Relative Luciferase Activity Research Articles

Binding site redundancy is critical for the regulation of fas by miR-30c in blunt snout bream (Megalobrama amblycephala)

MiR-30c and fatty acid synthase (fas) both play important roles in physiological processes such as lipid synthesis and fat metabolism. Predictive analysis revealed that fas is a target gene of miR-30c with multiple seed sites. Seed sites are useful to predict miRNA targeting relationships; however, detailed analyses of seed sites in fish genomes remain poorly studied. In this study, the regulatory relationship between miR-30c and fas, number and effect of seed regions, and mechanism by which miR-30c regulates lipid metabolism were evaluated in blunt snout bream (Megalobrama amblycephala). Four miR-30c target sites for fas were identified using various prediction tools. miR-30c mimics were transfected into 293 T cells, and dual-luciferase reporter assays were used to evaluate the roles of different fas target sites. When a single target site was mutated, relative luciferase activity was higher than that in the control group, with different activity levels depending on the mutation site. When multiple target sites were mutated, relative luciferase activity increased significantly as the number of mutation sites increased and was the highest when the four sites were mutated simultaneously. The miR-30c agomir was injected into the abdominal cavity of M. amblycephala at various concentrations for analyses of physiological and biochemical parameters in the liver and blood and the expression of genes related to lipid metabolism in the liver. Total cholesterol, free fatty acid, triglyceride, and low density lipoprotein levels were significantly lower after miR-30c agomir injection comparing to the control (P < 0.05). Additionally, the expression levels of genes related to lipid metabolism were significantly lower after miR-30c agomir injection than in the control (P < 0.05). In summary, this study identified four specific miR-30c target sites in the 3′ UTR of fas mRNA; the effects of these sites are cumulative, and the redundancy ensures the accurate regulation of fas during evolution. In addition, miR-30c has a negative regulatory effect on fas and regulates lipid metabolism via various genes related to this process. Therefore, the regulation of miR-30c can effectively ameliorate the side effects of a high-fat diet on liver function in M. amblycephala.

Screening of the lncRNA SNHG3 regulating CART expression in the bovine hypothalamus

This study aimed to screen for the long non-coding RNA (lncRNA) small nucleolar RNA host gene 3 (SNHG3) capable of regulating the expression of cocaine- and amphetamine-regulated transcriptional peptide (CART) in the bovine hypothalamus and elucidate the underlying mechanism. StarBase v2.0, NCBI, and DIANA tools were used to predict the lncRNAs targeting miR-381 and miR-491, which were responsible for inhibiting CART expression. The binding sites were analyzed, and the endogenous expression of the selected lncRNAs was determined by semi-quantitative RT-PCR of the hypothalamus tissue from three healthy adult Simmental cows. The dual-luciferase reporter gene assay was employed to detect the targeted binding relationship between miR-381/491 and lncRNAs. The over-expression vectors of lncRNAs, CART, and miR-381/491 mimics were constructed and transfected into 293T cells to reveal the mechanism of lncRNAs in regulating the CART expression. Animal experiments were conducted to analyze the regulatory function of the strongest lncRNA at the cellular level. The results showed that lncRNAs TUG1, SNHG3, H19, SNHG12, and DANCR were expressed in the bovine hypothalamus. The lncRNAs TUG1 and SNHG3 had binding sites for miR-381, and H19, SNHG12, and DANCR had binding sites for miR-491. The dual-luciferase reporter gene assay showed that miR-381 inhibited the relative luciferase activities of TUG1-WT (P < 0.05) and SNHG3-WT (P < 0.01), and miR-491 inhibited the luciferase activities of DANCR-WT (P < 0.05), H19-WT (P < 0.05), and SNHG12-WT (P < 0.01). SNHG3 and SNHG12 up-regulated the CART expression by specifically binding to miR-381 (P < 0.001) and miR-491 (P < 0.01), respectively, and SNHG3 had the strongest effect of regulating CART expression. The results from animal experiments showed that SNHG3 significantly up-regulated the mRNA and protein levels of CART by specifically binding to miR-381. This study confirmed that the lncRNA SNHG3, acting as a competing endogenous RNA of miR-381, significantly up-regulated CART expression at the transcriptional and post-transcriptional levels, laying a foundation for deciphering the mechanism of the molecular network regulation of CART in the bovine hypothalamus.

Exosome microRNA-125a-5p derived from epithelium promotes M1 macrophage polarization by targeting IL1RN in chronic obstructive pulmonary disease

BackgroundThe interplay between airway epithelium and macrophages plays a pivotal role in Chronic Obstructive Pulmonary Disease (COPD) pathogenesis. Exosomes, which transport miRNA cargo, have emerged as novel mediators of intercellular communication. MicroRNA-125a-5p (miR-125a-5p) has been implicated in macrophage polarization.This study aims to investigate the role of exosomal miR-125a-5p in the dysfunctional epithelium-macrophage cross-talk in cigarette smoke (CS)-induced COPD. MethodsIn cell models, THP-1 monocytic cells were differentiated into macrophages (M0). Human bronchial epithelial cells treated with CS extract (CSE) were co-cultured with M0. Exosomes were isolated from culture media using commercial kits and characterized using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Exosomes labeled with PKH26 red fluorescent cell linker kits were incubated with macrophages. Luciferase reporter assay was used to confirm the target gene of miR-125a-5p. In mouse experiments, inhibiting miR-125a-5p was utilized to examine its role in macrophage polarization. Furthermore, the underlying mechanism was explored. ResultsIn vitro results indicated that CSE treatment led to upregulation of miR-125a-5p in HBE cells, and exosomes contained miR-125a-5p. PKH26-labeled exosomes were internalized by macrophages. Co-culture experiments between bronchial epithelial cells and miR-125a-5p mimic resulted in significant increase in M1 macrophage markers (TNF-α, iNOS-2, IL-1β) and decrease in M2 markers (IL-10 and Arg-1). In COPD mouse models, miR-125a-5p inhibitor reduced levels of TNF-α, IL-1β, and IL-6. Luciferase assays revealed that miR-125a-5p inhibitors enhanced the relative luciferase activity of IL1RN. Mechanistic experiments demonstrated that HBE-derived exosomes transfected with miR-125a-5p mimics promoted upregulation of MyD88, TRAF6, p65, iNOS-2, and downregulation of Arg-1. ConclusionThis study suggests that exosomal miR-125a-5p may act as a mediator in the cross-talk between airway epithelium and macrophage polarization in COPD. Exosomal miR-125a-5p targeting IL1RN may promote M1 macrophage polarization via the MyD88/NF-κB pathway.

Vernalization promotes bolting in sugar beet by inhibiting the transcriptional repressors of BvGI.

Sugar beet (Beta vulgaris L.), a biennial sugar crop, contributes about 16% of the world's sugar production. The transition from vegetative growth, during which sugar accumulated in beet, to reproductive growth, during which sugar exhausted in beet, is determined by vernalization and photoperiod. GIGANTEA (GI) is a key photoperiodic flowering gene that is induced by vernalization in sugar beet. To identify the upstream regulatory factors of BvGI, candidate transcription factors (TF) that were co-expressed with BvGI and could bind to the BvGI promoter were screened based on weighted gene co-expression network analysis (WGCNA) and TF binding site prediction. Subsequently, their transcriptional regulatory role on the BvGI was validated through subcellular localization, dual-luciferase assays and yeast transformation tests. A total of 7,586 differentially expressed genes were identified after vernalization and divided into 18 co-expression modules by WGCNA, of which one (MEcyan) and two (MEdarkorange2 and MEmidnightblue) modules were positively and negatively correlated with the expression of BvGI, respectively. TF binding site predictions using PlantTFDB enabled the screening of BvLHY, BvTCP4 and BvCRF4 as candidate TFs that negatively regulated the expression of BvGI by affecting its transcription. Subcellular localization showed that BvLHY, BvTCP4 and BvCRF4 were localized to the nucleus. The results of dual-luciferase assays and yeast transformation tests showed that the relative luciferase activity and expression of HIS3 was reduced in the BvLHY, BvTCP4 and BvCRF4 transformants, which suggested that the three TFs inhibited the BvGI promoter. In addition, real-time quantitative reverse transcription PCR showed that BvLHY and BvTCP4 exhibited rhythmic expression characteristics similar to that of BvGI, while BvCRF4 did not. Our results revealed that vernalization crosstalked with the photoperiod pathway to initiate bolting in sugar beet by inhibiting the transcriptional repressors of BvGI.

BMSC-derived exosomes attenuate persistent corneal epithelial injury by affecting retinal neural-like cells

Persistent corneal epithelial defect (PED) is a challenge to human which is difficultly cured, normally requiring long-term follow-up. Herein, this study explored the potential role of bone marrow mesenchymal stem cell-derived exosomes (BMSC-exos) in persistent corneal epithelial injury and the underlying mechanism. After characterization of BMSC-exos, RGC-5 cell viability was determined and surface markers of BMSCs were analyzed. Additionally, RT-qPCR and Western blot measured miR-150-5p, Brn3, Islet-1, and PCNA expression. Dual luciferase assays were conducted to evaluate the targeting relationship between long non-coding Ribonucleic Acid (lncRNA) MIAT and miR-150-5p. Presented in ellipsoidal or cup shape, BMSC-exos were positive to BMSC-specific markers CD29 and CD90, and cell surface markers CD9 and CD63 were detected on exosomes. Importantly, treatment with BMSC-exos significantly promoted proliferation of retinal ganglion cells (RGCs) and hindered cell differentiation. Interestingly, down-regulating lncRNA MIAT exerted the same effect as BMSC-exos, increasing cell viability and decreasing the expression of differentiation-related proteins Brn3 and ISL1. Bioinformatics software predicted miR-150-5P as target of lncRNA MIAT, and the relative luciferase activity of miR-150-5P+MIAT-WT co-transfection group was lower. Conclusively, BMSC-exos can improve PED by targeting and regulating miR-150-5p expression through lncRNA MIAT, which can up-regulate PCNA in RGC-5 cells and down-regulate Brn3 and ISL1.

Abstract 4021: The development of CAR T cell therapeutics targeting tumor specific CD146

Abstract Chimeric antigen receptor (CAR) T cells represent a promising class of "living drugs" for cancer treatment. These primary T cells are genetically engineered to express a synthetic antigen receptor, incorporating various domains to enhance their effector functions. While CAR T cells have shown success against liquid cancers, their application to solid cancers is hindered by significant "on-target/off-tumor" toxicities due to the absence of specific target proteins. In this study, we aim to enhance the tumor specificity of CAR T cells by targeting a tumor-specific version of CD146. CD146 is a glycoprotein widely expressed in several solid tumors, particularly melanoma and ovarian cancer. However, it is also expressed on benign cells, such as endothelial cells, pericytes, and smooth muscle cells. Nevertheless, a CD146 isoform exists that is enriched in cancer cells. We assessed CAR activity using flow cytometry to measure surface CD69 expression (an early activation marker) on CAR-expressing Jurkat cells co-cultured with various CD146-positive and CD146-negative human melanoma and ovarian cancer cell lines. The killing ability of primary CAR-T cells was evaluated by measuring the relative luciferase activity of target cells, and their effector functions were further analyzed by ELISA for the secretion of IFN-g and TNF-a. Through multiple rounds of protein engineering, we have developed a CAR construct capable of reprogramming T cells to respond to a tumour-enriched CD146 isoform. Our CAR demonstrated antigen-dependent activation of Jurkat cells when co-cultured with CD146-positive human melanoma and ovarian cancer cell lines, comparable to a CAR targeting pan-CD146. Moreover, primary CAR T cells exhibited proportional killing activity and other effector functions. We have successfully engineered a CAR with specificity for a tumour-enriched isoform of CD146. Our future work will involve rigorously evaluating the tumor selectivity of our CAR, as well as assessing the ability of primary CAR T cells expressing this CAR to control tumor growth in vivo. Citation Format: Erez Uzuner, Holly Mole, Chun W. Wong, Macarena L. Fernandez Carro, Rotem Leshem, Kieran Sefton, Helen Tovey, Andrew Picken, Marcel Blot-Chabaud, Adam F. Hurlstone. The development of CAR T cell therapeutics targeting tumor specific CD146 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4021.

Myogenin Regulates DUSP13 to Inhibit Apoptosis Induced by Reactive Oxygen Species.

Myogenin is well known as a crucial transcription factor in skeletal muscle development, yet its other biological functions remain unexplored. Previous research showed that myogenin suppresses apoptosis induced by angiotensin II in human induced pluripotent stem cell-derived cardiomyocytes, and offered a new perspective on myogenin's role in cardioprotection. However, the detailed mechanism of this cardioprotection, especially under oxidative stress, is still unclear. In this study, hydrogen peroxide (H2O2) was used to generate reactive oxygen species in myogenin-overexpressing cardiomyocytes. The apoptosis was examined by flow cytometry. Transcriptome sequencing (RNA-seq) was performed to identify genes regulated by myogenin. Western blotting was used to detect the protein level of DUSP13 and the phosphorylation level of p38 mitogen-activated protein kinase (MAPK). The dual-luciferase reporter assay and ChIP assay were used to confirm the binding of myogenin to the promoter region of DUSP13. DUSP13 overexpression and knockdown assays were performed to study its anti-apoptotic role. Flow cytometry analysis of apoptosis showed that overexpressing myogenin for 24 and 48 hours decreased the apoptotic ratio by 47.9% and 63.5%, respectively, compared with untreated controls. Transcriptome sequencing performed on cardiomyocytes that expressed myogenin for different amounts of time (6, 12, 24, and 48 hours) identified DUSP13 as being up-regulated by myogenin. Western blotting showed that overexpression of myogenin increased the expression of DUSP13 and decreased the phosphorylation level of p38 MAPK. A dual-luciferase reporter assay proved that myogenin bound directly to the promoter region of DUSP13 and led to strong relative luciferase activity. Direct expression of DUSP13A and DUSP13B significantly reduced the rates of apoptosis and necrosis in cells treated with H2O2. Knockdown of DUSP13B significantly increased the rate of apoptosis in cells treated with H2O2. The present findings suggest that myogenin might attenuate apoptosis induced by reactive oxygen species by up-regulating DUSP13 and inactivating the p38 MAPK pathway.

MiR-182 promotes apoptosis of neural cells in cerebral infarction rats by PI3K/AKT signaling pathway.

To explore the effects of micro ribonucleic acid (miR)-182 on the proliferation and apoptosis of neural cells in cerebral infarction rats and its underlying mechanism. The rat model of cerebral infarction was established, and neural cells were extracted accordingly. The cell proliferation ability was detected via cell counting kit-8 (CCK8) assay. In addition, the apoptosis rate was determined through flow cytometry and the activity of active caspase-3. Furthermore, the interaction between miR-182 and PI3K was explored via dual luciferase reporter assay, and the protein expression levels were observed via Western blotting. The neural cells in mouse brain tissues significantly decreased in the model group compared with that in the control group via HE stain. Additionally, the expression level of miR-182 was significantly increased in the model group compared with that in the control group. Furthermore, overexpression of miR-182 could inhibit the proliferation of neural cells through inducing cell apoptosis. Besides, the results of the luciferase reporter assay showed that the relative luciferase activity in neural cells could be inhibited by the transfection with miR-182 (P<0.05). Overexpression of miR-182 significantly reduced the protein expression levels of phosphatidylinositol 3-hydroxy kinase (PI3K) and p-AKT. MiR-182 induces apoptosis of neural cells through inhibiting the PI3K/AKT signaling pathway, which plays an important regulatory role in the apoptosis of neural cells in cerebral infarction rats.

LncRNA Small Nucleolar RNA Host Gene 11 Modulates Ferroptosis in Renal Tubular Epithelial Cells via miR-324-3p/GPX4 Axis in Acute Kidney Injury

Role of ferroptosis in acute kidney injury (AKI) is not fully uncovered. We aim to explore a novel role that SNHG11/miR-324-3p modulated ferroptosis in AKI via modulating GPX4. To mimic AKI in vivo, 6-week male C57BL/6 mice were administrated with lipopolysaccharide (LPS). shRNA (sh-NC or sh-SNHG11), miRNA antagomir (antagomir-NC or miR-324-3p antagomir), miRNA agomir (agomir-NC and miR-324-3p agomir) were injected in mice to regulate SNHG11 and miR-324-3p, respectively. To stimulate the in vitro model of AKI, HK-2 cells were incubated with LPS for 6 h, followed by the transfection with shRNA (sh-NC or sh-SNHG11), miRNA mimics (mimics-NC or miR-324-3p mimics), miRNA inhibitor (inhibitor-NC and miR-324-3p inhibitor), respectively. Co-transfection of miR-324-3p mimics and SNHG11-wt decreased the relative luciferase activity, suggesting miR-324-3p was the target of SNHG11. SNHG11 silence increased miR-324-3p expression in LPS-stimulated HK-2 cells. Both of SNHG11 silence and miR-324-3p upregulation aggravated LPS-induced ferroptosis and kidney injury, and decreased GPX4 whereas downregulation of miR-324-3p inhibited LPS-caused impairment, and increased GPX4 in AKI models. In AKI models with SNHG11 silence, upregulation of miR-324-3p further enhanced ferroptosis and kidney injury, and resulted in the lower expression of GPX4. Decreased SNHG11 caused miR-324-3p upregulation in renal tubular epithelial cells, which led to GPX4 reduction that trigger ferroptosis in AKI.

MicroRNA-411-3p motivates methotrexate's cellular uptake and cytotoxicity via targeting Yin-yang 1 in leukemia cells.

This study aimed to figure out how microRNA (miR)-411-3p's impacts on methotrexate (MTX)'s cellular uptake and cytotoxicity in acute lymphoblastic leukaemia (ALL) CEM-C1 cells by targeting Yin-yang 1 (YY1). miR-411-3p and YY1 were detected by RT-qPCR or Western blot. Intracellular MTX concentration was measured by enzyme-linked immunosorbent assay. Cell viability and apoptosis were evaluated by CCK-8, clonal formation assay, and flow cytometry. Verification of miR-411-3p and YY1's targeting link was manifested. It came out that miR-411-3p mimic or si-YY1 elevated intracellular MTX, MTX-induced cytotoxicity and apoptosis rate in CEM-C1. However, the inverse results were noticed in cells introduced with miR-411-3p inhibitor or oe-YY1. Meanwhile, it was found that cell relative luciferase activity was reduced after co-transfection of miR-411-3p mimic with YY1-WT, indicating that miR-411-3p targeted YY1. Elevation of YY1 could turn around elevating miR-411-3p's impacts on MTX's cellular uptake and cytotoxicity in CEM-C1 cells. These findings convey that miR-411-3p motivated MTX's cellular uptake and cytotoxic impacts via targeting YY1 in leukemia cells. This study is helpful for learning about the mechanisms underlying MTX responses in ALL patients.

MicroRNA hsa-miR-320a-3p and Its Targeted mRNA FKBP5 Were Differentially Expressed in Patients with HIV/TB Co-Infection.

Among the PLWH (people living with HIV) population, the risk of developing active tuberculosis (TB) is increasing. Active TB also accelerates the deterioration of PLWH's immune function and is one of the leading causes of death in the PLWH population. So far, accurate diagnosis of active TB in the PLWH population remains challenging. Through data analysis of HIV/TB co-infection in the GEO database, the differentially expressed genes as well as their related microRNA (miRNA) were acquired and were further verified through clinical blood samples. Dual-luciferase assay was used to verify the mechanism of miRNA on mRNA. The enrichment of immune cells in database patient samples was analyzed by bioinformatics and finally verified by blood routine data. Our study found that FKBP5 (FK506 binding protein 5) was highly expressed in the HIV/TB co-infection group; hsa-miR-320a-3p was highly expressed in the HIV infection group but decreased in the HIV/TB co-infection group. Dual-luciferase assay results showed that hsa-miR-320a-3p mimics significantly reduced the relative luciferase activity of the WT-FKBP5 group; however, this phenomenon was not observed in the MUT-FKBP5 group. At the same time, as a key molecule of the immune-related pathway, FKBP5 is highly correlated with the amount of neutrophils, which provides a new suggestion for the treatment of the HIV/TB co-infection population. Our study found that hsa-miR-320a-3p can decrease FKBP5 expression, suggesting a potential regulatory role for FKBP5. The involvement of FKBP5 and its related molecule hsa-miR-320a-3p in HIV/TB co-infection proposes them as potential biomarkers for the diagnosis of active TB in the PLWH population.

Single‑nucleotide polymorphism rs6592645 confers asthma risk through regulating LRRC32 expression.

Asthma is a complex disease, often with evident genetic predisposition; for example, the single-nucleotide polymorphism (SNP) rs7130588 was significantly associated with asthma by genome-wide association study (GWAS). Analysis of 1000 Genomes Project data suggests that there is another SNP, rs6592645, in complete linkage disequilibrium with rs7130588 and should present the same signal in GWAS. However, the causal SNP and the mechanism for the association between rs7130588 and asthma remain to be elucidated. In the presents study, results from dual-luciferase assays indicated that the A/G alleles of rs7130588 failed to present significantly different reporter gene expression. By contrast, A allele of rs6592645 presented a significant increase in relative luciferase activity than G allele, thus suggesting that rs6592645 may be a causal SNP. Using chromosome conformation capture, the enhancer region containing rs6592645 was observed to interact with promoter region of leucine-rich repeat-containing 32 (LRRC32). Gene expression quantification suggested that LRRC32 expression is significantly increased in lung tissue of patients with asthma and is dependent on the genotype of this locus, thus verifying that LRRC32 may be involved in asthma onset and that rs6592645 can regulate LRRC32 expression. Through chromatin immunoprecipitation, transcription factor 3 (TCF3) was identified to bind to rs6592645 surrounding region and the interaction between TCF3 and rs6592645 surrounding region was investigated. Results from the present study may improve our understanding of the mechanism by which the genetic variation in this locus might influence asthma susceptibility.

Extracellular Vesicles Derived from Human Umbilical Cord Mesenchymal Stem Cells Promote Trophoblast Cell Proliferation and Migration by Targeting TFPI2 in Preeclampsia.

Preeclampsia is a pregnancy disorder characterized by systemic organ damage and high blood pressure. It has been reported that microRNA-195 (miR-195) is associated with preeclampsia. In this study, we discovered the target of miR-195 in regulating human extravillous cytotrophoblast-derived transformed cell proliferation and migration. We analyzed the clinicopathological factors of preeclampsia and normal pregnancies. The messenger ribonucleic acid (mRNA) levels of miR-195 and tissue factor pathway inhibitor 2 (TFPI2) were measured in placental tissues derived from normal and preeclampsia patients by real-time polymerase chain reaction (PCR). Human umbilical cord mesenchymal stem cell (hUC-MSC)-derived extracellular vesicles were verified by western blot. HTR8-S/Vneo cell proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, and cell migration rate was assessed by the transwell assay. Relative luciferase activities were measured in TFPI2 wild-type (WT) and mutant cells. miR-195 expression was negatively correlated with TFPI2 mRNA levels in preeclampsia patients. Extracellular vesicles derived from hUC-MSCs enhanced HTR8-S/Vneo cell proliferation and migration. In addition, miR-195 isolated from hUC-MSCs enhanced HTR8-S/Vneo cell proliferation and migration by targeting TFPI2. Our findings demonstrate that the upregulation of miR-195 in extracellular vesicles derived from hUC-MSCs promotes HTR8-S/Vneo cell proliferation and migration by targeting TFPI2.

MiR-101–5p overexpression suppresses the proliferation of goat spermatogonial stem cells by targeting EZH2

MicroRNAs (miRNAs), as post-transcriptional gene mediators, regulate the biological characteristics of spermatogonial stem cells (SSCs), including proliferation, differentiation and apoptosis. However, the potential roles and mechanisms by which miR-101–5p affected the biological characters of goat SSCs have not been fully elucidated. Herein, we reported that miR-101–5p overexpression decreased cell viability (P < 0.01), arrested cell cycle in the G1 phase (P < 0.05), and aggravated apoptosis of goat SSCs (P < 0.01) compared with negative control (NC), as determined by CCK-8 assay and flow cytometry analysis. Additionally, PCNA protein expression was attenuated by miR-101–5p overexpression (P < 0.05). Notably, the expression of SSCs specific genes Oct4 (P < 0.05), PLZF (P < 0.01) and DAZL (P < 0.01) were decreased in miR-101–5p overexpressed SSCs. Furthermore, the dual luciferase reporter assay showed that, when co-transfected with miR-101–5p mimics, the relative luciferase activity of EZH2 wide-type (WT) was inhibited (P < 0.05) compared with the transfection of EZH2 mutant (MUT). EZH2 expression was negatively correlated with miR-101–5p expression in goat SSCs. Collectively, our data implicates that miR-101–5p overexpression aggravates cell apoptosis, and suppresses cell proliferation of goat SSCs via targeting EZH2, which may impair spermatogenesis.

Epigenetic and genetic inactivation of tumor suppressor miR-135a in non-small-cell lung cancer.

Despite therapeutic advances, lung cancer prognosis remains poor. Loss of heterozygosity (LOH) in the 3p21 region is well documented in lung cancer, but the specific causative genes have not been identified. Here, we aimed to examine the clinical impact of miR-135a, located in the 3p21 region, in lung cancer. miR-135a expression was assessed using quantitative real-time polymerase chain reaction. LOH was analyzed at microsatellite loci D3S1076 and D3S1478, and promoter methylation status was determined by pyrosequencing of resected samples of primary non-small-cell lung cancer (NSCLC). The regulation of telomerase reverse transcriptase (TERT) was evaluated in lung cancer cells H1299 by luciferase report assays after treatment with miR-135a mimics. miR-135a was significantly downregulated in squamous cell cancer (SCC) tumor tissues compared to normal tissues (p=0.001). Low miR-135a expression was more frequent in patients with SCC (p=2.9 × 10-4 ) and smokers (p=0.01). LOH and hypermethylation were detected in 27.8% (37/133) and 17.3% (23/133) of the tumors, respectively. Overall, 36.8% (49/133) of the NSCLC cases harbored either miR-135a LOH or promoter hypermethylation. The frequencies of LOH and hypermethylation were significantly associated with SCCs (p=2× 10-4 ) and late-stage (p=0.04), respectively. MiR-135a inhibited the relative luciferase activity of psiCHECK2-TERT-3'UTR. These results suggest that miR-135a may act as a tumor suppressor to play an important role in lung cancer carcinogenesis, which will provide a new insight into the translational value of miR-135a. Further large-scale studies are required to confirm these findings.

Rs77283072 influences breast cancer susceptibility by regulating CDKN2A expression.

Breast cancer is the cancer type with the highest morbidity rates in women, and previous genome-wide association studies (GWASs) have suggested that the single nucleotide polymorphism (SNP) rs1011970 is significantly associated with this disease. An analysis of data from the 1000 Genomes Project demonstrated that there is an SNP, rs77283072, in almost complete linkage disequilibrium with rs1011970, which should therefore present the same signal in a GWAS. However, the actual causal SNP and its associated underlying mechanism have yet to be elucidated. Therefore, the present study evaluated the role of rs77283072 in terms of its association with breast cancer. A dual-luciferase assay was performed, which demonstrated that the two alleles of rs1011970 did not exhibit significantly different reporter gene activity. However, the A allele of rs77283072 exhibited a significant increase in relative luciferase activity compared with the G allele, which suggested that rs77283072 was the causal SNP for breast cancer. Chromosome conformation capture demonstrated that the enhancer containing rs77283072 interacted with the promoter of cyclin-dependent kinase inhibitor 2A (CDKN2A). Furthermore, expression quantitative trait locus analysis demonstrated that the expression of CDKN2A was dependent on the genotype of rs77283072. Taken together, the findings of the present study provided novel insights into the mechanism underlying how the genetic variation in this locus was able to influence breast cancer susceptibility and further the treatment for this disease.

Goat FADS2 controlling fatty acid metabolism is directly regulated by SREBP1 in mammary epithelial cells.

Goat milk provides benefits to human health due to its richness in bioactive components, such as polyunsaturated fatty acids (PUFAs). The fatty acid desaturase 2 (FADS2) is the first rate-limiting enzyme in PUFAs biosynthesis. However, its role and transcriptional regulation mechanisms in fatty acid metabolism in dairy goat remain unclear. Here, our study revealed that the FADS2 gene was highly expressed during the peak lactation compared with the dry period, early lactation, and late lactation. The content of triacylglycerol (TAG) was enhanced with the increasing mRNA expression of TAG synthesis genes (diacylglycerol acyltransferase 1/2, DGAT1/2) in FADS2-overexpressed goat mammary epithelial cells (GMECs). Overexpression of FADS2 was positively correlated with the elevated concentrations of dihomo-gamma-linolenic acid (DGLA) and docosahexaenoic acid (DHA) in GMECs. BODIPY staining showed that FADS2 promoted lipid droplet accumulation in GMECs. To clarify the transcriptional regulatory mechanisms of FADS2, 2,226 bp length of FADS2 promoter was obtained. Deletion mutation assays revealed that the core region of FADS2 promoter was located between the -375 and -26 region, which contained SRE1 (-361/-351) and SRE2 (-191/-181) cis-acting elements of transcription factor sterol regulatory element-binding protein 1 (SREBP1). Overexpression of SREBP1 enhanced relative luciferase activity of the single mutant of SRE1 or SRE2, vice versa, and failed to alter the relative luciferase activity of the joint mutant of SRE1 and SRE2. Chromatin immunoprecipitation (ChIP) and site-directed mutation assays further demonstrated that SREBP1 regulated the transcription of the FADS2 gene by binding to SRE sites in vivo and in vitro. In addition, the mRNA levels of FADS2 were significantly decreased by targeting SRE1 and SRE2 sites in the genome via the CRISPR interference (CRISPRi) system. These findings establish a direct role for FADS2 regulating TAG and fatty acid synthesis by SREBP1 transcriptional regulation in dairy goat, providing new insights into fatty acid metabolism in mammary gland of ruminants.

LncRNA RP11-551L14.4 suppresses breast cancer development by inhibiting the expression of miR-4472.

Previous studies have been reported that long non-coding RNA (lncRNA) can regulate the expression of genes which are involved in many important cellular processes The potential role of lncRNA RP11-551L14.4 in the development of breast cancer and the possible regulatory mechanisms was investigated. Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to analyze RP11-551L14.4 expression in 36 paired breast cancer tissues and adjacent tissues. The expression of RP11-551L14.4 in multiple breast cancer cell lines was detected by qRT-PCR. Meanwhile, overexpression of RP11-551L14.4 models was established using lentivirus in BT474 and T47D breast cancer cells. Cell counting kit-8 (CCK-8), cell colony formation and cell cycle assays were performed to detect the effects of RP11-551L14.4 on the biological function of breast cancer cells. Besides, bioinformatics techniques, dual luciferase reporter gene assay and rescue experiments were used to investigate the potential mechanisms. RP11-551L14.4 expression was negatively associated with the advanced tumor stage. Breast cancer patients with low RP11-551L14.4 expression manifested a poorer prognosis. The results of qRT-PCR showed that RP11-551L14.4 expression in breast cancer tissues was significantly lower than in adjacent tissues. Meanwhile, overexpression of RP11-551L14.4 significantly decreased the cell proliferation and cell cycle. Bioinformatics technology showed that RP11-551L14.4 could complementarily bind to miR-4472. qRT-PCR results indicated that the expression levels of miR-4472 and RP11-551L14.4 in breast cancer were negatively correlated. Luciferase reporter gene assay showed that miR-4472 remarkably decreased the relative luciferase activity of the wild-type RP11-551L14.4 vector. miR-4472 is a direct target gene of RP11-551L14.4. miR-4472 levels were reduced, and repulsive guidance molecule A (RGMA) mRNA or protein levels were increased after overexpression of RP11-551L14.4 in the breast cancer cells. miR-4472 reversed the effects caused by RP11-551L14.4 in breast cancer cells. RP11-551L14.4 expression was remarkably decreased in breast cancer tissues and cells. RP11-551L14.4 may inhibit the malignant progression of breast cancer by regulating miR-4472 expression.

Implications of differential transcription start site selection on chronic myeloid leukemia and prostate cancer cell protein expression.

The relevance of minor transcription start sites in broad promoters is not well understood. We have studied AGAP2 expression in prostate cancer and chronic myeloid leukemia (CML), showing transcription is initiated from alternative transcription start sites (TSSs) within a single TSS cluster, producing cancer-type-specific AGAP2 mRNAs with small differences in their 5' UTR length. Interestingly, in the CML cell lines where the 5' UTR is longer, AGAP2 protein levels are lower. We demonstrate that the selection of an upstream TSS involved the formation of a G quadruplex in the 5' UTR, decreasing polysome formation. After developing a bioinformatics pipeline to query data from the FANTOM project and the NCl-60 human tumor cell lines screen, we found HK1 expression can also be regulated by the same mechanism. Overall, we present compelling data supporting TSS selection within a TSS cluster play a role in protein expression and should not be ignored.

The tRNA-Cys-GCA Derived tsRNAs Suppress Tumor Progression of Gliomas via Regulating VAV2.

The tsRNAs (tRNA-derived small RNAs) are new types of small noncoding RNAs derived from tRNAs. Gliomas are well-known malignant brain tumors. The study focused on tsRNA characterizations within gliomas. Datasets processing, bioinformatics analyses, and visualizations were performed with the packages of Python and R. Cell proliferations were demonstrated via CCK8 assays and colony formation assays, and in vivo xenograft experiments. Dual-luciferase reporter assay was performed to confirm the binding of tsRNA with its targets. Via using bioinformatics approaches, the hundreds of tsRNAs with available expression abundance were identified in gliomas dataset, most of them derived from D-loop or T-loop fragments of tRNAs. Among tsRNAs derived from tRNA-Cys-GCA, tRFdb-3003a and tRFdb-3003b (tRFdb-3003a/b) were remarkably down-regulated in gliomas. The survival outcome of gliomas patients with low tRFdb-3003a/b expressions was notably worse than that of high-expression patients. In glioma cells, tRFdb-3003a could suppress cells proliferation and colony formation ability. In vivo, tRFdb-3003a suppressed the tumor growth of xenograft gliomas. Enrichment analyses displayed the tRFdb-3003a-related mRNAs were enriched in the specific GO terms, spliceosome and autophagy pathways, and three GSEA molecular signatures. Mechanically, 3'-UTR regions of VAV2 mRNA were predicted to contain the binding positions of tRFdb-3003a/b, tRFdb-3003a and tRFdb-3003b was effective to reduce the relative luciferase activity of cells with VAV2 wild-type reporter. Overexpression of tRFdb-3003a/b could down-regulated the expression levels of VAV2 protein and mRNA in glioma cells. The tRNA-Cys-GCA derived tRFdb-3003a and tRFdb-3003b might act as key player in tumor progressions of gliomas; tRFdb-3003a/b might directly bind to VAV2 and regulate VAV2 expressions in gliomas.