Ptch1 is essential for cochlear marginal cell differentiation and stria vascularis formation.

A common cause of deafness in humans is dysregulation of the endocochlear potential generated by the stria vascularis (SV). Thus, proper formation of the SV is critical for hearing. Using single-cell transcriptomics and a series of Shh signaling mutants, we discovered that the Shh receptor Patched1 (Ptch1) is essential for marginal cell (MC) differentiation and SV formation. Single-cell RNA sequencing analyses revealed that the cochlear roof epithelium is already specified into discrete domains with distinctive gene expression profiles at embryonic day 14, with Gsc as a marker gene of the MC lineage. Ptch1 deficiency leads to defective specification of MC precursors along the cochlear basal-apical regions. We demonstrated that elevated Gli2 levels impede MC differentiation through sustaining Otx2 expression and maintaining the progenitor state of MC precursors. Our results uncover an early specification of cochlear non-sensory epithelial cells and establish a crucial role of the Ptch1-Gli2 axis in regulating the development of SV.

Salidroside promotes angiogenesis after cerebral ischemia in mice through Shh signaling pathway.

AIMS: The purpose of this study was to explore the impacts of salidroside on vascular regeneration, vascular structural changes and long-term neurological recuperation following cerebral ischemia and its possible mechanism. MAIN METHODS: From Day 1 to Day 28, young male mice with middle cerebral artery blockage received daily doses of salidroside and measured neurological deficits. On the 7th day after stroke, the volume of cerebral infarction was determined using TTC and HE staining. Microvascular density, astrocyte coverage, angiogenesis and the expression of the Shh signaling pathway were detected by IF, qRTPCR and WB at 7, 14 and 28 days after stroke. Changes in blood flow, blood vessel density and diameter from stroke to 28 days were measured by the LSCI and TPMI. KEY FINDINGS: Compared with the dMACO group, the salidroside treatment group significantly promoted the recovery of neurological function. Salidroside was found to enhance cerebral blood flow perfusion and reduce the infarct on the 7th day after stroke. From the 7th to the 28th day after stroke, salidroside treatment boosted the expression of CD31, CD31+/BrdU+, and GFAP in the cortex around the infarction site. On the 14th day after stroke, salidroside significantly enhanced the width and density of blood vessels. Salidroside increased the expression of histones and genes in the Shh signaling pathway during treatment, and this effect was weakened by the Shh inhibitor Cyclopamine. SIGNIFICANCE: Salidroside can restore nerve function, improve cerebral blood flow, reduce cerebral infarction volume, increase microvessel density and promote angiogenesis via the Shh signaling pathway.

Molecular mechanism of angiogenesis for cerebral infarction rats by acupuncture intervention based on sonic hedgehog signaling pathway.

BACKGROUND: To study the factors of the Sonic Hedgehog (Shh) signaling pathway after permanent cerebral ischemic and the effects by acupuncture. METHODS: Male Wistar rats were divided into Electro-acupuncture (EA) group, Model Control (MC) group, and blank control (Control) group. EA and MC were divided into 9 phases, namely 1 h, 3 h, 6 h, 9 h, 12 h, 24 h, 3 d, 7 d, and 12 d after the operation. The neurological deficits and permanent cerebral ischemic volume were observed. The immunofluorescence method was used to examine the angiogenesis. (Polymerase Chain Reaction) PCR and (Immunohistochemistry) IHC were used to test the changes in Shh, Ptch, Smo, and Gli2 mRNA and proteins. RESULTS: The neurological severity scores (NSS) of the Control was 0, the score of the EA group was less than that of the MC. The cerebral permanent ischemic volume of the Control was 0 %, and the EA group's was smaller than that of the MC. The expression of copositive cells in the EA group was higher than the MC's from 12 h to 12 d, and the EA group had more peripheral blood vessels. The rat brain expressions of Shh, Ptch, Smo and Gli2 mRNA and proteins in the MC was higher than that of the Control, the rat brain expression of the EA group was higher than that of the MC. CONCLUSIONS: EA can upregulate the expression of the Shh signaling pathway factors, thereby promoting angiogenesis.

A de novo variant in RERE causes autistic behavior by disrupting related genes and signaling pathway.

Autism spectrum disorder (ASD) is a highly variable neurodevelopmental disorder that typically manifests childhood, characterized by a triad of symptoms: impaired social interaction, communication difficulties, and restricted interests with repetitive behaviors. De novo variants in related genes can cause ASD. We present the case of a 6-year-old Chinese boy with autistic behavior, including language communication impairments, intellectual disabilities, stunted development, and irritability in social interactions. Using Sanger sequencing, we confirmed a pathogenic in the RERE gene (NM_012102.4) (c.3732delC, p.Tyr1245Thrfs*12; EX21; Het). Subsequently, we generated an RERE point mutation cell line (ReMut) using CRISPR/Cas9 Targeted Genome Editing. Immunofluorescence was conducted to determine the location of the mutant RERE. RNA-sequencing and mass spectrometry analyses were performed to elucidate the ASD-related genes and signaling pathways disrupted by this variant in RERE. We identified 3790 differentially expressed genes and 684 differentially expressed proteins. The SHH signaling pathway was found to be downregulated, and the Hippo pathway was upregulated in ReMut. Genes implicated in autism, such as CNTNAP2, STX1A, FARP2, and GPC1, were significantly downregulated. Simultaneously, we noted alterations in HDAC1 and HDAC2, which are members of the WHHERE complex, suggesting their role in the pathogenesis of this patient. In conclusion, we report a de novo variant in RERE associated with autistic behavior. The finding that ASD is associated with RERE variants underscore the role of genetic factors in ASD and provides insights regarding the mechanisms underlying RERE variants in disease onset.

Rutaecarpine Ameliorates Murine N-Methyl-N'-Nitro-N-Nitrosoguanidine-Induced Chronic Atrophic Gastritis by Sonic Hedgehog Pathway.

CAG is a burdensome and progressive disease. Numerous studies have shown the effectiveness of RUT in digestive system diseases. The therapeutic effects of RUT on MNNG-induced CAG and the potential mechanisms were probed. MNNG administration was employed to establish a CAG model. The HE and ELISA methods were applied to detect the treatment effects. WB, qRT-PCR, immunohistochemistry, TUNEL, and GES-1 cell flow cytometry approaches were employed to probe the mechanisms. The CAG model was successfully established. The ELISA and HE staining data showed that the RUT treatment effects on CAG rats were reflected by the amelioration of histological damage. The qRT-PCR and WB analyses indicated that the protective effect of RUT is related to the upregulation of the SHH pathway and downregulation of the downstream of apoptosis to improve gastric cellular survival. Our data suggest that RUT induces a gastroprotective effect by upregulating the SHH signaling pathway and stimulating anti-apoptosis downstream.

Total saponins from Trillium tschonoskii Maxim promote neurological recovery in model rats with post-stroke cognitive impairment.

Total saponins from Trillium tschonoskii Maxim (TSTT), a bioactive component of local natural herbs in the Enshi area, China, have been demonstrated to have functions of restoring cognitive capacity and promoting axonal regeneration post-stroke, but the mechanism of this process remains unclear. The hippocampus is a critical tissue for controlling learning and memory capacity, and the sonic hedgehog (Shh) signaling pathway plays a major role in the patterning and synaptic plasticity of hippocampal neural circuits. Therefore, we aimed to investigate whether TSTT could restore learning and cognitive functions by modulating the Shh pathway in rats with post-stroke cognitive impairment (PSCI). The ischemia model was established by permanent middle cerebral artery occlusion (MCAO) in 100 Sprague-Dawley (SD) rats, and the model rats were administered using TSTT (100 mg/kg) or donepezil hydrochloride as the positive control (daily 0.45 mg/kg, DON) for 4 weeks after the operation. As assessed by the Morris water maze test, the cognitive function of PSCI rats was significantly improved upon TSTT treatment. Meanwhile, the cerebral infarct volume reduced with TSTT, as shown by HE and TTC staining, and the number of Nissl bodies and dendritic spine density were significantly increased, as shown by Nissl and Golgi staining. In addition, TSTT upregulated PSD-95, SYN, and GAP-43, and inhibited neuronal apoptosis, as evidenced by increased Bcl-2 levels along with decreased Bax and caspase-3 expression. TSTT could also significantly upregulate Shh, Ptch1, Smo, and Gli1 proteins, indicating the activation of the Shh signaling pathway. Therefore, TSTT can protect PSCI rats by inhibiting apoptosis and promoting neuronal synaptic remodeling. The Shh pathway is also involved.

The ALKBH5/SOX4 axis promotes liver cancer stem cell properties via activating the SHH signaling pathway.

Hepatocellular carcinoma (HCC), featured with high prevalence and poor prognosis, is the major cause of cancer-related deaths worldwide. As a subgroup of liver cancer cells capable of differentiation, tumorigenesis and self-renewal, liver cancer stem cells (LCSCs) serve as one of the reasons leading to HCC progression and therapeutic resistance. Therefore, in-depth exploration of novel molecular biomarkers related to LSCSs is of great necessity. In our study, we found that human AlkB homolog H5 (ALKBH5) expression was enriched in LCSCs, which could foster proliferation, invasion and migration of the HCC cells. Mechanically, ALKBH5 positively mediated the expression of SOX4 via demethylation, and SOX4 promoted SHH expression at the transcriptional level to activate sonic hedgehog (SHH) signaling pathway. Furthermore, exosomes derived from CD133+ HCC cells could transmit ALKBH5 into THP-1 cells, which might be associated with M2 polarization of macrophages. In summary, the ALKBH5/SOX4 axis plays a significant role in exacerbating LCSC properties via activating SHH signaling pathway, and ALKBH5 could be a critical effector related to macrophage M2 polarization. These findings might provide a promising new biomarker for HCC diagnosis and treatment.

Genomic alterations of oligodendrogliomas at distant recurrence.

BACKGROUND: Oligodendroglioma is known for its relatively better prognosis and responsiveness to radiotherapy and chemotherapy. However, little is known about the evolution of genetic changes as oligodendroglioma progresses. METHODS: In this study, we evaluated gene evolution invivo during tumor progression based on deep whole-genome sequencing data (ctDNA). We analyzed longitudinal genomic data from six patients during tumor evolution, of which five patients developed distant recurrence. RESULTS: Whole-exome sequencing demonstrated that the rate of shared mutations between the primary and recurrent samples was relatively low. In two cases, even well-known major driver mutations in CIC and FUBP1 that were detected in primary tumors were not detected in the relapse samples. Among these cases, two patients had a conversion from the IDH mutation in the originating state to the IDH1 wild state during the process of gene evolution under chemotherapy treatment, indicating that the cell phenotype and genetic characteristics of oligodendroglioma may change during tumor evolution. Two patients received long-term temozolomide (TMZ) treatment before the operation, and we found that recurrence tumors harbored mutations in the PI3K/AKT and Sonic hedgehog (SHh) signaling pathways. Hypermutation occurred with mutations in MMR genes in one patient, contributing to the rapid progression of the tumor. CONCLUSION: Oligodendroglioma displayed great spatial and temporal heterogeneity during tumor evolution. The PI3K/AKT and SHh signaling pathways may play an important role in promoting treatment resistance and distant relapse during oligodendroglioma evolution. In addition, there was a tendency to increase the degree of tumor malignancy during evolution. Distant recurrence may be a later event duringoligodendroglioma progression. CLINICALTRIALS: gov, Identifier: NCT05512325.

Recent advances in the molecular understanding of medulloblastoma.

Medulloblastoma is the most common pediatric malignant brain tumor composed of four molecular subgroups. Recent intensive genomics has greatly contributed to our understanding of medulloblastoma pathogenesis. Sequencing studies identified novel mutations involved in the cyclic AMP-dependent pathway or RNA processing in the Sonic Hedgehog (SHH) subgroup, and core-binding factor subunit alpha (CBFA) complex in the group 4 subgroup. Likewise, single-cell sequencing provided detailed insights into the cell of origin associated with brain development. In this review, we will summarize recent findings by sequencing analyses for medulloblastoma.

ABTC-0904: targeting glioma stem cells in GBM: a phase 0/II study of hedgehog pathway inhibitor GDC-0449.

PURPOSE: Gliomagenesis and resistance of glioblastoma (GBM) are believed to be mediated by glioma stem cells (GSC). Evidence suggests that SHH signaling promotes GSC proliferation and self-renewal. METHODS: ABTC-0904 was a two-arm, multicenter phase 0/II study of GDC-0449, an oral inhibitor of Smoothened (SMO) in patients undergoing resection for recurrent GBM. All patients (Arms I and II) had surgery and received drug post-operatively. Only patients in Arm I received drug prior to surgery. The primary objective was to determine 6-month progression free survival (PFS-6). Secondary endpoints include median PFS (mPFS) and overall survival (mOS), response rate, and toxicity. Correlative studies included bioanalysis of GDC-0449, and inhibition of SHH signaling, GSC proliferation and self-renewal. RESULTS: Forty-one patients were enrolled. Pharmacokinetics of GDC-0449 in plasma demonstrated levels within expected therapeutic range in 75% of patients. The proportion of tumorcells producing CD133+ neurospheres, neurosphere proliferation, self-renewal, and expression of the SHh downstream signaling was significantly decreased in Arm I following GDC-0449 treatment (p < 0.005; p < 0.001 respectively) compared to Arm II (no drug pre-op). Treatment was well tolerated. There were no objective responders in either arm. Overall PFS-6 was 2.4% (95% CI 0.9-11.1%). Median PFS was 2.3 months (95% CI 1.9-2.6) and mOS was 7.8 months (95% CI 5.4-10.1). CONCLUSIONS: GDC-0449 was well tolerated, reached tumor, and inhibited CD133+ neurosphere formation, but had little clinical efficacy as a single agent in rGBM. This suggests growth and maintenance of rGBM is not solely dependent on the SHH pathway thus targeting SMO may require combined approaches.

SHH/GLI2-TGF-ß1 feedback loop between cancer cells and tumor-associated macrophages maintains epithelial-mesenchymal transition and endoplasmic reticulum homeostasis in cholangiocarcinoma.

BACKGROUND: Tumor-associated macrophages (TAMs) play a dual role in tumors. However, the factors which drive the function of TAMs in cholangiocarcinoma remain largely undefined. METHODS: SHH signaling pathway and endoplasmic reticulum stress (ERS) indicators were detected in clinical tissues and cholangiocarcinoma cell lines. TAMs were co-cultured with cholangiocarcinoma cells under conditions of hypoxia/normoxia. Polarized TAMs were counted by flow cytometry, and TGF-ß1 levels in cell supernatants were detected by ELISA. The effects of glioma-associated oncogene GLI2 on TAMs themselves and cholangiocarcinoma cells were examined by conducting interference and overexpression assays. RESULTS: The SHH signaling pathway and ERS were both activated in tumor tissues or tumor cell lines under conditions of hypoxia. In co-culture experiments, the presence of cholangiocarcinoma cells increased the proportion of M2-polarized TAMs and the secretion of TGF-ß1 by TAMs, while knockdown of SHH expression reversed those increases. Overexpression of GLI2 in TAMS or stimulation of TAMS with Hh-Ag1.5 increased their levels of TGF-ß1 expression. Furthermore, under co-culture conditions, interference with GLI2 expression in TAMs reduced the tumor cell migration, invasion, and ER homeostasis induced by Hh-Ag1.5-pretreated TAMs. Under conditions of hypoxia, the presence of cholangiocarcinoma cells promoted the expression of GLI2 and TGF-ß1 in Tams, and in turn, TAMs inhibited the apoptosis and promoted the migration and invasion of cholangiocarcinoma cells. In vivo, an injection of cholangiocarcinoma cells plus TAMs contributed to the growth, EMT, and ER homeostasis of tumor tissue, while an injection of TAMs with GLI2 knockdown had the opposite effects. CONCLUSION: Cholangiocarcinoma cells regulated TAM polarization and TGF-ß1 secretion via a paracrine SHH signaling pathway, and in turn, TAMs promoted the growth, EMT, and ER homeostasis of cholangiocarcinoma cells via TGF-ß1.

The Role of GLI in the Regulation of Hepatic Epithelial-Mesenchymal Transition in Biliary Atresia.

Objective: To study the regulatory role of GLI1/GLI2, a nuclear transcription factor of the Sonic hedgehog (Shh) signaling pathway, in epithelial-mesenchymal transition (EMT) related to hepatic fibrosis in patients with biliary atresia (BA). Methods: The messenger RNA (mRNA) and protein expression levels of GLI1/GLI2, Snail/Slug, and other Shh- and EMT-related cytokines were tested in the liver tissues of BA patients and animals. Then, GLI1/GLI2 was silenced and overexpressed in mouse intrahepatic bile duct epithelial cells (mIBECs) and BA animals to investigate changes in the mRNA and protein expression of EMT key factors and liver fibrosis indicators. After silencing and overexpression of GLI1/GLI2, immunofluorescence was used to detect the expression of cytokeratin-19 (CK19) and α-smooth muscle actin (α-SMA) in mIBECs, and hematoxylin and eosin (HE) staining and Masson staining were used to observe the degree of liver fibrosis in the BA animals. Results: Compared with the control, the mRNA and protein expression levels of GLI2, Snail, vimentin, and α-SMA were significantly increased and those of E-cadherin were significantly decreased in liver tissue from BA patients and animals. Overexpression of GLI2 increased the mRNA and protein expression levels of Snail, vimentin, and α-SMA and that of E-cadherin was significantly decreased in mIBECs and BA animals. After GLI2 silencing, the opposite pattern was observed. Immunofluorescence detection showed enhanced expression of the bile duct epithelial cell marker CK19 in mIBECs after GLI2 silencing and enhanced expression of the mesenchymal cell marker α-SMA after GLI2 overexpression. HE and Masson staining suggested that the GLI2-overexpressing group had a significantly higher degree of fibrosis. Conclusion: The Shh signaling pathway plays an important role in fibrogenesis in BA. GLI2 can significantly regulate EMT in mIBECs and livers of BA mice.

Deleterious Rare Mutations of GLI1 Dysregulate Sonic Hedgehog Signaling in Human Congenital Heart Disease.

The Glioma-associated oncogene (Gli) family members of zinc finger DNA-binding proteins are core effectors of Sonic hedgehog (SHH) signaling pathway. Studies in model organisms have identified that the Gli genes play critical roles during organ development, including the heart, brain, kidneys, etc. Deleterious mutations in GLI genes have previously been revealed in several human developmental disorders, but few in congenital heart disease (CHD). In this study, the mutations in GLI1-3 genes were captured by next generation sequencing in human cohorts composed of 412 individuals with CHD and 213 ethnically matched normal controls. A total of 20 patient-specific nonsynonymous rare mutations in coding regions of human GLI1-3 genes were identified. Functional analyses showed that GLI1 c.820G> T (p.G274C) is a gain-of-function mutation, while GLI1 c.878G>A (p.R293H) and c.1442T>A (p.L481X) are loss-of-function mutations. Our findings suggested that deleterious rare mutations in GLI1 gene broke the balance of the SHH signaling pathway regulation and may constitute a great contribution to human CHD, which shed new light on understanding genetic mechanism of embryo cardiogenesis regulated by SHH signaling.

Metformin regulates macrophage polarization via the Shh signaling pathway to improve pulmonary vascular development in bronchopulmonary dysplasia.

Metformin has potential anti-inflammatory properties and accelerates wound healing by enhancing vascular development. In this study, we aimed to investigate the effects of metformin on pulmonary vascular development and the underlying mechanism. Newborn mice were subcutaneously injected with metformin from day 2 after exposure to hyperoxia. Pulmonary vascular development, inflammation, and Shh signaling pathway-related protein expression were evaluated by western blotting and immunofluorescence staining. M2 macrophage polarization was measured by flow cytometry. The effect of metformin on macrophage polarization was determined using RAW264.7 macrophages exposed to 90% oxygen in vitro. The role of metformin and purmorphamine on M1 and M2 polarization was observed by flow cytometry. M2 polarization of pulmonary macrophages was inhibited after hyperoxic exposure, and metformin increased the number of M2 macrophages in the lung on postnatal day 14. Metformin upregulated CD31 expression and suppressed inflammation in the lung of mice exposed to hyperoxia on postnatal days 7 and 14. Metformin downregulated the Gli1 expression in macrophages in the lung after exposure to hyperoxia on postnatal day 14. In vitro studies showed that metformin inhibited the Gli1 expression in RAW264.7 macrophages exposed to 90% oxygen, which was reversed after purmorphamine pretreatment. Exposure to 90% oxygen inhibited the polarization of M2 macrophages, whereas metformin increased the number of M2 macrophages. Purmorphamine reversed the effects of metformin on M2 polarization and vascular endothelial growth factor (VEGF) upregulation in RAW264.7 macrophages exposed to hyperoxia. In conclusion, metformin regulates macrophage polarization via the Shh signaling pathway to improve pulmonary vascular development in bronchopulmonary dysplasia.

BBS7-SHH Signaling Activity Regulates Primary Cilia for Periodontal Homeostasis.

Objectives: Mechanical stimuli are essential for the maintenance of periodontal ligament (PDL) homeostasis. Although there are several studies on atrophic changes in PDL due to occlusal hypofunction, the underlying mechanism is still unknown. Here, we aimed to explore the changes of gene expression in occlusal hypofunctional PDL and elucidate the related role in maintaining the PDL homeostasis. Methods: To investigate the transcriptomic difference between control and hypofunctional PDL tissue from patients, RNA sequencing was performed on 34 human teeth. The atrophic changes in PDL were evaluated by histological analysis. The effect of the Bardet-Biedl syndrome 7 (BBS7) knockdown was evaluated by the RT-qPCR, Western blot, wound healing, and tubule formation assay. Results: We detected that the expression of BBS7 was downregulated in occlusal hypofunctional PDL through RNA sequencing. Dynamic changes, including the number of periodontal ligament cells, alignment of collagen fibers, diameter of blood vessels, appearance of primary cilia, and torturous oxytalan fibers, were observed following occlusal hypofunction. Furthermore, Sonic hedgehog signaling (Shh) activity was closely associated with BBS7 expression in PDL cells. In addition, the cell migration and angiogenesis were also suppressed by BBS7 knockdown in vitro. Conclusion: We suggest that BBS7 plays an essential role in maintaining Shh signaling activity for PDL homeostasis.

A high level of KLF12 causes folic acid-resistant neural tube defects by activating the Shh signaling pathway in mice†.

Although adequate periconceptional folic acid (FA) supplementation has reduced the occurrence of pregnancies affected by neural tube defects (NTDs), the mechanisms underlying FA-resistant NTDs are poorly understood, and thus NTDs still remain a global public health concern. A high level of Krüppel-like factor 12 (KLF12) exerts deleterious effects on heath in most cases, but evidence for its roles in development has not been published. We observed KLF12-overexpressing mice showed disturbed neural tube development. KLF12-overexpressing fetuses died in utero at approximately 10.5 days post-coitus, with 100% presenting cranial NTDs. Neither FA nor formate promoted normal neural tube closure in mutant fetuses. The RNA-seq results showed that a high level of KLF12 caused NTDs in mice via overactivating the sonic hedgehog (Shh) signaling pathway, leading to the upregulation of patched 1, GLI-Krüppel family member GLI1, hedgehog-interacting protein, etc., whereas FA metabolism-related enzymes did not express differently. PF-5274857, an antagonist of the Shh signaling pathway, significantly promoted dorsolateral hinge point formation and partially rescued the NTDs. The regulatory hierarchy between a high level of KLF12 and FA-resistant NTDs might provide new insights into the diagnosis and treatment of unexplained NTDs in the future.

Cyclosporin A impairs neurogenesis and cognitive abilities in brain development via the IFN-γ-Shh-BDNF pathway.

A wealth of evidence indicate that the peripheral immune activation alters brain development. However, it is still largely unclear whether and how peripheral immunosuppression affects neurodevelopment. Here, we found that the immunosuppressant cyclosporin A (CsA) decreased the number of BrdU+, BrdU+/DCX+, BrdU+/NeuN + cells in the hippocampus, impaired learning and memory and inhibited protein levels of the shh signaling pathway, including Shh, Smo and Gli1. However, the shh pathway receptor agonist SAG could block the impairment of cognitive ability and the decrease of hippocampal neurogenesis and brain-derived neurotrophic factor (BDNF) level induced by CsA. We also found that CsA decreased the level of interferon-gamma (IFN-γ), while up-regulation of IFN-γ altered the inhibitory effect of the shh signaling pathway and the decrease of BDNF induced by CsA. Collectively, these data indicate that peripheral CsA impairs neurogenesis and cognition in brain development through downregulating the IFN-γ-Shh-BDNF pathway. The present study guides us to correctly apply immunomodulatory drugs in early life and suggests that the IFN-γ-Shh-BDNF pathway may represent a novel protective target for neurodevelopment under the condition of immunosuppression.

ALKAL1 gene silencing prevents colorectal cancer progression via suppressing Sonic Hedgehog (SHH) signaling pathway.

Anaplastic lymphoma kinase (ALK) has been described in a range of human cancers and is involved in cancer initiation and progression via activating multiple signaling pathways, such as the PI3K-AKT, CRKL-C3G, MEKK2/3-MEK5-ERK5, JAK-STAT and MAPK signal pathways. Recently ALK and LTK ligand 1 (ALKAL1) also named "augmentor-ß" or "FAM150A" is identified as a potent activating ligands for human ALK that bind to the extracellular domain of ALK. However, due to its poor stability, the mechanisms of ALKAL1 underlying the tumor progression in the human cancers including colorectal cancer have not been well documented. Herein, ALKAL1 expression was evaluated by RNA sequencing datasets from The Cancer Genome Atlas (TCGA) of 625 cases colorectal cancer, immunohistochemical analysis of 377 cases colorectal cancer tissues, and Western blotting even Real-time PCR of 10 pairs of colorectal cancer tissues and adjacent normal tissues, as well as 8 colorectal cancer cell lines. Statistical analysis was performed to explore the correlation between ALKAL1 expression and clinicopathological features in colorectal cancer. Univariate and multivariate Cox regression analysis were performed to examine the association between ALKAL1 expression and overall survival. In vitro and in vivo assays were performed to assess the biological roles of ALKAL1 in colorectal cancer. Gene set enrichment analysis (GSEA), Western blotting and luciferase assays were used to identify the underlying signal pathway involved in the tumor progression role of ALKAL1. As a result, we showed that ALKAL1 was upregulated in colorectal cancer tissues and cell lines. Upregulation of ALKAL1 correlated with tumor malignancy and poor prognosis in colorectal cancer. ALKAL1 silencing inhibited tumorigenesis, metastasis and invasion of colorectal cancer cells, and inhibited SHH signaling pathway, which is essential for ALKAL1 induced migration. Our findings reveal a new mechanism by which ALKAL1 participates in colorectal cancer migration and invasion via activating the SHH signaling pathway.

Protective effect of sonic hedgehog signaling pathway activation on acute myocardial infarction.

To study changes in the sonic hedgehog (Shh) signaling pathway in acute myocardial infarction (AMI) and the protective effect of changes in Shh signaling pathway activity on AMI, specific pathogen-free (SPF) C57BL/6 mice were treated with left anterior descending (LAD) ligation to establish an AMI model. The samples were collected on the 1st, 3rd, 14th, and 21st days after AMI induction. After the operations, the mice were administered the Shh signaling pathway receptor agonist SAG1.3 (5 mg/kg/d) and antagonist SANT-1 (3.3 mg/kg/d) by intraperitoneal injection. The myocardial ischemia model was established by oxygen glucose deprivation (OGD) in vitro. The AMI mouse model and the in vitro OGD-induced myocardial ischemia model were established. The Smo agonist SAG1.3 was used to activate the Shh signaling pathway, thereby reducing the expression of Bcl-2 and Bax. The number of apoptotic cells was reduced. Administration of the antagonist SANT-1 inhibited Shh signaling pathway activity by increasing the expression of Bcl-2 and Bax, and the number of apoptotic cells increased. In conclusion, activation of the Shh signaling pathway improved cardiac functions and myocardial remodeling and reduced the apoptosis of myocardial cells.

MED12-Related Disease in a Chinese Girl: Clinical Characteristics and Underlying Mechanism.

The RNA polymerase II transcription subunit 12 homolog (MED12) is a member of the mediator complex, which plays a critical role in RNA transcription. Mutations in MED12 cause X-linked intellectual disability and other anomalies collectively grouped as MED12-related disorders. While MED12 mutations have been most commonly reported in male patients, we present the case of a 1-year-old girl with clinical characteristics similar to MED12-related disorders. To explore the clinical characteristics of the condition and its possible pathogenesis, we analyzed the patient's clinical data; genetic testing by whole-exome sequencing revealed a de novo heterozygous mutation (c.1249-1G > C) in MED12. Further cDNA experiments revealed that the patient had an abnormal splicing at the skipping of exon9, which may have produced a truncated protein. qPCR showed decreased MED12 gene expression level in the patient, and an X-chromosome inactivation test confirmed a skewed inactivation of the X-chromosome. The lymphoblast transcription levels of the genes involved in the Gli3-dependent sonic hedgehog (SHH) signaling pathway, namely, CREB5, BMP4, and NEUROG2, were found to be significantly elevated compared with those of her parents and sex- and age-matched controls. Our results support the view that MED12 mutations may dysregulate the SHH signaling pathway, which may have accounted for the aberrant craniofacial morphology of our patient.