Gli1 mRNA Expression Levels Research Articles

Construction of a Lentiviral Vector for Fgfr2 Overexpression and its Impact on the Biological Behavior of Cranial Suture Mesenchymal Stem Cells.

Suture mesenchymal stem cells (SuSCs), possessing self-renewal and multilineage differentiation abilities, play a crucial role in cranial bone growth. However, the impact of the disease-causing fibroblast growth factor receptor 2 (FGFR2) mutation on SuSCs in Crouzon syndrome has not been explored. This study aims to employ a lentivirus to overexpress Fgfr2 and investigate its role in the pathogenesis of Crouzon syndrome. Starting with the prevalent FGFR2 mutation site in patients with Crouzon syndrome, a lentiviral vector carrying the Fgfr2.C361Y mutation was developed and transfected into SuSCs, with a determined multiplicity of infection values. The experimental group, SuSCs+Fgfr2.C361Y, was compared with the empty vector and normal SuSC groups. Cell proliferation, cycle, apoptosis, and osteogenic functionality were assessed using CCK-8 assays, flow cytometry, ALP activity assays, and real-time quantitative polymerase chain reaction. The lentiviral vector effectively infected SuSCs, leading to heightened Fgfr2 expression, with optimal multiplicity of infection values of 80. The experimental group demonstrated decreased proliferation activity and a higher apoptosis rate compared with controls (P < 0.05). After osteogenic induction, the experimental group showed significantly higher ALP activity than controls (P < 0.05). Real-time quantitative polymerase chain reaction indicated lower mRNA expression levels of Gli1, Axin2, Pcna, Cdk2, and Bcl-2 in the experimental group than controls, whereas Bax, Runx2, and Bmp-2 showed higher expression (P < 0.05). This study constructed a lentivirus vector to upregulate Fgfr2 expression in SuSCs, suppressing stem cell stemness by inhibiting proliferation, promoting apoptosis, and accelerating premature osteogenic differentiation, resulting in premature suture closure. These findings establish the groundwork for further understanding the pathogenesis of Crouzon syndrome.

GLI1 activation is a key mechanism of erlotinib resistance in human non-small cell lung cancer.

Lung cancer is the leading cause of cancer-associated death worldwide. In recent years, the advancement of epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) targeted therapies has provided clinical benefits for lung cancer patients with EGFR mutations. The response to EGFR-TKI varies in patients with lung cancer, and resistance typically develops during the course of the treatment. Therefore, understanding biomarkers which can predict resistance to EGFR-TKI is important. Overexpression of GLI causes activation of the Hedgehog (Hh) signaling pathway and plays a critical role in oncogenesis in numerous types of cancer. In the present study, the role of GLI1 in erlotinib resistance was investigated. GLI1 mRNA and protein expression levels were determined using reverse transcription-quantitative PCR and immunohistochemistry (IHC) in lung cancer cell lines and tumor specimens, respectively. GLI1 mRNA expression levels were found to be positively correlated with the IC50 of erlotinib in 15 non-small cell lung cancer (NSCLC) cell lines. The downregulation of GLI1 using siRNA sensitized lung cancer cells to the erlotinib treatment, whereas the overexpression of GLI1 increased the survival of lung cancer cells in the presence of erlotinib, indicating that Hh/GLI activation may play a critical role in the development of TKI resistance in lung cancer. Combined treatment with erlotinib and a GLI1 inhibitor reduced the cell viability synergistically. A retrospective study of patients with NSCLC treated with erlotinib revealed that those with a high IHC score for GLI1 protein expression had a poorer prognosis. These results indicated that GLI1 is a key regulator for TKI sensitivity, and patients with lung cancer may benefit from the combined treatment of TKI and GLI1 inhibitor.

Aberrant activation of hedgehog signaling promotes cell proliferation via the transcriptional activation of forkhead Box M1 in colorectal cancer cells

BackgroundRecent evidence suggests that the aberrant activation of Hedgehog (Hh) signaling by Gli transcription factors is characteristic of a variety of aggressive human carcinomas, including colorectal cancer (CRC). Forkhead box M1 (FoxM1) controls the expression of a number of cell cycle regulatory proteins, and FoxM1 expression is elevated in a broad range of human malignancies, which suggests that it plays a crucial role in tumorigenesis. However, the mechanisms underlying FoxM1 expression are not fully understood. Here, we aim to further investigate the molecular mechanism by which Gli1 regulates FoxM1 in CRC.MethodsWestern blotting and immunohistochemistry (IHC) were used to evaluate FoxM1 and Gli1 protein expression, respectively, in CRC tissues and matched adjacent normal mucosa. BrdU (5-bromo-2′-deoxyuridine) and clone formation assays were used to clarify the influence of FoxM1 on CRC cell growth and proliferation. Chromatin immunoprecipitation (ChIP) and luciferase experiments were performed to explore the potential mechanisms by which Gli1 regulates FoxM1. Additionally, the protein and mRNA expression levels of Gli1 and FoxM1 in six CRC cell lines were measured using Western blotting and real-time PCR. Finally, the effect of Hh signaling on the expression of FoxM1 was studied in cell biology experiments, and the effects of Hh signaling activation and FoxM1 inhibition on the distribution of CRC cells among cell cycle phases was assessed by flow cytometry.ResultsGli1 and FoxM1 were abnormally elevated in human CRC tissues compared with matched adjacent normal mucosa samples, and FoxM1 is a downstream target gene of the transcription factor Gli1 in CRC and promoted CRC cell growth and proliferation. Moreover, the aberrant activation of Hh signaling promoted CRC cell proliferation by directly binding to the promoter of FoxM1 and transactivating the activity of FoxM1 in CRC cells.ConclusionThe dysregulation of the Hh-Gli1-FoxM1 axis is essential for the proliferation and growth of human CRC cells and offers a potent target for therapeutic intervention in CRC.

In vitro and in vivo inhibition of breast cancer cell growth by targeting the Hedgehog/GLI pathway with SMO (GDC-0449) or GLI (GANT-61) inhibitors.

Aberrant Hedgehog (Hh)/glioma-associated oncogene (GLI) signaling has been implicated in cancer progression. Here, we analyzed GLI1, Sonic Hedgehog (Shh) and NF-κB expression in 51 breast cancer (ductal carcinoma) tissues using immunohistochemistry. We found a positive correlation between nuclear GLI1 expression and tumor grade in ductal carcinoma cases. Cytoplasmic Shh staining significantly correlated with a lower tumor grade. Next, the in vitro effects of two Hh signaling pathway inhibitors on breast cancer cell lines were evaluated using the Smoothened (SMO) antagonist GDC-0449 and the direct GLI1 inhibitor GANT-61. GDC-0449 and GANT-61 exhibited the following effects: a) inhibited breast cancer cell survival; b) induced apoptosis; c) inhibited Hh pathway activity by decreasing the mRNA expression levels of GLI1 and Ptch and inhibiting the nuclear translocation of GLI1; d) increased/decreased EGFR and ErbB2 protein expression, reduced p21-Ras and ERK1/ERK2 MAPK activities and inhibited AKT activation; and e) decreased the nuclear translocation of NF-κB. However, GANT-61 exerted these effects more effectively than GDC-0449. The in vivo antitumor activities of GDC-0449 and GANT-61 were analyzed in BALB/c mice that were subcutaneously inoculated with mouse breast cancer (TUBO) cells. GDC-0449 and GANT-61 suppressed tumor growth of TUBO cells in BALB/c mice to different extents. These findings suggest that targeting the Hh pathway using antagonists that act downstream of SMO is a more efficient strategy than using antagonists that act upstream of SMO for interrupting Hh signaling in breast cancer.

AB0107 Sonic hedgehog promotes survival and apoptosis resistance through smoothened protein in human umbilical vein endothelial cell

BackgroundRheumatoid arthritis (RA) is characterized by chronic synovitis and pannus formation, leading to destruction of cartilage and bone. Sonic Hedgehog (Shh) signaling pathway plays significant role in regulating the morphogenesis...