Target Genes Research Articles

Gene Targeting via CRISPR/Cas9-Mediated DNA Double-Strand Break Induction in Rice.

Gene targeting (GT) is a precise genome editing tool to achieve desired modification of a target gene, e.g., introduction of point mutations, knock-in of a reporter gene, or swapping of a functional domain, through homologous recombination. However, due to its low frequency, it has proved difficult to establish a universal GT system. The availability of the CRISPR/Cas9 system for genome editing in plants has opened up possibilities to apply GT successfully to some plant species. Here, we provide protocols for CRISPR/Cas9-mediated DNA double-strand break (DSB)-induced GT in rice.

CD137 Protein Expression Pattern Determines the Functional Role of Galectin-9 in Colorectal Cancer.

The rapid advancement of single-cell sequencing technology has generated extensive data, providing critical resources for colorectal cancer (CRC) research. This study conducts a detailed analysis of CRC single-cell sequencing data to develop a novel clinical prognostic tool and explore potential therapeutic targets for the LGALS9 gene. Using the Scissor algorithm, we created a CRC prognostic scoring system (SDRS) based on 13 key genes, with particular focus on LGALS9 and its protein, Galectin-9, in mice CRC model with altered CD137 expression. Our findings demonstrate that the SDRS accurately reflects clinical and pathological features of CRC patients, acting as an independent predictor of outcomes. LGALS9 expression is generally reduced in CRC tissues and is associated with poorer prognosis. We also observed a strong positive correlation between LGALS9 and CD137 expression, with CD137 showing significant variability in CRC tissues. In mouse models with CD137 overexpression, Galectin-9 treatment led to notable antitumor effects and increased infiltration of activated T cells. In contrast, in CD137-deficient models, Galectin-9 promoted tumor growth with limited T cell presence. These results suggest that the role of LGALS9 in CRC may depend on CD137 expression, highlighting the potential of LGALS9 as a therapeutic target. CD137 levels may serve as a key indicator for predicting the effectiveness of this treatment strategy.

Superior target genes and pathways for RNAi-mediated pest control revealed by genome-wide analysis in the beetle Tribolium castaneum.

An increasing human population, the emergence of resistances against pesticides and their potential impact on the environment call for the development of new eco-friendly pest control strategies. RNA interference (RNAi)-based pesticides have emerged as a new option with the first products entering the market. Essentially, double-stranded RNAs targeting essential genes of pests are either expressed in the plants or sprayed on their surface. Upon feeding, pests mount an RNAi response and die. However, it has remained unclear whether RNAi-based insecticides should target the same pathways as classic pesticides or whether the different mode-of-action would favor other processes. Moreover, there is no consensus on the best genes to be targeted. We performed a genome-wide screen in the red flour beetle to identify 905 RNAi target genes. Based on a validation screen and clustering, we identified the 192 most effective target genes in that species. The transfer to oral application in other beetle pests revealed a list of 34 superior target genes, which are an excellent starting point for application in other pests. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analyses of our genome-wide dataset revealed that genes with high efficacy belonged mainly to basic cellular processes such as gene expression and protein homeostasis - processes not targeted by classic insecticides. Our work revealed the best target genes and target processes for RNAi-based pest control and we propose a procedure to transfer our short list of superior target genes to other pests. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Reducing competition between msd and genomic DNA improves retron editing efficiency.

Retrons, found in bacteria and used for defense against phages, generate a unique molecule known as multicopy single-stranded DNA (msDNA). This msDNA mimics Okazaki fragments during DNA replication, making it a promising tool for targeted gene editing in prokaryotes. However, existing retron systems often exhibit suboptimal editing efficiency. Here, we identify the msd gene in Escherichia coli, which encodes the noncoding RNA template for msDNA synthesis and carries the homologous sequence of the target gene to be edited, as a critical bottleneck. Sequence homology causes the msDNA to bind to the msd gene, thereby reducing its efficiency in editing the target gene. To address this issue, we engineer a retron system that tailors msDNA to the leading strand of the plasmid containing the msd gene. This strategy minimizes msd gene editing and reduces competition with target genes, significantly increasing msDNA availability. Our optimized system achieves very high retron editing efficiency, enhancing performance and expanding the potential for in vivo techniques that rely on homologous DNA synthesis.

YTHDF2 upregulation and subcellular localization dictate CD8 T cell polyfunctionality in anti-tumor immunity.

RNA methylation is an important regulatory process to determine immune cell function but how it affects the anti-tumor activity of CD8 T cells is not fully understood. Here we show that the N6-methyladenosine (m6A) RNA reader YTHDF2 is highly expressed in early effector or effector-like CD8 T cells. We find that YTHDF2 facilitates nascent RNA synthesis, and m6A recognition is fundamental for this distinctively nuclear function of the protein, which also reinforces its autoregulation at the RNA level. Loss of YTHDF2 in T cells exacerbates tumor progression and confers unresponsiveness to PD-1 blockade in mice and in humans. In addition to initiating RNA decay that is necessary for mitochondrial fitness, YTHDF2 orchestrates chromatin changes that promote T cell polyfunctionality. YTHDF2 interacts with IKZF1/3, which is important for sustained transcription of their target genes. Accordingly, immunotherapy-induced efficacy could be largely restored in YTHDF2-deficient T cells through combinational use of IKZF1/3 inhibitor lenalidomide in a mouse model. Thus, YTHDF2 coordinates epi-transcriptional and transcriptional networks to potentiate T cell immunity, which could inform therapeutic intervention.

Comparison Between Electroporation at Different Voltage Levels and Microinjection to Generate Porcine Embryos with Multiple Xenoantigen Knock-Outs

In the context of xenotransplantation, the production of genetically modified pigs is essential. For several years, knock-out pigs were generated through somatic cell nuclear transfer employing donor cells with the desired genetic modifications, which resulted in a lengthy and cumbersome procedure. The CRISPR/Cas9 system enables direct targeting of specific genes in zygotes directly through microinjection or electroporation. However, these techniques require improvement to minimize mosaicism and low mutation rates without compromising embryo survival. This study aimed to determine the gene editing potential of these two techniques to deliver multiplexed ribonucleotide proteins (RNPs) to generate triple-knock-out porcine embryos with a multi-transgenic background. We designed RNP complexes targeting the major porcine xenoantigens GGTA1, CMAH, and B4GALNT2. We then compared the development of mosaicism and gene editing efficiencies between electroporation and microinjection. Our results indicated a significant effect of voltage increase on molecule intake in electroporated embryos, without it notably affecting the blastocyst formation rate. Our gene editing analysis revealed differences among delivery approaches and gene loci. Notably, employing electroporation at 35 V yielded the highest frequency of biallelic disruptions. However, mosaicism was the predominant genetic variant in all RNP delivery methods, underscoring the need for further research to optimize multiplex genome editing in porcine zygotes.

Effects of changes in SHP2 expression on liver fibrosis by influencing the apoptosis of hepatic stellate cells.

Accumulating research has revealed that src-homology domain 2-containing protein tyrosine phosphatase-2 (SHP2), an oncogenic protein tyrosine phosphatase, is associated with liver fibrosis. Currently, it is still unclear whether SHP2 affects liver fibrosis by influencing the apoptosis of hepatic stellate cells (HSC). In present study, we investigate effects of SHP2 expression changes on liver fibrosis, with special emphasis on the apoptosis of HSC. Using adenovirus vector, wild-type SHP2 gene and short hairpin RNA targeting SHP2 were introduced into rats with liver fibrosis and LX-2 cells in vitro. The expressions of type I and III collagen, pathological and functional changes, collagen deposition in rat liver and apoptosis of LX-2 cells were detected by immunohistochemical and HE staining, automated biochemical analyzer, Masson trichrome staining, and TUNEL. This study showed that overexpression of SHP2 exacerbated dysfunction, inflammatory damage, collagen deposition and increased expression of type I and III collagen in rat liver reduced apoptosis of LX-2 cells. On the contrary, low expression of SHP2 alleviated the aforementioned detection indicators of rats and promoted apoptosis of LX-2 cells. In conclusion, the downregulation of SHP2 expression alleviates liver fibrosis by inducing the apoptosis of HSC, while overexpressed SHP2 exacerbates liver fibrosis by inhibiting the apoptosis of HSC.

High-throughput sequencing reveals twelve cell death pattern prognostic target genes as potential drug-response-associated genes in the treatment of colorectal cancer cells with palmatine hydrochloride

Abstract Objectives Palmatine Hydrochloride (PaH), an isoquinoline alkaloid from Phellodendron amurense and Coptis chinensis, has analgesic, anti-inflammatory, and anticancer properties. This study aimed to assess PaH’s effectiveness against SW480 colorectal cancer (CRC) cells and explore its molecular mechanisms. Methods PaH’s effects on SW480 CRC cells were evaluated using MTT assays for proliferation, scratch assays for migration, and flow cytometry for apoptosis. Differentially expressed genes (DEGs) were identified through high-throughput sequencing. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses assessed DEG roles. Prognostic significance related to programmed cell death (PCD) was analyzed using R-Package with TCGA data. RT-qPCR validated key genes identified. Results PaH significantly inhibited SW480 cell growth, invasion, and apoptosis. The MTT assay showed inhibition rates increased from 5.49 % at 25 μg/mL to 52.48 % at 400 μg/mL. Scratch assays indicated reduced cell invasion over 24, 48, and 72 h. Apoptosis rose from 12.36 % in controls to 45.54 % at 400 μg/mL. Sequencing identified 3,385 significant DEGs, primarily in cancer pathways (p=0.004). Among 35 PCD-related DEGs, Lasso Cox regression highlighted 12 key genes, including TERT, TGFBR1, WNT4, and TP53. RT-qPCR confirmed TERT and TGFBR1 downregulation (0.614-fold, p=0.008; 0.41-fold, p<0.001) and TP53 and WNT4 upregulation (5.634-fold, p<0.001; 5.124-fold, p=0.002). Conclusions PaH inhibits CRC cell proliferation, migration, and invasion by modulating key PCD genes, suggesting its potential as a CRC therapeutic agent.

MiR-301b-3p promotes breast cancer development through inhibiting the expression of transforming growth factor-beta receptor 2

Background Breast cancer (BC) is a serious health threat to the patients. The present work explored the mechanism of miR-301b-3p and transforming growth factor-beta receptor 2 (TGFBR2 ) in affecting BC progression. Methods The miR-301b-3p-inhibitor and si-TGFBR2 solution were added to the DEME/F12 medium to culture the BC and normal breast epithelial cell lines to prepare negative control, miR-301b-3p-IN and miR-301b-3p-IN+si-TGFBR2 in the two types of cell lines. The relative expression of target genes and the interference effect were analyzed by quantitative real-time PCR (qRT- PCR). Cell viability was detected applying cell counting kit-8 (CCK-8) assay. Transwell and wound healing assay were conducted to evaluate the invasion and migration of BC cells after miR-301b-3p inhibition. Additionally, cell apoptosis and the expression STAT protein were measured by flow cytometry and Western blot, respectively Results The qRT-PCR results showed that miR-301b-3p were high-expressed but the level of TGFBR2 was significantly inhibited in BC cells. The miR-301b-3p-inhibitor significantly downregulated the expression of miR-301b-3p and upregulated that of TGFBR2. Meanwhile, inhibition of miR-301b-3p suppressed the cell viability, invasion, and migration of BC cells, which, however, were restored by the inhibition of TGFBR2. MiR-301b-3p conferred anti-apoptosis ability to BC cells, while TGFBR2 promoted apoptosis of BC cells through producing an antagonistic effect with miR-301b-3p. We found that miR-301b-3p played a crucial role in the phosphorylation of STAT1 and STAT3 to promote BC progression. Conclusion The present findings demonstrated that miR-301b-3p played a crucial role in promoting BC cell growth, invasion and migration and anti-apoptosis, and that targeting TGFBR2 could inhibit the tumor-promoting effect of miR-301b-3p.

TMEM206 Contributes to Cancer Hallmark Functions in Colorectal Cancer Cells and Is Regulated by p53 in a p21-Dependent Manner

Acid-induced ion flux plays a role in pathologies where tissue acidification is prevalent, including cancer. In 2019, TMEM206 was identified as the molecular component of acid-induced chloride flux. Localizing to the plasma membrane, TMEM206 contributes to cellular processes like acid-induced cell death. Since over 50% of human cancers carry loss of function mutations in the p53 gene, we aimed to analyze how TMEM206 is regulated by p53 and its role in cancer hallmark function and acid-induced cell death in HCT116 colorectal cancer (CRC) cells. We generated p53-deficient HCT116 cells and assessed TMEM206-mediated Cl− currents and transcriptional regulation using the patch-clamp and a dual-luciferase reporter assay, respectively. To investigate the contribution of TMEM206 to cancer hallmark functions, we performed migration and metabolic activity assays. The role of TMEM206 in p53-mediated acid-induced cell death was assessed with cell death assays. The TMEM206 mRNA level was significantly elevated in human primary CRC tumors. TMEM206 knockout increased acid-induced cell death and reduced proliferation and migration, indicating a role for TMEM206 in these cancer hallmark functions. Furthermore, we observed increased TMEM206 mRNA levels and currents in HCT116 p53 knockout cells. This phenotype can be rescued by transient overexpression of p53 but not by overexpression of dysfunctional p53. In addition, our data suggest that TMEM206 may mediate cancer hallmark functions within p53-associated pathways. TMEM206 promoter activity is not altered by p53 overexpression. Conversely, knockout of p21, a major target gene of p53, increased TMEM206-mediated currents, suggesting expression control of TMEM206 by p21 downstream signaling. Our results show that in colorectal cancer cells, TMEM206 expression is elevated, contributes to cancer hallmark functions, and its regulation is dependent on p53 through a p21-dependent mechanism.

Integrative analysis based on ATAC-seq and RNA-seq reveals a novel oncogene PRPF3 in hepatocellular carcinoma.

Assay of Transposase Accessible Chromatin Sequencing (ATAC-seq) is a high-throughput sequencing technique that detects open chromatin regions across the genome. These regions are critical in facilitating transcription factor binding and subsequent gene expression. Herein, we utilized ATAC-seq to identify key molecular targets regulating the development and progression of hepatocellular carcinoma (HCC) and elucidate the underlying mechanisms. We first compared chromatin accessibility profiles between HCC and normal tissues. Subsequently, RNA-seq data was employed to identify differentially expressed genes (DEGs). Integrating ATAC-seq and RNA-seq data allowed the identification of transcription factors and their putative target genes associated with differentially accessible regions (DARs). Finally, functional experiments were conducted to investigate the effects of the identified regulatory factors and corresponding targets on HCC cell proliferation and migration. Enrichment analysis of DARs between HCC and adjacent normal tissues revealed distinct signaling pathways and regulatory factors. Upregulated DARs in HCC were enriched in genes related to the MAPK and FoxO signaling pathways and associated with transcription factor families like ETS and AP-1. Conversely, downregulated DARs were associated with the TGF-β, cAMP, and p53 signaling pathways and the CTCF family. Integration of the datasets revealed a positive correlation between specific DARs and DEGs. Notably, PRPF3 emerged as a gene associated with DARs in HCC, and functional assays demonstrated its ability to promote HCC cell proliferation and migration. To the best of our knowledge, this is the first report highlighting the oncogenic role of PRPF3 in HCC. Furthermore, ZNF93 expression positively correlated with PRPF3, and ChIP-seq data indicated its potential role as a transcription factor regulating PRPF3 by binding to its promoter region. This study provides a comprehensive analysis of the epigenetic landscape in HCC, encompassing both chromatin accessibility and the transcriptome. Our findings reveal that ZNF93 promotes the proliferation and motility of HCC cells through transcriptional regulation of a novel oncogene, PRPF3.

Development and Application of a TaqMan RT-qPCR for the Detection of Foot-and-Mouth Disease Virus in Pigs

The global livestock industry is facing a serious threat from a widespread foot-and-mouth disease virus (FMDV) epidemic. The timely detection of FMDV can significantly mitigate its harmful effects. This study aimed to establish and evaluate a TaqMan fluorescence quantitative PCR assay to assess its sensitivity, specificity, reproducibility, and stability. The standard curve equation range is 6.43 × 109–6.43 × 101 copies/µL, with an R2 value of 0.996 and a standard curve equation of y = −3.586x + 36.245. The method successfully detected 64.3 copies/µL of the target gene for FMDV and exhibited high specificity for FMDV. Repeatability tests demonstrated low coefficients of variation within and between groups (<2%), indicating good reproducibility. The clinical samples analyzed using this method showed results consistent with those of the SYBR Green I RT-qPCR assay, confirming the reliability of the method. Overall, the developed test method displayed high sensitivity, specificity, reproducibility, and stability, making it suitable for the rapid diagnosis of foot-and-mouth disease in clinical settings.

The role of lncRNA TSIX in osteoarthritis pathogenesis: mechanistic insights and clinical biomarker potential

BackgroundThis study seeks to elucidate the expressions of lncRNA TSIX in Osteoarthritis (OA) and to explore its mechanisms in regulating OA progression.MethodsRT-qPCR was employed to analyze the expression of TSIX in OA patients classified by Kellgren-Lawrence (K-L) grades. Receiver operator characteristic (ROC) was conducted to evaluate the diagnostic value of TSIX. Correlation between TSIX levels and clinical scores such as Lysholm and visual analogue scale (VAS) score was evaluated using Pearson method. IL-1β-induced SW1353 cells served as an in vitro model. The cell function were assessed by flow cytometry and cell counting kit-8 (CCK-8) assay. The relationship between TSIX and miR-320a was verified by luciferase reporting system, while bioinformatics approaches were utilized to predict the downstream target genes of miR-320a.ResultsThe findings revealed that TSIX level in OA patients was elevated compared to that of the control group, with a notable progressive increase in TSIX expression correlated with higher K-L grades. In OA patients, the Lysholm score showed a negative correlation with TSIX expression, while the VAS score displayed a positive correlation with TSIX levels. Cell studies demonstrated that inhibition of TSIX enhanced cell viability and mitigated IL-1β-induced apoptosis by targeting miR-320a, in addition to promoting Aggrecan and Collagen II secretion. Luciferase reporter assay further validated the targeting interaction among TSIX, miR-320a, and PTEN.ConclusionsThis study demonstrated an increased expression of TSIX in OA patients. It suggests that TSIX may play a role in chondrocyte dysfunction during OA by modulating the miR-320a/PTEN axis.

High-Yield-Related Genes Participate in Mushroom Production

In recent years, the increasing global demand for mushrooms has made the enhancement of mushroom yield a focal point of research. Currently, the primary methods for developing high-yield mushroom varieties include mutation- and hybridization-based breeding. However, due to the long breeding cycles and low predictability associated with these approaches, they no longer meet the demands for high-yield and high-quality varieties in the expansive mushroom market. Modern molecular biology technologies such as RNA interference (RNAi) and gene editing, including via CRISPR-Cas9, can be used to precisely modify target genes, providing a new solution for mushroom breeding. The high-yield genes of mushrooms can be divided into four categories based on existing research results: the genes controlling mycelial growth are very suitable for genetic modification; the genes controlling primordium formation are directly or indirectly regulated by the genes controlling mycelial growth; the genes controlling button germination are more difficult to modify; and the genes controlling fruiting body development can be regulated during the mycelial stage. This article reviews the current research status for the four major categories of high-yield-related genes across the different stages of mushroom growth stages, providing a foundation and scientific basis for using molecular biology to improve mushroom yield and promote the economic development of the global edible-mushroom industry.

The long non-coding RNA lncRNA-DRNR enhances infectious bronchitis virus replication by targeting chicken JMJD6 and modulating interferon-stimulated genes expression via the JAK-STAT signalling pathway.

Infectious bronchitis virus (IBV) is the causative agent of infectious bronchitis (IB), a severe disease that primarily affects young chickens and poses a significant challenge to the global poultry industry. Understanding the complex interaction between the virus and its host is vital for developing innovative antiviral strategies. Long non-coding RNA (lncRNA) plays a crucial role in regulating host antiviral immune responses. Our previous studies have shown that IBV infection disrupts the stability of lncRNA in host cells, indicating a potential regulatory role for lncRNA in IBV pathogenesis. It is still not clear how lncRNA precisely modulates IBV replication. In this study, we observed down-regulation ofMSTRG.26120.58 (named lncRNA-DRNR) expression in various chicken cell lines upon IBV infection. We demonstrated that silencing lncRNA-DRNR using siRNA enhances intracellular replication of IBV. Through exploring genes encoding proteins upstream and downstream of lncRNA-DRNR within a 100kb range, we identified chJMJD6 (chicken JMJD6) as a potential target gene negatively regulated by lncRNA-DRNR expression levels. Furthermore, chJMJD6 inhibits STAT1 methylation, thereby affecting the induction of interferon-stimulated genes (ISGs) through the activation of the IFN-β-mediated JAK-STAT signalling pathway, ultimately promoting the intracellular replication of IBV. In summary, our findings reveal the critical role played by lncRNA-DRNR during IBV infection, providing novel insights into mechanisms underlying coronavirus-induced disruption in lncRNA stability.

Genome-Wide Characterization of Class III Peroxidases and Their Expression Profile During Mycorrhizal Symbiosis and Phosphorus Deprivation in Lettuce (Lactuca sativa L.)

Lettuce cultivation requires high fertilizer inputs, which impact the environment and costs. Arbuscular mycorrhizal symbiosis (AMS) can reduce fertilizer use, enhance plant nutrition (especially phosphorus), and promote healthier plants. Class III peroxidases (PRXs) play crucial roles in various physiological processes and stress responses. However, their role in AMS and phosphorous (P) deficiency is still unclear. Our study identified 91 PRX genes in the lettuce genome (LsPRXs) and clustered them into eight subfamilies based on phylogenetic relationships. Evolutionary analysis indicated that tandem duplication was the main driver for LsPRX gene family expansion. Synteny analysis showed orthologous relationships of the PRX gene family between lettuce and potato, Arabidopsis, and maize, identifying 39, 28, and 3 shared PRXs, respectively. Transcriptomic data revealed that most LsPRX genes were more expressed in roots than in leaves and differentially expressed LsPRXs were found in response to AMS and P supply. Notably, 15% of LsPRX genes were differentially expressed in roots during mycorrhization. Gene expression network analysis highly correlated five LsPRXs (LsPRX17, LsPRX23, LsPRX24, LsPRX64, and LsPRX79) with genes involved in cell wall remodeling and reorganization during mycorrhization. Our results provide insights into the evolutionary history and functional roles of PRX genes in lettuce and identify candidate gene targets that may enhance the bio-stimulant effects of AMS and help to cope with P deficiency.

The molecular consequences of FOXF1 missense mutations associated with alveolar capillary dysplasia with misalignment of pulmonary veins

BackgroundAlveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV) is a fatal congenital lung disorder strongly associated with genomic alterations in the Forkhead box F1 (FOXF1) gene and its regulatory region. However, little is known about how FOXF1 genomic alterations cause ACD/MPV and what molecular mechanisms are affected by these mutations. Therefore, the effect of ACD/MPV patient-specific mutations in the FOXF1 gene on the molecular function of FOXF1 was studied.MethodsEpitope-tagged FOXF1 constructs containing one of the ACD/MPV-associated mutations were expressed in mammalian cell lines to study the effect of FOXF1 mutations on protein function. EMSA binding assays and luciferase assays were performed to study the effect on target gene binding and activation. Immunoprecipitation followed by SDS‒PAGE and western blotting were used to study protein‒protein interactions. Protein phosphorylation was studied using phos-tag western blotting.ResultsAn overview of the localization of ACD/MPV-associated FOXF1 mutations revealed that the G91-S101 region was frequently mutated. A three-dimensional model of the forkhead DNA-binding domain of FOXF1 showed that the G91-S101 region consists of an α-helix and is predicted to be important for DNA binding. We showed that FOXF1 missense mutations in this region differentially affect the DNA binding of the FOXF1 protein and influence the transcriptional regulation of target genes depending on the location of the mutation. Furthermore, we showed that some of these mutations can affect the FOXF1 protein at the posttranscriptional level, as shown by altered phosphorylation by MST1 and MST2 kinases.ConclusionMissense mutations in the coding region of the FOXF1 gene alter the molecular function of the FOXF1 protein at multiple levels, such as phosphorylation, DNA binding and target gene activation. These results indicate that FOXF1 molecular pathways may be differentially affected in ACD/MPV patients carrying missense mutations in the DNA-binding domain and may explain the phenotypic heterogeneity of ACD/MPV.

Analysis of Polyphyllin II alters microRNA expression profile in lung cancer A549 cells

Non-small cell lung cancer (NSCLC) is a malignant tumor that threatens the whole world, previous studies found that Polyphyllin II (PPII) can suppress NSCLC cells growth, exhibits significant antitumor activity. MicroRNAs (miRNAs) can regulate the expression of other genes and play a crucial role in the prevention and development of cancer. However, the miRNA portrait of ginsenoside PPII-treated NSCLC A549 cells has not yet been studied. In this work, miRNA-seq analysis was used to determine the changes in miRNA expression profile of NSCLC A549 cells PPII-treaded. In addition, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed on differentially expressed miRNA target genes. Then, predicted the target genes of miRNA and performed functional enrichment analysis on them. We identified 58 up-regulated and 24 down-regulated miRNAs displaying changes their expression in PPII treated A549 cells were greater than 2-fold. In addition, the expression levels of miRNAs were validated by real-time PCR. Therefore, this work has a certain promoting effect on the study of the anti-cancer mechanism of PPII in lung cells.

Clinical significance and potential mechanism of hsa_circ_0006892 in acute respiratory distress syndrome complicated with pulmonary fibrosis.

Acute respiratory distress syndrome (ARDS) is a serious acute lung injury, and can develop into pulmonary fibrosis (PLF). Circular RNAs (circRNAs) regulatory network in ARDS is important. The study explored the role of hsa_circ_0006892 in the occurrence of ARDS and the development of PLF. Hsa_circ_0006892 levels were verified in serum samples of 203 ARDS patients with or without PLF, and the diagnostic value was evaluated through ROC. Cox regression analysis was performed to identify PLF-related factors. The downstream target genes were predicted online. The function and pathway of key genes were annotated through GO and KEGG pathway analysis. Protein-protein interaction (PPI) analysis was performed for the examination of protein interactions. qRT-PCR determined the downregulation of hsa_circ_0006892 in the serum of both ARDS and PLF patients. Hsa_circ_0006892 can differentiate ARDS from controls, and independently related to the development of PLF. Nine targeted related miRNAs were integrated with dysregulated miRNAs from GSE27430 dataset. Clinically, miR-486-3p was the only miRNA that was significantly different in both ARDS and PLF groups, and was determined to be the target of hsa_circ_0006892. 180 target genes of miR-486-3p were predicted, which were integrated with ARDS and PLF-related GSE84439 and GSE38958 datasets. Go and KEGG pathway analysis identified Ras signaling pathway as the most commonly enriched pathway in the overlapped genes. The present results identified the differentially expressed hsa_circ_0006892 in ARDS and PLF, and suggested a possible molecular mechanism of hsa_circ_0006892/miR-486-3p axis.

Recruitment of homodimeric proneural factors by conserved CAT-CAT E-boxes drives major epigenetic reconfiguration in cortical neurogenesis.

Proneural factors of the basic helix-loop-helix family coordinate neurogenesis and neurodifferentiation. Among them, NEUROG2 and NEUROD2 subsequently act to specify neurons of the glutamatergic lineage. Disruption of these factors, their target genes and binding DNA motifs has been linked to various neuropsychiatric disorders. Proneural factors bind to specific DNA motifs called E-boxes (hexanucleotides of the form CANNTG, composed of two CAN half sites on opposed strands). While corticogenesis heavily relies on E-box activity, the collaboration of proneural factors on different E-box types and their chromatin remodeling mechanisms remain largely unknown. Here, we conducted a comprehensive analysis using chromatin immunoprecipitation followed bysequencing (ChIP-seq) data for NEUROG2 and NEUROD2, along with time-matched single-cell RNA-seq, ATAC-seq and DNA methylation data from the developing mouse cortex. Our findings show that these factors are highly enriched in transiently active genomic regions during intermediate stages of neuronal differentiation. Although they primarily bind CAG-containing E-boxes, their binding in dynamic regions is notably enriched in CAT-CAT E-boxes (i.e. CATATG, denoted as 5'3' half sites for dimers), which undergo significant DNA demethylation and exhibit the highest levels of evolutionary constraint. Aided by HT-SELEX data reanalysis, structural modeling and DNA footprinting, we propose that these proneural factors exert maximal chromatin remodeling influence during intermediate stages of neurogenesis by binding as homodimers to CAT-CAT motifs. This study provides an in-depth integrative analysis of the dynamic regulation of E-boxes during neuronal development, enhancing our understanding of the mechanisms underlying the binding specificity of critical proneural factors.