<i>LINC00609</i> inhibits A549 cells progression through the regulation of <i>miR-128-3p/RND3</i> axis

Background: Long-chain non-coding RNA (lncRNA) LINC00609 is a potential tumor suppressor, but the mechanism of action in non-small cell lung cancer (NSCLC) is yet to be understood.Objectives: The effects of LINC00609 on A549 cell proliferation, apoptosis, and cell cycle arrest were investigated. Methods: The LINC00609 levels in NSCLC and normal tissues were analyzed by bioinformatics. Expressions of LINC00609, miR-128-3p, and Rho family GTPase 3 (RND3) in NSCLC cells (A549) were determined by qRT-PCR. Bioinformatics analysis predicted target genes and dual-luciferase reporter assays to ensure that LINC00609 targeted miR-128-3p and miR-128-3p targeted RND3. The proliferation of cells was determined using EDU and CCK-8. Flow cytometry was used to evaluate cell apoptosis rate and cell cycle. The western blotting assay identified proteins related to proliferation and apoptosis. Results: In NSCLC tissues, LINC00609 was expressed in low levels, while its high expression was associated with a higher survival rate. LINC00609 affected cell proliferation, apoptosis, cell cycle arrest, and expression of related proteins. Dual-luciferase reporter assay showed that LINC00609 binds specifically to miR-128-3p, and miR-128-3p binds to RND3. MiR-128-3p overexpression could neutralize the effects of LINC00609. A siRNA targeting RND3 could reverse the effect of the miR-128-3p inhibitor. Silencing RND3 resulted in a decrease in apoptosis rate and the number of cells in the S-phase and an increase in the number of cells in the G1-phase. Furthermore, phosphorylation levels of the AKT protein and mTOR protein, and Bcl2 expression, increased; however, the expression of RND3, Bax, and caspase3 decreased. Conclusions: LINC00609 regulated miR-128-3p/RND3 axis to modulate A549 cell proliferation, apoptosis, and cell cycle arrest. In the case of NSCLC, LINC00609 could be a potential target for therapy.     

LncRNA <i>ZFAS1</i> regulates cardiomyocyte differentiation of human embryonic stem cells

Background: Cardiomyocytes derived from human embryonic stem cells (hESCs) are regulated by complex and stringent gene networks during differentiation. Long non-coding RNAs (lncRNAs) exert critical epigenetic regulatory functions in multiple differentiation processes. However, the involvement of lncRNAs in the differentiation of hESCs into cardiomyocytes has not yet been fully elucidated. Here, we identified the key roles of ZFAS1 (lncRNA zinc finger antisense 1) in the differentiation of cardiomyocytes from hESCs. Methods: A model of cardiomyocyte differentiation from stem cells was established using the monolayer differentiation method, and the number of beating hESCs-derived cardiomyocytes was calculated. Gene expression was analyzed by quantitative real-time PCR (qRT-PCR). Immunofluorescence assays were performed to assess the expression of cardiac troponin T (cTnT) and α-actinin protein in cardiomyocytes. Results: qRT-PCR showed that ZFAS1 expression in the mesoderm was significantly higher than that in embryonic stem cells, cardiac progenitor cells, and cardiomyocytes. Knockdown of ZFAS1 inhibited cardiomyocyte differentiation from hESCs, which was characterized by reduced expression of the cardiac-specific markers cTnT, α-actinin, myosin heavy chain 6 (MYH6), and myosin heavy chain 7 (MYH7). In contrast, ZFAS1 overexpression remarkably increased the percentage of spontaneously beating cardiomyocytes. In terms of the mechanism, we found that ZFAS1 is an antisense lncRNA at the 5′ end of the protein-coding gene ZNFX1. Knockdown of ZFAS1 could increase the mRNA expression level of ZNFX1. Furthermore, qRT-PCR demonstrated that the silencing of ZNFX1 led to an increase in cardiac-specific markers that predicted the promotion of cardiomyocyte differentiation. Conclusion: Altogether, these data suggest that lncRNA-ZFAS1 is required for cardiac differentiation by functionally inhibiting the expression of ZNFX1, which may provide a reference for the treatment of heart disease to a certain extent.     

The extracellular secretion of <i>miR-1825</i> wrapped by exosomes increases <i>CLEC5A</i> expression: A potential oncogenic mechanism in ovarian cancer

Background: Ovarian cancer (OC) is a leading cause of gynecological cancer-linked deaths worldwide. Exosomal miR-1825 and its target gene C-type lectin domain family 5 member A (CLEC5A) are associated with tumorigenesis in cancers that was further probed. Methods: Exosomal miR-1825 expression in exosomes and its impact on overall survival (OS) prediction were determined using Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) data. Target genes of miR-1825 were searched in five prediction databases and prognostically significant differentially expressed genes were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were carried out. The ability of CLEC5A to predict OS was evaluated using univariate and multivariate Cox regression analyses and Kaplan-Meier curves. The CLEC5A expression pattern in OC was validated using immunohistochemistry. The CIBERSORT algorithm was used to compare the immune cell landscape, and the results were validated in a GEO cohort. Finally, the predicted half maximal inhibitory concentration (IC50) values for five commonly used chemotherapy agents were also compared. Results: MiR-1825 level was higher in exosomes derived from OC cells and served as a tumor suppressor. The CLEC5A gene was found to be a target of miR-1825, the upregulation of which was correlated with a poor prognosis. M2 macrophage infiltration was significantly enhanced in the CLEC5A high expression group, while T follicular helper cell infiltration was reduced in it. While the predicted IC50 for cisplatin and doxorubicin was higher in the CLEC5A high expression group, that of docetaxel, gemcitabine, and paclitaxel was lower. Conclusion: MiR-1825, a promising OC biomarker, may promote OC progression by increasing CLEC5A expression via exosome-mediated efflux from tumor cells.     

Expression analysis of <i>OsSERK</i>, <i>OsLEC1</i> and <i>OsWOX4</i> genes in rice (<i>Oryza</i> sativa L.) callus during somatic embryo development

Somatic embryogenesis is an asexual reproduction process that occurs in many plant species, including rice. This process contains several totipotency markers such as Somatic Embryogenesis Receptor-like Kinase (SERK), Leafy Cotyledon1 (LEC1) and WUSCHEL-Related Homeobox4 (WOX4) and also a helpful model for embryo development and clones and transformations. Here, we report the gene expression during somatic embryo development correlates with regeneration frequency in 14 Javanica rice (pigmented and non-pigmented) using modifified N6 media supplemented with Kinetin (2.0 mg/L) and NAA (1.0 mg/L). Although there have been advances in understanding the genetic basis of somatic embryogenesis in other varieties, rice is still unexplored, especially during somatic embryo development. Moreover, for the formation of callus induction from immature embryos, 2,4-D (2.0 mg/L, 3.0 mg/L) was used. This study analysed the gene expression of OsSERK, OsWOX4 and OsLEC1 genes through RT-PCR analysis. Higher expression of the OsLEC1 gene indicates that their function may correlate in the in vitro with the high response of rice after transfer to regeneration media. This study found that rice varieties of pigmented rice (MS Pendek and Gogoniti II) and non-pigmented rice (Pandan Ungu) showed high regeneration frequency, showing higher OsLEC1 expression than other varieties because OsLEC1 promotes the maturation of somatic embryos in plant regeneration on day 14. However, the contrast with Genjah nganjuk may be effective because of other regulatory genes. RT-PCR analysis showed OsSERK had less expression level than OsLEC1 and OsWOX4 in the varieties, which correlate with the percentage of plant regeneration, but not for Gogoniti II. In conclusion, the higher percentage of plant regeneration correlates with the higher expression level of OsLEC1 at day 14 of media regeneration of rice.     

<i>miR-103-3p</i> regulates the differentiation of bone marrow mesenchymal stem cells in myelodysplastic syndrome

The pathogenesis of myelodysplastic syndrome (MDS) may be related to the abnormal expression of microRNAs (miRNAs), which could influence the differentiation capacity of mesenchymal stem cells (MSCs) towards adipogenic and osteogenic lineages. In this study, exosomes from bone marrow plasma were successfully extracted and identified. Assessment of miR-103-3p expression in exosomes isolated from BM in 34 MDS patients and 10 controls revealed its 0.52-fold downregulation in patients with MDS compared with controls (NOR) and was downregulated 0.55-fold in MDS-MSCs compared with NOR-MSCs. Transfection of MDS-MSCs with the miR-103-3p mimic improved osteogenic differentiation and decreased adipogenic differentiation in vitro, while inhibition of miR-103-3p showed the opposite results in NOR-MSCs. Thus, the expression of miR-103-3p decreases in MDS BM plasma and MDS-MSCs, significantly impacting MDS-MSCs differentiation. The miR-103-3p mimics may boost MDS-MSCs osteogenic differentiation while weakening lipid differentiation, thereby providing possible target for the treatment of MDS pathogenesis.     

Targeting the “undruggable” cancer driver genes: <i>Ras</i>, <i>myc</i>, and <i>tp53</i>

The term “undruggable” is to describe molecules that are not targetable or at least hard to target pharmacologically. Unfortunately, some targets with potent oncogenic activity fall into this category, and currently little is known about how to solve this problem, which largely hampered drug research on human cancers. Ras, as one of the most common oncogenes, was previously considered “undruggable”, but in recent years, a few small molecules like Sotorasib (AMG-510) have emerged and proved their targeted anti-cancer effects. Further, myc, as one of the most studied oncogenes, and tp53, being the most common tumor suppressor genes, are both considered “undruggable”. Many attempts have been made to target these “undruggable” targets, but little progress has been made yet. This article summarizes the current progress of direct and indirect targeting approaches for ras, myc, two oncogenes, and tp53, a tumor suppressor gene. These are potential therapeutic targets but are considered “undruggable”. We conclude with some emerging research approaches like proteolysis targeting chimeras (PROTACs), cancer vaccines, and artificial intelligence (AI)-based drug discovery, which might provide new cues for cancer intervention. Therefore, this review sets out to clarify the current status of targeted anti-cancer drug research, and the insights gained from this review may be of assistance to learn from experience and find new ideas in developing new chemicals that directly target such “undruggable” molecules.     

Increased <i>MAD2L2</i> expression predicts poor clinical outcome in Colon Adenocarcinoma

Background: Colon adenocarcinoma (COAD) is the second leading cause of cancer death worldwide thus, identification of COAD biomarkers is critical. Mitotic Arrest Deficient 2 Like 2 (MAD2L2) is a key factor in mammalian DNA damage repair and is highly expressed in many malignant tumors. This is a comprehensive study of MAD2L2 expression, its diagnostic value, prognostic analysis, potential biological function, and impact on the immune system of patients with COAD. Methods: Gene expression, clinical relevance, prognostic analysis, diagnostic value, GO/KEGG cluster analysis, data obtained from TCGA, and bioinformatics statistical analysis were performed using the R package. Immune responses to MAD2L2 expression in COAD were analyzed using TIMER. The expression of MAD2L2 in HCT116 cells induced by the inflammatory factor TNF-α was detected using Western blot. Results: Our results underscore the clinical diagnostic value and potential biological significance of MAD2L2 in patients with COAD. A high level of MAD2L2 expression has been found in COAD and correlated with tumor status and colon polyps. ROC curve analysis showed that MAD2L2 expression has high diagnostic value in COAD. Analysis of immune infiltration results showed that MAD2L2 expression was positively correlated with neutrophil levels. The western blot results demonstrated that MAD2L2 was dose-dependently present with TNF-α. GO/KEGG revealed that MAD2L2 overexpressed and coexpressed genes were mostly involved in biological functions, including hypoxia response, response to reduced oxygen levels, mitochondrial translation elongation, and other processes. Conclusion: MAD2L2 as a new COAD biomarker contributes to our understanding of how alterations in gene expression and the immunological environment contribute to the development of colon cancer. Following further investigation, MAD2L2 may prove to be a viable target factor for clinical diagnosis and therapy of COAD.     

Co-ordinated combination of Embden-Meyerhof-Parnas pathway and pentose phosphate pathway in <i>Escherichia coli</i> to promote L-tryptophan production

In this study, phosphoenolpyruvate and erythrose-4-phosphate are efficiently supplied by collaborative design of Embden-Meyerhof-Parnas (EMP) pathway and pentose phosphate (PP) pathway in Escherichia coli, thus increasing the L-tryptophan production. Firstly, the effects of disrupting EMP pathway on L-tryptophan production were studied, and the results indicated that the strain with deletion of phosphofructokinase A (i.e., E. coli JW-5 ΔpfkA) produced 23.4 ± 2.1 g/L of L-tryptophan production. However, deletion of phosphofructokinase A and glucosephosphate isomerase is not conducive to glucose consumption and cell growth, especially deletion of glucosephosphate isomerase. Next, the carbon flux in PP pathway was enhanced by introduction of the desensitized glucose-6-phosphate dehydrogenase (zwf) and 6-phosphogluconate dehydrogenase (gnd) and thus increasing the L-tryptophan production (i.e., 26.5 ± 3.2 g/L vs. 21.7 ± 1.3 g/L) without obviously changing the cell growth (i.e., 0.41 h⁻¹ vs. 0.44 h⁻¹) as compared with the original strain JW-5. Finally, the effects of co-modifying EMP pathway and PP pathway on L-tryptophan production were investigated. It was found that the strain with deletion of phosphofructokinase A as well as introduction of the desensitized zwf and gnd (i.e., E. coli JW-5 zwf243 gnd361 ΔpfkA) produced 31.9 ± 2.7 g/L of L-tryptophan, which was 47.0% higher than that of strain JW-5. In addition, the glucose consumption rate of strain JW-5 zwf243 gnd361 ΔpfkA was obviously increased despite of the bad cell growth as compared with strain JW-5. The results of this study have important reference value for the following application of metabolic engineering to improve aromatic amino acids producing strains.     

A 66-kDa protein of bovine hypophyseal <i>Pars tuberalis</i> induces luteinizing hormone release from rat <i>Pars distalis</i>

In this study, evidence for a factor secreted by bovine hypophyseal pars tuberalis that stimulates luteinizing hormone (LH) release from rat pars distalis cells is shown. The secretion products of bovine pars tuberalis cells into the culture medium were assayed on dispersed rat pars distalis cells in 30 min incubations and superfusion experiments. The culture medium from pars tuberalis total cell populations, added at a dose of 6 μg per tube, induced the greater LH release from pars distalis cells, without effect on follicle stimulating hormone (FSH) release. After pars tuberalis cells separation on a discontinuos Percoll gradient, only the culture medium of cells from 50 and 60% strength Percoll were able to release LH from rat pars distalis cells. Therefore, cell fractions from 50 and 60% strenght Percoll were cultured together. To elicit maximal LH release (6 times the basal output), with the addition of 2 μg of pars tuberalis protein was required, suggesting that these cells produce the factor or factors which affect pars distalis gonadotrope cells. After applying the pars tuberalis culture medium on 12% SDS-PAGE, the band with biological activity was that of 66-kDal. Fifty ng protein of its eluate released almost 9 times the basal output of LH from pars distalis cells. Results suggest a modulating effect of a protein from the bovine pars tuberalis on rat cultured gonadotrope cells from the pars distalis.     

A novel prognostic gene signature,nomogram and immune landscape based on tanshinone IIA drug targets for hepatocellular carcinoma: Comprehensive bioinformatics analysis and <i>in vitro</i> experiments

Background: Tanshinone IIA, one of the main ingredients of Danshen, is used to treat hepatocellular carcinoma (HCC). However, potential targets of the molecule in the therapy of HCC are unknown. Methods: In this study, we collected the tanshinone IIA targets from public databases for investigation. We screened differentially expressed genes (DEGs) across HCC and normal tissues using mRNA expression profiles from The Cancer Genome Atlas (TCGA). Univariate Cox regression analysis and least absolute shrinkage and selection operator (LASSO) Cox regression models were used to identify and construct the prognostic gene signature. Results: Finally, we discovered common genes across tanshinone IIA targets and HCC DEGs. We reported Fatty acid binding protein-6 (FABP6), Polo-like Kinase 1 (PLK1), deoxythymidylate kinase (DTYMK), Uridine Cytidine Kinase 2 (UCK2), Enhancer of Zeste Homolog 2 (EZH2), and Cytochrome P450 2C9 (CYP2C9) as components of a gene signature. The six-gene signature’s prognostic ability was evaluated using the Kaplan-Meier curve, time-dependent receiver operating characteristic (ROC), multivariate Cox regression analysis, and the nomogram. The mRNA level and protein expression of UCK2 were experimentally validated after treatment with different concentrations of tanshinone IIA in HEPG2 cells. CIBERSORTx, TIMER2.0, and GEPIA2 tools were employed to explore the relationship between the prognostic signature and immune cell infiltration. Conclusion: We established a six-gene signature as a reliable model with significant therapeutic possibility for prognosis and overall survival estimation in HCC patients, which might also benefit medical decision-making for appropriate treatment.     

Immune strategies of phagoctytic cells stimulated <i>in vitro</i> with live and heat-inactivated <i>Streptococcus pyogenes</i>

Streptococcus pyogenes (group A Streptococcus) is frequently involved in a wide range of human diseases. Here we evaluated polymorphonuclear neutrophils and mononuclear cells from healthy subjects for their bactericidal function after stimulation with live and inactivated Streptococcus pyogenes (Streptococcus Group A). Mononuclear cells and Neutrophils were isolated from heparinized blood samples (n=18) using a Ficoll-Hypaque gradient and cultured in RPMI 1640 for 18 hours with a suspension of either live or inactivated Streptococcus pyogenes. Both the respiratory burst (flow cytometry) and nitrite, TNF and IL17 production (ELISA) were measured in the cell culture supernatants. An increased respiratory burst (expressed as R index) was induced by both live and inactivated bacteria. Also, increased nitrite, TNF and IL17 concentrations were found in cell culture supernatants in both cases. These findings may provide some explanation as to the roles played by neutrophils and mononuclear cells in Streptococcus pyogenes immunopathogenicity.     

Genome-wide identification of <i>U-box</i> gene family and expression analysis under abiotic stresses in <i>Salvia miltiorrhiza</i>

Plant U-box (PUB) E3 ubiquitin ligases play important roles in hormone signaling pathways and in response to different abiotic stresses, but little is known about U-box genes in Danshen (root of Salvia miltiorrhiza Bunge). Here, we identified and characterized 70 SmPUB genes based on its genome sequence. Phylogenetic analysis of U-box genes from S. miltiorrhiza and Arabidopsis suggested that they can be clustered into seven subgroups (I–VII). Typical U-box domains were found in all identified SmPUB genes through the analysis of conserved motifs. Moreover, qRT-PCR was applied to analyze the relative expression levels of U-box genes in S. miltiorrhiza roots and leaves under PEG-induced water deficit and salt stresses. Results revealed that the SmPUB genes exhibited stronger response to drought than to salt stress. To the best of our knowledge, this report is the first to perform genome-wide identification and analysis of the U-box gene family in S. miltiorrhiza, and the results provide valuable information for better understanding of the function of U-box in S. miltiorrhiza.     

The chloroplast genome comparative characteristic of artificial breeding tree,a case about <i>Broussonetia kazinoki</i> × <i>Broussonetia papyrifera</i>

Broussonetia kazinoki × Broussonetia papyrifera (ZJGS) is a hybrid species in Moraceae family, which has a very complicated hybrid origin. The excellent characteristics of fast growth, strong soil and water conservation ability, high leaf protein content and stem fiber content in ZJGS make it both ecological benefits in the mining area and economically valuable. This study aims to further understand ZJGS and other Moraceae taxa through the ZJGS chloroplast (cp) genome structure and the comparison with 12 closely related Moraceae species. Among the 13 Moraceae species, the cp genome length of seven Broussonetia species (ranges from 160,239 bp to 162,594 bp) is larger than that of six Morus species (ranges from 158,459 bp to 159,265 bp). Among the 77 shared protein-coding genes (PCGs) in Moraceae species, the obvious positive selection of Ka/Ks ratios acted on petD and rpl16 genes of B. kazinoki and B. papyrifera, respectively. Phylogenetic analysis based on shared PCGs from 28 species shows that ZJGS is closely related to maternal B. kazinoki. These findings provide data support for the origin of ZJGS hybridization and provide genomic resources for future ZJGS resource development and molecular breeding.     

<i>In vivo</i> protective effect of late embryogenesis abundant protein (ApSK₃ dehydrin) on <i>Agapanthus praecox</i> to promote post-cryopreservation survival

Dehydrins (DHNs), as members of the late embryogenesis abundant protein family, play critical roles in the protection of seeds from dehydration and plant adaptation to multiple abiotic stresses. Vitrification is a basic method in plant cryopreservation and is characterized by forming a glassy state to prevent lethal ice crystals produced during cryogenic storage. In this study, ApSK₃ type DHN was genetically transformed into embryogenic calluses (EC) of Agapanthus praecox by overexpression (OE) and RNA interference (RNAi) techniques to evaluate the in vivo protective effect of DHNs during cryopreservation. The cell viability showed a completely opposite trend in OE and RNAi cell lines, the cell relative death ratio was decreased by 20.0% in ApSK₃-OE EC and significantly increased by 66.15% in ApSK₃-RNAi cells after cryopreservation. Overexpression of ApSK₃ increased the content of non-enzymatic antioxidants (AsA and GSH) and up-regulated the expression of CAT, SOD, POD, and GPX genes, while ApSK₃-RNAi cells decreased antioxidant enzyme activities and FeSOD, POD, and APX genes expression during cryopreservation. These findings suggest that ApSK₃ affects ROS metabolism through chelating metal ions (Cu²⁺ and Fe³⁺), alleviates H₂O₂ and OH· excessive generation, activates the antioxidant system, and improves cellular REDOX balance and membrane lipid peroxidation damage of plant cells during cryopreservation. DHNs can effectively improve cell stress tolerance and have great potential for in vivo or in vitro applications in plant cryopreservation.