Search Results (425)

Anthocyanins are important secondary metabolites in plants, which contribute to fruit color and nutritional value. Anthocyanins can be regulated by environmental factors such as light, low temperature, water conditions, and nutrition limitations. Nitrogen (N) is an essential macroelement for plant development, its deficiency as a kind of nutrition limitation often induces anthocyanin accumulation in many plants. However, there is a lack of reports regarding the effect of nitrogen deficiency on anthocyanin biosynthesis in pears. In this study, we found that N deficiency resulted in anthocyanin accumulation in pear callus and upregulated the expression of anthocyanin biosynthesis pathway structural genes (PyPAL, PyCHS, PyCHI, PyF3H, PyDFR, PyANS, and PyUFGT) and key regulatory factors (PyMYB10, PyMYB114, and PybHLH3). Through analysis of transcriptome data of treated pear callus and RT-qPCR assay, a differentially expressed gene PyNAC42 was identified as significantly induced by the N deficiency condition. Overexpression of PyNAC42 promoted anthocyanin accumulation in “Zaosu” pear peels. Additionally, dual luciferase assay and yeast one-hybrid assay demonstrated that PyNAC42 could not directly activate the expression of PyDFR, PyANS, and PyUFGT. Furthermore, yeast two-hybrid and pull-down assays confirmed that PyNAC42 interacted with PyMYB10 both in vivo and in vitro. Co-expression of PyNAC42 and PyMYB10 significantly enhanced anthocyanin accumulation in “Zaosu” pear peels. Dual luciferase assay showed that PyNAC42 significantly enhanced the activation of PyDFR, PyANS, and PyUFGT promoters by interacting with PyMYB10, which suggests that PyNAC42 can form the PyNAC42-PyMYB10 complex to regulate anthocyanin biosynthesis in pear. Thus, the molecular mechanism underlying anthocyanin biosynthesis induced by N deficiency is preliminarily elucidated. Our finding has expanded the regulatory network of anthocyanin biosynthesis and enhanced our understanding of the mechanisms underlying nutrient deficiency modulates anthocyanin biosynthesis in pear. Full article

Cow milk possesses high nutritional value due to its rich array of beneficial fatty acids. It is important to understand the mechanisms involved in lipid metabolism in dairy cows. These mechanisms are driven by a complex molecular regulatory network. In addition, there are many regulatory factors involved in the process of fatty acid metabolism, including transcription factors and non-coding RNAs, amongst others. MicroRNAs (miRNAs) can regulate the expression of target genes and modulate various biological processes, including lipid metabolism. Specifically, miR-206 has been reported to impair lipid accumulation in nonruminant hepatocytes. However, the effects and regulatory mechanisms of miR-206 on lipid metabolism in bovine mammary cells remain unclear. In the present study, we investigated the effects of miR-206 on lipid-related genes and TAG accumulation. The direct downstream gene of miR-206 was subsequently determined via a dual-luciferase assay. Finally, the fatty acid content of bovine mammary epithelial cells (BMECs) upon ELOVL6 inhibition was examined. The results revealed that miR-206 overexpression significantly decreased triacylglycerol (TAG) concentration and abundances of the following: acetyl-coenzyme A carboxylase alpha (ACACA); fatty acid synthase (FASN); sterol regulatory element binding transcription factor 1 (SREBF1); diacylglycerol acyltransferase 1 (DGAT1); 1-acylglycerol-3-phosphate O-acyltransferase 6 (AGPAT6); lipin 1 (LPIN1); and fatty acid elongase 6 (ELOVL6). Overexpression of miR-206 was also associated with an increase in patatin-like phospholipase domain-containing 2 (PNPLA2), while inhibition of miR-206 promoted milk fat metabolism in vitro. In addition, we found that ELOVL6 is a direct target gene of miR-206 through mutation of the binding site. Furthermore, ELOVL6 intervention significantly decreased the TAG levels and elongation indexes of C16:0 and C16:1n-7 in BMECs. Finally, ELOVL6 siRNA partially alleviated the increased TAG accumulation caused by miR-206 inhibition. In summary, we found that miR-206 inhibits milk fatty acid synthesis and lipid accumulation by targeting ELOVL6 in BMECs. The results presented in this paper may contribute to the development of strategies for enhancing the quality of cow milk and its beneficial fatty acids, from the perspective of miRNA–mRNA networks. Full article

The prolactin (PRL) hormone is a major regulator of mammary gland development and lactation. However, it remains unclear whether and how PRL contributes to mammary epithelial cell proliferation and secretion. The Boer and Macheng black crossbred goats are superior in reproduction, meat, and milk, and are popular in Hubei province. To elucidate the mechanisms of PRL on mammary growth and lactation, to improve the local goat economic trade, we have performed studies on these crossbred goats during pregnancy and early lactation, and in goat mammary epithelial cells (GMECs). Here, we first found that the amino acid transporters of SNAT1 and SNAT2 expression in vivo and in vitro were closely associated with PRL levels, the proliferation and secretion of GMECs; knockdown and over-expression of SNAT1/2 demonstrated that PRL modulated the proliferation and lactation of GMECs through regulating SNAT1/2 expression. Transcriptome sequencing and qPCR assays demonstrated the effect of PRL on the transcriptional regulation of SNAT1 and SNAT2 in GMECs. Dual-luciferase reporter gene assays further verified that the binding of the potential PRL response element in the SNAT1/2 promoter regions activated SNAT1/2 transcription after PRL stimulation. Additionally, silencing of either PRLR or STAT5 nearly abolished PRL-stimulated SNAT1/2 promoter activity, suggesting PRLR–STAT5 signaling is involved in the regulation of PRL on the transcriptional activation of SNAT1/2. These results illustrated that PRL modulates the proliferation and secretion of GMECs via PRLR–STAT5-mediated regulation of the SNAT1/2 pathway. This study provides new insights into how PRL affects ruminant mammary development and lactation through regulation of amino acid transporters. Full article

Non-muscle myosin IIA (NM IIA) is a motor protein that belongs to the myosin II family. The myosin heavy chain 9 (MYH9) gene encodes the heavy chain of NM IIA. NM IIA is a hexamer and contains three pairs of peptides, which include the dimer of heavy chains, essential light chains, and regulatory light chains. NM IIA is a part of the actomyosin complex that generates mechanical force and tension to carry out essential cellular functions, including adhesion, cytokinesis, migration, and the maintenance of cell shape and polarity. These functions are regulated via light and heavy chain phosphorylation at different amino acid residues. Apart from physiological functions, NM IIA is also linked to the development of cancer and genetic and neurological disorders. MYH9 gene mutations result in the development of several autosomal dominant disorders, such as May-Hegglin anomaly (MHA) and Epstein syndrome (EPS). Multiple studies have reported NM IIA as a tumor suppressor in melanoma and head and neck squamous cell carcinoma; however, studies also indicate that NM IIA is a critical player in promoting tumorigenesis, chemoradiotherapy resistance, and stemness. The ROCK-NM IIA pathway regulates cellular movement and shape via the control of cytoskeletal dynamics. In addition, the ROCK-NM IIA pathway is dysregulated in various solid tumors and leukemia. Currently, there are very few compounds targeting NM IIA, and most of these compounds are still being studied in preclinical models. This review provides comprehensive evidence highlighting the dual role of NM IIA in multiple cancer types and summarizes the signaling networks involved in tumorigenesis. Furthermore, we also discuss the role of NM IIA as a potential therapeutic target with a focus on the ROCK-NM IIA pathway. Full article

The bHLH transcription factors are important plant regulators against abiotic stress and involved in plant growth and development. In this study, SlALC, a gene coding for a prototypical DNA-binding protein in the bHLH family, was isolated, and SlALC-overexpression tomato (SlALC-OE) plants were generated by Agrobacterium-mediated genetic transformation. SlALC transgenic lines manifested higher osmotic stress tolerance than the wild-type plants, estimated by higher relative water content and lower water loss rate, higher chlorophyll, reducing sugar, starch, proline, soluble protein contents, antioxidant enzyme activities, and lower MDA and reactive oxygen species contents in the leaves. In SlALC-OE lines, there were more significant alterations in the expression of genes associated with stress. Furthermore, SlALC-OE fruits were more vulnerable to dehiscence, with higher water content, reduced lignin content, SOD/POD/PAL enzyme activity, and lower phenolic compound concentrations, all of which corresponded to decreased expression of lignin biosynthetic genes. Moreover, the dual luciferase reporter test revealed that SlTAGL1 inhibits SlALC expression. This study revealed that SlALC may play a role in controlling plant tolerance to drought and salt stress, as well as fruit lignification, which influences fruit dehiscence. The findings of this study have established a foundation for tomato tolerance breeding and fruit quality improvement. Full article

Rad6 is a canonical ubiquitin-conjugating enzyme known for its role in regulating chromosome-related cellular processes in yeast and has been proven to have multiple functions in Beauveria bassiana, including insect-pathogenic lifestyle, UV damage repair, and conidiation. However, previous studies have only reported the key role of Rad6 in regulating conidial production in a nutrient-rich medium, without any deep mechanism analyses. In this study, we found that the disruption of Rad6 leads to a profound reduction in conidial production, irrespective of whether the fungus is cultivated in nutrient-rich or nutrient-poor environments. The absence of rad6 exerts a suppressive effect on the transcription of essential genes in the central developmental pathway, namely, brlA, abaA, and wetA, resulting in a direct downregulation of conidiation capacity. Additionally, mutant strains exhibited a more pronounced decline in both conidial generation and hyphal development when cultured in nutrient-rich conditions. This observation correlates with the downregulation of the central developmental pathway (CDP) downstream gene vosA and the upregulation of flaA in nutrient-rich cultures. Moreover, single-transcriptomics analyses indicated that irregularities in biotin metabolism, DNA repair, and tryptophan metabolism are the underlying factors contributing to the reduced conidial production. Comprehensive dual transcriptomics analyses pinpointed abnormal biotin metabolism as the primary cause of conidial production decline. Subsequently, we successfully restored conidial production in the Rad6 mutant strain through the supplementation of biotin, further confirming the transcriptomic evidence. Altogether, our findings underscore the pivotal role of Rad6 in influencing biotin metabolism, subsequently impacting the expression of CDP genes and ultimately shaping the asexual life cycle of B. bassiana. Full article

Overcoming temozolomide (TMZ)-resistance is a major challenge in glioblastoma therapy. Therefore, identifying the key molecular player in chemo-resistance becomes urgent. We previously reported the downregulation of PDCD10 in primary glioblastoma patients and its tumor suppressor-like function in glioblastoma cells. Here, we demonstrate that the loss of PDCD10 causes a significant TMZ-resistance during treatment and promotes a rapid regrowth of tumor cells after treatment. PDCD10 knockdown upregulated MGMT, a key enzyme mediating chemo-resistance in glioblastoma, accompanied by increased expression of DNA mismatch repair genes, and enabled tumor cells to evade TMZ-induced cell-cycle arrest. These findings were confirmed in independent models of PDCD10 overexpressing cells. Furthermore, PDCD10 downregulation led to the dedifferentiation of glioblastoma cells, as evidenced by increased clonogenic growth, the upregulation of glioblastoma stem cell (GSC) markers, and enhanced neurosphere formation capacity. GSCs derived from PDCD10 knockdown cells displayed stronger TMZ-resistance and regrowth potency, compared to their parental counterparts, indicating that PDCD10-induced stemness may independently contribute to tumor malignancy. These data provide evidence for a dual role of PDCD10 in tumor suppression by controlling both chemo-resistance and dedifferentiation, and highlight PDCD10 as a potential prognostic marker and target for combination therapy with TMZ in glioblastoma. Full article

Cucumber (Cucumis sativus L.) is a vital economic vegetable crop, and the TONNEAU1 Recruiting Motif (TRM) gene plays a key role in cucumber organ growth. However, the pan-genomic characteristics of the TRM gene family and their expression patterns under different stresses have not been reported in cucumber. In this study, we identified 29 CsTRMs from the pan-genomes of 13 cucumber accessions, with CsTRM29 existing only in PI183967. Most CsTRM proteins exhibited differences in sequence length, except five CsTRMs having consistent protein sequence lengths among the 13 accessions. All CsTRM proteins showed amino acid variations. An analysis of CsTRM gene expression patterns revealed that six CsTRM genes strongly changed in short-fruited lines compared with long-fruited lines. And four CsTRM genes strongly responded to salt and heat stress, while CsTRM14 showed responses to salt stress, powdery mildew, gray mold, and downy mildew. Some CsTRM genes were induced or suppressed at different treatment timepoints, suggesting that cucumber TRM genes may play different roles in responses to different stresses, with expression patterns varying with stress changes. Remarkably, the expression of CsTRM21 showed considerable change between long and short fruits and in responses to abiotic stresses (salt stress and heat stress), as well as biotic stresses (powdery mildew and gray mold), suggesting a dual role of CsTRM21 in both fruit shape determination and stress resistance. Collectively, this study provided a base for the further functional identification of CsTRM genes in cucumber plant growth and stress resistance. Full article

Porcine reproductive and respiratory syndrome (PRRS) has caused substantial damage to the pig industry. MicroRNAs (miRNAs) were found to play crucial roles in modulating the pathogenesis of PRRS virus (PRRSV). In the present study, we revealed that PRRSV induced let-7f-5p to influence lipid metabolism to regulate PRRSV pathogenesis. A transcriptome analysis of PRRSV-infected PK15^CD163 cells transfected with let-7f-5p mimics or negative control (NC) generated 1718 differentially expressed genes, which were primarily associated with lipid metabolism processes. Furthermore, the master regulator of lipogenesis SREBP2 was found to be directly targeted by let-7f-5p using a dual-luciferase reporter system and Western blotting. The findings demonstrate that let-7f-5p modulates lipogenesis by targeting SREBP2, providing novel insights into miRNA-mediated PRRSV pathogenesis and offering a potential antiviral therapeutic target. Full article

The highly abundant and stable antiviral small RNA derived from honeysuckle, known as miR2911, has been shown to play a key role in inhibiting influenza virus infection and SARS-CoV-2 infection. However, whether miR2911 inhibits the replication of porcine reproductive and respiratory syndrome virus (PRRSV) remains unknown. Hence, this study investigated the mechanisms underlying the action of miR2911 during PRRSV infection. Six targets of miR2911 within the PRRSV orf1 (Nsp2: 2459 to 2477, 1871 to 1892, 954 to 977, and 1271 to 1292; Nsp1: 274 to 296 and 822 to 841) were successfully identified by using the miRanda v1.0b software. The miR2911 target sequence was analyzed by target sequence comparison, and only individual base mutations existed in different prevalent strains, and the miR2911 target region was highly conserved among different strains. Subsequently, through the dual luciferase reporter gene assay and miR2911 overexpression assay, it was demonstrated that miR2911 significantly inhibits the replication of PRRSV by targeting regions of PRRSV Nsp1 and Nsp2. These findings offer new insights for the development of novel anti-PRRSV drugs. Full article

Background: The level of the regulator of G-protein signaling 4-1 (RGS4-1) isoform, the longest RGS4 isoform, is significantly reduced in the dorsolateral prefrontal cortex (DLPFC) of people with schizophrenia. However, the mechanism behind this has not been clarified. The 3′untranslated regions (3′UTRs) are known to regulate the levels of their mRNA splice variants. Methods: We constructed recombinant pmir-GLO vectors with a truncated 3′ regulatory region of the RGS4 gene (3R1, 3R2, 3R3, 3R4, 3R5, and 3R6). The dual-luciferase reporter assay was conducted to find functional regions in HEK-293, SK-N-SH, and U87cells and then predicted miRNA binding to these regions. We performed a dual-luciferase reporter assay and a Western blot analysis after transiently transfecting the predicted miRNAs. Results: The dual-luciferase reporter assay found that regions +401–+789, +789–+1152, and +1562–+1990 (with the last base of the termination codon being +1) might be functional regions. Hsa-miR-874-3p, associated with many psychiatric disorders, might target the +789–+1152 region in the 3′UTR of the RGS4 gene. In the dual-luciferase reporter assay, the hsa-miR-874-3p mimic, co-transfected with 3R1, down-regulated the relative fluorescence intensities. However, this was reversed when the hsa-miR-874-3p mimic was co-transfected with m3R1 (deletion of +853–+859). The hsa-miR-874-3p mimic significantly decreased the endogenous expression of the RGS4-1 isoform in HEK-293 cells. Conclusions: Hsa-miR-874-3p inhibits the expression of the RGS4-1 isoform by targeting +853–+859. Full article

In this study, the expression profiles of miR-148a were constructed in eight different ovine tissues, including mammary gland tissue, during six different developmental periods. The effect of miR-148a on the viability, proliferation, and milk fat synthesis of ovine mammary epithelial cells (OMECs) was investigated, and the target relationship of miR-148a with two predicted target genes was verified. The expression of miR-148a exhibited obvious tissue-specific and temporal-specific patterns. miR-148a was expressed in all eight ovine tissues investigated, with the highest expression level in mammary gland tissue (p < 0.05). Additionally, miR-148a was expressed in ovine mammary gland tissue during each of the six developmental periods studied, with its highest level at peak lactation (p < 0.05). The overexpression of miR-148a increased the viability of OMECs, the number and percentage of Edu-labeled positive OMECs, and the expression levels of two cell-proliferation marker genes. miR-148a also increased the percentage of OMECs in the S phase. In contrast, transfection with an miR-148a inhibitor produced the opposite effect compared to the miR-148a mimic. These results indicate that miR-148a promotes the viability and proliferation of OMECs in Small-tailed Han sheep. The miR-148a mimic increased the triglyceride content by 37.78% (p < 0.01) and the expression levels of three milk fat synthesis marker genes in OMECs. However, the miR-148a inhibitor reduced the triglyceride level by 87.11% (p < 0.01). These results suggest that miR-148a promotes milk fat synthesis in OMECs. The dual-luciferase reporter assay showed that miR-148a reduced the luciferase activities of DNA methyltransferase 1 (DNMT1) and peroxisome proliferator-activated receptor gamma coactivator 1-A (PPARGC1A) in wild-type vectors, suggesting that they are target genes of miR-148a. The expression of miR-148a was highly negatively correlated with PPARGC1A (r = −0.789, p < 0.001) in ovine mammary gland tissue, while it had a moderate negative correlation with DNMT1 (r = −0.515, p = 0.029). This is the first study to reveal the molecular mechanisms of miR-148a underlying the viability, proliferation, and milk fat synthesis of OMECs in sheep. Full article

The yak (Bos grunniens) is a valuable livestock animal endemic to the Qinghai–Tibet Plateau in China with low reproductive rates. Cryptorchidism is one of the primary causes of infertility in male yaks. Compared with normal testes, the tight junctions (TJs) of Sertoli cells (SCs) and the integrity of the blood–testis barrier (BTB) in cryptorchidism are both disrupted. MicroRNAs are hairpin-derived RNAs of about 19–25 nucleotides in length and are involved in a variety of biological processes. Numerous studies have shown the involvement of microRNAs in the reproductive physiology of yak. In this study, we executed RNA sequencing (RNA-seq) to describe the expression profiles of mRNAs and microRNAs in yaks with normal testes and cryptorchidism to identify differentially expressed genes. GO and KEGG analyses were used to identify the biological processes and signaling pathways which the target genes of the differentially expressed microRNAs primarily engaged. It was found that novel-m0230-3p is an important miRNA that significantly differentiates between cryptorchidism and normal testes, and it is down-regulated in cryptorchidism with p < 0.05. Novel-m0230-3p and its target gene CSF1 both significantly contribute to the regulation of cell adhesion and tight junctions. The binding sites of novel-m0230-3p with CSF1 were validated by a dual luciferase reporter system. Then, mimics and inhibitors of novel-m0230-3p were transfected in vitro into SCs, respectively. A further analysis using qRT-PCR, immunofluorescence (IF), and Western blotting confirmed that the expression of cell adhesion and tight-junction-related proteins Occludin and ZO-1 both showed changes. Specifically, both the mRNA and protein expression levels of Occludin and ZO-1 in SCs decreased after transfection with the novel-m0230-3p mimics, while they increased after transfection with the inhibitors, with p < 0.05. These were achieved via the CSF1/CSF1R/Ras signaling pathway. In summary, our findings indicate a negative miRNA-mRNA regulatory network involving the CSF1/CSF1R/Ras signaling pathway in yak SCs. These results provide new insights into the molecular mechanisms of CSF1 and suggest that novel-m0230-3p and its target protein CSF1 could be used as potential therapeutic targets for yak cryptorchidism. Full article

Cashmere goats possess two types of hair follicles, with the secondary hair follicles producing valuable cashmere fiber used for textiles. The growth of cashmere exhibits a seasonal pattern arising from photoperiod change. Transcription factors play crucial roles during this process. The transcription factor, cold-shock domain, containing C2 (Csdc2) plays a crucial role in modulating cell proliferation and differentiation. Our preceding research indicated that the expression of Csdc2 changes periodically during anagen to telogen. However, the mechanisms of Csdc2 in regulating SHF growth remain unclear. Here, we found that the knockdown of Csdc2 inhibits the proliferation of dermal papilla cells. ChIP-Seq analysis showed that Csdc2 had a unique DNA binding motif in SHFs. Through conjoint analysis of ChIP-Seq and RNA-Seq, we revealed a total of 25 candidate target genes of Csdc2. Notably, we discovered a putative Csdc2 binding site within roundabout guidance receptor 2 (Robo2) on chromosome 1 of the goat genome. Furthermore, qRT-PCR and dual-luciferase reporter assay confirmed Csdc2’s positive regulatory influence on Robo2. These findings expand the research field of hair follicle transcriptional regulatory networks, offering insights into molecular breeding strategies to enhance cashmere production in goats. Full article

Wool is generated by hair follicles (HFs), which are crucial in defining the length, diameter, and morphology of wool fibers. However, the regulatory mechanism of HF growth and development remains largely unknown. Dermal papilla cells (DPCs) are a specialized cell type within HFs that play a crucial role in governing the growth and development of HFs. This study aims to investigate the proliferation and induction ability of ovine DPCs to enhance our understanding of the potential regulatory mechanisms underlying ovine HF growth and development. Previous research has demonstrated that microRNA-181a (miR-181a) was differentially expressed in skin tissues with different wool phenotypes, which indicated that miR-181a might play a crucial role in wool morphogenesis. In this study, we revealed that miR-181a inhibited the proliferation and induction ability of ovine DPCs by quantitative Real-time PCR (qRT-PCR), cell counting Kit-8 (CCK-8), 5-ethynyl-2′-deoxyuridine (EdU), flow cytometry, and alkaline phosphatase staining. Then, we also confirmed G protein subunit alpha i2 (GNAI2) is a target gene of miR-181a by dual luciferase reporter assay, qRT-PCR, and Western blot, and that it could promote the proliferation and induction ability of ovine DPCs. In addition, GNAI2 could also activate the Wnt/β-Catenin signaling pathway in ovine DPCs. This study showed that miR-181a can inhibit the proliferation and induction ability of ovine DPCs by targeting GNAI2 through the Wnt/β-Catenin signaling pathway. Full article