LSD1 modulates the bone metastasis of breast cancer cells through hnRNPA2B1-mediated sorting of exosomal miRNAs.

Bone metastasis is a key contributor to morbidity and mortality of breast cancer patients. We have previously shown that exosomal miRNAs derived from LSD1 knockdown (KD) breast cancer cells inhibit osteoblast differentiation and promote osteoclast differentiation. However, how LSD1 regulates exosomal miRNAs and whether miRNAs promote bone metastasis through the formation of pre-metastatic niches remains unclear. In vivo experiments demonstrates that exosomes derived from LSD1 KD breast cancer cells significantly promoted bone metastasis. To explore the mechanism underlying the effect of LSD1 on exosomes in breast cancer cells, exosomal and cellular miRNAs from control, LSD1 KD, and rescue cells were sequenced. Interestingly, approximately 80% of LSD1-associated miRNAs were downregulated in exosomes from LSD1 KD cells. The consensus sequence UAGGGC, was identified in many miRNAs downregulated in LSD1 KD exosomes. We found that hnRNPA2B1 regulated the exosomal sorting of miR-6881-3p and some other miRNAs. LSD1 deficiency reduced hnRNPA2B1 expression in breast cancer cells by decreasing the level of H3K9me2 demethylation in the promoter region of the hnRNPA2B1 gene. Our study revealed that LSD1 plays a crucial role in the regulation of exosomal sorting of miRNA.

HaMYBA-HabHLH1 regulatory complex and HaMYBF fine-tune red flower coloration in the corolla of sunflower (Helianthus annuus L.).

Sunflowers are well-known ornamental plants, while sunflowers with red corolla are rare and the mechanisms underlying red coloration remain unclear. Here, a comprehensive analysis of metabolomics and transcriptomics on flavonoid pathway was performed to investigate the molecular mechanisms underlying the differential color formation between red sunflower Pc103 and two yellow sunflowers (Yr17 and Y35). Targeted metabolomic analysis revealed higher anthocyanin levels but lower flavonol content in Pc103 compared to the yellow cultivars. RNA-sequencing and phylogenetic analysis identified multiple genes involved in the flavonoid pathway, including series of structural genes and three MYB and bHLH genes. Specifically, HaMYBA and HabHLH1 were up-regulated in Pc103, whereas HaMYBF exhibited reduced expression. HaMYBA was found to interact with HabHLH1 in vivo and in vitro, while HaMYBF does not. Transient expression analysis further revealed that HabHLH1 and HaMYBA cooperatively regulate increased expression of dihydroflavonol 4-reductase (DFR), leading to anthocyanin accumulation. On the other hand, ectopic expression of HaMYBF independently modulates flavonol synthase (FLS) expression, but hindered anthocyanin production. Collectively, our findings suggest that the up-regulation of HaMYBA and HabHLH1, as well as the down-regulation of HaMYBF, contribute to the red coloration in Pc103. It offers a theoretical basis for improving sunflower color through genetic engineering.

Effect of co-fermentation system with isolated new yeasts on soymilk: microbiological, physicochemical, rheological, aromatic, and sensory characterizations.

The beany flavor adversely influences consumer acceptance of soymilk (SM) products. Thus, in this work, the co-fermentation of isolated new yeasts (Kluyveromyces marxianus SP-1, Candida ethanolica ATW-1, and Pichia amenthionina Y) and Kluyveromyces marxianus K (a commercial yeast) along with an XPL-1 starter (including five strains of lactic acid bacteria (LAB)) was utilized to mend the beany flavor of fermented SM (FSM) beverages. Probiotic count, pH, titratable acidity, syneresis, water holding capacity, rheological characteristics, and sensory attributes were investigated. Furthermore, the free amino acids, nucleotides, and volatile compounds (VCs) were analyzed, also presenting the collected VC data by exploiting a principal component analysis (PCA) and a heatmap with a hierarchical cluster analysis. The co-fermentation with Kluyveromyces marxianus SP-1 and K remarkably enhanced the LAB strain growth and acid production, improving the rheological attributes, whereas that of yeast along with XPL-1 as a mullite starter could reduce the beany odor. PCA chart displayed that higher amounts of alcohols, ketones, acids, and esters that significantly improved the flavor quality of FSM beverages were generated throughout the co-fermentation process. The co-fermentation with Pichia amenthionina Y generated the highest acetoin (36.19%) and diacetyl (2.02%), thus improving the overall acceptance of FSM, as well as the sensory characteristics of FSM beverages with the highest umami, sweet, odorless amino acids, and umami nucleotides, and the lowest content of alcohol and inosine. Taken together, the co-fermentation of Pichia amenthionina Y along with XPL-1 within SM provides novel insights regarding the development of FSM and fermented beverages.

The Identification of Broomcorn Millet bZIP Transcription Factors, Which Regulate Growth and Development to Enhance Stress Tolerance and Seed Germination.

Broomcorn millet (Panicum miliaceum L.) is a water-efficient and highly salt-tolerant plant. In this study, the salt tolerance of 17 local species of broomcorn millet was evaluated through testing based on the analysis of the whitening time and the germination rate of their seeds. Transcriptome sequencing revealed that PmbZIP131, PmbZIP125, PmbZIP33, PmABI5, PmbZIP118, and PmbZIP97 are involved in seed germination under salt stress. Seedling stage expression analysis indicates that PmABI5 expression was induced by treatments of high salt (200 mM NaCl), drought (20% W/V PEG6000), and low temperature (4 °C) in seedlings of the salt-tolerant variety Y9. The overexpression of PmABI5 significantly increases the germination rate and root traits of Arabidopsis thaliana transgenic lines, with root growth and grain traits significantly enhanced compared to the wild type (Nipponbare). BiFC showed that PmABI5 undergoes homologous dimerization in addition to forming a heterodimer with either PmbZIP33 or PmbZIP131. Further yeast one-hybrid experiments showed that PmABI5 and PmbZIP131 regulate the expression of PmNAC1 by binding to the G-box in the promoter. These results indicate that PmABI5 can directly regulate seed germination and seedling growth and indirectly improve the salt tolerance of plants by regulating the expression of the PmNAC1 gene through the formation of heterodimers with PmbZIP131.

A Comparative Study of Milk Fat Extracted from the Milk of Different Goat Breeds in China: Fatty Acids, Triacylglycerols and Thermal and Spectroscopic Characterization.

Goat milk (GM) is an excellent alternative to cow milk and has recently been used in commercial infant formula preparation due to its superior fat composition. Here, the fatty acid (FA) composition, triacylglycerol (TAG) molecular species, thermal behavior and infrared spectra of extracted milk fat from the milk of the two main breeds of dairy goat bred in China (Guanzhong GM (GZG) and Xinong Saanen GM (XSG)) are investigated. Gas chromatography, Fourier-transform infrared spectroscopy, differential scanning calorimetry and ultra-performance convergence chromatography with quadrupole time-of-flight mass spectrometry are applied. The obtained results evidence significant fat compositional differences based on the breed that produced the considered GM. The major FAs in both GM fats were capric (C10:0), myristic (C14:0), palmitic (C16:0), stearic (C18:0) and oleic (C18:1 n-9c). GZG presented a higher content of medium-chain saturated FAs, while XSG had higher unsaturated FAs with higher ratios of L/Ln and n-6/n-3. A total of 339 and 359 TAGs were detected and quantified in GZG and XSG, and the major TAGs were those of m/z 740.6712 (14.10 ± 0.27%) and m/z 684.6094 (10.94 ± 0.02%), respectively. Milk TAGs of GZG and XSG showed 24-54 and 26-54 total acyl carbon numbers with a 0-4 and 0-5 double bond number at 68 and 72 various retention times, respectively. Thermal analysis showed that all GM fat samples melted below normal body temperature. Infrared spectra revealed higher absorption values of GZG milk fat. This study provides valuable information to the dairy industry sector about GM fat produced in China, assessing the appropriateness of Chinese GM fat to be applied in Chinese infant formula.

Diterpenoids from Sauropus spatulifolius Leaves with Antimicrobial Activities.

As a plant used in both food and medicine, Sauropus spatulifolius is consumed widely as a natural herbal tea, food source, and Chinese medicine. Inspired by its extensive applications, we conducted a systematic phytochemical study of the leaves of S. spatulifolius. Thirteen new diterpenoids, sauspatulifols A-M (1-13), including four ent-cleistanthane-type diterpenoids (1-4), eight 15,16-di-nor-ent-cleistanthane-type diterpenoids (5-12), and one 17-nor-ent-pimarane-type diterpenoid (13) as well as one known diterpenoid, cleistanthol (14), were isolated. All of these diterpenoids feature a 2α,3α-dihydroxy unit within the A ring, and their structures were elucidated by spectroscopic data analysis, electronic circular dichroism calculations, and single-crystal X-ray diffraction analysis. Compound 14 displayed moderate inhibitory activity against Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, and Shigella flexneri with the same minimum inhibitory concentration value of 12 µg/mL as well as activity against vesicular stomatitis virus and influenza A virus.

Generation of immunodeficient pig with hereditary tyrosinemia type 1 and their preliminary application for humanized liver.

BACKGROUND: Mice with humanized livers are important models to study drug toxicology testing, development of hepatitis virus treatments, and hepatocyte transplantation therapy. However, the huge difference between mouse and human in size and anatomy limited the application of humanized mice in investigating human diseases. Therefore, it is urgent to construct humanized livers in pigs to precisely investigate hepatocyte regeneration and human hepatocyte therapy. CRISPR/Cas9 system and somatic cell cloning technology were used to generate two pig models with FAH deficiency and exhibiting severe immunodeficiency (FAH/RAG1 and FAH/RAG1/IL2RG deficiency). Human primary hepatocytes were then successfully transplanted into the FG pig model and constructed two pigs with human liver. RESULTS: The constructed FAH/RAG1/IL2RG triple-knockout pig models were characterized by chronic liver injury and severe immunodeficiency. Importantly, the FG pigs transplanted with primary human hepatocytes produced human albumin in a time dependent manner as early as 1 week after transplantation. Furthermore, the colonization of human hepatocytes was confirmed by immunochemistry staining. CONCLUSIONS: We successfully generated pig models with severe immunodeficiency that could construct human liver tissues.

Lacticaseibacillus rhamnosus C1 effectively inhibits Penicillium roqueforti: Effects of antimycotic culture supernatant on toxin synthesis and corresponding gene expression.

Recently, consumers are increasingly concerned about the contamination of food by molds and the addition of chemical preservatives. As natural and beneficial bacteria, probiotics are a prospective alternative in food conservation because of their antimycotic activities, although the mechanism has not been explained fully at the level of metabolites. This study aimed at investigating the antifungal activities and their mechanisms of five potential probiotic strains (Lacticaseibacillus rhamnosus C1, Lacticaseibacillus casei M8, Lactobacillus amylolyticus L6, Schleiferilactobacillus harbinensis M1, and Limosilactobacillus fermentum M4) against Penicillium roqueforti, the common type of mold growth on the bread. Results showed that C1 emerged the strongest effectiveness at blocking mycelium growth, damaging the morphology of hyphae and microconidia, decreasing DNA content and interfering in the synthesis of the fungal toxins patulin, roquefortine C and PR-toxin, as well as downregulating the expression of key genes associated with the toxin biosynthesis pathways. Further metabonomic investigation revealed that protocatechuic acid with the minimum inhibitory concentration of 0.40 mg/mL, may be most likely responsible for positively correlated with the antimycotic effects of C1. Thus, C1 is expected to be both a potentially greatly efficient and environmental antimycotic for controlling P. roqueforti contamination in foods.

BdFAR4, a root-specific fatty acyl-coenzyme A reductase, is involved in fatty alcohol synthesis of root suberin polyester in Brachypodium distachyon.

Suberin is a complex hydrophobic polymer of aliphatic and phenolic compounds which controls the movement of gases, water, and solutes and protects plants from environmental stresses and pathogenic infection. The synthesis and regulatory pathways of suberin remain unknown in Brachypodium distachyon. Here we describe the identification of a B. distachyon gene, BdFAR4, encoding a fatty acyl-coenzyme A reductase (FAR) by a reverse genetic approach, and investigate the molecular relevance of BdFAR4 in the root suberin synthesis of B. distachyon. BdFAR4 is specifically expressed throughout root development. Heterologous expression of BdFAR4 in yeast (Saccharomyces cerevisiae) afforded the production of C20:0 and C22:0 fatty alcohols. The loss-of-function knockout of BdFAR4 by CRISPR/Cas9-mediated gene editing significantly reduced the content of C20:0 and C22:0 fatty alcohols associated with root suberin. In contrast, overexpression of BdFAR4 in B. distachyon and tomato (Solanum lycopersicum) resulted in the accumulation of root suberin-associated C20:0 and C22:0 fatty alcohols, suggesting that BdFAR4 preferentially accepts C20:0 and C22:0 fatty acyl-CoAs as substrates. The BdFAR4 protein was localized to the endoplasmic reticulum in Arabidopsis thaliana protoplasts and Nicotiana benthamiana leaf epidermal cells. BdFAR4 transcript levels can be increased by abiotic stresses and abscisic acid treatment. Furthermore, yeast one-hybrid, dual-luciferase activity, and electrophoretic mobility shift assays indicated that the R2R3-MYB transcription factor BdMYB41 directly binds to the promoter of BdFAR4. Taken together, these results imply that BdFAR4 is essential for the production of root suberin-associated fatty alcohols, especially under stress conditions, and that its activity is transcriptionally regulated by the BdMYB41 transcription factor.

Association of interleukin-6 gene polymorphisms with the risk of hepatocellular carcinoma: An up-to-date meta-analysis.

BACKGROUND: This study was aimed to evaluate the association between interleukin-6 (IL-6) gene polymorphisms and the risk of hepatocellular carcinoma (HCC) in a meta-analysis. METHODS: A literature search was performed for case-control studies published during May, 1993 to May, 2020 focusing on IL-6 gene polymorphisms (-174G > C, -572G > C, and -597G > A) and HCC susceptibility by using PubMed, Cochrane Database, EMBASE, Web of science, and China National Knowledge Infrastructure. From 128 full-text articles, 11 were included in this meta-analysis. I index was used to assess heterogeneity and Newcastle-Ottawa Scale was utilized for quality assessment. RESULTS: For IL-6 -174G > C polymorphism, in codominant (GG vs CC: odds ratios [OR] = 2.78, 95% confidence intervals [CI] = 1.25-6.19, P = .01, I = 16%) and recessive (GG+GC vs CC: OR = 2.76, 95% CI = 1.29-5.90, P = .009, I = 3%) models, IL-6 -174G>C polymorphism was significantly associated with the risk of HCC. In dominant (GG vs CC+GC: OR = 1.80, 95% CI = 0.92-3.54, P = .09, I = 86%) and allele (G vs C: OR = 1.49, 95% CI = 0.95-2.32, P = .08, I = 68%) models, IL-6 -174G>C polymorphism had no impact on the risk of HCC. However, in non-Italian Caucasian population, IL-6 -174G>C polymorphism was significantly related to the occurrence of HCC in both dominant (GG vs CC+GC: OR = 3.26, 95% CI = 2.29-4.65, P < .00001, I = 0%) and allele (G vs C: OR = 2.48, 95% CI = 1.48-4.15, P = .0006) models. Such correlations also could be observed when healthy individuals were selected as controls. For IL-6 -572G>C and -597G>A polymorphisms, no significant association was observed in all models, regardless of the source of control and population subgroups. No publication bias could be calculated when Begg and Egger tests were employed. CONCLUSION: This meta-analysis indicated that IL-6 -174G>C polymorphism was significantly related with the risk for HCC, especially in non-Italian Caucasian population. No significant association was observed for the correlation between IL-6 -572G>C and -597G>A polymorphisms and HCC susceptibility.

IL2RG-deficient minipigs generated via CRISPR/Cas9 technology support the growth of human melanoma-derived tumours.

OBJECTIVES: Immunodeficient mice injected with human cancer cell lines have been used for human oncology studies and anti-cancer drug trials for several decades. However, rodents are not ideal species for modelling human cancer because rodents are physiologically dissimilar to humans. Therefore, anti-tumour drugs tested effective in rodents have a failure rate of 90% or higher in phase III clinical trials. Pigs are similar to humans in size, anatomy, physiology and drug metabolism rate, rendering them a desirable pre-clinical animal model for assessing anti-cancer drugs. However, xenogeneic immune rejection is a major barrier to the use of pigs as hosts for human tumours. Interleukin (IL)-2 receptor γ (IL2RG), a common signalling subunit for multiple immune cytokines including IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21, is required for proper lymphoid development. MATERIALS AND METHODS: IL2RG-/Y pigs were generated by CRISPR/Cas9 technology, and examined for immunodeficiency and ability to support human oncogenesis. RESULTS: Compared to age-matched wild-type pigs, IL2RG-/Y pigs exhibited a severely impaired immune system as shown by lymphopenia, lymphoid organ atrophy, poor immunoglobulin function, and T- and NK-cell deficiency. Human melanoma Mel888 cells generated tumours in IL2RG-/Y pigs but not in wild-type littermates. The human tumours grew faster in IL2RG-/Y pigs than in nude mice. CONCLUSIONS: Our results indicate that these pigs are promising hosts for modelling human cancer in vivo, which may aid in the discovery and development of anti-cancer drugs.

Generation of GGTA1-/-ß2M-/-CIITA-/- Pigs Using CRISPR/Cas9 Technology to Alleviate Xenogeneic Immune Reactions.

BACKGROUND: Xenogeneic organ transplantation has been proposed as a potential approach to fundamentally solve organ shortage problem. Xenogeneic immune responses across species is one of the major obstacles for clinic application of xeno-organ transplantation. The generation of glycoprotein galactosyltransferase α 1, 3 (GGTA1) knockout pigs has greatly contributed to the reduction of hyperacute xenograft rejection. However, severe xenograft rejection can still be induced by xenoimmune responses to the porcine major histocompatibility complex antigens swine leukocyte antigen class I and class II. METHODS: We simultaneously depleted GGTA1, ß2-microglobulin (ß2M), and major histocompatibility complex class II transactivator (CIITA) genes using clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins technology in Bamma pig fibroblast cells, which were further used to generate GGTA1ß2MCIITA triple knockout (GBC-3KO) pigs by nuclear transfer. RESULTS: The genotype of GBC-3KO pigs was confirmed by polymerase chain reaction and Sanger sequencing, and the loss of expression of α-1,3-galactose, SLA-I, and SLA-II was demonstrated by flow cytometric analysis using fluorescent-conjugated lectin from bandeiraea simplicifolia, anti-ß2-microglobulin, and swine leukocyte antigen class II DR antibodies. Furthermore, mixed lymphocyte reaction assay revealed that peripheral blood mononuclear cells from GBC-3KO pigs were significantly less effective than (WT) pig peripheral blood mononuclear cells in inducing human CD3CD4 and CD3CD8 T-cell activation and proliferation. In addition, GBC-3KO pig skin grafts showed a significantly prolonged survival in immunocompetent C57BL/6 mice, when compared with wild-type pig skin grafts. CONCLUSIONS: Taken together, these results demonstrate that elimination of GGTA1, ß2M, and CIITA genes in pigs can effectively alleviate xenogeneic immune responses and prolong pig organ survival in xenogenesis. We believe that this work will facilitate future research in xenotransplantation.

[Enhancing thermostability of xylanase from rumen microbiota by molecular cyclization].

The capacity for thermal tolerance is critical for industrial enzyme. In the past decade, great efforts have been made to endow wild-type enzymes with higher catalytic activity or thermostability using gene engineering and protein engineering strategies. In this study, a recently developed SpyTag/SpyCatcher system, mediated by isopeptide bond-ligation, was used to modify a rumen microbiota-derived xylanase XYN11-6 as cyclized and stable enzyme C-XYN11-6. After incubation at 60, 70 or 80 ℃ for 10 min, the residual activities of C-XYN11-6 were 81.53%, 73.98% or 64.41%, which were 1.48, 2.92 or 3.98-fold of linear enzyme L-XYN11-6, respectively. After exposure to 60-90°C for 10 min, the C-XYN11-6 remained as soluble in suspension, while L-XYN11-6 showed severely aggregation. Intrinsic and 8-anilino-1-naphthalenesulfonic acid (ANS)-binding fluorescence analysis revealed that C-XYN11-6 was more capable of maintaining its conformation during heat challenge, compared with L-XYN11-6. Interestingly, molecular cyclization also conferred C-XYN11-6 with improved resilience to 0.1-50 mmol/L Ca²âº or 0.1 mmol/L Cu²âº treatment. In summary, we generated a thermal- and ion-stable cyclized enzyme using SpyTag/SpyCatcher system, which will be of particular interest in engineering of enzymes for industrial application.

New insights into the alleviating role of Melaleuca alternifolia oil on metabolites pathway disorder of grapes caused by Aspergillus niger, verified by corresponding key genes expression.

Grape berries are susceptible to Aspergillus niger (A. niger) infection during storage, leading to a significant reduction in its nutritional quality. However, most alternations in nutrient contents and related gene expression during fungal infection or treated with antimycotics remain unexplored. This work aimed to monitor and verify the metabolic changes in berries caused by A. niger or Melaleuca alternifolia oil (MAO) by using UHPLC-ESI-MS2 and Quantitative Real-time PCR (RT-qPCR). Results showed that sucrose, glucose, fructose, trans-resveratrol and pterostilbene levels were down and pentose phosphate pathway, glycolysis pathway and phenylpropanoid pathway were significantly down-regulated compared with healthy berries due to A. niger infection, all of which were alleviated by MAO treated. A. niger also induced down-regulation of key genes expression associated with metabolic pathways and magnitude of down-regulation was reduced by MAO. These results provide a theoretical basis for MAO used to control the risk of A. niger-mediated diseases.

TaCER1-1A is involved in cuticular wax alkane biosynthesis in hexaploid wheat and responds to plant abiotic stresses.

To protect above-ground plant organs from excessive water loss, their surfaces are coated by waxes. The genes involved in wax formation have been investigated in detail in Arabidopsis but scarcely in crop species. Here, we aimed to isolate and characterize a CER1 enzyme responsible for formation of the very long-chain alkanes present in high concentrations especially during late stages of wheat development. On the basis of comparative wax and transcriptome analyses of various wheat organs, we selected TaCER1-1A as a primary candidate and demonstrated that it was located to the endoplasmic reticulum, the subcellular compartment for wax biosynthesis. A wheat nullisomic-tetrasomic substitution line lacking TaCER1-1A had significantly reduced amounts of C33 alkane, whereas rice plants overexpressing TaCER1-1A showed substantial increases of C25 -C33 alkanes relative to wild type control. Similarly, heterologous expression of TaCER1-1A in Arabidopsis wild type and the cer1 mutant resulted in increased levels of unbranched alkanes, iso-branched alkanes and alkenes. Finally, the expression of TaCER1-1A was found activated by abiotic stresses and abscisic acid treatment, resulting in increased production of alkanes in wheat. Taken together, our results demonstrate that TaCER1-1A plays an important role in wheat wax alkane biosynthesis and involved in responding to drought and other environmental stresses.

Determination of metabolites of Geotrichum citri-aurantii treated with peppermint oil using liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry.

Sour rot is a leading disease of citrus fruit caused by the postharvest pathogen Geotrichum citri-aurantii. It has been reported that essential oils can be used as substitutes for synthetic fungicides to control the pathogen. In this study, changes in metabolites and antifungal effects of G. citri-aurantii treated with peppermint oil (PO) were investigated. The inhibition rate of the mycelial growth increased as the PO concentration increased, and 6 µl PO/disk resulted in a radial growth inhibition of 79.2%. The electrical conductivity of G. citri-aurantii treated with PO increased compared to the control. By comparing the metabolic profiles of treated and untreated G. citri-aurantii cells, a total of 53 distinct metabolites 9 were up-regulated and 44 were down-regulated were found, including 16 lipid metabolites, 6 carbohydrate metabolites, 2 amino acid metabolites, 5 alcohols, 2 glycoside metabolites, and 3 ketone metabolites, etc, and these metabolites are involved in 25 major metabolic pathways. PRACTICAL APPLICATIONS: Chemical fungicides can effectively control G. citri-aurantii during fruit postharvest period. However, synthetic chemical fungicides have gradually led to buildup of resistance of fungil, which seriously causes the frequent of food-borne diseases. PO extracted from natural plants can be used as natural additive in many foods due to their antioxidant, antibacterial, and antifungal properties. Therefore, PO can be considered as a promising bacteriostatic agent for the defense of G. citri-aurantii during fruit postharvest period.

Sodium butyrate-induced histone hyperacetylation up-regulating WT1 expression in porcine kidney fibroblasts.

OBJECTIVES: Wilms' tumor 1 gene (WT1) is essential for the development of kidney and histone acetylation and is involved in its expression regulation in mice. However, whether WT1 expression is associated with histone acetylation in porcine kidney cells is unclear. Here, the effect of histone deacetylase inhibitor sodium butyrate (NaBu)-induced hyperacetylation on WT1 expression in porcine kidney fibroblasts (PKF) was examined. RESULTS: Treatments of NaBu (1, 3, 6 mM) for 24 h increased PKF viability, and 24, 48 h-treatments of 1 mM NaBu enhanced PKF proliferation. WT1 mRNA levels were significantly elevated in NaBu-treated (1, 3 mM for 24, 48 h, respectively) PKF samples. Consistently, strengthened expression of WT1 protein and histone acetylation level were detected in NaBu-treated PKF cells. CONCLUSION: Together, NaBu-induced hyperacetylation up-regulates WT1 expression in PKF, suggesting the involvement of histone acetylation in the transcriptional modulation of WT1 in porcine kidney cells.

Effects of WT1 down-regulation on oocyte maturation and preimplantation embryo development in pigs.

The Wilms' tumour 1 (WT1) gene originally identified as a tumour suppressor associated with WTs encodes a zinc finger-containing transcription factor that is expressed in multiple tissues and is an important regulator of cellular and organ growth, proliferation, development, migration and survival. However, there is a deficiency of data regarding the expression and function of WT1 during oocyte maturation and preimplantation embryonic development. Herein, we sought to define the expression characteristics and functions of WT1 during oocyte maturation and preimplantation embryonic development in pigs. We show that WT1 is expressed in porcine oocytes and at all preimplantation stages in embryos generated by ICSI. We then evaluated the effects of down-regulating WT1 expression at germinal vesicle and early ICSI stages using a recombinant plasmid (pGLV3-WT1-shRNA). Down-regulation of WT1 did not affect oocyte maturation but significantly decreased preimplantation embryonic development and increased apoptosis in blastocysts. These results indicate that WT1 plays important roles in the development of porcine preimplantation embryos.

Effects of WT1 gene downregulation on apoptosis in porcine fetal fibroblasts.

Wilms' tumor gene 1 (WT1) is located on chromosome 11p13. Besides a role in the development of Wilms' tumor, specific mutations in the Zn finger region are found in Denys-Drash syndrome and Frasier syndrome, both characterized by urogenital abnormalities, sometimes in combination with Wilms' tumor. Our past study shows that WT1 is expressed in porcine kidney fibroblasts (PKFs) and swine testis cells (ST cells) and is essential for the maintenance of the development and survival of PKFs and ST cells. But we do not know whether WT1 gene was expressed in porcine fetal fibroblasts or not. To further explore whether WT1 was expressed in porcine fetal fibroblasts (PFFs) and its contribution to cell apoptosis, RT-PCR, immunocytochemical staining, and Western blot were used to detect the expression of WT1, the recombinant plasmids of pLV3-WT1 short hairpin ribonucleic acid (shRNA) were used to downregulate the WT1 gene in porcine fetal fibroblasts, and the role of WT1 in cell proliferation was examined by apoptosis analysis also. Our results indicated that WT1 was expressed in PFFs, the pLV3-WT1 shRNA dramatically reduced the expression of WT1, and downregulation of WT1 directly led to early cell apoptosis by downregulating the expression of antiapoptotic gene Bcl-2 and upregulating the expression of proapoptotic gene Bax in PFFs. Our results demonstrate that WT1 is also essential for the maintenance of the survival of PFFs.

Effects of TET1 knockdown on gene expression and DNA methylation in porcine induced pluripotent stem cells.

TET1 is implicated in maintaining the pluripotency of embryonic stem cells. However, its precise effects on induced pluripotent stem cells (iPSCs), and particularly on porcine iPSCs (piPSCs), are not well defined. To investigate the role of TET1 in the pluripotency and differentiation of piPSCs, piPSCs were induced from porcine embryonic fibroblasts by overexpression of POU5F1 (OCT4), SOX2, KLF4, and MYC (C-MYC). siRNAs targeting to TET1 were used to transiently knockdown the expression of TET1 in piPSCs. Morphological abnormalities and loss of the undifferentiated state of piPSCs were observed in the piPSCs after the downregulation of TET1. The effects of TET1 knockdown on the expression of key stem cell factors and differentiation markers were analyzed to gain insights into the molecular mechanisms underlying the phenomenon. The results revealed that knockdown of TET1 resulted in the downregulated expression of pluripotency-related genes, such as LEFTY2, KLF2, and SOX2, and the upregulated expression of differentiation-related genes including PITX2, HAND1, GATA6, and LEF1. However, POU5F1, MYC, KLF4, and NANOG were actually not downregulated. Further analysis showed that the methylation levels of the promoters for POU5F1 and MYC increased significantly after TET1 downregulation, whereas there were no obvious changes in the promoters of SOX2, KLF4, and NANOG. The methylation of the whole genome increased, while hydroxymethylation slightly declined. Taken together, these results suggest that TET1 may play important roles in the self-renewal of piPSCs and the maintenance of their characteristics by regulating the expression of genes and the DNA methylation.