Dihydroartemisinin (DHA) inhibits myofibroblast differentiation through inducing ferroptosis mediated by ferritinophagy.

Idiopathic pulmonary fibrosis (IPF) is caused by persistent micro-injuries and aberrant repair processes. Myofibroblast differentiation in lung is a key event for abnormal repair. Dihydroartemisinin(DHA), a well-known anti-malarial drug, have been shown to alleviate pulmonary fibrosis, but its mechanism is not clear. Ferroptosis is involved in the pathgenesis of many diseases, including IPF. Ferritinophagy is a form of cellular autophagy which regulates intracellular iron homeostasis. The function of DHA on myofibroblasts differentiation of pulmonary and whether related with ferroptosis and ferritinophagy are unknown now. Using human fetal lung fibroblast 1(HFL1) cell line and the qRT-PCR, immunofluorescent and Western blotting techniques, we found that after TGF-ß1 treatment, the levels of ɑ-SMA expression and ROS increased; the mRNA and protein levels of FTH1 and NCOA4, the content of Fe2+ and 4-HNE increased significantly at 6h, then gradually reduced with time. After DHA treatment, FHL1 cells appeared ferroptosis; the levels of α-SMA mRNA and protein reduced and the levels of ROS and 4-HNE increased; the Fe2+ levels decreased sharply at 6h, then increased with time, and were higher than normal since 24h; the mRNA and protein levels of FTH1 and NCOA4 decreased, exhibited a downward trend. These results show that Fe2+, ROS and lipid peroxidation are involved in and ferritinophagy is inhibited during fibroblast-to-myofibroblast differentiation; The depletion of Fe2+ at early stage induced by DHA treatment triggers the ferritinophagy in HFL1 cells, leading to degradation of FTH1 and NCOA4 and following increase of Fe2+ levels. DHA may inhibit the fibroblast-to-myofibroblast differentiation through inducing ferroptosis mediated by ferritinophagy.

Clonorchis sinensis legumain promotes migration and invasion of cholangiocarcinoma cells via regulating tumor-related molecules.

BACKGROUND: Clonorchis sinensis infection causes serious pathological changes in the bile duct and is highly correlated with cholangiocarcinoma. The excretory-secretory products (ESP) of C. sinensis play a critical role in the oncogenesis and progression of cholangiocarcinoma, while the components and precise mechanism remain unclear. Here, we evaluated the function of C. sinensis legumain (Cslegumain) in promoting the invasion and migration of cholangiocarcinoma cells and the mechanism involved. METHODS: The structural and molecular characteristics of Cslegumain were predicted and analyzed using the online program Phyre2. Quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemical staining were performed to test the transcriptional level of Cslegumain and its localization in the adult. Native Cslegumain was detected by western blotting assay. The effects of Cslegumain on the proliferation, invasion and migration of cholangiocarcinoma cells were checked using CCK-8 assay, Matrigel transwell assay and scratch wound healing assay. Expression levels of tumor-related molecules regulated by Cslegumain were evaluated by qRT-PCR and western blotting assay. RESULTS: Cslegumain showed high similarity with human legumain in the secondary and tertiary structures and displayed higher transcriptional levels in the adult worm than in the metacercariae. Native Cslegumain was detected in a catalytic form and was localized mainly in the intestine of the C. sinensis adult and epithelial cells of the intrahepatic bile duct. After transfection into RBE cells, Cslegumain showed high ability in promoting the invasion and migration but not the proliferation of cholangiocarcinoma RBE cells. Furthermore, the expression levels of some molecules including E-cadherin and N-cadherin were downregulated, while the levels of α-actinin 4, ß-catenin and inducible nitric oxide synthase (iNOS) were upregulated. CONCLUSIONS: Our findings indicated that Cslegumain showed very similar structures as those of human legumain and could promote the invasion and migration of cholangiocarcinoma cells by regulating some tumor-related molecules.

[SOX2-OT/SOX2 axis regulates lung cancer H520 cell migration via Gli1-mediated epithelial-mesenchymal transition].

OBJECTIVE: To explore the regulatory role of SOX2-OT in migration of lung squamous cell carcinoma H520 cells and the underlying mechanisms. METHODS: Wound- healing and Transwell migration assays were performed to examine the changes in migration and invasion capacity of lung squamous cell line H520, which expressed higher levels of SOX2-OT than other lung cancer cell lines, following RNA interference-mediated SOX2-OT knockdown. The transcription levels of epithelial-mesenchymal transition (EMT)-related components was detected by qRT-PCR and immunoblotting. Gli1 gain-of-function analysis was performed in H520 cells with SOX2-OT knockdown and the changes in EMT phenotype of the cells were examined. miR-200c mimic and inhibitor were used to analyze the mechanism by which SOX2-OT positively regulates Gli1 and the mediating role of SOX2. RESULTS: SOX2-OT knockdown significantly lowered the invasiveness and migration capacity of H520 cells and caused changes in EMT phenotype of the cells. Overexpression of Gli1, which was positively regulated by SOX2-OT, reversed the inhibitory effect of SOX2-OT knockdown on migration of H520 cells. Transfection of the cells with miR-200c inhibitor effectively reversed SOX2-OT knockdown-induced down-regulation of SOX2. CONCLUSION: The SOX2-OT/SOX2 axis positively regulates migration of lung squamous H520 cells via Gli1-mediated EMT.

A Novel missense mutation of COL2A1 gene in a large family with stickler syndrome type I.

Stickler syndrome type I (STL1, MIM 108300) is characterized by ocular, auditory, skeletal and orofacial manifestations. Nonsyndromic ocular STL1 (MIM 609508) characterized by predominantly ocular features is a subgroup of STL1, and it is inherited in an autosomal dominant manner. In this study, a novel variant c.T100>C (p.Cys34Arg) in COL2A1 related to a large nonsyndromic ocular STL1 family was identified through Exome sequencing (ES). Bioinformatics analysis indicated that the variant site was highly conserved and the pathogenic mechanism of this variant may involve in affected structure of chordin-like cysteine-rich (CR) repeats of ColIIA. Minigene assay indicated that this variant did not change alternative splicing of exon2 of COL2A1. Moreover, the nonsyndromic ocular STL1 family with 16 affected members showed phenotype variability and certain male gender trend. None of the family members had hearing loss. Our findings would expand the knowledge of the COL2A1 mutation spectrum, and phenotype variability associated with nonsyndromic ocular STL1. Search for genetic modifiers and related molecular pathways leading to the phenotype variation warrants further studies.

Lipid Peroxidation, GSH Depletion, and SLC7A11 Inhibition Are Common Causes of EMT and Ferroptosis in A549 Cells, but Different in Specific Mechanisms.

Epithelial-mesenchymal transition (EMT) induced by transforming growth factor-ß1 (TGF-ß1) is thought to be involved in the pathogenesis of pulmonary fibrosis. Emerging evidence suggested that there are some common causes between ferroptosis and pulmonary fibrosis. The interaction of EMT and ferroptosis and its mechanism were investigated by detecting the expression levels of α-smooth muscle actin (α-SMA), E-cadherin, solute carrier family 7 member 11 (SLC7A11), and glutathione peroxidase 4 (GPX4) and measuring the contents of reactive oxygen species (ROS), malondialdehyde (MDA), and glutathione (GSH). The cellular morphology and ultrastructure of mitochondria were studied by microscope and transmission electron microscope (TEM), respectively. The results showed that ferroptosis in A549 cells was induced by Erastin, which decreased the expression levels of E-cadherin (E-Ca), α-SMA, and SLC7A11, accompanied with ROS and MDA increase, as well as GSH decrease. TGF-ß1 promoted ultrastructure variation of mitochondria similar to ferroptosis and mesenchymal changes in morphology during EMT of A549 cells, accompanied with reduced GSH content and expression of SLC7A11, as well as ROS and MDA increase. Ferrostatin-1 (Fer-1) recovered ferroptosis induced by Erastin, but had no effect on the morphological change caused by TGF-ß1. Furthermore, Fer-1 reduced ROS and MDA production, and increased SLC7A11 expression in the early subsequently increased GSH. However, the effects of Fer-1 on above indicators were different in time. The expression of GPX4 had no significant change during EMT induced by TGF-ß1 and ferroptosis induced by Erastin in A549 cells. It is indicating that Erastin promoted the de-epithelialization of lung epithelial cells, but inhibited the process of myofibroblast differentiation; Fer-1 could partially inhibit EMT induced by TGF-ß1, but reverse ferroptosis induced by Erastin. TGF-ß1 could delay the ferroptosis, but could not prevent it. Lipid peroxidation, GSH depletion, and SLC7A11 inhibition are common causes of EMT and ferroptosis in A549 cells, but different in specific mechanisms. The exact effects of GPX4 involved in EMT and ferroptosis in A549 cells need further study.

DX5+ NKT cells' increase was correlated with liver damage in FVB mice not in BALB/c mice infected by Clonorchis sinensis.

AIMS: DX5+ NKT cells' distribution and population change in BALB/c and FVB mice infected by C sinensis and their function in liver damage were investigated. METHODS AND RESULTS: Mice were infected by Clonorchis sinensis metacercariae, and lymphocytes were isolated from the livers, spleens and peripheral blood. NK, DX5+ NKT, INF-γ+ DX5+ NKT cells and liver fibrosis were analysed. The DX5+ NKT cells displayed the largest amount in normal BALB/c mice liver followed by peripheral blood and spleen. Although the hepatic DX5+ NKT cells of BALB/c mice were more than that of FVB mice, they did not show significant percentage change after C sinensis infection. The hepatic DX5+ NKT cells of FVB mice increased remarkably after infection accompanied with heavier liver injury and fibrosis than the BALB/c mice. And hydroxyproline content was also positively correlated with DX5+ NKT cells only in FVB mice. However, the increase of IFN-γ producing DX5+ NKT cells was lower in FVB mice than in BALB/c mice which showed sharp increase with mild liver damage after infection. The frequencies of anti-fibrotic NK cells were similar in both of the two mouse strains. CONCLUSIONS: C sinensis could induce different DX5+ NKT cells responses in different mouse strains which may play roles in liver injury and fibrosis in FVB mice.

Lipid Peroxidation and GPX4 Inhibition Are Common Causes for Myofibroblast Differentiation and Ferroptosis.

Ferroptosis is a new form of regulated cell death. Fibroblast-to-myofibroblast differentiation is known to be involved in the pathogenesis of idiopathic pulmonary fibrosis. Utilizing HFL1 cell line treated with transforming growth factor-ß1 (TGF-ß1), we investigated the relationship between ferroptosis and pulmonary fibrosis, and the function of glutathione peroxidase 4 (GPX4) in them. The results indicated that α-smooth muscle actin and collagen I (COL I) mRNA expression levels increased significantly from 24 h after TGF-ß1-treatment, and further rose after TGF-ß1+erastin treatment. The levels of reactive oxygen species (ROS), malondialdehyde were increased, and the levels of GPX4 mRNA and protein were reduced after treatment with TGF-ß1, and all these were magnified after TGF-ß1+erastin treatment. All these changes induced by TGF-ß1 and erastin can be recovered by Fer-1 treatment. The cell viability rate was decreased significantly when treated with TGF-ß1+erastin, but no obvious variation of cell viability was found in TGF-ß1-treated group and in other groups, suggesting that ROS, lipid peroxidation, and GPX4 inhibition are not sufficient conditions for ferroptosis. Collectively, our study reveals that ROS, lipid peroxidation, and GPX4 play important roles in pulmonary fibrosis and ferroptosis induced by erastin. Erastin promoted fibroblast-to-myofibroblast differentiation by increasing lipid peroxidation and inhibiting the expression of GPX4. Fer-1 may inhibit pulmonary fibrosis and ferroptosis through suppressing lipid peroxidation and enhancing GPX4 expression.

Characterization, expression, and response to stress of p8 gene in amphioxus.

The amphioxus Branchiostoma belcheri tsingtaunese homolog of p8, Bbp8, was identified from the gut cDNA library. The full-length Bbp8 cDNA consists of 1032 bp, which is clearly longer than those of p8 in human, mouse, rat, frog, zebrafish and fruit fly. The genomic DNA sequences of amphioxus p8 contain three exons and two introns, which is similar to the exon/intron organization of p8 homologues in vertebrates such as human, mouse and zebrafish, while it is sharply different to the organization of p8 gene in fruit fly, which has only one exon. Sequence alignment and phylogenetic analysis showed that the basic helix-loop-helix (bHLH) region of p8 is well conserved during the long process of evolution, and Bbp8 is more close to its homologous proteins in the invertebrates than to those in the vertebrates. RT-PCR and In situ hybridization histochemistry demonstrated the expression of Bbp8 in all the tissues assayed, with relatively higher expression in the gut, gill and ovaries. Quantitative real-time PCR assay revealed quick up-regulation of Bbp8 transcripts on lipopolysaccharide (LPS) challenge and starvation, implying a stress-related function for Bbp8.

Characterization and expression of gamma-interferon-inducible lysosomal thiol reductase (GILT) gene in amphioxus Branchiostoma belcheri with implications for GILT in innate immune response.

An amphioxus cDNA, AmphiGILT, encoding GILT protein was isolated from the gut cDNA library of Branchiostoma belcheri. It codes for a deduced protein of 254 amino acids, which has all the main features typical of GILT proteins including the signature sequence CQHGX(2)CX(2)NX(4)C, CXXC motif and 11 conserved cysteines. Phylogenetic analysis showed that AmphiGILT and sea urchin GILT clubbed together and positioned at the base of vertebrate GILT clade, suggesting that both AmphiGILT and sea urchin GILT might share some characteristics of the archetype of vertebrate GILT proteins. The genomic DNA sequence of B. floridae contains seven exons and six introns, which is similar to vertebrate GILT exon-intron organization. AmphiGILT was expressed in a tissue-specific manner with the most abundant mRNA in the digestive system including hepatic caecum and hind-gut. It was also found that mammalian IFN-gamma only exerted a slight effect on the expression of GILT gene in amphioxus, forming a contrast to the marked induction of human and mouse GILT expression by IFN-gamma. Taken the absence of the adaptive immune system including MHC class II molecules and lymphocytes into consideration, these results suggest that AmphiGILT is highly likely to play a role in the innate immune responses in amphioxus.

Identification and tissue-specific expression of amphioxus GM2 activator protein gene from amphioxus Branchiostoma belcheri.

An amphioxus cDNA, AmphiGM2AP, encoding GM2 activator protein was isolated from the gut cDNA library of Branchiostoma belcheri. It is 907 bp long, and its longest open reading frame codes for a precursor protein consisting of 242 amino acid residues with a signal peptide of 14 amino acids. The deduced amino acid sequence includes a conserved domain typical of GM2APs between residues 53 and 224, a single N-linked glycosylation site at position 65 and 8 conserved cysteines. Phylogenetic analysis showed that amphiGM2AP forms a club together with invertebrate GM2APs, indicating that AmphiGM2AP is evolutionarily closely related to invertebrate GM2APs rather than vertebrate ones. Both Northern blotting and in situ hybridization histochemistry analyses revealed a tissue-specific expression pattern of AmphiGM2AP in adult amphioxus with the strongest expression in the digestive system, which is in contrast to the widespread expression pattern of human, mouse and sheep GM2AP genes. It is suggested that AmphiGM2AP is possibly involved in the take-in of digested food components like lipid molecules.

Verification, characterization and tissue-specific expression of UreG, a urease accessory protein gene, from the amphioxus Branchiostoma belcheri.

UreG genes have been found in bacteria, fungi and plants but have not yet identified in animals, although a putative UreG-like gene has been documented in sea urchin. In the course of a large-scale sequencing of amphioxus gut cDNA library, we have identified a cDNA with high similarity to UreG genes. Both reverse transcription-polymerase chain reaction and nested polymerase chain reaction, as well as in situ hybridization histochemistry, verified that the cDNA represented an amphioxus UreG gene (AmphiUreG) rather than a microbial contaminant of the cDNA library. This is further supported by the presence of urease activity in amphioxus gut, gill and ovary. AmphiUreG encodes a deduced protein of 200 amino acid residues including a highly conserved P-loop, bearing approximately 46%-49%, 44%-48%, and 29%-37% similarity to fungal, plant and bacterial UreG proteins, respectively. It shows a tissue-specific expression pattern in amphioxus, and is especially abundant in the digestive system. This is the first UreG gene identified in animal species.

[Synergism of c-myc and p16 in oral squamous cell carcinoma].

BACKGROUND & OBJECTIVE: The study on the protein expression of c-myc and p16 in oral squamous cell carcinoma (OSCC) has been reported, and a few researches involved in their gene amplification and mRNA expression in OSCC, however, the synergism of c-myc and p16 in OSCC in multiple levels is still unclear. This study aims to probe the synergism of c-myc and p16 in OSCC in gene amplification, mRNA expression, and protein expression levels. METHODS: The gene amplification, expression of mRNA, and protein of c-myc and p16 in 30 cases of OSCC were determined by in situ hybridization and immunohistochemical techniques. RESULTS: The amplification rate of c-myc was 63.3%, but no amplification of p16 was found in OSCC. The mRNA expression rates of c-myc and p16 were 83.3% and 93.3%, and their protein expression rates were 60.0% and 86.7%, respectively. The correlation analysis showed that there was significant correlativity between p16 and c-myc both in mRNA expression (r=0.676 5, P=4.055 6E-05) and in protein expression (r=0.564 2, P=0.001 2). CONCLUSION: The gene amplification and overexpression of c-myc plays an important role in the tumorigenesis and development of OSCC. There is certain internal relationship between p16 and c-myc expression in OSCC.