Dual-Luciferase Reporter Assay for Prescreening CRISPR (d)Cas9-Mediated Epigenetic Editing on a Plant Promoter Using Human Cells.

Epigenetic editing, also known as EpiEdit, offers an exciting way to control gene expression without altering the DNA sequence. In this study, we evaluate the application of EpiEdit to plant promoters, specifically the MLO (mildew locus o) gene promoter. We use a modified CRISPR-(d)Cas9 system, in which the nuclease-deficient Cas9 (dCas9) is fused to an epigenetic modifier, to experimentally demonstrate the utility of this tool for optimizing epigenetic engineering of a plant promoter prior to in vivo plant epigenome editing. Guide RNAs are used to deliver the dCas9-epigenetic modifier fusion protein to the target gene sequence, where it induces modification of MLO gene expression. We perform preliminary experiments using a plant promoter cloned into the luciferase reporter system, which is transfected into a human system and analyzed using the dual-luciferase reporter assay. The results suggest that this approach may be useful in the early stages of plant epigenome editing, as it can aid in the selection of appropriate modifications to the plant promoter prior to conducting in vivo experiments under plant system conditions. Overall, the results demonstrate the potential of CRISPR (d)Cas9-based EpiEdit for precise and controlled regulation of gene expression.

Differential miRNA Profiling Reveals miR-4433a-5p as a Key Regulator of Chronic Obstructive Pulmonary Disease Progression via PIK3R2- mediated Phenotypic Modulation.

OBJECTIVE: In this study, a high-throughput sequencing technology was used to screen the differentially expressed miRNA in the patients with "fast" and "slow" progression of chronic obstructive pulmonary disease (COPD). Moreover, the possible mechanism affecting the progression of COPD was preliminarily analyzed based on the target genes of candidate miRNAs. METHODS: The "fast" progressive COPD group included 6 cases, "slow" and Normal progressive COPD groups included 5 cases each, and COPD group included 3 cases. The peripheral blood samples were taken from the participants, followed by total RNA extraction and high throughput miRNA sequencing. The differentially expressed miRNAs among the progressive COPD groups were identified using bioinformatics analysis. Then, the candidate miRNAs were externally verified. In addition, the target gene of this miRNA was identified, and its effects on cell activity, cell cycle, apoptosis, and other biological phenotypes of COPD were analyzed. RESULTS: Compared to the Normal group, a total of 35, 16, and 7 differentially expressed miRNAs were identified in the "fast" progressive COPD, "slow" progressive COPD group, and COPD group, respectively. The results were further confirmed using dual-luciferase reporter assay and transfection tests with phosphoinositide- 3-kinase, regulatory subunit 2 (PIK3R2) as a target gene of miR-4433a-5p; the result showed a negative regulatory correlation between the miRNA and its target gene. The phenotype detection showed that the activation of the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT) signaling pathway might participate in the progression of COPD by promoting the proliferation of inflammatory A549 cells and inhibiting cellular apoptosis. CONCLUSIONS: MiR-4433a-5p can be used as a marker and potential therapeutic target for the progression of COPD. As a target gene of miR-4433a-5p, PIK3R2 can affect the progression of COPD by regulating phenotypes, such as cellular proliferation and apoptosis.

Restoring microRNA-34a overcomes acquired drug resistance and disease progression in human breast cancer cell lines via suppressing the ABCC1 gene.

PURPOSE: Breast cancer is one of the leading types of cancer diagnosed in women. Despite the improvements in chemotherapeutic cure strategies, drug resistance is still an obstacle leading to disease aggressiveness. The small non-coding RNA molecules, miRNAs, have been implicated recently to be involved as regulators of gene expression through the silencing of mRNA targets that contributed to several cellular processes related to cancer metastasis. Hence, the present study aimed to investigate the beneficial role and mechanism of miRNA-34a-based gene therapy as a novel approach for conquering drug resistance mediated by ATP-binding cassette (ABC) transporters in breast cancer cells, besides exploring the associated invasive behaviors. MATERIAL AND METHODS: Bioinformatics tools were used to predict miRNA ABC transporter targets by tracking the ABC transporter pathway. After the establishment of drug-resistant breast cancer MCF-7 and MDA-MB-231 sublines, cells were transfected with the mimic or inhibitor of miRNA-34a-5p. The quantitative expression of genes involved in drug resistance was performed by QRT-PCR, and the exact ABC transporter target specification interaction was confirmed by dual-luciferase reporter assay. Furthermore, flow cytometric analysis was utilized to determine the ability of miRNA-34a-treated cells against doxorubicin uptake and accumulation in cell cycle phases. The spreading capability was examined by colony formation, migration, and wound healing assays. The apoptotic activity was estimated as well. RESULTS: Our findings firstly discovered the mechanism of miRNA-34a-5p restoration as an anti-drug-resistant molecule that highly significantly attenuates the expression of ABCC1 via the direct targeting of its 3'- untranslated regions in resistant breast cancer cell lines, with a significant increase of doxorubicin influx by MDA-MB-231/Dox-resistant cells. Additionally, the current data validated a significant reduction of metastatic potentials upon miRNA-34a-5p upregulation in both types of breast cancer-resistant cells. CONCLUSION: The ectopic expression of miRNA-34a ameliorates the acquired drug resistance and the migration properties that may eventually lead to improved clinical strategies and outcomes for breast cancer patients. Additionally, miRNA-34a could be monitored as a diagnostic/prognostic biomarker for resistant conditions.

Identifying potential pathogenesis and immune infiltration in diabetic foot ulcers using bioinformatics and in vitro analyses.

BACKGROUND: Diabetic foot ulcers (DFU) are among the fastest-growing diseases worldwide. Recent evidence has emphasized the critical role of microRNA (miRNA)-mRNA networks in various chronic wounds, including DFU. In this study, we aimed to clarify the miRNA-mRNA axes associated with the occurrence of DFU. METHODS: Expression profiles of miRNAs and mRNAs were extracted from the Gene Expression Omnibus. Differentially expressed genes and differentially expressed miRNAs were identified, and miRNA-mRNA regulatory axes were constructed through integrated bioinformatics analyses. We validated the miRNA-mRNA axes using quantitative real-time PCR (qPCR) and dual-luciferase reporter assays. We conducted an immune infiltration analysis and confirmed the bioinformatics results using immunofluorescence staining. Single-sample gene set enrichment analysis (ssGSEA) was used to analyze the metabolic mechanisms. RESULTS: miR-182-5p-CHL1/MITF and miR-338-3p-NOVA1 interactions were identified using in silico analysis. The qPCR results showed apparent dysregulation of these miRNA-mRNA axes in DFU. The dual-luciferase reporter assay confirmed that miR-182-5p targeted CHL1 and MITF, and miR-338-3p targeted NOVA1. We conducted an immune infiltration analysis and observed that key genes correlated with decreased infiltration of M1 macrophages and resting mast cells in DFU. Immunofluorescence staining verified the co-localization of CHL1 and tryptase, while MITF and CD68 showed weak positive correlations. Metabolic pathways related to these three genes were identified using ssGSEA. CONCLUSIONS: In summary, the miR-182-5p-CHL1/MITF and miR-338-3p-NOVA1 pathway interactions and decreased infiltration of M1 macrophages and resting mast cells may provide novel clues to the pathogenesis of DFU. TRIAL REGISTRATION: The clinical trial included in this study was registered in the Chinese Clinical Trial Registry ( ChiCTR2200066660 ) on December 13, 2022.

[miR-23b-3p regulates the differentiation of goat intramuscular preadipocytes by targeting the PDE4B gene].

This study aimed to explore the effect of miR-23b-3p on the differentiation of goat intramuscular preadipocytes, and to confirm whether miR-23b-3p plays its roles via targeting the PDE4B gene. Based on the pre-transcriptome sequencing data obtained previously, the miR-23b-3p, which was differentially expressed in goat intramuscular adipocytes before and after differentiation, was used as an entry point. real-time quantitative-polymerase chain reaction (qPCR) was used to detect the expression pattern of miR-23b-3p during the differentiation of goat intramuscular preadipocytes. The effects of miR-23b-3p on adipose differentiation and adipose differentiation marker genes were determined at the morphological and molecular levels. The downstream target genes of miR-23b-3p were determined using bioinformatics prediction as well as dual luciferase reporter assay to clarify the targeting relationship between miR-23b-3p and the predicted target genes. The results indicated that overexpression of miR-23b-3p reduced lipid droplet accumulation in goat intramuscular adipocytes, significantly down-regulated the expression levels of adipogenic marker genes AP2, C/EBPα, FASN, and LPL (P < 0.01). In addition, the expressions of C/EBPß, DGAT2, GLUT4 and PPARγ were significantly downregulated (P < 0.05). After interfering with the expression of miR-23b-3p, lipid droplet accumulation was increased in goat intramuscular adipocytes. The expression levels of ACC, ATGL, AP2, DGAT2, GLUT4, FASN and SREBP1 were extremely significantly up-regulated (P < 0.01), and the expression levels of C/EBPß, LPL and PPARγ were significantly up-regulated (P < 0.05). It was predicted that PDE4B might be a target gene of miR-23b-3p. The mRNA expression level of PDE4B was significantly decreased after overexpression of miR-23b-3p (P < 0.01), and the interference with miR-23b-3p significantly increased the mRNA level of PDE4B (P < 0.05). The dual luciferase reporter assay indicated that miR-23b-3p had a targeting relationship with PDE4B gene. MiR-23b-3p regulates the differentiation of goat intramuscular preadipocytes by targeting the PDE4B gene.

H19/Mir-130b-3p/Cyp4a14 potentiate the effect of praziquantel on liver in the treatment of Schistosoma japonicum infection.

BACKGROUND: Schistosomiasis is a prevalent infectious disease caused by the parasitic trematodes of the genus Schistosoma. Praziquantel (PZQ), a safe and affordable drug, is the recommended oral treatment for schistosomiasis. The main pathologic manifestation of schistosomiasis is liver injury. However, the role and interactions of various RNA molecules in the effect of PZQ on the liver after S. japonicum infection have not been elucidated. RESULTS: In this study, C57BL/6 mice were randomly divided into the control group, infection group, and PZQ treatment group. Total RNA was extracted from the livers of the mice. High-throughput whole transcriptome sequencing was performed to detect the RNA expression profiles in the three groups. A co-expression gene-interaction network was established based on the significant differentially expressed genes in the PZQ treatment group; messenger RNA (mRNA) Cyp4a14 was identified as a critical hub gene. Furthermore, competitive endogenous RNA networks were constructed by predicting the specific binding relations between mRNA and long noncoding (lnc) RNA and between lncRNA and microRNA (miRNA) of Cyp4a14, suggesting the involvement of the H19/miR-130b-3p/Cyp4a14 regulatory axis. Dual luciferase reporter assay result proved the specific binding of miR-130b-3p with Cyp4a14 3'UTR. CONCLUSIONS: Our findings indicate the involvement of the H19/miR-130b-3p/Cyp4a14 axis in the effect of PZQ on the liver after S. japonicum infection. Moreover, the expression of mRNA Cyp4a14 could be regulated by the bonding of miR-130b-3p with 3'UTR of Cyp4a14. The findings of this study could provide a novel perspective to understand the host response to PZQ against S. japonicum in the future.

Identification and Verification of Candidate miRNA Biomarkers with Application to Infection with Emiliania huxleyi Virus.

The interactions of Emiliania huxleyi and its specific lytic virus (EhV) have a profound influence on marine biogeochemical carbon-sulfur cycles and play a prominent role in global climate change. MicroRNAs (miRNAs) have emerged as promising candidates with extensive diagnostic potential due to their role in virus-host interactions. However, the application of miRNA signatures as diagnostic markers in marine viral infection has made limited progress. Based on our previous small-RNA sequencing data, one host miRNA biomarker that is upregulated in early infection and seven viral miRNA biomarkers that are upregulated in late infection were identified and verified using qRT-PCR and a receiver operating characteristic curve analysis in pure culture, mixed culture, and natural seawater culture. The host ehx-miR20-5p was able to significantly differentiate infection groups from the control in the middle (24 h post-infection, hpi) and late infection (48 hpi) phases, while seven virus-derived miRNA biomarkers could diagnose the early and late stages of EhV infection. Functional enrichment analysis showed that these miRNAs participated in numerous essential metabolic pathways, including gene transcription and translation, cell division-related pathways, protein-degradation-related processes, and lipid metabolism. Additionally, a dual-luciferase reporter assay confirmed the targeted relationship between a viral ehv-miR7-5p and the host dihydroceramide desaturase gene (hDCD). This finding suggests that the virus-derived miRNA has the ability to inhibit the host sphingolipid metabolism, which is a specific characteristic of EhV infection during the late stage. Our data revealed a cluster of potential miRNA biomarkers with significant regulatory functions that could be used to diagnose EhV infection, which has implications for assessing the infectious activity of EhV in a natural marine environment.

LncRNA LINC00847 accelerates melanoma progression by regulating miR-133a-3p/TGFBR1 axis.

AIMS: Growing evidence has suggested that lncRNAs play a regulatory role in tumorigenesis. Dysregulation of a newly identified lncRNA (LINC00847) has been involved in several tumors. Nevertheless, the expression and roles of lncRNAs in skin melanoma remain unclear. Therefore, we attempted to investigate the expressions and roles of lncRNAs in this study. MATERIALS AND METHODS: Expression levels of LINC00847 were quantified in tissue samples from the TCGA database and clinically recruited participants. LINC00847 was inhibited in cells by transfecting with si-LINC00847 or si-NC. Expressions of LINC00847 and miR-133a-3p were determined using RT-qPCR, and the TGFBR1 level was determined using Western blotting. Targeting sites of LINC00847 with miR-133a-3p and miR-133a-3p with TGFBR1 were predicted by bioinformatic tools and proved by dual-luciferase reporter system and RNA immunoprecipitation. Cell proliferation, invasion, and migration abilities were assessed using CCK8, cell colony formation, cell wound scratch, and transwell assay, respectively. RESULTS: In both TCGA and clinical cohorts, the expression of LINC00847 was abnormally upregulated in skin melanoma tissues than that of benign nevus. Besides, LINC00847 expression increased more markedly in A375 and SK-MEL-28 cells than in normal epidermal melanocytes (HEMa-LP cells). LINC00847 knockdown remarkably restrained skin melanoma cell proliferation, metastasis, and wound healing rate. Furthermore, miR-133a-3p/TGFBR1 was the downstream target for LINC00847. LINC00847 negatively regulated miR-133a-3p expression in skin melanoma cells. Both miR-133a-3p inhibitors and TGFBR1 vector transfection reversed the effect of LINC00847 silence in skin melanoma cells. CONCLUSION: LINC00847 was highly expressed in skin melanoma, and its overexpression accelerated the malignant tumor behavior of skin melanoma cells. The miR-133a-3p /TGFBR1 axis was involved in the roles of LINC00847 in skin melanoma.

Anthocyanin accumulation and transcriptional regulation in purple flowering stalk (Brassica campestris L. var. purpurea Bailey).

KEY MESSAGE: 1. Purple flowering stalk (Brassica campestris L. ssp. chinensis L. var. purpurea Bailey) is a crop with the high-level anthocyanin. 2. Increased abundance of LBGs promoted the synthesis of anthocyanin. 3. TTG2 (WRKY) interacted with TTG1 (WD40), probably regulating anthocyanin accumulation by shaping a MBWW complex. Brassica crops are a class of nutrient-rich vegetables. Here, two Brassica Crops-Flowering Stalk cultivars, purple flowering stalk (Brassica campestris L. var. purpurea Bailey) and pakchoi (Brassica campestris ssp. chinensis var. communis) were investigated. HPLC-ESI-MS/MS analysis demonstrated that Cy 3-p-coumaroylsophoroside-5-malonylglucoside and Cy 3-diferuloylsophoroside-5-malonylglucoside were identified as the major anthocyanin in peel of purple flowering stalk. The transcript level of structural genes including C4H, CHS, F3H, DFR, ANS and UFGT, and regulatory genes such as TT8, TTG1, Bra004162, Bra001917 and TTG2 in peel of purple flowering stalk were significantly higher than that in peel of pakchoi. In addition, the TTG2(WRKY) interacted only with TTG1(WD40) and the interaction between TT8 (bHLH) and TTG1/Bra004162(MYB)/Bra001917(MYB) were identified. Else, the WD40-WRKY complex (TTG1-TTG2) could activate the transcript of TT12. Our study laid a foundation for the research on the anthocyanin accumulation in Brassica crops.

miR-23b-3p regulates the differentiation of goat intramuscular preadipocytes by targeting the PDE4B gene / 生物工程学报

This study aimed to explore the effect of miR-23b-3p on the differentiation of goat intramuscular preadipocytes, and to confirm whether miR-23b-3p plays its roles via targeting the PDE4B gene. Based on the pre-transcriptome sequencing data obtained previously, the miR-23b-3p, which was differentially expressed in goat intramuscular adipocytes before and after differentiation, was used as an entry point. real-time quantitative-polymerase chain reaction (qPCR) was used to detect the expression pattern of miR-23b-3p during the differentiation of goat intramuscular preadipocytes. The effects of miR-23b-3p on adipose differentiation and adipose differentiation marker genes were determined at the morphological and molecular levels. The downstream target genes of miR-23b-3p were determined using bioinformatics prediction as well as dual luciferase reporter assay to clarify the targeting relationship between miR-23b-3p and the predicted target genes. The results indicated that overexpression of miR-23b-3p reduced lipid droplet accumulation in goat intramuscular adipocytes, significantly down-regulated the expression levels of adipogenic marker genes AP2, C/EBPα, FASN, and LPL (P < 0.01). In addition, the expressions of C/EBPβ, DGAT2, GLUT4 and PPARγ were significantly downregulated (P < 0.05). After interfering with the expression of miR-23b-3p, lipid droplet accumulation was increased in goat intramuscular adipocytes. The expression levels of ACC, ATGL, AP2, DGAT2, GLUT4, FASN and SREBP1 were extremely significantly up-regulated (P < 0.01), and the expression levels of C/EBPβ, LPL and PPARγ were significantly up-regulated (P < 0.05). It was predicted that PDE4B might be a target gene of miR-23b-3p. The mRNA expression level of PDE4B was significantly decreased after overexpression of miR-23b-3p (P < 0.01), and the interference with miR-23b-3p significantly increased the mRNA level of PDE4B (P < 0.05). The dual luciferase reporter assay indicated that miR-23b-3p had a targeting relationship with PDE4B gene. MiR-23b-3p regulates the differentiation of goat intramuscular preadipocytes by targeting the PDE4B gene.

miR-137 modulates coelomocytes autophagy by targeting Atg13 in the sea cucumber Apostichopus japonicus.

MicroRNAs (miRNAs), as important regulators of host immune responses, play an crucial position in the interaction between host and pathogen by inhibiting the target gene's transcriptional and post-transcriptional expression. A well-validated tumor suppressor, Previously, miR-137 was found to be variably expressed in the sick sea cucumber Apostichopus japonicus specimens by high-throughput sequencing. To further investigate the mechanism of miR-137 regulation of SUS, we identified Atg13 from sea cucumber by dual luciferase reporter assay and RACE (designated as AjAtg13) and was able to serve as a target gene for miR-137. The full-length cDNA of AjAtg13 is a 2197 bp fragment containing an ORF (open reading frame) of 1149 bp and encodes a total of 382 amino acid polypeptides with a predicted molecular weight of 41.7 kDa. Further expression profiling analysis showed increased mRNA levels of AjAtg13 and reduced expression levels of miR-137 in LPS-stimulated sea cucumber coelomocytes, hinting that miR-137 may negatively regulate AjAtg13. MiR-137 targets AjAtg13 through binding to the 3'UTR region by dual-luciferase reporter gene analysis. MiR-137 overexpression in coelomocytes repressed the expression of autophagy related genes, such as AjAtg13, AjLC3, at the same time, it significantly inhibited autophagy and reduced the ability to clear Vibrio splendidus. Conversely, inhibition of miR-137 significantly upregulated the expression of AjAtg13, promoted autophagy and increased clearance of V. splendidus. Subsequent interference with AjAtg13 also significantly inhibits autophagy. In summary, our results suggested that miR-137 could promote coelomocytes autophagy to restrict bacterial invasion by aiming at AjAtg13 in pathogen-stimulated sea cucumbers.

Influence of the rs6736 Polymorphism on Ischemic Stroke Susceptibility in Han Chinese Individuals via the Disruption of miR-7-1 Binding to the C14orf119 Gene.

This study investigates the association between the C14orf119 gene rs6736 polymorphism and ischemic stroke (IS) susceptibility, and explores the influence of the rs6736 polymorphism on the binding between miR-7-1 and the C14orf119 gene. mRNA expression levels were determined in 45 IS patients and 45 matched controls via real-time quantitative PCR. A total of 774 IS patients and 793 matched controls were recruited from a Han Chinese population for genotyping, performed with the Sequenom MassARRAY iPLEX platform. A dual-luciferase reporter assay was used for the analysis of miRNA-mRNA binding. The results showed that the mRNA expression of C14orf119 differed significantly between IS patients and controls (t = -2.235, P = 0.030). Significant associations were noted between the C14orf119 gene rs6736 polymorphism and IS susceptibility in Han Chinese individuals under the additive model [ORadj (95% CI) = 0.87 (0.76-1.00) Padj = 0.048] and dominant model [ORadj (95% CI) = 0.76 (0.61-0.94), Padj = 0.014], with adjustment for age and sex. Mutations in the rs6736 polymorphism disrupted the binding of miR-7-1 and the C14orf119 gene. The results of this study show that the rs6736 polymorphism in the 3'-untranslated region of the C14orf119 gene not only is associated with IS but also modifies the binding between miR-7-1 and the C14orf119 gene. The C14orf119 gene may participate in the relationship between IS and miR-7-1.

Genetic marker identification of SEC13 gene for milk production traits in Chinese holstein.

SEC13 homolog, nuclear pore and COPII coat complex component (SEC13) is the core component of the cytoplasmic COPII complex, which mediates material transport from the endoplasmic reticulum to the Golgi complex. Our preliminary work found that SEC13 gene was differentially expressed in dairy cows during different stages of lactation, and involved in metabolic pathways of milk synthesis such as citric acid cycle, fatty acid, starch and sucrose metabolisms, so we considered that the SEC13 might be a candidate gene affecting milk production traits. In this study, we detected the polymorphisms of SEC13 gene and verified their genetic effects on milk yield and composition traits in a Chinese Holstein cow population. By sequencing the whole coding and partial flanking regions of SEC13, we found four single nucleotide polymorphisms (SNPs). Subsequent association analysis showed that these four SNPs were significantly associated with milk yield, fat yield, protein yield or protein percentage in the first and second lactations (p ≤.0351). We also found that two SNPs in SEC13 formed one haplotype block by Haploview4.2, and the block was significantly associated with milk yield, fat yield, fat percentage, protein yield or protein percentage (p ≤ .0373). In addition, we predicted the effect of SNP on 5'region on transcription factor binding sites (TFBSs), and found that the allele A of 22:g.54362761A>G could bind transcription factors (TFs) GATA5, GATA3, HOXD9, HOXA10, CDX1 and Hoxd13; and further dual-luciferase reporter assay verified that the allele A of this SNP inhibited the fluorescence activity. We speculate that the A allele of 22:g.54362761A>G might inhibit the transcriptional activity of SEC13 gene by binding the TFs, which may be a cause mutation affecting the formation of milk production traits in dairy cows. In summary, we proved that SEC13 has a significant genetic effect on milk production traits and the identified significant SNPs could be used as candidate genetic markers for GS SNP chips development; on the other hand, we verified the transcriptional regulation of 22:g.54362761A>G on SEC13 gene, providing research direction for further function validation tests.

Chemical constituent from the peel of Trichosanthes kirilowii Maxim and their NF-κB inhibitory activity.

The peel of Trichosanthes kirilowii Maxim is clinically used to treat cardiovascular diseases in China. In this study, the NF-κB inhibitory activity of the peel of T. kirilowii Maxim extracts was determined by Dual-Luciferase Reporter Assay System, and the results showed 70% ethanol extract could significantly inhibit the activation of NF-κB (p < 0.001). Further, 21 compounds were isolated from 70% ethanol extract. One new compound, namely (2 R)-(2-amino-2-hydroxymethyl-3-[(4-hydroxy-3-methoxybenzoyl)-O-]-propanoic acid (1), and 20 known compounds were elucidated by comprehensive spectroscopic analyses. The isolated compounds were tested in the anti-inflammatory assay, and the results indicated compounds 5, 8, 11, 14, 15, 17 and 21 could inhibit the activation of NF-κB (p <0 .05, p < 0.001) at concentration of 1 µM.

MicroRNA-141-3p expression in serum of patients with cerebral hemorrhage and its mechanism / 解剖学报

Objective To analyze the expression level of microRNA-141-3p (miR-141-3) in patients with intracerebral hemorrhage (ICH), and explore the effect and mechanism of miR-141-3p on cerebral hemorrhage injury in rats. Methods Forty patients with ICH and 40 healthy controls in total were enrolled in this study. The expression of miR- 141-3p in peripheral blood serum was determined by the Real-time PCR method. The target relationship between miR-141- 3p and NOD-like receptor 3 (NLRP3) 3′ UTR was confirmed by dual luciferase reporter assay. miR-141-3p agonist and agonist NC were injected into rats via the lateral ventricle, respectively. On day 7 after treatment, the neurological function score was evaluated, and then all rats were killed to obtain brain tissue. Brain water content was examined by the dried and wet mass. HE staining was conducted to observe the pathological changes of cerebral tissue. The mRNA expressions of NLRP3 and miR-141-3p were detected by Real-time PCR. The protein expression of interleukin (IL)-lβ, IL-6 and IL-18 were detected by Western blotting analysis. Results The expression of miR-141-3p in serum of ICH patients was significantly down-regulated compared to healthy controls and negatively correlated with the severity of edema around the hematoma [(0.068±0.038) vs (0.520±0.028), t = 15.93, P<0.001; r =-0.8948, -0.9434 to-0.8087, P<0.001 ]. The result of luciferase reporter assay showed that miR-141-3p was related to the regulation of NLRP3 gene expression. The relative expression levels of miR-141-3p in agonist group were significantly higher than those in the agonist NC group (P< 0.001), while the expression levels of NLRP3, IL-lβ, IL-6 and IL-18 were significantly lower than those in the agonist NC group (P< 0.001). Compared with the agonist NC group, the cerebral water content reduced significantly (P< 0.001), and the neurological function score was significantly improved on the day 7 after treatment in agonist group (P< 0.001). The result of HE staining showed that injection of miR-141-3p in ICH rats could reduced the severity of edema around the hematoma. Conclusion MiR-141-3p alleviates ICH-induced inflammatory injury in rat possibly by modulating miR-141-3p.

MicroRNA-214 Inhibits Chicken Myoblasts Proliferation, Promotes Their Differentiation, and Targets the TRMT61A Gene.

The proliferation and differentiation of myoblasts is an important process of skeletal muscle development. In this process, microRNAs (miRNAs) play an important role in the proliferation and differentiation of chicken primary myoblasts (CPMs). Our previous study found that miR-214 and the tRNA methyltransferase 61A (TRMT61A) gene were differentially expressed in different stages of proliferation and differentiation. Therefore, this study aimed to explore the effect of miR-214 on the proliferation and differentiation of CPMs and the functional relationship between miR-214 and TRMT61A. In this study, we detected the effect of miR-214 on the proliferation of CPMs by qPCR, flow cytometry, CCK-8, and EdU after the overexpression and interference of miR-214. qPCR, Western blotting, and indirect immunofluorescence were used to detect the effect of miR-214 on the differentiation of the CPMs. The expression patterns of miR-214 and TRMT61A were observed at different time points of differentiation induced by the CPMs. The results show that miR-214 inhibited the proliferation of the CPMs and promoted the differentiation of the CPMs. The Dual-Luciferase Reporter assay and the expression pattern of miR-214 and TRMT61A suggested that they had a negative regulatory target relationship. This study revealed the function and regulatory mechanism of miR-214 in the proliferation and differentiation of CPMs.

MiR-210 regulates coelomocyte proliferation through targeting E2F3 in Apostichopus japonicus.

MiR-210 plays a crucial role in cell survival, migration, and regeneration in vertebrates. In our previous work, the expression of miR-210 was considerably induced in diseased Apostichopus japonicus with skin ulcer syndrome (SUS). To further explore the mechanism of miR-210 in regulating the SUS, this study identified E2F transcription factor 3 (E2F3), a candidate target of miR-210, from the sea cucumber A. japonicus via RNA-seq and RACE (designated as AjE2F3). A 1992 bp fragment representing the full-length cDNA of AjE2F3 was obtained, which includes an ORF of 1194 bp encoding a polypeptide of 398 amino acids with a molecular weight of 44.43 kDa. Expression profiling analysis suggested that the expression of AjE2F3 decreased while that of miR-210 increased in Vibrio splendidus-challenged sea cucumber coelomocytes. Dual-luciferase reporter assay revealed that miR-210 targeted AjE2F3 via binding to the 3'UTR region from 108 nt to 128 nt. MiR-210 overexpression in cultured coelomocytes repressed AjE2F3 at the mRNA level and reduced cell proliferation in vitro. Consistently, AjE2F3 overexpression significantly promoted coelomocyte proliferation, as assessed by MTT in vitro. Overall, our results indicated that miR-210 can suppress coelomocyte proliferation by targeting AjE2F3 in pathogen-challenged sea cucumbers.

MiR-26 regulates ddx3x expression in medaka (Oryzias latipes) gonads.

MicroRNAs (miRNAs) participate in the reproductive process by regulating the expression of target genes, however, there are few studies on the regulation of mRNA target by miRNAs in medaka (Oryzias latipes). Our previous data showed that miR-202-5p has a sex-biased expression and miR-26 is abundantly expressed in both ovary and testis of medaka. In this study, DEAD-box helicase 3, X-linked (ddx3x) was proposed to be a putative target of miR-26 and miR-202-5p through bioinformatic analysis. Here, medaka ddx3x (Olddx3x) was cloned and sequence alignment analysis indicated that Olddx3x is highly conserved and has more than 90% identity with some other teleosts. Reverse transcription polymerase chain reaction (RT-PCR) showed that Olddx3x was highly expressed in testis and ovary. Meanwhile, chromogenic in situ hybridization manifested that Olddx3x was abundantly expressed in the early oocytes in the ovary and in various stages of spermatogenesis, especially the obvious signal in the testis of spermatocytes and sperm. Dual-luciferase reporter assay and the injection of miR-26 agomir and antagomir demonstrated that the expression of Olddx3x was regulated by miR-26 but not miR-202-5p. The overall results revealed that miR-26 may perform a vital role in medaka reproduction development by regulating the expression of ddx3x.

Quantitative Evaluation of the Transcriptional Activity of Steroid Hormone Receptor Mutants and Variants Using a Single Vector With Two Reporters and a Receptor Expression Cassette.

Although the rapid development of high-throughput sequencing has led to the identification of a large number of truncated or mutated steroid hormone receptor (SHR) variants, their clinical relevance remains to be defined. A platform for functional analysis of these SHR variants in cells would be instrumental for better assessing their impact on normal physiology and SHR-associated diseases. Here we have developed a new reporter system that allows rapid and accurate assessment of the transcriptional activity of SHR variants in cells. The reporter is a single construct containing a firefly luciferase reporter gene, whose expression is under the control of a promoter with multiple steroid hormone responsive elements, and a Renilla luciferase reporter gene, that is constitutively expressed under the control of an internal ribosome entry site (IRES) and is not regulated by steroid hormones. The corresponding SHR (wildtype or mutant/variant) is also expressed from the same construct. Using this improved reporter system, we revealed a large spectrum of transactivation activities within a set of previously identified mutations and variations of the androgen receptor (AR), the estrogen receptor α (ERα) and the glucocorticoid receptor (GR). This novel reporter system enables functional analysis of SHR mutants and variants in physiological and pathological settings, offering valuable preclinical, or diagnostic information for the understanding and treatment of associated diseases.

Clinicopathological value and underlying molecular mechanism of annexin A2 in 992 cases of thyroid carcinoma.

BACKGROUND: Thyroid carcinoma (THCA) is one of the most frequent endocrine cancers and has increasing morbidity. Annexin A2 (ANXA2) has been found to be highly expressed in various cancers; however, its expression level and potential mechanism in THCA remain unknown. This study investigated the clinicopathological value and primary molecular machinery of ANXA2 in THCA. MATERIAL AND METHODS: Public RNA-sequencing and microarray data were obtained and analyzed with ANXA2 expression in THCA and corresponding non-cancerous thyroid tissue. A Pearson correlation coefficient calculation was used for the acquisition of ANXA2 coexpressed genes, while edgR, limma, and Robust Rank Aggregation were employed for differentially expressed gene (DEG) in THCA. The probable mechanism of ANXA2 in THCA was predicted by gene ontology and pathway enrichment. A dual-luciferase reporter assay was employed to confirm the targeting relationships between ANXA2 and its predicted microRNA (miRNA). RESULTS: Expression of ANXA2 was significantly upregulated in THCA tissues with a summarized standardized mean difference of 1.09 (P < 0.0001) based on 992 THCA cases and 589 cases of normal thyroid tissue. Expression of ANXA2 was related to pathologic stage. Subsequently, 1442 genes were obtained when overlapping 4542 ANXA2 coexpressed genes with 2248 DEGs in THCA; these genes were mostly enriched in pathways of extracellular matrix-receptor interaction, cell adhesion molecules, and complement and coagulation cascades. MiR-23b-3p was confirmed to target ANXA2 by dual-luciferase reporter assay. CONCLUSIONS: Upregulated expression of ANXA2 may promote the malignant biological behavior of THCA by affecting the involving pathways or being targeted by miR-23b-3p.