Toxic effects of sodium dodecyl sulfate on planarian Dugesia japonica.

Sodium dodecyl sulfate (SDS) is an anionic surfactant, which is widely used in various fields in human life. However, SDS discharged into the water environment has a certain impact on aquatic organisms. In this study, planarian Dugesia japonica (D. japonica) was used to identify the toxic effects of SDS. A series of SDS solutions with different concentrations were used to treat planarians for the acute toxicity test , and the results showed that the semi-lethal concentration (LC50) of SDS to D. japonica at 24 h, 48 h, 72 h, and 96 h were 4.29 mg/L, 3.76 mg/L, 3.45 mg/L, and 3.20 mg/L respectively. After the planarians were exposed to 0.5 mg/L and 1.0 mg/L SDS solutions for 1, 3, and 5 days, the activities of superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) content were measured to detect the oxidative stress and lipid peroxidation in planarians. Random amplified polymorphic DNA (RAPD) analysis was performed to detect the genotoxicity caused by SDS to planarians. The results showed that the activities of SOD, CAT, and MDA content increased after the treatment, indicating that SDS induced oxidative stress in planarians. RAPD analysis showed that the genomic template stability (GTS) values of planarians treated by 0.5 mg/L and 1.0 mg/L SDS for 1, 3, and 5 days were 67.86%, 64.29%, 58.93%, and 64.29%, 60.71%, 48.21%, respectively. GTS values decreased with the increasing of SDS concentration and exposure time, indicating that SDS had genotoxicity to planarians in a time and dose-related manner. Fluorescent quantitative PCR (qPCR) was used to investigate the effects of SDS on gene expression of planarians. After the planarians were exposed to 1.0 mg/L SDS solution for 1, 3, and 5 days, the expression of caspase3 was upregulated, and that of piwiA, piwiB, PCNA, cyclinB, and RAD51 were downregulated. These results suggested that SDS might induce apoptosis, affect cell proliferation, differentiation, and DNA repair ability of planarian cells and cause toxic effects on planarian D. japonica.

Genotoxicity Evaluation of Metformin in Freshwater Planarian Dugesia japonica by the Comet Assay and RAPD Analysis.

Objective: To investigate the genotoxicity of metformin on planarian with different concentrations and exposure times. Methods: The planarians were treated, respectively, with 10 mmol/L and 50 mmol/L metformin for 1, 3, and 5 days, and then, the comet assay and random amplified polymorphic DNA (RAPD) analysis were performed. 13 random primers were used for PCR amplification with the genomic DNAs as templates. Planarians cultured in clear water were used as the control. Genomic template stability (GTS) was calculated by comparing and analyzing the RAPD patterns of the control group and the treatment groups. Results: In the comet assay, DNA damage of planarians treated with 10 mmol/L metformin for 1, 3, and 5 days was 10.2%, 25.4%, and 36.8%, respectively, and that of planarians treated with 50 mmol/L metformin was 40.6%, 62.8%, and 65.4%, respectively. GTS values of planarians exposed to 10 mmol/L metformin for 1, 3, and 5 days were 64.1%, 62.8%, and 52.6%, respectively, and those of planarians exposed to 50 mmol/L metformin for 1, 3, and 5 days were 52.6%, 51.3%, and 50%, respectively. DNA damage increased and GTS values decreased with the increasing metformin exposure concentration and exposure time. Conclusion: Metformin has certain genotoxicity on planarian in a dose- and time-related manner. The comet assay and RAPD analysis are highly sensitive methods for detecting genotoxicity with drugs.

Exposure to polystyrene microplastics reduces regeneration and growth in planarians.

The potential adverse effects of microplastics (MPs) on ecosystems and human health have received much attention in recent years. However, only limited data are available on the mechanisms for the uptake, distribution, and effects of MPs in freshwater organisms, especially with respect to tissue repair, regeneration and impairment of stem cell functions. To address this knowledge gap, we conducted exposure experiments in which planarians (Dugesia japonica) were exposed to polystyrene (PS)-MPs mixed in liver homogenate and examined the tissue growth and regeneration, stem cell functions, and oxidative stress. The body and blastema areas decreased upon exposure to PS-MPs, indicating that the growth and regeneration of planarians were delayed. The proliferation and differentiation processes of stem cells were inhibited, and the proportion of mitotic stem cells decreased, which may be related to the activation of the TGFß/SMAD4 and Notch signaling pathways. The enhancement of antioxidant enzyme activities and malondialdehyde on the first day of exposure to PS-MPs confirmed the oxidative stress response of planarians to PS-MPs. The present study demonstrated the likelihood of biotoxicity induced by PS-MPs. These results will provide clues for further investigations into the potential risks of PS-MPs to human stem cells.

Molecular cloning and characterization of DjRac1, a novel small G protein gene from planarian Dugesia japonica.

Rac proteins are classified as a subfamily of the Rho family of small G proteins. They are important molecular switches which act as key signal transducers regulating a wide variety of processes in the cell. DjRac1, a novel Rac gene from planarian Dugesia japonica was cloned by RACE method and characterized. This cDNA contains 851 bp with a putative open reading frame of 190 amino acids. It has a predicted molecular mass of 21.12 kDa and an isoelectric point of 8.42. Whole-mount in situ hybridization and relative quantitative real-time PCR were used to study the spatial and temporal expression pattern of DjRac1 from 1 to 7 days in the regenerating planarians. Results showed that the expression of DjRac1 was concentrated in the blastema and the transcription level of DjRac1 was significantly upregulated after amputation within three days, suggesting DjRac1 might participate in the process of regeneration in planarian.

Effects of Fe3+ on Acute Toxicity and Regeneration of Planarian (Dugesia japonica) at Different Temperatures.

OBJECTIVE: To investigate the effects of different concentrations of Fe3+ on the acute toxicity and regeneration of planarian at different temperatures. METHOD: The planarians were treated with 40 mg/l, 50 mg/l, 60 mg/l, and 70 mg/l Fe3+ solution and placed in 15°C, 20°C, and 25°C, respectively, to observe the mortality and the poisoning pattern of the planarian. In addition, the planarians were cut into three parts of head, trunk, and tail, then placed in Fe3+ solution at concentrations of 10 mg/l, 15 mg/l, 20 mg/l, and 30 mg/l, and placed in 15°C, 20°C, and 25°C respectively, and the regeneration rate of the planarian was investigated. RESULTS: At the same temperature, in the concentration of Fe3+ from 40 mg/l to 70 mg/l, the mortality of the planarian increased with the increasing of the concentration of Fe3+; at the same concentration and different temperatures, the death speed of the planarian is the fastest at 20°C, the next at 25°C, and the lowest at 15°C, indicating that the toxic effect of Fe3+ can be accelerated at a suitable temperature of 20°C. At the same temperature, in the low concentration of Fe3+ from 10 mg/l to 30 mg/l, the regeneration rate of the planarian gradually decreased with the increasing of the concentration of Fe3+; at the same concentration and different temperature, the regeneration rate of planarian was faster at 20°C and 25°C, but the difference between 20°C and 25°C was small, and the slowest at 15°C, indicating that the low temperature significantly affects the planarian regeneration speed. The study also found the regeneration rates of the head, trunk, and tail of the planarian were different; the head regeneration was the fastest, the trunk was the second, and the tail was the slowest. CONCLUSION: Fe3+ had obvious toxic effects on the survival and regeneration of planarian; the planarian is sensitive to Fe3+ and may be used to detect Fe3+ water pollution; in addition, temperature can affect the toxic effects of Fe3+ and thus affect the survival and regeneration of the planarian. Therefore, the temperature should be taken into consideration when detecting water Fe3+ pollution.

Molecular cloning and characterization of a novel Y-box gene from planarian Dugesia japonica.

Y-box binding protein (YB protein) is an ancient conserved multifunctional DNA/RNA-binding protein. A novel YB protein DjY2 gene from planarian Dugesia japonica was cloned by RACE method and characterized. This cDNA contains 689 bp with a putative open reading frame of 197 amino acids. It has a predicted molecular mass of 22.14 kDa and an isoelectric point of 9.67. Whole-mount in situ hybridization and relative quantitative real-time PCR were used to study the spatial and temporal expression pattern of DjY2 in the process of planarian regeneration. Results showed that DjY2 was expressed in many parts of the body in intact planarian, but the expression level was low in head and pharynx. The transcripts of DjY2 was significantly increased both at the head parts and the tail parts after amputation, especially at the site of cutting. The spatial expression gradually recovered to the state of intact planarian with the time of regeneration. Our results indicated that DjY2 might participate in the process of regeneration in planarian.

Mmu-miR-125b overexpression suppresses NO production in activated macrophages by targeting eEF2K and CCNA2.

BACKGROUND: MicroRNAs have been shown to be important regulators of the immune response and the development of the immune system. It was reported that microRNA-125b (miR-125b) was down-regulated in macrophages challenged with endotoxin. However, little is known about the function and mechanism of action of miR-125b in macrophage activation. Macrophages use L-arginine to synthesize nitric oxide (NO) through inducible NO synthase (iNOS), and the released NO contributes to the tumoricidal activity of macrophages. METHODS: Luciferase reporter assays were employed to validate regulation of a putative target of miR-125b. The effect of miR-125b on endogenous levels of this target were subsequently confirmed via Western blot. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed to determine the expression level of miR-125b in macrophage. MTS assays were conducted to explore the impact of miR-125b overexpression on the cell viability of 4T1 cells. RESULTS: Here, we demonstrate that mmu-miR-125b overexpression suppresses NO production in activated macrophages and that LPS-activated macrophages with overexpressed mmu-miR-125b promote 4T1 tumor cell proliferation in vitro and 4T1 tumor growth in vivo. CCNA2 and eEF2K are the direct and functional targets of mmu-miR-125b in macrophages; CCNA2 and eEF2K expression was knocked down, which mimicked the mmu-miR-125b overexpression phenotype. CONCLUSIONS: These data suggest that mmu-miR-125b decreases NO production in activated macrophages at least partially by suppressing eEF2K and CCNA2 expression.

A double feedback loop mediated by microRNA-23a/27a/24-2 regulates M1 versus M2 macrophage polarization and thus regulates cancer progression.

In response to microenvironmental signals, macrophages undergo different types of activation, including the "classic" pro-inflammatory phenotype (also called M1) and the "alternative" anti-inflammatory phenotype (also called M2). Macrophage polarized activation has profound effects on immune and inflammatory responses, but mechanisms underlying the various types of macrophage is still in its infancy. In this study, we reported that M1-type stimulation could down-regulate miR-23a/27a/24-2 cluster transcription through the binding of NF-κB to this cluster's promoter and that miR-23a in turn activated the NF-κB pathway by targeting A20 and thus promoted the production of pro-inflammatory cytokines. Furthermore, STAT6 occupied the miR-23a/27a/24-2 cluster promoter and activated their transcription in IL-4-stimulated macrophages. In addition, miR-23a in turn suppressed the JAK1/STAT-6 pathway and reduced the production of M2 type cytokines by targeting JAK1 and STAT-6 directly, while miR-27a showed the same phenotype by targeting IRF4 and PPAR-γ. The miR-23a/27a/24-2 cluster was shown to be significantly decreased in TAMs of breast cancer patients, and macrophages overexpressing the miR-23a/27a/24-2 cluster inhibited tumor growth in vivo. Taken together, these data integrated microRNA expression and function into macrophage polarization networks and identified a double feedback loop consisting of the miR-23a/27a/24-2 cluster and the key regulators of the M1 and M2 macrophage polarization pathway. Moreover, miR-23a/27a/24-2 regulates the polarization of tumor-associated macrophages and thus promotes cancer progression.

Functions of miR-146a and miR-222 in Tumor-associated Macrophages in Breast Cancer.

Tumor-associated macrophages (TAMs) play critical roles in promoting tumor progression and invasion. However, the molecular mechanisms underlying TAM regulation remain to be further investigated and may make significant contributions to cancer treatment. Mammalian microRNAs (miRNAs) have recently been identified as important regulators of gene expression that function by repressing specific target genes mainly at the post-transcriptional level. However, systematic studies of the functions and mechanisms of miRNAs in TAMs in tumor tissues are rare. In this study, miR-146a and miR-222 were shown to be significantly decreased in TAMs associated with the up-regulated NF-κB p50 subunit. miR-146a promoted the expression of some M2 macrophage phenotype molecules, and miR-146a antagomir transfected RAW264.7 monocyte-macrophage cells inhibited 4T1 tumor growth in vivo. Meanwhile, overexpression of miR-222 inhibited TAM chemotaxis, and miR-222 in TAMs inhibited 4T1 tumor growth by targeting CXCL12 and inhibiting CXCR4. These data revealed that miRNAs influence breast tumor growth by promoting the M2 type polarization or regulating the recruitment of TAMs. These observations suggest that endogenous miRNAs may exert an important role in controlling the polarization and function of TAMs in breast cancer.

Dysregulated expression of miR-101b and miR-26b lead to age-associated increase in LPS-induced COX-2 expression in murine macrophage.

Aging is the natural process of decline in physiological structure and function of various molecules, cells, tissues, and organs. Growing evidence indicates that increased immune genetic diversity and dysfunction of immune system cause aging-related pathophysiological process with the growth of age. In the present study, we observed that LPS-induced higher activation of cyclooxygenase (COX)-2 promoter is associated with the upregulated binding activity of nuclear factor kappa B (NF-κB) in peritoneal macrophages of aged mice than young ones. Additionally, COX-2 is a direct target of miR-101b and miR-26b in the macrophages. Significant upregulation of miR-101b and miR-26b effectively prevented LPS-induced excessive expression of COX-2 in the young mice. Because these negative regulatory factors were unresponsive to LPS stimulation, the levels of COX-2 were markedly higher in the macrophages of aged mice. Further study showed that NF-κB activation contributed to the increase in the expression of miR-101b and miR-26b in the LPS-stimulated macrophages of young mice, but not aged ones. Moreover, histone deacetylase (HDAC) inhibitor trichostatin A (TSA) upregulated expression of miR-101b and miR-26b in the aged mouse macrophages only, but not the young cells. This demonstrated that HDAC suppressed the expression of miR-101b and miR-26b in the LPS-treated macrophages of aged mice and contributed to the aging process. TSA-induced increased expression of miR-101b and miR-26b could further suppress COX-2 expression. These findings provide novel evidence on the regulation of immune senescence and miR-101b and miR-26b, which might be promising targets in treating aged-related inflammatory diseases. Epigenetic regulation of the microRNAs (miRNAs) provides an important evidence for the treatment of innate inflammatory disease with HDAC inhibitors in elderly.

Deep sequencing identifies regulated small RNAs in Dugesia japonica.

MicroRNAs (miRNAs), which are 18 ~ 24 nucleotides length, play important roles in regulating the expression of gene at the post-transcription level. Dugesia japonica is a branch of planarian organism. It is a model organism for studying the role of miRNAs in stem cell function. Next generation sequencing technology was used to identify the miRNAs of D. japonica. Bioinformatic analysis showed that 262 miRNA and miRNA* sequences were discovered, of which 102 miRNAs were the same as Schmidtea mediterranea and 160 miRNAs were related to other animals. There were 21 miRNAs expressed differentially after amputation. Results also revealed that some key miRNAs might play essential roles in the regeneration progress and some miRNAs might take part in the regulation progress of polarity regeneration in D. japonica.

Molecular cloning and characterization of a phosphoglycerate mutase gene from Clonorchis sinensis.

Phosphoglycerate mutase (PGM) is a widely distributed glycolytic enzyme. Two known distinct classes of PGM enzymes were identified, a cofactor-dependent one (dPGM) and a cofactor-independent one (iPGM). A complementary DNA (cDNA) encoding a PGM was cloned from a Clonorchis sinensis cDNA library by large-scale sequencing. This new cDNA contains 955 bp with a putative open reading frame of 256 amino acids, which has a high homology with dPGMs from a number of species. The putative peptide was produced in E. coli and was purified to electrophoretic homogeneity. Enzymatic assays showed that the product of this gene could catalyze the conversion of 3-phosphoglycerate to 2-phosphoglycerate when the cofactor was present and the enzyme activities could be inhibited by vanadate.