Luciferase Activity Research Articles

A novel splice variant of the human MSTN gene encodes a myostatin-specific myostatin inhibitor.

Myostatin, encoded by the MSTN gene comprising 3 exons, is a potent negative regulator of skeletal muscle growth. Although a variety of myostatin inhibitors have been invented for increasing muscle mass in muscle wasting diseases, no effective inhibitor is currently available for clinical use. Myostatin isoforms in several animals have been reported to inhibit myostatin, but an isoform has never been identified for the human MSTN gene, a conserved gene among animals. Here, a splice variant of the human MSTN gene was explored. Transcripts and proteins were analysed by reverse transcription-PCR amplification and western blotting, respectively. Proteins were expressed from expression plasmid. Myostatin signalling was assayed by the SMAD-responsive luciferase activity. Cell proliferation was assayed by the Cell Counting Kit-8 (CCK-8) assay and cell counting. Cell cycle was analysed by the FastFUCCI system. Reverse transcription-PCR amplification of the full-length MSTN transcript in CRL-2061 rhabdomyosarcoma cells revealed two bands consisting of a thick expected-size product and a thin additional small-size product. Sequencing of the small-size product showed a 963-bp deletion in the 5' end of exon 3, creating exon 3s, which contained unusual splice acceptor TG dinucleotides. The novel variant was identified in other human cell lines, although it was not identified in skeletal muscle. The 251-amino acid isoform encoded by the novel variant (myostatin-b) was identified in CRL-2061 rhabdomyosarcoma cells. Transfection of a myostatin-b expression plasmid into CRL-2061 and myoblast cells inhibited endogenous myostatin signalling (44%, P<0.001 and 63%, P<0.001, respectively). Furthermore, myostatin-b inhibited myostatin signalling induced by recombinant myostatin (68.8%, P<0.001). In remarkable contrast, myostatin-b did not inhibit the myostatin signalling induced by recombinant growth differentiation factor 11 (9.2%, P=0.70), transforming growth factor β (+3.1%, P=0.83) or activin A (+1.1%, P=0.96). These results indicate the myostatin-specific inhibitory effect of myostatin-b. Notably, the expression of myostatin-b in myoblasts significantly enhanced cell proliferation higher than the mock-transfected cells by the CCK-8 and direct cell counting assays (60%, P<0.05 and 39%, P<0.05, respectively). Myostatin-b increased the percentage of S-phase cells significantly higher than that of the mock-transfected cells (53% vs. 80%, P<0.05). We cloned a novel human MSTN variant produced by unorthodox splicing. The variant encoded a novel myostatin isoform, myostatin-b, that inhibited myostatin signalling by myostatin-specific manner and enhanced myoblast proliferation by shifting cell cycle. Myostatin-b, which has myostatin-specific inhibitory activity, could be developed as a natural myostatin inhibitor.

MiRNA-103-3p-Hlf regulates apoptosis and autophagy by targeting hepatic leukaemia factor in heart failure.

Cardiomyocyte apoptosis is an important factor leading to the occurrence and development of heart failure (HF), which is associated with high mortality of patients with cardiovascular diseases. This study aims to investigate the underlying mechanisms of HF in terms of expression and regulation patterns using bioinformatics and experimental validation. Two HF datasets were collected: a dataset GSE112056 downloaded from the GEO database (including mRNA and miRNA sequencing data) and another is the laboratory-owned mRNA dataset. Differential mRNAs and miRNAs in the two datasets were screened using the raw Bayesian approach method. Gene Ontology was used to perform functional enrichment analysis of the differential mRNAs and co-expression network analysis of the differential mRNAs, combined with nuclear transcription factors in the differential miRNAs and mRNAs for target gene prediction. A HF cell model was constructed using mouse cardiomyocytes (HL-1), and the role and mechanism of miRNA-103-3p-Hlf (hepatic leukaemia factor) in the process of HF was verified by cell transfection, luciferase reporter gene, WB, and qPCR. We found that Hlf gene expression was decreased in the HF model group and strongly correlated with FYCO1 (FYVE and coiled-coil domain-containing protein 1) gene, a phenomenon enriched in apoptotic autophagy-related pathways. MiR-103-3p expression was up-regulated in the HF model group, and its targeting correlation with Hlf was confirmed by luciferase activity assay. In the HL-1 cell model, miR-103-3p significantly promoted apoptosis and inhibited autophagy in HL-1 cells (all P<0.05), and overexpression of the Hlf gene reversed this phenomenon, inhibiting apoptosis and promoting autophagy in HL-1 cells (all P<0.05). MiR-103-3p affects myocardial cells apoptosis and autophagy by targeting Hlf, playing as a potential therapeutic biomarker for HF progression.

Intronic microRNA-directed regulation of mitochondrial reactive oxygen species enhances plant stress tolerance in Arabidopsis.

MicroRNAs (miRNAs) play crucial roles in regulating plant development and stress responses. However, the functions and mechanism of intronic miRNAs in plants are poorly understood. This study reports a stress-responsive RNA splicing mechanism for intronic miR400 production, whereby miR400 modulates reactive oxygen species (ROS) accumulation and improves plant tolerance by downregulating its target expression. To monitor the intron splicing events, we used an intronic miR400 splicing-dependent luciferase transgenic line. Luciferase activity was observed to decrease after high cadmium concentration treatment due to the retention of the miR400-containing intron, which inhibited the production of mature miR400. Furthermore, we demonstrated that under Cd treatments, Pentatricopeptide Repeat Protein 1 (PPR1), the target of miR400, acts as a positive regulator by inducing ROS accumulation. Ppr1 mutation affected the Complex III activity in the electron transport chain and RNA editing of the mitochondrial gene ccmB. This study illustrates intron splicing as a key step in intronic miR400 production and highlights the function of intronic miRNAs as a 'signal transducer' in enhancing plant stress tolerance.

Functional analysis of CTLA4 promoter variant and its possible implication in colorectal cancer immunotherapy.

Colorectal cancer (CRC) is a prevalent cancer, ranking as the third most common. Recent advances in our understanding of the molecular causes of this disease have highlighted the crucial role of tumor immune evasion in its initiation and progression. CTLA4, a receptor that acts as a negative regulator of T cell responses, plays a pivotal role in this process, and genetic variations in CTLA4 have been linked to CRC susceptibility, prognosis, and response to therapy. We conducted a case-control study involving 98 CRC patients and 424 controls. We genotyped the CTLA4 c.-319C > T variant (rs5742909) and performed an association analysis by comparing allele frequencies between the patients and controls. To assess the potential functional impact of this variant, we first performed an In Silico analysis of transcription factor binding sites using Genomatix. Finally, to validate our findings, we conducted a luciferase reporter gene assay using different cell lines and an electrophoretic mobility shift assay (EMSA). The case-control association analysis revealed a significant association between CTLA4 c.-319C > T and CRC susceptibility (p = 0.023; OR 1.89; 95% CI = 1.11-3.23). Genomatix analysis identified LEF1 and TCF7 transcription factors as specific binders to CTLA4 c.-319C. The reporter gene assay demonstrated notable differences in luciferase activity between the c.-319 C and T alleles in COS-7, HCT116, and Jurkat cell lines. EMSA analysis showed differences in TCF7 interaction with the CTLA4 C and T alleles. CTLA4 c.-319C > T is associated with CRC susceptibility. Based on our functional validation results, we proposed that CTLA4 c.-319C > T alters gene expression at the transcriptional level, triggering a stronger negative regulation of T-cells and immune tumoral evasion.

MiR-147b is an oncomiR acting synergistically with HIPK2 to promote pancreatic carcinogenesis

MicroRNAs (miRs, miRNAs) are known players in the regulatory network of pancreatic tumorigenesis, but the downstream effectors remain poorly characterized. This study addressed this issue based on in silico prediction, in vitro experiments, and in vivo validation. The differentially expressed PCa-related miRNAs and bioinformatics tools predicted downstream regulators. The expression of miR-147b was examined in PCa cell lines. Putative targets of miR-147b were predicted by a publicly available database and confirmed by luciferase activity assay. Mimic/inhibitor, siRNA/overexpression plasmid, or pifithrin-α (p53 inhibitor) were delivered into PCa cells to assess the effect of miR-147b, HIPK2, and p53 on malignant phenotypes of PCa cells. AntagomiR-147b and shRNA targeting HIPK2 were introduced to xenograft-bearing nude mice for in vivo experiments. The expression of miR-147b was significantly increased in PCa cell lines. Ectopic expression of miR-147b promoted the malignant phenotypes of PCa cells and inhibited their apoptosis. HIPK2 was confirmed as a target gene of miR-147b. Inhibiting miR-147b could promote HIPK2 expression and potentially activate the p53 pathway, inhibiting PCa cell growth. In vivo experiments suggested that miR-147b inhibition suppressed the growth of xenograft tumors in nude mice, while HIPK2 knockdown counteracted its effect. Collectively, our work reveals a novel miR-147b-mediated carcinogenic regulatory network in PCa that may be a viable target for PCa treatment.

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.

MiR-122-5p is involved in posttranscriptional regulation of the mitochondrial thiamin pyrophosphate transporter (SLC25A19) in pancreatic acinar cells.

Thiamin (vitamin B1) plays a vital role in cellular energy metabolism/ATP production. Pancreatic acinar cells (PACs) obtain thiamin from circulation and convert it to thiamin pyrophosphate (TPP) in the cytoplasm. TPP is then taken up by the mitochondria via a carrier-mediated process that involves the mitochondrial TPP transporter (MTPPT; encoded by the gene SLC25A19). We have previously characterized different aspects of the mitochondrial carrier-mediated TPP uptake process, but nothing is known about its possible regulation at the posttranscriptional level. We address this issue in the current investigations focusing on the role of miRNAs in this regulation. First, we subjected the human (and rat) 3'-untranslated region (3'-UTR) of the SLC25A19 to three in-silico programs, and all have identified putative binding sites for miR-122-5p. Transfecting pmirGLO-hSLC25A19 3'-UTR into rat PAC AR42J resulted in a significant reduction in luciferase activity compared with cells transfected with pmirGLO-empty vector. Mutating as well as truncating the putative miR-122-5p binding sites in the hSLC25A19 3'-UTR led to abrogation of inhibition in luciferase activity in PAC AR42J. Furthermore, transfecting/transducing PAC AR42J and human primary PACs with mimic of miR-122-5p led to a significant inhibition in the level of expression of the MTPPT mRNA and protein as well as in mitochondrial carrier-mediated TPP uptake. Conversely, transfecting PAC AR42J with an inhibitor of miR-122-5p increased MTPPT expression and function. These findings show, for the first time, that expression and function of the MTPPT in PACs are subject to posttranscriptional regulation by miR-122-5p.NEW & NOTEWORTHY This study shows that the expression and function of mitochondrial TPP transporter (MTPPT) are subject to posttranscriptional regulation by miRNA-122-5p in pancreatic acinar cells.

Assessment of mechanism of Ganoderma lucidum neutral triterpenes mediating bone marrow mesenchymal stem cells with high expression of miR-125b targeting Bak1 gene in leukemia cell proliferation and apoptosis

This study focused on assessing mechanism of bone marrow mesenchymal stem cells (BMSCs) with high miR-125b level on leukemia cells. Cultured MSC cells were identified, transfected with miR-125b, and miR-125b level was tested by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). After transfection, cells were divided into NC group, miR-125b mimics group, miR-125b inhibitor group, BMSC group and BMSCs group with high miR-125b expression. The miR-125b target gene was further explored by luciferase activity. Morphology of BMSC cell line P3 was stable, and CD90, CD71 and CD29 surface markers were positive, while CD45 was negative. miR-125b overexpression of BMSC in this study was successfully transfected, and the BMSCs with transfected miR-125b notably suppressed THP-1 cells proliferation, further enhancing cell apoptosis and levels of apoptosis-related proteins. Moreover, the BMSCs with miR-125b notably inhibited colony formation ability, and induced G1 phase arrest. miR-125b showed targeted relationship to Bak1, while the BMSCs with miR-125b targeted Bak1 gene, leading to up-regulation of Bakl, p53 and Puma protein expressions and inhibition of proliferation and apoptosis of leukemia THP-1 cells. miR-125b can therefore be used as a therapeutic target for acute myeloid leukemia.

Suppression of P2X7R by Local Treatment Alleviates Acute Gouty Inflammation.

Gout is the most common inflammatory arthritis associated with interleukin-1β (IL-1β) accumulation during exacerbation. In this study, we aimed to clarify whether potassium channel antagonists attenuate local inflammation in mice with monosodium urate (MSU)-induced gout. We cultured human macrophage THP-1 cells and evaluated the molecular levels of both IL-1β and potassium channels stimulated with MSU and/or potassium channel antagonists. Acute gout models were generated in IL-1β luciferase transgenic male mice using synovium-like subcutaneous air pouches with MSU injection. Their luciferase activities were monitored following potassium channel blocker treatment using the IVIS Spectrum CT imaging system. The lavages and tissues were extracted from their air pouches, followed by cell counting and pathological analysis. MSU stimulation increased the gene expression levels of pro-IL-1β, P2x7r and Kv1.3, whereas the expression of Kcnq1 was decreased in phorbol 12-myristate 13-acetate-induced THP-1 cells. Both high and low concentrations of the P2x7 receptor inhibitor adenosine 5'-triphosphate (ATP) derivative periodate oxidized ATP (oATP) decreased the production of IL-1β in the supernatant of THP-1 cells. The sixth hour was the peak time of IL-1β luciferase activity after MSU intervention in vivo. oATP ameliorated the synovial IL-1β luciferase activity, reduced inflammatory cell infiltration and alleviated the erosive damage in the cartilage. The anti-inflammatory properties of potassium channel inhibitors, especially of oATP, might point to new strategies for local anti-inflammatory therapy for acute gout.

SEMA4G targeted by miR‐363‐5p regulates the proliferation of granulosa cells in Yunshang black goats

AbstractProliferation of granular cells (GCs) plays an important role in ovary development, providing energy and a microenvironment for oocyte ovulation. In this study, we explored the spatiotemporal expression of SEMA4G and its effects on the growth and development of goat GCs using primary GCs cultured in vitro as a model. The results showed that the expression level of SEMA4G was significantly higher in the ovaries of high‐fertility goats than in those of low‐fertility goats (p &lt; 0.05). The mRNA and protein expression levels of the cell proliferation markers of GCs were significantly increased after the overexpression of SEMA4G in goat primary GCs. The EdU and CCK8 results showed that cell viability was elevated in goat GCs and that proliferation was promoted by an increase in the number of proliferating cells. The proliferation of goat GCs was significantly inhibited by SEMA4G inhibition (p &lt; 0.05). The results of online miRNA and target gene prediction software and dual luciferase activity analysis confirmed that SEMA4G could bind to mi‐363‐5p and was one of its target genes. The RT‒qPCR results showed that the expression level of miR‐363‐5p was significantly lower in the ovaries of high‐fertility goats than in those of low‐fertility goats in contrast to the expression level of SEMA4G (p &lt; 0.05). After the overexpression of miR‐363‐5p in goat GCs, the expression of SEMA4G was significantly suppressed (p &lt; 0.05). Collectively, the results of this study could lay the foundation for exploring the molecular mechanisms by which SEMA4G and miR‐363‐5p regulate the growth and development of goat GCs and provide targets for breeding high‐fertility goats.

Downregulation of miR-92a in Decidual Stromal Cells Suppresses Migration Ability of Trophoblasts by Promoting Macrophage Polarization.

Preeclampsia (PE) is a severe pregnancy complication that accounts for about 14% of maternal deaths. Its clinical manifestations commonly include hypertension and proteinuria. However, it is largely limited in understanding its pathogenetic mechanism. In this study, we used bioinformatics to compare differential gene expressions in decidual stromal cells from PE patients and healthy donors. The result indicated that higher levels of CCL5 and CXCL2 were expressed in decidual stromal cells of PE patients compared with healthy pregnancy. The bioinformatics analysis confirmed that decidual stromal cells derived from PE patients expressed significantly lower miR-92a compared with those derived from healthy donors. Transfection of miR-92a inhibitors upregulated IL-6, CXCL2, CXCL3, CCL5, and CXCL8 expressions in decidual stromal cells. Luciferase activity assay confirmed that miR-92a directly targeted the mRNA of IRF3 whose overexpression could promote the secretion of cytokines. The flow cytometric analysis demonstrated that M1 macrophage infiltration was higher in the placentas of PE patients than in those of healthy donors. We also observed that after transfection of miR-92a inhibitor, condition medium (CM) derived from decidual stromal cells significantly promoted M1 polarization of macrophages. In addition, the transwell migration assay and flow cytometric analysis together showed that decidual stromal cell-derived CM induced macrophages to suppress the trophoblast migration and proliferation. Taken together, our result indicates that downregulation of miR-92a in decidual stromal cells promotes the macrophage polarization and suppresses the trophoblast migration and proliferation.

The cotton MYB33 gene is a hub gene regulating the trade-off between plant growth and defense in Verticillium dahliae infection

IntroductionSessile plants engage in trade-offs between growth and defense capacity in response to fluctuating environmental cues. MYB is an important transcription factor that plays many important roles in controlling plant growth and defense. However, the mechanism behind how it keeps a balance between these two physiological processes is still largely unknown. ObjectivesOur work focuses on the dissection of the molecular mechanism by which GhMYB33 regulates plant growth and defense. MethodsThe CRISPR/Cas9 technique was used to generate mutants for deciphering GhMYB33 functions. Yeast two-hybrid, luciferase complementary imaging, and co-immunoprecipitation assays were used to prove that proteins interact with each other. We used the electrophoretic mobility shift assay, yeast one-hybrid, and luciferase activity assays to analyze GhMYB33 acting as a promoter. A β-glucuronidase fusion reporter and 5′ RNA ligase mediated amplification of cDNA ends analysis showed that ghr-miR319c directedly cleaved the GhMYB33 mRNA. ResultsOverexpressing miR319c-resistant GhMYB33 (rGhMYB33) promoted plant growth, accompanied by a significant decline in resistance against Verticillium dahliae. Conversely, its knockout mutant, ghmyb33, demonstrated growth restriction and concomitant augmentation of V. dahliae resistance. GhMYB33 was found to couple with the DELLA protein GhGAI1 and bind to the specific cis-elements of GhSPL9 and GhDFR1 promoters, thereby modulating internode elongation and plant resistance in V. dahliae infection. The ghr-miR319c was discovered to target and suppress GhMYB33 expression. The overexpression of ghr-miR319c led to enhanced plant resistance and a simultaneous reduction in plant height. ConclusionOur findings demonstrate that GhMYB33 encodes a hub protein and controls the expression of GhSPL9 and GhDFR1, implicating a pivotal role for the miR319c-MYB33 module to regulate the trade-offs between plant growth and defense.

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.

Role of miR‑let‑7c‑5p/c‑myc signaling axis in the committed differentiation of leukemic THP‑1 cells into monocytes/macrophages.

In a preliminary experiment, it was found that c-myc expression was decreased following the differentiation of THP-1 cells into monocytes/macrophages induced by phorbol 12-myristate 13 acetate (PMA) + lipopolysaccharide (LPS) + interferon (IFN)-γ. The expression of miR-let-7c-5p was then found to be elevated by cross-sectional analysis using TargetScan and PubMed and differential microarray analysis. The present study aimed to investigate the role of the miR-let-7c-5p/c-myc signaling axis in the committed differentiation of THP-1 leukemic cells into monocytes/macrophages induced by PMA + LPS + IFN-γ. Human THP-1 leukemic cells were induced to differentiate into monocytes/macrophages by PMA + LPS + IFN-γ. Following induction for 48 h, the growth density of the THP-1 cells was observed directly under an inverted microscope, cell proliferation was measured using Cell Counting Kit-8 assay and the cell cycle and the expression of differentiation-related antigens (CD11b and CD14) were measured using flow cytometry. The mRNA expression of miR-let-7c-5p and c-myc was detected using reverse transcription-quantitative PCR and the protein expression of c-myc was detected using western blot analysis. Dual luciferase reporter gene analysis was used to detect the targeted binding of miR-let-7c-5p on the 3'UTR of c-myc. The relative expression of miR-let-7c-5p and c-myc genes in THP-1 cells induced by PMA + LPS + IFN-γ was found to be up- and downregulated respectively, and expression of miR-let-7c-5p was negatively correlated with the expression of c-myc gene. Dual luciferase reporter gene assays confirmed that miR-let-7c-5p targeted the 3'UTR of c-myc and inhibited luciferase activity. Following transfection with miR-let-7c-5p mimics, the expression of c-myc was markedly downregulated and the proliferative ability of the THP-1 cells was decreased, while the expression rate of CD11b and CD14 was significantly increased. The rescue experiment revealed that the effects of miR-let-7c-5p mimics on the proliferation and differentiation of THP-1 cells were attenuated by transfection with c-myc overexpression vector. Together, the findings of the present study demonstrated that miR-let-7c-5p can target the 3'UTR region of c-myc and that the miR-let-7c-5p/c-myc signaling axis is one of the critical pathways involved in the directional differentiation of leukemic cells into monocytes/macrophages.

Genetic variants in key necroptosis regulators predict prognosis of non-small cell lung cancer after surgical resection.

Necroptosis is a regulated inflammatory cell death which plays a significant role in cancer development and progression. In this study, we evaluated whether genetic variants in key regulators of necroptosis may affect survival outcome of non-small cell lung cancer (NSCLC) patients after surgical resection. A total of 674 patients who underwent curative surgery were included. Fifteen genetic variants in key regulators of necroptosis (RIPK1, RIPK3, and MLKL) were selected. The association of these variants with survival outcomes was evaluated. Two variants, RIPK1 rs17548629C > T and MLKL rs877375G > C, were associated with better overall survival and disease-free survival in multivariate analyses. When the patients were divided according to histology, the associations were significant only in adenocarcinoma, but not in squamous cell carcinoma. RIPK1 rs17548629 C-to-T change was associated with significantly increased luciferase activity by modulating the binding of miR-642a. Promoter assays showed a significantly increased promoter activity in MLKL rs877375C allele compared to G allele. Consistently, the mRNA expression level of RIPK1 and MLKL showed significant positive correlation with RIPK1 rs17548629C-to-T and MLKL rs877375G-to-C changes. Two genetic variants in key regulators in necroptosis, RIPK1 rs17548629C > T and MLKL rs877375G > C, may be used as biomarkers to predict survival outcomes in surgically resected NSCLC patients.

EGCG attenuated acute myocardial infarction by inhibiting ferroptosis via miR-450b-5p/ACSL4 axis

BackgroundEpigallocatechin gallate (EGCG) has multiple biological effects such as anti-tumor multiple drug resistance, antioxidation and anti-inflammatory properties. Ferroptosis is the main driving factor of ischemic heart injury, thus inhibiting ferroptosis may prove to be an effective treatment strategy for cardiovascular diseases. However, the role of EGCG on ferroptosis in ischemic myocardium and underlying mechanisms remain uncertain. PurposeThis study was aimed to investigate the effects and potential mechanisms of EGCG on myocardial ischemic-induced ferroptosis both in vitro and in vivo. MethodsCardiomyocyte hypoxia model and mouse acute myocardial infarction (AMI) model were established in vitro and in vivo. MiR-450b-5p and ACSL4 silencing or overexpression plasmids were transfected, with or without EGCG pretreatment. Cell viability was determined by the CCK-8 assay. Hematoxylin and eosin (HE) staining and transmission electron microscopy (TEM) were used to evaluate the morphologic alterations. TTC staining was used to observe the infarction area, and echocardiography was adopted to appraise the heart function. Using flow cytometry, the presence of reactive oxygen species (ROS) was assessed. The content of cardiac troponin I (cTn I), glutathione (GSH), malondialdehyde (MDA), divalent iron ions (Fe2+) and superoxide dismutase (SOD) were detected using reagent kits. A luciferase activity assay was performed to assess the binding ability of miR-450b-5p to ACSL4. Expressions of related genes and proteins were measured by RT-qPCR and western blotting respectively. ResultsEGCG attenuated AMI-induced ferroptosis and improved myocardial ischemia injury, which was associated with reducing iron deposition and cTn I, inhibition of lipid peroxidation, decreasing TFR1 and ACSL4, and upregulating SLC7A11, FTH1 and GPX4. Meanwhile, EGCG pretreatment increased miR-450b-5p expression in ischemic myocardium. Further researches discovered that knockdown of miR-450b-5p partially compromised EGCG-generated protective effect in hypoxia HL-1 cells, while combination with miR-450b-5p mimic could strengthen the potency of EGCG on ischemic myocardium. The dual-luciferase test demonstrated that miR-450b-5p has binding to ACSL4. Furthermore, silencing of ACSL4 synergistically increased the cardioprotective effect of EGCG. More significantly, EGCG treatment regulated the ferroptosis-related proteins expression via miR-450b-5p/ACSL4 axis. ConclusionIn summary, the present study evidently demonstrated that EGCG attenuates myocardial ischemia injury by targeting ferroptosis. Our work revealed the role of miR-450b-5p/ACSL4 axis in AMI for the first time. Further, it also elucidated the molecular mechanisms of EGCG on inhibiting ferroptosis greatly depend on the miR-450b-5p/ACSL4 axis, suggesting that EGCG may act as a novel anti-ferroptosis agent and exert a therapeutic role in AMI.

Small molecule heat shock protein 27 inhibitor J2 decreases ovarian cancer cell proliferation via induction of apoptotic pathways.

Heat shock protein 27 (Hsp27) is an important member of the chaperone protein family and its overexpression promotes cancer cell survival. Here, we investigated the apoptosis inducer role of the J2 compound (Hsp27 inhibitor) in human ovarian cancer cell lines (SKOV3 and OVCAR-3). Cell proliferation was measured by MTT assay. The parameters of J2-Hsp27 interaction were determined with molecular docking calculation. The inhibitory effect of the J2 compound on Hsp27 chaperone activity was investigated by luciferase activity assay. Finally, the apoptotic inducer role of the J2 compound on SKOV3 and OVCAR-3 cells was determined by RT-PCR and caspase-3 activity assay. J2 compound decreased SKOV3 and OVCAR-3 cell proliferation in a dose-dependent manner at 48h with IC50 values of 17.34 µM and 12.63 µM, respectively. J2 inhibited the refolding process of denatured luciferase as an Hsp27 inhibitor. Molecular docking calculation was carried out to determine the interaction between Hsp27 and J2. The results indicated that J2 selectively binds to the phosphorylation site of the Hsp27 and inhibits the phosphorylation process of Hsp27. To determine the apoptotic potential of the J2 compound against ovarian cancer cells, the mRNA expression levels of apoptotic and antiapoptotic markers (Bax, Bcl-2, Bcl-xL, Cyt-c, p53, Apaf-1, Cas-3, Cas-8, Cas-9, TNF-α, DAXX, and Ask-1) were measured using RT-PCR. While J2 increased the expressions of apoptotic genes, it decreased the expressions of anti-apoptotic genes. Further, the J2 compound increased Cas-3 activity in SKOV3 and OVCAR-3 at 5.52 and 4.12 folds, respectively. These results confirm that J2 has great potential and significance in the stimulation of apoptosis in ovarian cancer cells as an Hsp27 inhibitor.

LncRNA UCA1 regulates miR-132/Lrrfip1 axis to promote vascular smooth muscle cell proliferation.

UCA1 is predicted to bind to miR-132, which is a key player in the proliferation of vascular smooth muscle cells (VSMCs). This research studied the role of lncRNA UCA1 in atherosclerosis. The binding of UCA1 to miR-132 was proved by dual luciferase activity assay and RNA immunoprecipitation. UCA1 and miR-132 failed to affect each other's expression in VSMCs. UCA1 was upregulated and miR-132 was decreased in atherosclerosis plasma. However, they are not closely correlated across atherosclerosis and control plasma sample. Interestingly, UCA1 suppressed the role of miR-132 in downregulating Lrrfip1 expression and promoting VSMC proliferation. Therefore, UCA1 is downregulated in atherosclerosis and may regulate miR-132/Lrrfip1 axis to promote VSMC proliferation.

Resveratrol Promotes Autophagy to Improve neuronal Injury in Parkinson's Disease by Regulating SNHG1/miR-128-3p/SNCA Axis.

Parkinson's disease (PD) is seriously threatening the health and life quality of the elderly, who have a high incidence and high disability rate. Resveratrol (RES) was reported to play a protective role in PD. However, the functions and potential mechanism of RES in PD remain unclear, which need to be further explored. Human neuroblastoma cells (SH-SY5Y and SK-N-SH) were subjected to 1-Methyl-4-phenylpyridium (MPP+) induction to construct a cell model of PD. Cell viability was evaluated using CCK-8. The gene expression was evaluated using qRT-PCR and western blot. Luciferase activity assay and RIP were performed to validate interactions among SNHG1, miR-128-3p and SNCA. Our results exhibited that RES reduced SNHG1 and SNCA expression but elevated miR-128-3p expression in human neuroblastoma cells upon MPP+ induction. Functionally, RES resulted in the promotion of cell autophagy in MPP+-induced human neuroblastoma cells, while these influences were abolished by SNHG1 overexpression. Mechanistically, SNHG1 could indirectly elevate SNCA expression via sponging miR-128-3p. Moreover, SNCA overexpression reversed SNHG1 silencing-induced cell autophagy in MPP+-induced human neuroblastoma cells upon RES pre-incubation. RES prevented MPP+-induced repression of cell autophagy through inhibiting the SNHG1/miR-128-3p/SNCA axis, suggesting that RES might play a preventive effect on PD progression.

Mutation of the ETS1 3′UTR interacts with miR-216a-3p to regulate granulosa cell apoptosis in sheep

ETS1, an important member of the ETS transcription factor family, is involved in a variety of physiological processes in living organisms, such as cell development, differentiation, proliferation and apoptosis, and is thought to be associated with embryonic development and reproduction. However, the polymorphism of ETS1 has been rarely studied, and its potential impact on the formation of reproductive traits in sheep remains unclear. Here, we first analyzed polymorphisms of ETS1 in a population of 382 small-tailed Han sheep with a lambing number record using the Kompetitive Allele Specific PCR (KASP) technique. The results showed the presence of a SNP locus rs161611767 (T > C) in the 3′UTR of ETS1. The association analysis showed the lambing number of first, second and third parity in the individuals with the CC genotype (2.51 ± 0.108, 2.51 ± 0.179, 1.27 ± 0.196) was higher than that of individuals with the TT genotype (1.79 ± 0.086, 1.56 ± 0.102, 0.56 ± 0.100) (P < 0.05). Then, molecular biotechnologies were used to investigate the effects of the EST1 rs161611767 mutant locus on host gene expression in sheep and the underlying mechanism of its effect on sheep reproduction. The RT‒qPCR results showed that the expression of ETS1 was higher in individuals with the CC genotype than in those with the TT genotype (P < 0.05). The dual luciferase reporter assay showed that the luciferase activity of ETS1 in sheep with the TT genotype was decreased compared to CC genotype (P < 0.05), confirming the existence of EST1 rs161611767 in the 3′UTR as a functional SNP. Given that the 3′UTR is an important regulatory region of gene transcription and translation, we performed bioinformatics prediction and confirmed that the SNP rs161611767 of ETS1 was a direct functional target of miR-216a-3p using dual luciferase activity assay, and the binding capacity of allele T was stronger than that of allele C. Subsequently, the cell transfection results showed that miR-216a-3p suppressed the endogenous expression of ETS1 in sheep primary granulosa cells (GCs). Finally, CCK-8, EdU, WB detection of marker proteins and flow cytometry were used to detect the effects of miR-216a-3p on GCs viability and proliferation/apoptosis, respectively. The results showed that miR-216a-3p inhibited the proliferation of GCs while promoting apoptosis of GCs. In conclusion, these results demonstrate that the SNP rs161611767 of ETS1 is associated with lambing number in small-tailed Han sheep, and miR-216a-3p can act as a regulatory element binding to the T mutation in rs161611767 to regulate ETS1 expression and affect GCs development, which may indirectly affect the number of lambs in sheep. These studies provide evidence for the involvement of ETS1 polymorphisms in sheep reproduction and are expected to provide new insights to elucidate the molecular genetic mechanisms of lambing traits in sheep.