) were conducted for each EBUS-TBNA case. The sensitivity and specificity of cytology, CB histopathology, SHOX2, and RASSF1A methylation in diagnosing EBUS-TBNA samples were determined based on follow-up data. Results: Among the 72 cases confirmed as pulmonary carcinomas, the methylation test yielded positive results in 24 adenocarcinoma cases, 10 squamous cell carcinoma cases, and 14 small cell carcinoma cases. The sensitivity of the comprehensive diagnosis (combining LBC, CB, and methylation detection) in distinguishing metastatic pulmonary epithelial malignancies in mediastinal and hilar lymph nodes or masses from benign lesions was higher (97.22%, 70/72) than that of morphological diagnosis alone (LBC and CB) (88.89%, 64/72; P < 0.05). Conclusion: SHOX2 and RASSF1A methylation detection demonstrates a high sensitivity and negative predictive value in the identification of pulmonary epithelial malignancies and holds promise as a valuable ancillary approach to enhance morphological diagnosis of EBUS-TBNA.

A novel multimodal prediction model based on DNA methylation biomarkers and low-dose computed tomography images for identifying early-stage lung cancer.

Objective: DNA methylation alterations are early events in carcinogenesis and immune signalling in lung cancer. This study aimed to develop a model based on short stature homeobox 2 gene (SHOX2)/prostaglandin E receptor 4 gene (PTGER4) DNA methylation in plasma, appearance subtype of pulmonary nodules (PNs) and low-dose computed tomography (LDCT) images to distinguish early-stage lung cancers. Methods: We developed a multimodal prediction model with a training set of 257 individuals. The performance of the multimodal prediction model was further validated in an independent validation set of 42 subjects. In addition, we explored the association between SHOX2/PTGER4 DNA methylation and driver gene mutations in lung cancer based on data from The Cancer Genome Atlas (TCGA) portal. Results: There were significant differences between the early-stage lung cancers and benign groups in the methylation levels. The area under a receiver operator characteristic curve (AUC) of SHOX2 in patients with solid nodules, mixed ground-glass opacity nodules and pure ground-glass opacity nodules were 0.693, 0.497 and 0.864, respectively, while the AUCs of PTGER4 were 0.559, 0.739 and 0.619, respectively. With the highest AUC of 0.894, the novel multimodal prediction model outperformed the Mayo Clinic model (0.519) and LDCT-based deep learning model (0.842) in the independent validation set. Database analysis demonstrated that patients with SHOX2/PTGER4 DNA hypermethylation were enriched in TP53 mutations. Conclusions: The present multimodal prediction model could more efficiently distinguish early-stage lung cancer from benign PNs. A prognostic index based on DNA methylation and lung cancer driver gene alterations may separate the patients into groups with good or poor prognosis.

Detection of short stature homeobox 2 and RAS-associated domain family 1 subtype A DNA methylation in interventional pulmonology.

One of the most important aspects of interventional pulmonology is to obtain tissue or liquid samples of the chest to diagnose a respiratory disease; however, it is still possible to obtain insufficient tissue or cytologic specimens. Indeed, methylation detection is an effective method by which to establish a diagnosis. This review focuses on the clinical application of short stature homeobox 2 and RAS-associated domain family 1 subtype A DNA methylation detection in interventional pulmonology, including bronchoscopic fluid biopsy, transbronchial needle aspiration, and pleural effusion.

Long non-coding RNA LINC00460 promotes proliferation and inhibits apoptosis of cervical cancer cells by targeting microRNA-503-5p.

Long non-coding RNAs are associated with the pathogenesis of cancers. Moreover, LINC00460 is involved in the development of multiple cancers. However, the function of LINC00460 in cervical cancer (CC) remains inconclusive. Herein, CC tissues and tumor-adjacent tissues were collected from patients. The effect of LINC00460 silencing in cell proliferation and apoptosis in CC was explored in vitro and in vivo. Additionally, the interaction between LINC00460 and miR-503-5p was analyzed using dual luciferase reporter assay. The expression of genes and proteins was assayed using quantitative real-time PCR, western blotting and immunohistochemistry, cell viability using MTT assay, cell cycle distribution using flow cytometry, cell apoptosis using Annexin V staining, Hoechst staining and TUNEL assay. LINC00460 levels in CC tissues were higher than tumor-adjacent tissues. LINC00460 silencing suppressed proliferation and promoted apoptosis of CC cells as evidenced by decreased cell viability, inhibited proliferation-related protein and cell cycle protein expressions and G1/S transition, increased apoptotic cells and Hoechst-positive cells, and enhanced apoptosis-related protein expressions. LINC00460 could bind to miR-503-5p and LINC00460 silencing enhanced miR-503-5p expression and inhibited its target gene expressions in CC cells. MiR-503-5p inhibition reversed LINC00460 silencing-caused inhibition of cell proliferation and miR-503-5p target gene expressions, and promotion of cell apoptosis. LINC00460 silencing also attenuated tumor growth, promoted miR-503-5p levels and cell apoptosis, and inhibited cell proliferation and miR-503-5p target gene expressions in tumor tissues. Hence, LINC00460 functioned as an oncogene in CC that affected cell proliferation and apoptosis via sponging miR-503-5p. This study provides a novel therapeutic target for CC.

The transcriptional regulator MEIS2 sets up the ground state for palatal osteogenesis in mice.

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.

Shox2 regulates osteogenic differentiation and pattern formation during hard palate development in mice.

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.

Mice lacking the transcription factor SHOX2 display impaired cerebellar development and deficits in motor coordination.

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.