Prognostication of colorectal cancer liver metastasis by CE-based radiomics and machine learning.

The liver is the most common organ for the formation of colorectal cancer metastasis. Non-invasive prognostication of colorectal cancer liver metastasis (CRLM) may better inform clinicians for decision-making. Contrast-enhanced computed tomography images of 180 CRLM cases were included in the final analyses. Radiomics features, including shape, first-order, wavelet, and texture, were extracted with Pyradiomics, followed by feature engineering by penalized Cox regression. Radiomics signatures were constructed for disease-free survival (DFS) by both elastic net (EN) and random survival forest (RSF) algorithms. The prognostic potential of the radiomics signatures was demonstrated by Kaplan-Meier curves and multivariate Cox regression. 11 radiomics features were selected for prognostic modelling for the EN algorithm, with 835 features for the RSF algorithm. Survival heatmap indicates a negative correlation between EN or RSF risk scores and DFS. Radiomics signature by EN algorithm successfully separates DFS of high-risk and low-risk cases in the training dataset (log-rank test: p < 0.01, hazard ratio: 1.45 (1.07-1.96), p < 0.01) and test dataset (hazard ratio: 1.89 (1.17-3.04), p < 0.05). RSF algorithm shows a better prognostic implication potential for DFS in the training dataset (log-rank test: p < 0.001, hazard ratio: 2.54 (1.80-3.61), p < 0.0001) and test dataset (log-rank test: p < 0.05, hazard ratio: 1.84 (1.15-2.96), p < 0.05). Radiomics features have the potential for the prediction of DFS in CRLM cases.

ER membrane complex (EMC): Structure, functions, and roles in diseases.

The endoplasmic reticulum (ER) is the largest membrane system in eukaryotic cells and is the primary site for the biosynthesis of lipids and carbohydrates, as well as for the folding, assembly, modification, and transport of secreted and integrated membrane proteins. The ER membrane complex (EMC) on the ER membrane is an ER multiprotein complex that affects the quality control of membrane proteins, which is abundant and widely preserved. Its disruption has been found to affect a wide range of processes, including protein and lipid synthesis, organelle communication, endoplasmic reticulum stress, and viral maturation, and may lead to neurodevelopmental disorders and cancer. Therefore, EMC has attracted the attention of many scholars and become a hot field. In this paper, we summarized the main contributions of the research of EMC in the past nearly 15 years, and reviewed the structure and function of EMC as well as its related diseases. We hope this review will promote further progress of research on EMC.

The m6A reader YTHDC2 maintains visual function and retinal photoreceptor survival through modulating translation of PPEF2 and PDE6B.

Inherited retinal dystrophies (IRDs) are major causes of visual impairment and irreversible blindness worldwide, while the precise molecular and genetic mechanisms are still elusive. N6-methyladenosine (m6A) modification is the most prevalent internal modification in eukaryotic mRNA. YTH domain containing 2 (YTHDC2), an m6A reader protein, has recently been identified as a key player in germline development and human cancer. However, its contribution to retinal function remains unknown. Here, we explore the role of YTHDC2 in the visual function of retinal rod photoreceptors by generating rod-specific Ythdc2 knockout mice. Results show that Ythdc2 deficiency in rods causes diminished scotopic ERG responses and progressive retinal degeneration. Multi-omics analysis further identifies Ppef2 and Pde6b as the potential targets of YTHDC2 in the retina. Specifically, via its YTH domain, YTHDC2 recognizes and binds m6A-modified Ppef2 mRNA at the coding sequence and Pde6b mRNA at the 5'-UTR, resulting in enhanced translation efficiency without affecting mRNA levels. Compromised translation efficiency of Ppef2 and Pde6b after YTHDC2 depletion ultimately leads to decreased protein levels in the retina, impaired retinal function, and progressive rod death. Collectively, our finding highlights the importance of YTHDC2 in visual function and photoreceptor survival, which provides an unreported elucidation of IRD pathogenesis via epitranscriptomics.

Excessive SOX8 reprograms energy and iron metabolism to prime hepatocellular carcinoma for ferroptosis.

Lipid peroxidation and redox imbalance are hallmarks of ferroptosis, an iron-dependent form of cell death. Growing evidence suggests that dysregulation in glycolipid metabolism and iron homeostasis substantially contribute to the development of hepatocellular carcinoma (HCC). However, there is still a lack of comprehensive understanding regarding the specific transcription factors that are capable of coordinating glycolipid and redox homeostasis to initiate the onset of ferroptosis. We discovered that overexpression of SOX8 leads to impaired mitochondria integrate, increased oxidative stress, and enhanced lipid peroxidation. These effects can be attributed to the inhibitory impact of SOX8 on de novo lipogenesis, glycolysis, the tricarboxylic acid cycle (TCA), and the pentose phosphate pathway (PPP). Additionally, upregulation of SOX8 results in reduced synthesis of NADPH, disturbance of redox homeostasis, disruption of mitochondrial structure, and impairment of the electron transport chain. Furthermore, the overexpression of SOX8 enhances the process of ferroptosis by upregulating the expression of genes associated with ferroptosis and elevating intracellular levels of ferrous ion. Importantly, the overexpressing of SOX8 has been observed to inhibit the proliferation of HCC in immunodeficient animal models. In conclusion, the findings suggest that SOX8 has the ability to alter glycolipid and iron metabolism of HCC cells, hence triggering the process of ferroptosis. The results of our study present a novel strategy for targeting ferroptosis in the therapy of HCC.

Knockdown of TMEM30A in renal tubular epithelial cells leads to reduced glucose absorption.

The kidney reabsorbs large amounts of glucose through Na+-glucose cotransporter 2 (SGLT2). P4-ATPase acts together with the ß-subunit TMEM30A to mediate the asymmetric distribution of phosphatidylserine (PS), phosphatidylethanolamine (PE), and other amino phospholipids, promoting plasma membrane and internal vesicle fusion, and facilitating vesicle protein transport. We observed reduced TMEM30A expression in renal tubules of DKD and IgA patients, suggesting a potential role of TMEM30A in renal tubular cells. To investigate the role of TMEM30A in renal tubules, we constructed a TMEM30A knockdown cell model by transfecting mouse kidney tubular epithelium cells (TCMK-1) with TMEM30A shRNA. Knockdown of TMEM30A in TCMK-1 cells attenuated vesicle transporter protein synthesis, resulting in reduced transport and expression of SGLT2, which in turn reduced glucose absorption. These data suggested that TMEM30A plays a crucial role in renal tubules.

Atp8a1 deletion increases the proliferative activity of hematopoietic stem cells by impairing PTEN function.

PURPOSE: The eukaryotic cell plasma membrane contains several asymmetrically distributed phospholipids, which is maintained by the P4-ATPase flippase complex. Herein, we demonstrated the biological effects and mechanisms of asymmetrical loss in hematopoietic stem cells (HSCs). METHODS: An Atp8a1 knockout mouse model was employed, from which the HSC (long-term HSCs and short-term HSCs) population was analyzed to assess their abundance and function. Additionally, competitive bone marrow transplantation and 5-FU stress assays were performed. RNA sequencing was performed on Hematopoietic Stem and Progenitor Cells, and DNA damage was assayed using immunofluorescence staining and comet electrophoresis. The protein abundance for members of key signaling pathways was confirmed using western blotting. RESULTS: Atp8a1 deletion resulted in slight hyperleukocytosis, associated with the high proliferation of HSCs and BCR/ABL1 transformed leukemia stem cells (LSCs). Atp8a1 deletion increased the repopulation capability of HSCs with a competitive advantage in reconstitution assay. HSCs without Atp8a1 were more sensitive to 5-FU-induced apoptosis. Moreover, Atp8a1 deletion prevented HSC DNA damage and facilitated DNA repair processes. Genes involved in PI3K-AKT-mTORC1, DNA repair, and AP-1 complex signaling were enriched and elevated in HSCs with Atp8a1 deletion. Furthermore, Atp8a1 deletion caused decreased PTEN protein levels, resulting in the activation of PI3K-AKT-mTORC1 signaling, further increasing the activity of JNK/AP-1 signaling and YAP1 phosphorylation. CONCLUSION: We identified the role of Atp8a1 on hematopoiesis and HSCs. Atp8a1 deletion resulted in the loss of phosphatidylserine asymmetry and intracellular signal transduction chaos.

Downregulated miRNA-491-3p accelerates colorectal cancer growth by increasing uMtCK expression.

Colorectal carcinoma (CRC) is the second most frequent cancer worldwide. MiR-491-3p, a tumor-suppressive microRNA (miRNA, miR), has been revealed to be abnormally expressed in CRC tissues. Meanwhile, up-regulated ubiquitous mitochondrial creatine kinase (uMtCK) contributes to CRC cell proliferation. Here we aim to explore whether aberrant miR-491-3p expression promotes CRC progression through regulating uMtCK. To this end, miR-491-3p and uMtCK levels were assessed in CRC tissues using quantitative real-time PCR (qRT-PCR). The biological roles of miR-491-3p and uMtCK in regulating CRC growth were evaluated using colony formation assay and mouse Xenograft tumour model. We found that miR-491-3p expression was decreased in CRC tissues compared with matched para-cancerous tissues, whereas uMtCK expression was increased. Functionally, miR-491-3p overexpression repressed SW480 cell growth, whereas miR-491-3p depletion accelerated SW620 cell proliferation and growth. Inversely, uMtCK positively regulated CRC cell proliferation. Mechanistically, miR-491-3p post-transcriptionally downregulated uMtCK expression by binding to 3'-UTR of uMtCK. Consequently, restoring uMtCK expression markedly eliminated the role of miR-491-3p in suppressing CRC growth. Collectively, miR-491-3p functions as a tumour suppressor gene by repressing uMtCK, and may be a potential target for CRC treatment.

Septin 9 methylation analysis of lymph node micrometastases for predicting relapse of colorectal cancer.

BACKGROUND: Molecular markers for the detection of lymph node micrometastases of malignant tumors have been extensively investigated. However, epigenetic signatures have rarely been reported for identification of metastatic lymph nodes and disease relapse. Septin 9 is the most frequently reported hypermethylated gene in colorectal cancer (CRC). This study aimed to assess the clinical relevance of Septin 9 methylation in regional lymph nodes in recurrence/metastases of CRC. METHODS: We analyzed Septin 9 methylation of DNA from resected lymph nodes in 75 CRC patients with or without tumor recurrence using quantitative methylation-sensitive PCR (qMS-PCR). RESULTS: Of the 30 histologically negative lymph node CRC patients without recurrence (group 1), methylated Septin 9 was detected in 3 (10 %) cases. The positivity rate of methylated Septin 9 in group 2 containing 30 histologically node-negative CRC patients with recurrence was 30 % (9/30). For group 3, lymphatic invasion as well as tumor recurrence, 11 (73 %) out of 15 subjects had Septin 9 methylation-positive lymph nodes. Moreover, patients in group 3 had a higher level of methylated Septin 9 compared to subjects in group 1 and group 2 (p < 0.05). In addition, CRC patients with Septin 9 methylation in lymph nodes had significantly reduced survival (Log-rank P < 0.0001). CONCLUSION: Our data support the predictive role of Septin 9 methylation analysis of lymph node micrometastases for tumor relapse after surgery.

Establishment and validation of a prognostic model based on HRR-related lncRNAs in colon adenocarcinoma.

BACKGROUND: Colon cancer (CRC) is the second leading cause of cancer-related death, and its 5-year survival rate is very low. Homologous recombination repair (HRR) is deficient in most colon cancer. Some long non-coding RNAs (lncRNAs) participate in tumorigenesis of colon cancer through the HRR pathway. We aim to establish a prognostic model based on the HRR-related lncRNAs, expecting to provide a new strategy for precision treatment development in colon cancer. METHODS: Pearson's correlation was used to identify the HRR-related prognostic lncRNAs in the TCGA-COAD cohort. The TCGA-COAD cohort was randomized into the training set and the testing set. LASSO Cox regression was used to establish the model which was analyzed in the training set and validated in the testing set and the entire TCGA-COAD cohort. Finally, we explored the potential biological function of our model. RESULTS: A prognostic model was established based on nineteen HRR-related lncRNAs in the training set. COAD patients were scored by the uniform formula and divided into high-risk and low-risk groups based on the median risk score. Patients with high-risk scores indicated poor prognosis in the training set, and the result was confirmed in the testing set and the entire TCGA-COAD cohort (all p < 0.01). Multivariable analysis suggested that our model was an independent factor for overall survival in COAD. The area under the curve (AUC) and C-index indicated that our model had better predictive efficiency than other indicators in the TCGA-COAD cohort. Functional enrichment analysis suggested that our model was associated with the MAPK pathway in COAD. Besides, our model was positively correlated with the HRD scores. CONCLUSION: A new prognostic model was established based on nineteen HRR-related lncRNAs which had excellent predictive efficiency on the prognosis of COAD. This prognostic model may provide a new strategy for prognostic prediction of COAD patients.

Dysregulated m6A modification promotes lipogenesis and development of non-alcoholic fatty liver disease and hepatocellular carcinoma.

Type 2 diabetes mellitus (DM2) is associated closely with non-alcoholic fatty liver disease (NAFLD) by affecting lipid metabolism, which may lead to non-alcoholic steatohepatitis (NASH), fibrosis, and hepatocellular carcinoma (HCC). N6-methyladenosine (m6A) RNA methylation is an important epigenetic regulation for gene expression and is related to HCC development. We developed a new NAFLD model oriented from DM2 mouse, which spontaneously progressed to histological features of NASH, fibrosis, and HCC with high incidence. By RNA sequencing, protein expression and methylated RNA immunoprecipitation (MeRIP)-qPCR analysis, we found that enhanced expression of ACLY and SCD1 in this NAFLD model and human HCC samples was due to excessive m6A modification, but not elevation of mature SREBP1. Moreover, targeting METTL3/14 in vitro increases protein level of ACLY and SCD1 as well as triglyceride and cholesterol production and accumulation of lipid droplets. m6A sequencing analysis revealed that overexpressed METTL14 binds to mRNA of ACLY and SCD1 and alters their expression pattern. Our findings demonstrate a new NAFLD mouse model that provides a study platform for DM2-related NAFLD and reveals a unique epitranscriptional regulating mechanism for lipid metabolism via m6A-modified protein expression of ACLY and SCD1.

Overexpression of Twist1 in vascular endothelial cells promotes pathological retinal angiogenesis in mice.

Retinal angiogenesis is a critical process for normal retinal function. However, uncontrolled angiogenesis can lead to pathological neovascularization (NV), which is closely related to most irreversible blindness-causing retinal diseases. Understanding the molecular basis behind pathological NV is important for the treatment of related diseases. Twist-related protein 1 (TWIST1) is a well-known transcription factor and principal inducer of epithelial-mesenchymal transition (EMT) in many human cancers. Our previous study showed that Twist1 expression is elevated in pathological retinal NV. To date, however, the role of TWIST1 in retinal pathological angiogenesis remains to be elucidated. To study the role of TWIST1 in pathological retinal NV and identify specific molecular targets for antagonizing pathological NV, we generated an inducible vascular endothelial cell (EC)-specific Twist1 transgenic mouse model ( Tg-Twist1 iEC+ ). Whole-mount retinas from Tg-Twist1 iEC+ mice showed retarded vascular progression and increased vascular density in the front end of the growing retinal vasculature, as well as aneurysm-like pathological retinal NV. Furthermore, overexpression of Twist1 in the ECs promoted cell proliferation but disturbed cell polarity, thus leading to uncontrolled retinal angiogenesis. TWIST1 promoted pathological NV by activating the Wnt/ß-catenin signaling pathway and inducing the expression of NV formation-related genes, thereby acting as a 'valve' in the regulation of pathological angiogenesis. This study identified the critical role of TWIST1 in retinal pathological NV, thus providing a potential therapeutic target for pathological NV.

Upregulation of HSPA1A/HSPA1B/HSPA7 and Downregulation of HSPA9 Were Related to Poor Survival in Colon Cancer.

The human HSP70 family is a type of heat shock protein (HSP), consisting of 13 members encoded by the HSPA genes. HSPs play important roles in regulating cellular responses and functions during carcinogenesis, but their relationship with colon cancer is unclear. In our study, we found that the expressions of HSPA1B, HSPA4, HSPA5, HSPA6, HSPA8, HSPA9, HSPA13, and HSPA14 were significantly increased, while those of HSPA1A, HSPA2, HSPA7, and HSPA12B were significantly decreased in colon cancer tissues. The expression of HSPA gene family members was associated with some clinicopathological characteristics, including age, gender, TNM stage, pathological stage, and CEA level. Furthermore, the Kaplan-Meier method and Cox regression analysis showed that high HSPA1A, HSPA1B, and HSPA7 expressions were related to unfavorable survival, and high HSPA9 was associated with favorable survival. The relationships between HSPA1A and HSPA9 expression and survival were validated in the GEO dataset, and the HSPA1A and HSPA9 protein expression differences between colon cancer tissues and normal tissues were validated in the UALCAN database. Methylation of HSPA1A and HSPA9 was also analyzed, and it was found that the methylation of the HSPA1A promoter was significantly increased, and the methylation of the HSPA9 promoter was significantly decreased in colon cancer tissues. Increasing the methylation level of the HSPA1A gene and decreasing the methylation level of HSPA9 were related to favorable prognosis. The expression difference of HSPA1A/HSPA1B/HSPA7/HSPA9 was verified in colon cancer cell lines and colonic epithelial cells. Gene ontology analysis was used to screen signal pathways related to HSPA1A-, HSPA1B-, HSPA7-, and HSPA9- high phenotype. In summary, the increased expressions of HSPA1A1, HSPA1B, and HSPA7 were associated with poor prognosis, while that of HSPA9 was related to favorable prognosis for colon cancer patients.

A missense mutation in Pitx2 leads to early-onset glaucoma via NRF2-YAP1 axis.

Glaucoma is a leading cause of blindness, affecting 70 million people worldwide. Owing to the similarity in anatomy and physiology between human and mouse eyes and the ability to genetically manipulate mice, mouse models are an invaluable resource for studying mechanisms underlying disease phenotypes and for developing therapeutic strategies. Here, we report the discovery of a new mouse model of early-onset glaucoma that bears a transversion substitution c. G344T, which results in a missense mutation, p. R115L in PITX2. The mutation causes an elevation in intraocular pressure (IOP) and progressive death of retinal ganglion cells (RGC). These ocular phenotypes recapitulate features of pathologies observed in human glaucoma. Increased oxidative stress was evident in the inner retina. We demonstrate that the mutant PITX2 protein was not capable of binding to Nuclear factor-like 2 (NRF2), which regulates Pitx2 expression and nuclear localization, and to YAP1, which is necessary for co-initiation of transcription of downstream targets. PITX2-mediated transcription of several antioxidant genes were also impaired. Treatment with N-Acetyl-L-cysteine exerted a profound neuroprotective effect on glaucoma-associated neuropathies, presumably through inhibition of oxidative stress. Our study demonstrates that a disruption of PITX2 leads to glaucoma optic pathogenesis and provides a novel early-onset glaucoma model that will enable elucidation of mechanisms underlying the disease as well as to serve as a resource to test new therapeutic strategies.

Boosting Antitumor Sonodynamic Therapy Efficacy of Black Phosphorus via Covalent Functionalization.

Sonodynamic therapy (SDT) triggered by ultrasound represents an emerging tumor therapy approach with minimally invasive treatment featuring nontoxicity and deep tissue-penetration, and its efficacy sensitively depends on the sonosensitizer which determines the generation of reactive oxygen species (ROS). Herein, for the first time covalently functionalized few-layer black phosphorus nanosheets (BPNSs) are applied as novel sonosensitizers in SDT, achieving not only boosted SDT efficacy but also inhibited cytotoxicity relative to the pristine BPNSs. Three different covalently functionalized-BPNSs are synthesized, including the first fullerene-functionalized BPNSs with C60 covalently bonded onto the surface of BPNSs (abbreviated as C60 -s-BP), surface-functionalized BPNSs by benzoic acid (abbreviated as BA-s-BP), and edge-functionalized BPNSs by C60 (abbreviated as C60 -e-BP), and the role of covalent functionalization pattern of BPNSs on its SDT efficacy is systematically investigated. Except C60 -e-BP, both surface-functionalized BPNSs (C60 -s-BP, BA-s-BP) exhibit higher SDT efficacies than the pristine BPNSs, while the highest SDT efficacy is achieved for BA-s-BP due to its strongest capability of generating the hydroxyl (·OH) radicals, which act as the dominant ROS to kill the tumor cells.

Autosomal Recessive Rod-Cone Dystrophy Associated With Compound Heterozygous Variants in ARL3 Gene.

Purpose: ARL3 (ADP-ribosylation factor-like 3) variants cause autosomal dominant retinitis pigmentosa (RP) or autosomal recessive Joubert syndrome. We found a family with rod-cone dystrophy (RCD) and verified it was associated with compound heterozygous variants in ARL3 gene. Methods: Ophthalmic examinations including optical coherence tomography and electroretinogram (ERG) were performed. Targeted next generation sequencing (NGS) was performed for the proband using a custom designed panel. Sanger sequencing and co-segregation were conducted in the family members. Changes of protein structure mediated by the variants were studied in vitro. ARL3 protein stability and its interaction with RP2 protein were assessed by cycloheximide chase assay and co-immunoprecipitation (Co-IP) assay. Results: Visual acuity of the 18-year-old male proband was 0.25 in the right and 0.20 in the left eye, while his non-consanguineous parents and sister was normal. The proband showed signs of RCD, including nyctalopia, peripheral field loss, bone-spicule deposits in the retina, and reduced ERG responses. The father, aged 50 years old, showed visual acuity of 1.0 in both eyes. Unlike the proband, he presented late onset and mild cone-rod dystrophy (CRD), including macular atrophy, central scotomata, moderate reduction in photopic ERG responses. None of all the family members had hearing abnormality, mental dysplasia or gait instability. We identified two novel compound heterozygous variants (c.91A>G, p.T31A; c.353G>T, p.C118F) in ARL3 in the proband, while his father only had variant c.91A>G. Bioinformatics analysis indicated amino acid positions of the two variants are highly conserved among species. The in silico tools predicted the variants to be harmful. Protein structure analysis showed the two variants had potential to alter the protein structure. Based on the ACMG guidelines, the two variants were likely pathogenic. In addition, the ARL3 mutations destabilized ARL3 protein, and the mutation c.353G>T disrupted the interaction between ARL3 and RP2 in HEK293T cells. Conclusions: We showed novel compound heterozygous variants in ARL3 were associated with early onset of autosomal recessive RCD, while c.91A>G along may be associated with a late onset of dominant CRD. The two variants in ARL3 could be causative by destabilizing ARL3 protein and impairing its interaction with RP2 protein.

G-Protein-Coupled Estrogen Receptor 1 Promotes Gender Disparities in Hepatocellular Carcinoma via Modulation of SIN1 and mTOR Complex 2 Activity.

Due to its intricate heterogeneity and limited treatment, hepatocellular carcinoma (HCC) has been considered a major cause of cancer-related mortality worldwide. Increasing evidence indicates that G-protein-coupled estrogen receptor 1 (GPER1) can promote estrogen-dependent hepatocellular proliferation by activating AKT signaling. The mTOR complex 2 (mTORC2), whose integrity and activity are modulated by its subunit Sin1, controls the activation of AKT by phosphorylation at position S473. In this study, we investigate the modulation of Sin1 and how estrogen signaling may influence the mTORC2-AKT cascade in HCC cells and a DEN-induced mouse model. We have found that estradiol-dependent Sin1 expression is transcriptionally modulated by GPER1 as well as ERα. GPER1 is able to regulate Sin1 stability via nuclear translocation, therefore increasing Sin1-mTORC2-AKT activation. Moreover, Sin1 interacts with ERα and further enhances its transcriptional activity. Sin1 is highly expressed in acute liver injury and in cases of HCC harboring high expression of GPER1 and constitutive activation of mTORC2-AKT signaling. GPER1 inhibition using the antagonist G-15 reverses DEN-induced acute liver injury by suppressing Sin1 expression and mTORC2-AKT activation. Notably, SIN1 expression varies between male and female mice in the context of both liver injury and liver cancer. In addition, high SIN1 expression is predictive of good prognosis in both male and female patients with HCC who are free from hepatitis virus infection and who report low alcohol consumption. Hence, here we demonstrate that Sin1 can be regulated by GPER1 both through nongenomic and indirect genomic signaling. IMPLICATIONS: This study suggests that Sin1 may be a novel HCC biomarker which is gender-dependent and sensitive to particular risk factor.

Nuclear MYH9-induced CTNNB1 transcription, targeted by staurosporin, promotes gastric cancer cell anoikis resistance and metastasis.

Rationale: Peritoneal metastasis predicts poor prognosis of gastric cancer (GC) patients, and the underlying mechanisms are poorly understood. Methods: The 2-DIGE, MALDI-TOF/TOF MS and single-cell transcriptome were used to detect differentially expressed proteins among normal gastric mucosa, primary GC and peritoneal metastatic tissues. Lentiviruses carrying shRNA and transcription activator-like effector nuclease technology were used to knock down myosin heavy chain 9 (MYH9) expression in GC cell lines. Immunofluorescence, immune transmission electron microscopy, chromatin fractionation, co-immunoprecipitation, and assays for chromatin immunoprecipitation, dual luciferase reporter, agarose-oligonucleotide pull-down, flow cytometry and cell anoikis were performed to uncover nuclear MYH9-induced ß-catenin (CTNNB1) transcription in vitro. Nude mice and conditional transgenic mice were used to investigate the findings in vivo. Results: We observed that MYH9 was upregulated in metastatic GC tissues and was associated with a poor prognosis of GC patients. Mechanistically, we confirmed that MYH9 was mainly localized in the GC cell nuclei by four potential nuclear localization signals. Nuclear MYH9 bound to the CTNNB1 promoter through its DNA-binding domain, and interacted with myosin light chain 9, ß-actin and RNA polymerase II to promote CTNNB1 transcription, which conferred resistance to anoikis in GC cells in vitro and in vivo. Staurosporine reduced nuclear MYH9 S1943 phosphorylation to inhibit CTNNB1 transcription, Wnt/ß-catenin signaling activation and GC progression in both orthotropic xenograft GC nude mouse and transgenic GC mouse models. Conclusion: This study identified that nuclear MYH9-induced CTNNB1 expression promotes GC metastasis, which could be inhibited by staurosporine, indicating a novel therapy for GC peritoneal metastasis.

The anti-tumor effect of taxifolin on lung cancer via suppressing stemness and epithelial-mesenchymal transition in vitro and oncogenesis in nude mice.

BACKGROUND: Taxifolin is a natural flavonoid with anti-oxidant and anti-proliferative properties. In this study, we investigated the stemness-related inhibitory effects of taxifolin in two lung cancer cell lines, A549 and H1975, as well as in A549 xenografts. METHODS: A549 and H1975 cells, as well as A549 xenograft BALB/c mice were treated with taxifolin. Cell viability, stemness, mobility and protein expression were tested with Cell counting kit-8 (CCK-8), Colony formation assay, Flow cytometry, Transwell, Western blot and Immunohistochemistry, respectively. RESULTS: CCK-8 exhibited an obvious toxicity of taxifolin to both cell lines at higher dose. Then taxifolin of 0, 25, 50, and 100 µM/L were subsequently used. Taxifolin exhibited inhibitory effects on stemness and sphere formation, reduced protein expression of SOX2 and OCT4, and reduced CD133-positive cells. Furthermore, taxifolin decreased invasive cells, reduced N-cadherin and vimentin while increased E-cadherin expression, indicating that epithelial-mesenchymal transition (EMT) was inhibited. All of the effects observed were exhibited in a dose-dependent manner, and A549 cells proved to be more sensitive to taxifolin than H1975 cells. Taxifolin inactivated PI3K and TCF4 protein phosphorylation; however, taxifolin was not observed to have an effect on NF-κB P65 or STAT3. Taxifolin also suppressed tumor growth in A549 xenograft BALB/c mice, with decreased SOX2 and OCT4 expression and inhibited PI3K and TCF4. CONCLUSIONS: In summary, taxifolin inhibited stemness and EMT in lung cancer cells possibly via the inactivation of PI3K and OCT4. Taxifolin could be a potential prodrug or serve as an adjuvant in lung cancer treatment.

scAAV2-Mediated C3 Transferase Gene Therapy in a Rat Model with Retinal Ischemia/Reperfusion Injuries.

Glaucoma is characterized by retinal ganglion cell (RGC) death and axonal loss. Therefore, neuroprotection is important in treating glaucoma. In this study, we explored whether exoenzyme C3 transferase (C3)-based gene therapy could protect retinas in an ischemia/reperfusion (I/R) injury rat model. Self-complementary adeno-associated virus 2 (scAAV2) vectors encoding either C3 protein (scAAV2-C3) or enhanced green fluorescence protein (scAAV2-EGFP) were intravitreally delivered into both eyes of rats, and I/R models (acute ocular hypertension) were made in one eye of each rat at day 7 after the injection. The rats were divided into six groups: scAAV2-C3, scAAV2-C3 with I/R, scAAV2-EGFP, scAAV2-EGFP with I/R, blank control, and blank control with I/R. TUNEL (terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling), immunohistochemistry of cleaved caspase-3, NeuN and Brn-3a, and H&E staining were used to detect apoptotic cells and other changes in the retina. The results showed that scAAV2-C3 significantly reduced the number of apoptotic RGCs and decreased cell loss in the ganglion cell layer after I/R injury, and the I/R-injured retinas treated with scAAV2-C3 were the thickest in all I/R groups. These results suggest that scAAV2-mediated C3 gene therapy is able to protect the rat retina from I/R injury and has potential in the treatment of glaucoma in the future.

Identification of Novel Mutations in the FZD4 and NDP Genes in Patients with Familial Exudative Vitreoretinopathy in South India.

Background: Familial exudative vitreoretinopathy (FEVR) is an inheritable retinal vascular disease, which often leads to severe vision loss and blindness in children. However, reported mutations can only account for 50-60% of patients with FEVR. The purpose of this study was to identify novel frizzled class receptor 4 (FZD4) and Norrin cystine knot growth factor NDP (NDP) mutations in a cohort of Indian patients with FEVR by whole-exome sequencing. Methods: We performed data filtering and bioinformatic analyses. Results: Two novel heterozygous mutations in FZD4 gene were identified, each in two different families: c.1499_1500del [p.500_500del] and c.G296C [p.C99S]. One novel mutation in NDP in another family was identified: c.A256G [p.K86E]. All FZD4 mutations affected conserved amino acid residues and were absent in 1000 control individuals. To assess the effect of these FZD4 mutations on the biological activity of the protein, we introduced each FZD4 mutation into FZD4 cDNA by the site-directed mutagenesis techniques. A Norrin/beta-catenin pathway-based luciferase reporter assay revealed that the c.1499_1500del failed to activate the luciferase reporter; in contrast, compared with the wild-type FZD4 protein, the, c.G296C [p.C99S] mutation exhibited increased luciferase reporter activity. Conclusion: Our study found two novel FZD4 mutations, with opposite effects regarding functional expression levels in Indian patients with FEVR and expands on the mutational spectrum of FZD4 in Indian FEVR patients.