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.     

<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.     

<i>Agrobacterium rhizogenes</i> vs auxinic induction for <i>in vitro</i> rhizogenesis of <i>Prosopis chilensis</i> and <i>Nothofagus alpina</i>

The induction and improvement of in vitro rhizogenesis of microshoots of Prosopis chilensis (Mol.) Stuntz and Nothofagus alpina (Poep. et Endl. Oerst.) were compared using Agrobacterium rhizogenes (Ar) versus indole-3-butyric acid (IBA) in the culture media. Microshoots of P. chilensis (1-2 cm length), coming from in vitro grown seedlings, were cultivated in a modified Broadleaved Tree Medium (BTMm) containing half salt concentration of macronutrients and 0.05 mg.L^-1 benzilaminopurine (BAP). After 30 days, microshoots with 2-4 leaves were selected and cultured in BTMm-agar in presence or abscense of Ar and in combination with IBA. For N. alpina, the apical shoots with the first 2 true leaves, from 5 weeks old seedlings, were cultured in the abovementioned medium, but with 0.15 mg.L^-1 of BAP. After 2 months, microshoots with 2-3 leaves were selected and cultured in BTMm-agar, supplemented with 5 mg.L^-1 IBA or in liquid BTMm on perlite and, in the presence or absence of A. rhizogenes (Ar) and in combination with 3 mg.L^-1 IBA. Rooting in P. chilensis reached 100.0% when Ar infection was produced in the presence of IBA, increasing both, the number and dry weight of roots. In N. alpina, 90.0% of rooting efficiency was obtained when Ar infection was produced in liquid culture and in the absence of auxin.     

Tanshinone IIA protects intestinal epithelial cells from ferroptosis through the upregulation of <i>GPX4</i> and <i>SLC7A11</i>

Background: Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract. The destruction of the intestinal epithelial barrier is one of the major pathological processes in IBD pathology. Growing evidence indicated that epithelial cell ferroptosis is linked to IBD and is considered a target process. Methods: RAS-selective lethal 3 (RSL3) was used to induce ferroptosis in intestinal epithelial cell line No. 6 (IEC-6) cells, and cell ferroptosis and the effects of tanshinone IIA (Tan IIA) were determined by cell counting kit-8 (CCK-8), reactive oxygen species (ROS) staining, Giemsa staining and transmission electron microscope (TEM). The cell viability of natural product library compounds was determined by CCK-8. The expression of ferroptosis-related genes were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and western blot. Results: Treatment of IEC-6 cells results in the accumulation of ROS and typical morphological characteristics of ferroptosis. RSL3 treatment caused rapid cellular cytotoxicity which could be reversed by ferrostatin-1 (Fer-1) in IEC-6 cells. Natural product library screening revealed that Tan IIA is a potent inhibitor of IEC-6 cell ferroptosis. Tan IIA could significantly protect the RSL3-induced ferroptosis of IEC-6 cells. Furthermore, the ferroptosis suppressors, glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11), and miR-17-92 were found to be early response genes in RSL3-treated cells. Treatment of IEC-6 cells with Tan IIA resulted in upregulation of GPX4, SLC7A11, and miR-17-92. Conclusion: Our study demonstrated that Tan IIA protects IEC-6 cells from ferroptosis through the upregulation of GPX4, SLC7A11, and miR-17-92. The findings might provide a theoretical grounding for the future application of Tan IIA to treat or prevent IBD.     

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.     

Synergistic combination of colistin with imipenem,amikacine or ciprofloxacin against <i>Acinetobacter baumannii</i> and <i>Pseudomonas aeruginosa</i> carbapenem-resistant isolated in Annaba hospital Algeria

Objective: The aim of this study is to detect in vitro the synergetic activity of colistin in combination with imipenem, amikacin or ciprofloxacin, at sub-inhibitory concentrations, against carbapenems-resistant (CR) Acinetobacter baumannii and Pseudomonas aeruginosa strains isolated from various wards in Annaba teaching hospital in eastern Algeria.
Materials and Methods: The minimal inhibitory concentrations (MIC) were determined by broth macrodilution (BMD). Carbapenemase encoding genes were screened using polymerase chain reaction (PCR). The activity of colistin in combination with second antibiotic was evaluated by the Checkerboard Technique.
Results: 39 CR P. aeruginosa and 21 CR A. baumanni strains where collected. The MIC values ranging from (0.25 to 4 µg/ml) to colistin, ≥16 µg/ml for imipenem, ≥4 µg/ml to amikacin and ≥8 µg/ml ciprofloxacin. The PCR reveals the presence of the genes bla_OXA23 (n = 12), blaOXA24 (n = 6), blaNDM1 (n = 3) in A. baumannii and blaVIM2 (n = 12) in P. aeruginosa. The combination of colistin with imipenem showed synergistic effect on 57.14% and 46.15% of A. baumannii and P. aeruginosa isolates, respectively. For colistin and amikacin, the synergistic effect is detected in 28.6% of A. baumannii and 30.8% of P. aeruginosa. While colistin and ciprofloxacin showed synergy on 14.29% and 15.38% of A. baumannii and P. aeruginosa isolates, respectively.
Conclusion: CR A. baumannii and P. aeruginosa remain the most prevalent infection agents in patients from high-risk wards at Annaba Hospital. Colistin associated with imipenem or with amikacin at sub-inhibitory concentrations gives very encouraging results allowing better management of infections caused by this type of bacteria.     

Selection and validation of reference genes for quantitative real-time polymerase chain reaction analyses of <i>Serratia ureilytica</i> DW2

Background: Serratia ureilytica DW2 is a highly efficient phosphate-solubilizing bacteria isolated from Codonopsis pilosula rhizosphere soil that can promote the growth of C. pilosula; nonetheless, until now, no validated reference genes from the genus Serratia have been reported that can be used for the normalization of quantitative real-time polymerase chain reaction (RT–qPCR) data. Methods: To screen stable reference genes of S. ureilytica DW2, the expression of its eight candidate reference genes (16S rRNA, ftsZ, ftsA, mreB, recA, slyD, thiC, and zipA) under different treatment conditions (pH, temperature, culture time, and salt content) was assayed by RT–qPCR. The expression stability of these genes was analyzed using different algorithms (geNorm, NormFinder, and BestKeeper). To verify the reliability of the data, the expression of the glucose dehydrogenase (gdh) gene under different soluble phosphate levels was quantified using the most stably expressed reference gene. Results: The results showed that the zipA and 16S rRNA genes were the most stable reference genes, and the least stable genes were thiC and recA. The expression of gdh was consistent with the phosphate solubilization ability on plates containing the National Botanical Research Institute phosphate growth medium. Conclusion: Therefore, this study provides a stable and reliable reference gene of Serratia for the accurate quantification of functional gene expression in future studies.     

<i>Trypanosoma rangeli:</i> growth in mammalian cells <i>in vitro</i> and action of a repositioned drug (17-AAG) and a natural extract (<i>Artemisia sp</i>. essential oil)

Trypanosoma rangeli and T. cruzi are both parasitic unicellular species that infect humans. Unlike T. cruzi, the causative agent of Chagas disease, T. rangeli is an infective and non-pathogenic parasite for humans, but pathogenic for vectors from the Rhodnius genus. Because both species can coexist in different hosts and overlap their infective cycles but very little is known about the infection of T. rangeli in mammalian cells, we decided to characterize both the development of this parasite in cell culture and the effect of therapeutic agents with potential trypanocidal action on it. We found that T. rangeli exhibits a cycle of infection in Vero cells similar to that for T. cruzi and that the repurposed drug, 17-AAG, and the natural extract Artemisia sp. essential oil produce a toxic effect on epimastigotes showing a trypanocidal action from the fifth day of culture. Both treatments also affected the infection of trypomastigotes and reduced the capacity of replication of amastigotes of T. rangeli. Since T. cruzi / T. rangeli coinfection cases have been reported, the finding of drugs with potential activity against both species could be significant in the future. Furthermore, studies of susceptibility of both species to drugs could also help to know the different mechanisms of pathogenicity in humans displayed by T. cruzi that are absent in T. rangeli     

Antiproliferative effect of extracts of <i>Sida rhombifolia</i> L. (Malvaceae) on the <i>Allium cepa</i> cell cycle

Field collected roots of four populations of Sida rhombifolia were used for preparing aqueous decoctions at two concentrations: 4g/L; and 16g/L. Afterwards, we used three groups of six onion (Allium cepa) bulbs for testing each population. Slides were made with all bulbs through the smashing technique. Cells in all phases of the cell cycle of A. cepa were analyzed. The mitotic index (% of cells in mitosis) was calculated, and the statistical analysis through the χ2 test was carried out at 5% probability. The results showed that the aqueous extracts of S. rhombifolia have antiproliferative activity at high concentrations. Practically no chromosomal aberrations were induced by treatments.     

MicroRNA-382 inhibits the proliferation of mouse spermatogonia by targeting <i>Kmt5a</i>

Spermatogenesis is a highly efficient and intricate process in the testis by which mature spermatozoa are produced daily to maintain lifelong male fertility. Essential to this process are spermatogonia capable of both proliferation and differentiation. Nevertheless, the underlying mechanisms for spermatogonial proliferation and differentiation remain poorly understood. MicroRNAs (miRNAs) are a category of non-coding small RNAs with regulatory functions by binding to the 3’ untranslated region (UTR) of the target mRNA. Previous studies have demonstrated that miRNAs are capable of modulating cell proliferation, differentiation and apoptosis, but the roles of individual miRNAs in spermatogonial fate determination remain largely elusive. Here, by using a mouse spermatogonial cell line (GC-1), we investigated the role for miRNA-382 in spermatogonial proliferation. We found that pre-miRNA-382 was expressed in spermatogonia. The luciferase reporter assay demonstrated Kmt5a but not Top1 as a target gene of miRNA-382. Overexpression of miRNA-382 by transfecting a miRNA mimic downregulated Kmt5a at both RNA and protein levels, and further reduced the proliferation and viability of spermatogonia. Knockdown of Kmt5a by RNA interference (RNAi) resulted in a uniform phenotype in spermatogonia. We therefore conclude that miRNA-382 inhibits the proliferation of mouse spermatogonia by targeting Kmt5a. Our finding extends the knowledge about the regulatory roles of miRNAs in spermatogonia and lays the groundwork for diagnosis and treatment of male infertility.     

Exploration of the oxidative-inflammatory potential targets of <i>Coicis Semen</i> in osteoarthritis: Data mining and systematic pharmacology

Objective: On the basis of data mining, systematic pharmacology, molecular docking, and experiment validation, the oxidative-inflammatory molecular targets of Coicis Semen in the therapy of osteoarthritis (OA) were explored. Methods: The association rule analysis was effectively applied to highlight the correlation between Coicis Semen and oxidative inflammation indices. The random walk model was subsequently used to evaluate the clinical efficacy of Coicis Semen. Network pharmacology was used to predict network targets. The binding affinity of the active ingredient in Coicis Semen to the key target of OA was also successfully predicted. Results: Coicis Semen showed a significant reduction in oxidative-inflammatory indicators of OA. A total of 108 promising targets were predicted for the 24 bioactive compounds in Coicis Semen. Eight target genes were considered core target genes. The enrichment analysis predicts that Coicis Semen may activate the interleukin (IL)-17, mitogen-activated protein kinase (MAPK), and nuclear factor kappa B (NF-kappa B) signaling pathways. Molecular docking demonstrated that stigmasterol, 2-monoolein, sitosterol, and sitosterol alpha1 had free binding energies to oxidative and inflammatory targets (MAPK1, Estrogen Receptor 1 [ESR1], and Peroxisome Proliferator-Activated Receptor Alpha [PPARA]). Both clinical trials and in vitro cell experiments revealed that Coicis Semen could increase ESR1 and PPAR-α levels while decreasing MAPK1 levels. Conclusions: Coicis Semen has a remarkable anti-OA effect. Precisely, the major components of Coicis Semen, including stigmasterol, sitosterol alpha1, sitosterol, and 2-monoolein, specifically inhibit MAPK1, ESR1, and PPARA to reduce the inflammatory response and oxidative damage in OA.     

Response of <i>MaHMA2</i> gene expression and stress tolerance to zinc stress in mulberry (<i>Morus alba</i> L.)

HMA2 (heavy metal ATPase 2) plays a crucial role in extracellular and intracellular Zn²⁺ transport across biomembranes, maintaining ion homeostasis, and playing an important role in the normal physiological metabolism, growth, and development of plants. In our study, a novel HMA2 gene, named MaHMA2, was isolated and cloned from white mulberry (Morus alba L.). The gene sequence obtained was 1,342 bp long, with an open reading frame of 1,194 bp, encoding a protein of 397 amino acids, with a predicted molecular mass of 42.852 kD and an isoelectric point of 7.53. This protein belonged to the PIB-type ATPase transport protein family. We analyzed the expression of the MaHMA2 gene by quantitative real-time PCR. The results showed that the level of MaHMA2 gene expression decreased to a Zn concentration of 800 mg/kg. Malondialdehyde and proline levels increased and responded to increasing Zn when the MaHMA2 gene was silenced, whereas the activities of peroxidase and superoxide dismutase tended to increase in response to increasing Zn²⁺ ion stress concentrations but were lower in the gene-silenced plants. These findings suggested that the MaHMA2 gene played an active role in the tolerance response of mulberry to Zn stress.     

<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.     

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.