Glioma-associated Oncogene Research Articles

Curcumin and Methotrexate: A Promising Combination for Osteosarcoma Treatment via Hedgehog Pathway Inhibition

Osteosarcoma (OS) is the most severe bone tumor in children. A chemotherapy regimen includes a combination of high-dose Methotrexate (MTX), doxorubicin, and cisplatin. These drugs cause acute and chronic side effects, such as infections, thrombocytopenia, neutropenia, DNA damage, and inflammation. Therefore, to identify new therapeutic strategies, effective and with a safety profile, is necessary. The Hedgehog (Hh) signaling pathway involved in tumorigenesis is active in OS. Hh components Patched receptor 1 (PTCH1), Smoothened (SMO), and glioma-associated oncogene homolog transcription factors (GLI1 and GLI2) are overexpressed in OS cell lines and patient samples. Curcumin (CUR)—with antioxidant and anti-cancer properties—downregulates Hh components in cancer, inhibiting progression. This study investigates CUR effects on the MG-63 OS cell line, alone and combined with MTX, to propose a novel therapeutic approach. Our study suggests CUR as a novel therapeutic agent in OS, particularly when combined with MTX. Targeting the Hh signaling pathway, CUR and MTX showed significant pro-apoptotic effects, increasing the BAX/Bcl-2 ratio and total apoptotic cell percentage. They reduced the expression of Hh pathway components (PTCH1, SMO, GLI1, and GLI2), inhibiting OS cell proliferation, survival, and invasion. CUR and MTX combined determined a β-Catenin decrease and a trend toward reducing NF-kB and matrix metalloproteinases (MMP-2 and MMP-9). Our findings suggest CUR as a support to OS treatment, improving outcomes and reducing the adverse effects of current therapies.

Collaborative role of two distinct cilium-specific cytoskeletal systems in driving Hedgehog-responsive transcription factor trafficking.

Calibrated transcriptional outputs in cellular signaling require fine regulation of transcription factor activity. In vertebrate Hedgehog (Hh) signaling, the precise output of the final effectors, the GLI (Glioma-associated-oncogene) transcription factors, depends on the primary cilium. In particular, the formation of the activator form of GLI upon pathway initiation requires its concentration at the distal cilium tip. However, the mechanisms underlying this critical step in Hh signaling are unclear. We developed a real-time imaging assay to visualize GLI2, the primary GLI activator isoform, at single particle resolution within the cilium. We observed that GLI2 is a cargo of Intraflagellar Transport (IFT) machinery and is transported with anterograde bias during a restricted time window following pathway activation. Our findings position IFT as a crucial mediator of transcription factor transport within the cilium for vertebrate Hh signaling, in addition to IFT's well-established role in ciliogenesis. Surprisingly, a conserved Hh pathway regulator, the atypical non-motile kinesin KIF7, is critical for the temporal control of GLI2 transport by IFT and the spatial control of GLI2 localization at the cilium tip. This discovery underscores the collaborative role of a motile and a non-motile cilium-specific cytoskeletal system in determining the transcriptional output during Hh signaling.

Exosomal LINC00958 maintains ovarian cancer cell stemness and induces M2 macrophage polarization via Hedgehog signaling pathway and GLI1 protein

Long non-coding RNA (lncRNA) LINC00958 has been reported to promote many gynecological cancers, but its detailed function in OC remains unclear. Cancer stem cells (CSCs) and tumor-associated macrophages (TAMs) have been reported to participate in the occurrence and metastasis of cancers. We want to explore the effects of exosomal LINC00958 on cell stemness and macrophage polarization in OC. LINC00958 expression was first verified in OC cells and its function on cell stemness was verified by subcellular fractionation analysis, sphere formation assay and so on. Exosomal LINC00958 was secreted from OC cells and the model of M2 macrophage polarization was established to further verify the impact of exosomal LINC00958 on the cell stemness and macrophage polarization of OC cells using several mechanism experiments including flow cytometry, RNA pulldown, luciferase reporter assays and so on. LINC00958 was up-regulated in OC cells and exosomal LINC00958 enhanced the stem cell-like properties of OC cells and M2 macrophage polarization. Furthermore, LINC00958 combined with glioma-associated oncogene homolog 1 (GLI1) to activate Hedgehog pathway, thereby promoting M2 polarization. Exosomal LINC00958 maintained OC cell stemness and induced M2 polarization via the Hedgehog signaling pathway.

The silent information regulator 1 agonist SRT1720 reduces experimental intracerebral hemorrhagic brain injury by regulating the blood-brain barrier integrity.

Intracerebral hemorrhage (ICH) is a significant public health matter that has no effective treatment. ICH-induced destruction of the blood-brain barrier (BBB) leads to neurological deterioration. Astrocytic sonic hedgehog (SHH) alleviates brain injury by maintaining the integrity of the BBB after ICH. Silent information regulator 1 (SIRT1) is neuroprotective in several central nervous system diseases via BBB regulation. It is also a possible influential factor of the SHH signaling pathway. Nevertheless, the role of SIRT1 on BBB and the underlying pathological process associated with the SHH signaling pathway after ICH remain unclear. We established an intracerebral hemorrhagic mouse model by collagenase injection. SRT1720 (a selective agonist of SIRT1) was used to evaluate the effect of SIRT1 on BBB integrity after ICH. SIRT1 expression was reduced in the mouse brain after ICH. SRT1720 attenuated neurobehavioral impairments and brain edema of ICH mouse. After ICH induction, SRT1720 improved BBB integrity and tight junction expressions in the mouse brain. The SHH signaling pathway-related factors smoothened and glioma-associated oncogene homolog-1 were increased with the intervention of SRT1720, while cyclopamine (a specific inhibitor of the SHH signaling pathway) reversed these effects. These findings suggest that SIRT1 protects from ICH by altering BBB permeability and tight junction expression levels. This process is associated with the SHH signaling pathway, suggesting that SIRT1 may be a potential therapeutic target for ICH.

Glioma-associated oncogene (GLI)-specific decoy oligodeoxynucleotide induces apoptosis and attenuates proliferation, colony formation, and migration in liver cancer cells

Aim: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-associated death. The Sonic Hedgehog (SHH) signaling pathway participates in the initiation, progression, migration, and recurrence of HCC cancer stem cells. Furthermore, SHH regulates various cellular behaviors such as proliferation, differentiation, survival, self-renewal, epithelial-mesenchymal transition (EMT), and SHH autoregulation. Glioma-associated oncogene (GLI) family zinc finger are key transcription factors in the development of many organs and are deregulated in cancer. In this study, Huh-7 cells were treated with GLI-specific decoy oligodeoxynucleotide (ODN) to evaluate its anticancer impact. Methods: The transfection efficiency of GLI-specific decoy ODN was measured using fluorescent microscopy. Then, the effects of GLI-specific decoy ODN on apoptosis, viability, proliferation rate, colony formation, and migration capacities of Huh-7 cells were assessed. Furthermore, the expression of genes associated with the alteration of SHH was assessed. Results: Treatment of Huh-7 cells with GLI-specific decoy ODN decreased cell viability (56.36% ± 3%). Expression of certain genes such as c-MYC , SNAI2 , ZEB1 , and PROM1 decreased dramatically, while the expression of CDH1 increased significantly. Furthermore, the treated cells’ proliferation, colony formation, and migration capacity decreased considerably. This treatment induced apoptosis in the Huh-7 cells. Conclusion: Inhibition of the SHH signaling pathway using GLI-specific decoy ODN led to a decline in the growth rate of HCC cells, decreased migration, and attenuated EMT progression.

"GLI1 Subcellular Localization and Overexpression as Prognostic Factors for Disease-Free Survival in Colorectal Carcinoma".

Glioma-associated oncogene homolog-1 (GLI1) is amplified in human glioblastoma, and there is growing evidence suggesting its significant role in tumor development and metastasis. Our aim was to investigate the role of the GLI-1 gene in the progression of colorectal cancer (CRC) and its correlation with various clinicopathological features. Additionally, we examined the impact of the GLI-1 gene and other factors on the prognosis of CRC. We analyzed a total of 98 confirmed CRC cases and adjacent normal tissue controls. Patients suspected of having colon cancer underwent a colonoscopy and targeted biopsy, while those with rectal cancer underwent CT scans and MRI. GLI1 expression was detected using real-time PCR assay, Western blotting, and immunohistochemistry. The GLI1 gene was observed to be overexpressed in tumor tissues at both the protein and mRNA levels (p < 0.05). In addition, GLI1 overexpression was significantly associated with various factors such as tumor invasion (T3/T4), presence of lymph nodes, lymph node metastasis (LNM), stage (III/IV), tumor site (colon), tumor size (≥ 3cm), localization (nucleocytoplasmic), strong staining intensity and recurrence (p < 0.05). The results of survival analysis showed that the patients with overexpression of GLI1 had a significantly lower DFS rate which was 21months compared to those with normal expression who had 31months (p < 0.05). Moreover, individuals with early onset disease (15months) were more likely to have cytoplasmic localization of the GLI1 gene as opposed to nucleo-cytoplasmic localization of GLI1 which presented late-onset disease( 23months) (p < 0.05). Finally, Stage and PNI (p < 0.05) were found to independently affect outcomes of CRC according to Cox regression analysis. High expression of GLI-1 in CRC is associated with adverse pathology and poor prognosis for patients. The correlation between cytoplasmic localization of GLI-1 and reduced disease-free survival holds potential for guiding prognosis and treatment. Further research is needed to develop strategies targeting GLI-1 for improved outcomes.

P4HA2 hydroxylates SUFU to regulate the paracrine Hedgehog signaling and promote B-cell lymphoma progression

Aberrations in the Hedgehog (Hh) signaling pathway are significantly prevailed in various cancers, including B-cell lymphoma. A critical facet of Hh signal transduction involves the dynamic regulation of the suppressor of fused homolog (SUFU)-glioma-associated oncogene homolog (GLI) complex within the kinesin family member 7 (KIF7)-supported ciliary tip compartment. However, the specific post-translational modifications of SUFU-GLI complex within this context have remained largely unexplored. Our study reveals a novel regulatory mechanism involving prolyl 4-hydroxylase 2 (P4HA2), which forms a complex with KIF7 and is essential for signal transduction of Hh pathway. We demonstrate that, upon Hh pathway activation, P4HA2 relocates alongside KIF7 to the ciliary tip. Here, it hydroxylates SUFU to inhibit its function, thus amplifying the Hh signaling. Moreover, the absence of P4HA2 significantly impedes B lymphoma progression. This effect can be attributed to the suppression of Hh signaling in stromal fibroblasts, resulting in decreased growth factors essential for malignant proliferation of B lymphoma cells. Our findings highlight the role of P4HA2-mediated hydroxylation in modulating Hh signaling and propose a novel stromal-targeted therapeutic strategy for B-cell lymphoma.

Impaired Sonic Hedgehog Responsiveness of Induced Pluripotent Stem Cell-Derived Floor Plate Cells Carrying the LRRK2-I1371V Mutation Contributes to the Ontogenic Origin of Lower Dopaminergic Neuron Yield.

Lower population of dopaminergic (DA) neurons is known to increase susceptibility to Parkinson's disease (PD), and our earlier study showed a lower yield of DA neurons in Leucine-Rich Repeat Kinase Isoleucine 1371 Valine (LRRK2-I1371V) mutation-carrying PD patient-derived induced Pluripotent Stem Cells (iPSCs). Although the role of Sonic Hedgehog (SHH) in DA neurogenesis of floor plate cells (FPCs) is known, the effect of LRRK2 mutations on SHH responsiveness of FPCs impacting DA neuronal yield has not been studied. We investigated SHH responsiveness of FPCs derived from LRRK2-I1371V PD patient iPSCs with regard to the expression of SHH receptors Patched1 (Ptch1) and Smoothened (Smo), in conjunction with nuclear Gli1 (glioma-associated oncogene 1) expression, intracellular Ca2+ rise, and cytosolic cyclic adenosine monophosphate (cAMP) levels upon SHH induction. In addition, we examined the mechanistic link with LRRK2-I1371V gain-of-function by assessing membrane fluidity and Rab8A and Rab10 phosphorylation in SH-SY5Y cells and healthy control (HC) FPCs overexpressing LRRK2-I1371V as well as FPCs. Although total expression of Ptch1 and Smo was comparable, receptor expression on cell surface was significantly lower in LRRK2-I1371V FPCs than in HC FPCs, with distinctly lower nuclear expression of the downstream transcription factor Gli1. HC-FPCs transfected with LRRK2-I1371V exhibited a similarly reduced cell surface expression of Ptch1 and Smo. Intracellular Ca2+ response was significantly lower with corresponding elevated cAMP levels in LRRK2-I1371V FPCs compared with HC FPCs upon SHH stimulation. The LRRK2-I1371V mutant FPCs and LRRK2-I1371V-transfected SH-SY5Y and HC FPCs too exhibited higher autophosphorylation of phospho LRRK2 (pLRRK2) serine1292 and serine935, as well as substrate phosphorylation of Rab8A and Rab10. Concurrent increase in membrane fluidity, accompanied by a decrease in membrane cholesterol, and lower expression of lipid raft marker caveolin 1 were also observed in them. These findings suggest that impaired SHH responsiveness of LRRK2-I1371V PD FPCs indeed leads to lower yield of DA neurons during ontogeny. Reduced cell surface expression of SHH receptors is influenced by alteration in membrane fluidity owing to the increased substrate phosphorylation of Rab8A and reduced membrane protein trafficking due to pRab10, both results of the LRRK2-I1371V mutation.

PDLIM3 knockdown promotes ferroptosis in endometriosis progression via inducing Gli1 degradation and blocking Hedgehog signaling pathway.

Current evidence suggests that there is no completely effective method for endometriosis (EMS) without trauma due to diverse adverse effects. Reliable evidence illustrates that inhibiting ferroptosis is a potential strategy for EMS. We sufficiently verified that the expression of endogenous protein PDZ and LIM domain 3 (PDLIM3) was significantly increased in EMS. PDLIM3 knockdown reduced primary ectopic endometrial stromal cells' (EESCs) viability and migration, and elevated ferroptosis signaling indicators including Fe2+, malondialdehyde (MDA), and reactive oxygen species (ROS) in EESCs. Mechanistic studies revealed that inhibition of PDLIM3 accelerated glioma-associated oncogene-1 (Gli1) degradation and further deactivated Hedgehog signaling. Gli1 inhibitor, GANT61, abrogated the impact of PDLIM3 deletion on EESC growth, migration, and ferroptosis. In vivo experiments suggested that PDLIM3 reduction repressed the growth of endometrial lesions. Likewise, repression of PDLIM3 promoted ferroptosis and attenuated Hedgehog signaling in endometrial lesions. Collectively, silencing of PDLIM3 facilitates ferroptosis in EMS by inducing Gli1 degradation and blocking Hedgehog signaling. It may provide an alternative strategy for developing therapeutic agents of EMS in the future.

Transcription factor EHF drives cholangiocarcinoma development through transcriptional activation of glioma-associated oncogene homolog 1 and chemokine CCL2.

Cholangiocarcinoma (CCA) is characterized by rapid onset and high chance of metastasis. Therefore, identification of novel therapeutic targets is imperative. E26 transformation-specific homologous factor (EHF), a member of the E26 transformation-specific transcription factor family, plays a pivotal role in epithelial cell differentiation and cancer progression. However, its precise role in CCA remains unclear. In this study, through in vitro and in vivo experiments, we demonstrated that EHF plays a profound role in promoting CCA by transcriptional activation of glioma-associated oncogene homolog 1 (GLI1). Moreover, EHF significantly recruited and activated tumor-associated macrophages (TAMs) through the C-C motif chemokine 2/C-C chemokine receptor type 2 (CCL2/CCR2) axis, thereby remodeling the tumor microenvironment. In human CCA tissues, EHF expression was positively correlated with GLI1 and CCL2 expression, and patients with co-expression of EHF/GLI1 or EHF/CCL2 had the most adverse prognosis. Furthermore, the combination of the GLI1 inhibitor, GANT58, and CCR2 inhibitor, INCB3344, substantially reduced the occurrence of EHF-mediated CCA. In summary, our findings suggest that EHF is a potential prognostic biomarker for patients with CCA, while also advocating the therapeutic approach of combined targeting of GLI1 and CCL2/CCR2-TAMs to inhibit EHF-driven CCA development.

Blastomas of the digestive system in adults: A review.

Blastomas, characterized by a mixture of mesenchymal, epithelial, and undifferentiated blastematous components, are rare malignant neoplasms originating from precursor blast cells. This review focuses on digestive system blastomas in adult patients, including gastroblastoma, hepatoblastoma, and pancreatoblastoma. Gastroblastoma is a biphasic, epitheliomesenchymal tumor, with only sixteen cases reported to date. In addition to the characteristic histology, metastasis-associated lung adenocarcinoma transcript 1 - glioma-associated oncogene homolog 1 gene fusion is typical, although recently novel ewing sarcoma breakpoint region 1 - c-terminal binding protein 1 and patched 1 - glioma-associated oncogene homolog 2 fusions have been described. Hepatoblastoma is exceptionally rare in adults and can show a variety of histologic patterns which may cause diagnostic difficulty. Pancreatoblastoma, primarily a pediatric tumor, displays acinar differentiation and squamoid nests with other lines of differentiation also present, especially neuroendocrine. Diagnostic approaches for these blastomas include a combination of imaging modalities, histopathological examination, and molecular profiling. The treatment generally involves surgical resection, which may be supplemented by chemotherapy or radiotherapy in some cases. Prognoses vary with gastroblastoma generally showing favorable outcomes post-surgery whereas hepatoblastoma and pancreatoblastoma often have poorer outcomes, particularly in the setting of metastases. This review highlights the complexity of diagnosing and managing these rare adult blastomas as well as the need for ongoing research to better understand their pathogenesis and improve treatment strategies.

GLI1+ Cells Contribute to Vascular Remodeling in Pulmonary Hypertension.

The precise origin of newly formed ACTA2+ (alpha smooth muscle actin-positive) cells appearing in nonmuscularized vessels in the context of pulmonary hypertension is still debatable although it is believed that they predominantly derive from preexisting vascular smooth muscle cells (VSMCs). Gli1Cre-ERT2; tdTomatoflox mice were used to lineage trace GLI1+ (glioma-associated oncogene homolog 1-positive) cells in the context of pulmonary hypertension using 2 independent models of vascular remodeling and reverse remodeling: hypoxia and cigarette smoke exposure. Hemodynamic measurements, right ventricular hypertrophy assessment, flow cytometry, and histological analysis of thick lung sections followed by state-of-the-art 3-dimensional reconstruction and quantification using Imaris software were used to investigate the contribution of GLI1+ cells to neomuscularization of the pulmonary vasculature. The data show that GLI1+ cells are abundant around distal, nonmuscularized vessels during steady state, and this lineage contributes to around 50% of newly formed ACTA2+ cells around these normally nonmuscularized vessels. During reverse remodeling, cells derived from the GLI1+ lineage are largely cleared in parallel to the reversal of muscularization. Partial ablation of GLI1+ cells greatly prevented vascular remodeling in response to hypoxia and attenuated the increase in right ventricular systolic pressure and right heart hypertrophy. Single-cell RNA sequencing on sorted lineage-labeled GLI1+ cells revealed an Acta2high fraction of cells with pathways in cancer and MAPK (mitogen-activated protein kinase) signaling as potential players in reprogramming these cells during vascular remodeling. Analysis of human lung-derived material suggests that GLI1 signaling is overactivated in both group 1 and group 3 pulmonary hypertension and can promote proliferation and myogenic differentiation. Our data highlight GLI1+ cells as an alternative cellular source of VSMCs in pulmonary hypertension and suggest that these cells and the associated signaling pathways represent an important therapeutic target for further studies.

Diterpenoids from Scutellaria barbata D. Don Exert the Antiproliferative Effect on Colorectal Cancer Cells Via Hedgehog Signaling Pathway

Background: Scutellaria barbata D. Don, also known as “Banzhilian” in China, the diterpenes of Banzhilian (Bzl-D) is a commonly used herbal medicine for treating various tumors including colorectal cancer (CRC). Objectives: This study aimed to examine the function of Bzl-D in inhibiting CRC development and explore its potential targets and mechanisms involving the Hedgehog (HH) pathway. Materials and methods: To determine the major components in Bzl-D, we performed ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-qTOF-MS) analysis. The proliferation, migration, invasion, and apoptosis abilities of the CRC HT-29 cell line were examined, and the related protein expression was tested. Tumorigenesis after treatment was detected by in vivo experiments. Results: Nineteen diterpenoids were detected, Bzl-D significantly inhibited the cell proliferation and metastasis and induced the apoptosis of HT-29 cells through binding to Sonic HH (SHH) and down-regulating the key proteins (SHH, patched, smoothened, and glioma-associated oncogene) of the HH pathway, thus. Of note, Bzl-D remarkably suppressed the growth of HT-29 xenografts in nude mice. Conclusion: This research demonstrates that Bzl-D is a promising anti-rectal cancer drug, providing a new entry point for targeted therapy of CRC.

Gli1-mediated tumor cell-derived bFGF promotes tumor angiogenesis and pericyte coverage in non-small cell lung cancer

BackgroundTumor angiogenesis inhibitors have been applied for non-small cell lung cancer (NSCLC) therapy. However, the drug resistance hinders their further development. Intercellular crosstalk between lung cancer cells and vascular cells was crucial for anti-angiogenenic resistance (AAD). However, the understanding of this crosstalk is still rudimentary. Our previous study showed that Glioma-associated oncogene 1 (Gli1) is a driver of NSCLC metastasis, but its role in lung cancer cell-vascular cell crosstalk remains unclear.MethodsConditioned medium (CM) from Gli1-overexpressing or Gli1-knockdown NSCLC cells was used to educate endothelia cells and pericytes, and the effects of these media on angiogenesis and the maturation of new blood vessels were evaluated via wound healing assays, Transwell migration and invasion assays, tube formation assays and 3D coculture assays. The xenograft model was conducted to establish the effect of Gli1 on tumor angiogenesis and growth. Angiogenic antibody microarray analysis, ELISA, luciferase reporte, chromatin immunoprecipitation (ChIP), bFGF protein stability and ubiquitination assay were performed to explore how Gli1 regulate bFGF expression.ResultsGli1 overexpression in NSCLC cells enhanced the endothelial cell and pericyte motility required for angiogenesis required for angiogenesis. However, Gli1 knockout in NSCLC cells had opposite effect on this process. bFGF was critical for the enhancement effect on tumor angiogenesis. bFGF treatment reversed the Gli1 knockdown-mediated inhibition of angiogenesis. Mechanistically, Gli1 increased the bFGF protein level by promoting bFGF transcriptional activity and protein stability. Importantly, suppressing Gli1 with GANT-61 obviously inhibited angiogenesis.ConclusionThe Gli1-bFGF axis is crucial for the crosstalk between lung cancer cells and vascular cells. Targeting Gli1 is a potential therapeutic approach for NSCLC angiogenesis.

An improved medium for in vitro studies of female reproduction and oviposition in Schistosoma japonicum

BackgroundSchistosomiasis is a disease primarily caused by eggs laid by pathogens called schistosomes. Among the schistosome species infecting humans, Schistosoma japonicum possesses the largest fecundity; each adult female produces an average of 3500 eggs per day. The lack of proper culture conditions supporting continuous oviposition in vitro has precluded detailed investigation of mechanisms regulating sexual maturation and egg production in Schistosoma japonicum.MethodsWe optimized in vitro culture conditions by replacing reagents that are part of the classical ABC169 medium. Fast Blue BB staining and 4′,6-diamidino-2-phenylindole (DAPI) labeling were applied to observe the sexual development status of the females. In vitro RNA interference (RNAi) technology was used to validate the capability of the modified medium. The detection of male β-alanyl-tryptamine (BATT) was conducted using liquid chromatography–mass spectrometry (LC–MS).ResultsBoth m-AB169 (1640) and AB169 (1640) media are capable of facilitating the sexual development of paired virgin female S. japonicum, as well as sustaining the mature reproductive organs and egg production of adult S. japonicum for at least 22 days in vitro. M-AB169 (1640) provided a more stable condition for supporting the sexual maturity of female S. japonicum, as evidenced by the consistent initiation of egg production compared with AB169 (1640). Through a comparative analysis of S. japonicum and S. mansoni in diverse media, we demonstrated that these closely related species display distinct demands for their sexual development and egg production, suggesting a potential influence of nutritional factors on the observed variations in host ranges among different schistosome species. Importantly, we successfully identified the presence of the pheromone β-alanyl-tryptamine (BATT) in S. japonicum, previously identified in S. mansoni, highlighting its conserved role in schistosome reproductive development. Through the employment of double-stranded RNA (dsRNA) treatment to silence two genes that are involved in either the male (gli1, glioma-associated oncogene homolog 1) or female (vf1, vitellogenic factor 1) side in male-induced female reproductive development of S. mansoni, we confirmed that the combination of m-AB169 (1640) and RNAi technology has the capacity to facilitate in vitro studies of S. japonicum’s reproductive and oviposition processes.ConclusionsWe developed a novel medium, m-AB169 (1640), that not only maintains the mature reproductive organs and continuous oviposition of adult female Schistosoma japonicum for up to 22 days but also supports the reproductive development and subsequent egg-laying of virgin females after pairing with male worms. This study provides a valuable in vitro platform for functional studies of the mechanisms underlying the fascinating biology of the female sexual development and egg production of S. japonicum, which may accelerate the development of new strategies targeting schistosome egg production.Graphical

En1 promotes cell proliferation and migration via Hedgehog signaling pathway in esophageal squamous cell carcinoma

Objective: To explore the function and mechanism of transcription factor En1 in esophageal squamous cell carcinoma (ESCC). Methods: The correlations of En1 with prognosis were analyzed using the overall survival data of 9 397 pan-cancer patients and progression-free survival data of 4 349 pan-cancer patients from The Cancer Genome Atlas (TCGA) database. The En1 expression data in 53 and 155 cases of ESCC and their paired adjacent tissues were from Gene Expression Omnibus (GEO) database and National Genomics Data Center-Genome Sequence Archive(NGDC-GSA)database. Lentivirus was used to generate En1 stable knockout cell lines KYSE180 and KYSE450. The proliferation ability of the cells was detected by cell counting kit 8 and clone formation assay. The migration ability of the cells was detected by Transwell assay. The effect of En1 on the proliferation of ESCC was detected by xenograft experiment in BALB/c-nu/nu mice. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was used to detect the expressions of En1, glioma-associated oncogene family zinc finger 1 (GLI1), glioma-associated oncogene family zinc finger 2 (GLI2) and smoothened (SMO). Results: Pan-cancer data from TCGA showed that patients with low En1 expression had longer overall survival and progression-free survival than patients with high En1 expression (P< 0.001). Data from GEO and GSA databases also showed a high expression level of En1 in ESCC tissues compared with paired tissues (P＜0.001). Proliferation was inhibited after knockout of En1 in KYSE180 and KYSE450 cells (P＜0.001). The colony formation numbers decreased. The colony formation numbers of KYSE180 cells in the shEn1#1 group and the shEn1#2 group were 138.33±23.07 and 127.00±19.70, respectively, significantly lower than that of the shNC group 340.67±12.06 (P<0.001). The colony formation numbers of KYSE450 cells in the shEn1#1 group and the shEn1#2 group were 65.33±2.52 and 9.00±3.00, respectively, significantly lower than that of the shNC group 139.00±13.00 (P<0.001). The migration numbers was inhibited after knockout of En1 [the Transwell numbers of KYSE180 cells in the shEn1#1 group and the shEn1#2 group were 66.67±12.66 and 71.33±11.02, respectively, significantly lower than that of the shNC group 334.67±16.56 (P<0.001). The Transwell numbers of KYSE450 cells in the shEn1#1 group and the shEn1#2 group were 112.33±14.57 and 54.33±5.51, respectively, significantly lower than that of the shNC group 253.33±21.03 (P<0.001)]. Xenograft model showed a slower growth rate of shEn1#1 and shEn1#2 cell lines (P＜0.001). The tumor weights of KYSE450 cells in the shEn1#1 group and the shEn1#2 group were (0.046±0.026)g and (0.047±0.025)g, respectively, significantly lower than that of the shNC group (0.130±0.038)g (P＜0.001). After knockdown of En1, the relative expression levels of GLI1 in KYSE180 cells of the shEn1#1 group and the shEn1#2 group were 0.326±0.162 and 0.322±0.133, and the relative expression levels of GLI1 in KYSE450 cells of the shEn1#1 and shEn1#2 groups were 0.131±0.006 and 0.352±0.050, respectively, which were all lower than that in the shNC group (P＜0.01). After knockdown of En1, overexpression of GLI1 attenuated the inhibitory effect of knockdown of En1 on cell proliferation (P＜0.001), colony formation[the colony formation numbers of the shEn1#1-GLI1 group were 151.00±9.54, higher than 102.33±10.02 (P=0.004) of the shEn1#1-vector group] and migration [the migration numbers of the shEn1#1-GLI1 group were 193.67±10.07, higher than 109.33±11.50 (P<0.001) in the shEn1#1-vector group]. In clinical samples of ESCC, major regulatory factors of the Hedgehog pathway were up-regulated and the pathway was activated. Conclusion: En1 promotes the proliferation and migration of ESCC cells by regulating the Hedgehog pathway and can be used as a new potential target for targeted therapy of ESCC.

Photobiomodulation Recovers the Submandibular Gland in Vismodegib-Treated Rats.

Objective: The submandibular gland (SMG) produces the most saliva, and factors such as aging and chemotherapy can affect its structure and function. However, there are only temporary treatments available for salivary hypofunction. This study aimed to evaluate the effects of photobiomodulation (PBM) on the function of SMG by using a rat animal model and vismodegib, an antagonist of the sonic hedgehog (SHH) pathway. Methods: Vismodegib (10 mg/kg) drug was gavaged orally for 14 days in rats to significantly decrease the SHH signaling proteins [SHH, protein patched homolog 1 (PTCH1), smoothened protein (SMO), glioma-associated oncogene homolog 1 (GLI1)], induce damage in SMG tissue, and affect salivary functional markers AQP5 and Keratin5. After that, in conjunction with vismodegib administration, PBM was performed using an 850 nm high-power light-emitting diode (LED) device treated daily for 6 days at varying total energy densities of 60, 120, and 180 J/cm2 in at least 3 rats per group. The test results were confirmed by Western blot, immunofluorescence staining, and hematoxylin and eosin staining, and the statistics were t-test or one-way analysis of variance (ANOVA) with Tukey's multiple comparisons tests. Results: Significant decreases in the expression of SHH-related proteins (PTCH1, SMO, GLI1, p < 0.05) with damage of SMG ductal cells were observed with vismodegib administration. However, a significant increase in the expression levels of SHH-related proteins (SHH, SMO, GLI1, p < 0.05) and recovery of SMG ductal cells damaged after vismodegib administration were observed for PBM-treated groups. Salivary functional marker AQP5 also showed the same increase or decrease. Conclusions: This study found that vismodegib damages SMG ductal cells and decreases SHH-related proteins and associated salivary functional markers. Also, 850 nm high-power LED recovered the damaged structure of SMG and increased SHH-related proteins and salivary functional markers. The study results suggest that PBM can restore SMG structure and function through SHH signaling.

Genome-wide association identifies novel ROP risk loci in a multiethnic cohort

We conducted a genome-wide association study (GWAS) in a multiethnic cohort of 920 at-risk infants for retinopathy of prematurity (ROP), a major cause of childhood blindness, identifying 1 locus at genome-wide significance level (p < 5×10-8) and 9 with significance of p < 5×10-6 for ROP ≥ stage 3. The most significant locus, rs2058019, reached genome-wide significance within the full multiethnic cohort (p = 4.96×10-9); Hispanic and European Ancestry infants driving the association. The lead single nucleotide polymorphism (SNP) falls in an intronic region within the Glioma-associated oncogene family zinc finger 3 (GLI3) gene. Relevance for GLI3 and other top-associated genes to human ocular disease was substantiated through in-silico extension analyses, genetic risk score analysis and expression profiling in human donor eye tissues. Thus, we identify a novel locus at GLI3 with relevance to retinal biology, supporting genetic susceptibilities for ROP risk with possible variability by race and ethnicity.

Impact of Disease-Associated Mutations on the Deaminase Activity of ADAR1.

The innate immune system relies on molecular sensors to detect distinctive molecular patterns, including viral double-stranded RNA (dsRNA), which triggers responses resulting in apoptosis and immune infiltration. Adenosine Deaminases Acting on RNA (ADARs) catalyze the deamination of adenosine (A) to inosine (I), serving as a mechanism to distinguish self from non-self RNA and prevent aberrant immune activation. Loss-of-function mutations in the ADAR1 gene are one cause of Aicardi Goutières Syndrome (AGS), a severe autoimmune disorder in children. Although seven out of the eight AGS-associated mutations in ADAR1 occur within the catalytic domain of the ADAR1 protein, their specific effects on the catalysis of adenosine deamination remain poorly understood. In this study, we carried out a biochemical investigation of four AGS-causing mutations (G1007R, R892H, K999N, and Y1112F) in ADAR1 p110 and truncated variants. These studies included adenosine deamination rate measurements with two different RNA substrates derived from human transcripts known to be edited by ADAR1 p110 (glioma-associated oncogene homologue 1 (hGli1), 5-hydroxytryptamine receptor 2C (5-HT2cR)). Our results indicate that AGS-associated mutations at two amino acid positions directly involved in stabilizing the base-flipped conformation of the ADAR-RNA complex (G1007R and R892H) had the most detrimental impact on catalysis. The K999N mutation, positioned near the RNA binding interface, altered catalysis contextually. Finally, the Y1112F mutation had small effects in each of the assays described here. These findings shed light on the differential effects of disease-associated mutations on adenosine deamination by ADAR1, thereby advancing our structural and functional understanding of ADAR1-mediated RNA editing.

Glioma-associated oncogene homolog 1 in breast invasive carcinoma: a comprehensive bioinformatic analysis and experimental validation.

Breast cancer, despite significant advancements in treatment, remains a major cause of cancer-related deaths among women. Immunotherapy, an emerging therapeutic strategy, offers promise for better outcomes, particularly through the modulation of immune functions. Glioma-Associated Oncogene Homolog 1 (GLI1), a transcription factor implicated in cancer biology, has shown varying roles in different cancers. However, its immunoregulatory functions in breast invasive carcinoma (BRCA) remain elusive. The current study aimed to unravel the expression patterns and immune-regulatory roles of GLI1 in BRCA. Utilizing multiple bioinformatic platforms (TIMER2.0, GEPIA2, and R packages) based on The Cancer Genome Atlas (TCGA) and/or Genotype-Tissue Expression (GTEx) databases, we analyzed the expression of GLI1 in BRCA and its pan-cancer expression profiles. We further validated these findings by conducting qPCR and immunohistochemical staining on clinical BRCA samples. Kaplan-Meier analysis and Cox proportional hazards regression were performed to assess the prognostic value of GLI1. Additionally, the association between GLI1 expression and immune infiltration within the tumor immune microenvironment (TMIE) was examined. The findings reveal dysregulated expression of GLI1 in numerous cancers, with a significant decrease observed in BRCA. High GLI1 expression indicated better survival outcomes and was correlated with the age and stage of BRCA patients. GLI1 was involved in immune status, as evidenced by its strong correlations with immune and stromal scores and the infiltration levels of multiple immune cells. Meanwhile, GLI1 was co-expressed with multiple immune-related genes, and high GLI1 expression was associated with the activation of immune-related pathways, such as binding to proteasome and mismatch repair and retinol metabolism signaling pathways. Additionally, the differential expression of GLI1 may be related to the effect of immunotherapy on CTLA-4, PD-1, and other signals, and can effectively predict the immune efficacy. Our study underscores the critical role of GLI1 in BRCA, both as a potential tumor suppressor and an immune regulator. The association between GLI1 expression and favorable prognosis suggests its potential as a prognostic biomarker and immunotherapeutic target in BRCA.