Reporter Assay Research Articles

Single nucleotide polymorphisms in the KRT82 promoter region modulate irregular thickening and patchiness in the dorsal skin of New Zealand rabbits

BackgroundWhile rabbits are used as models in skin irritation tests, the presence of irregular patches and thickening on the dorsal skin can affect precise evaluation. In this study, genes associated with patchiness or non-patchiness on the dorsal skin of New Zealand rabbits were investigated to identify potential regulators of the patchiness phenotype.ResultsThe results showed that parameters associated with hair follicles (HFs), such as HF density, skin thickness, and HF depth, were augmented in rabbits with the patchiness phenotype relative to the non-patchiness phenotype. A total of 592 differentially expressed genes (DEGs) were identified between the two groups using RNA-sequencing. These included KRT72, KRT82, KRT85, FUT8, SOX9, and WNT5B. The functions of the DEGs were investigated by GO and KEGG enrichment analyses. A candidate gene, KRT82, was selected for further molecular function verification. There was a significant positive correlation between KRT82 expression and HF-related parameters, and KRT82 overexpression and knockdown experiments with rabbit dermal papilla cells (DPCs) showed that it regulated genes related to skin and HF growth and development. Investigation of single nucleotide polymorphisms (SNPs) in the exons and promoter region of KRT82 identified four SNPs in the promoter region but none in the exons. The G.-631G > T, T.-696T > C, G.-770G > T and A.-873 A > C alleles conformed to the Hardy − Weinberg equilibrium, and three identified haplotypes showed linkage disequilibrium. Luciferase reporter assays showed that the core promoter region of KRT82 was located in the − 600 to − 1200 segment, in which the four SNPs were located.ConclusionsThe morphological characteristics of the patchiness phenotype were analyzed in New Zealand rabbits and DEGs associated with this phenotype were identified by RNA-sequencing. The biological functions of the gene KRT82 associated with this phenotype were analyzed, and four SNPs were identified in the promoter region of the gene. These findings suggest that KRT82 may be a potential biomarker for the breeding of experimental New Zealand rabbits.

Helicobacter pylori disrupts gastric mucosal homeostasis by stimulating macrophages to secrete CCL3

BackgroundHelicobacter pylori (H. pylori) is the predominant etiological agent of gastritis and disrupts the integrity of the gastric mucosal barrier through various pathogenic mechanisms. After H. pylori invades the gastric mucosa, it interacts with immune cells in the lamina propria. Macrophages are central players in the inflammatory response, and H. pylori stimulates them to secrete a variety of inflammatory factors, leading to the chronic damage of the gastric mucosa. Therefore, the study aims to explore the mechanism of gastric mucosal injury caused by inflammatory factors secreted by macrophages, which may provide a new mechanism for the development of H. pylori-related gastritis.MethodsThe expression and secretion of CCL3 from H. pylori infected macrophages were detected by RT-qPCR, Western blot and ELISA. The effect of H. pylori-infected macrophage culture medium and CCL3 on gastric epithelial cells tight junctions were analyzed by Western blot, immunofluorescence and transepithelial electrical resistance. EdU and apoptotic flow cytometry assays were used to detect cell proliferation and apoptosis levels. Dual-luciferase reporter assays and chromatin immunoprecipitation assays were used to study CCL3 transcription factors. Finally, gastric mucosal tissue inflammation and CCL3 expression were analyzed by hematoxylin and eosin staining and immunohistochemistry.ResultsAfter H. pylori infection, CCL3 expressed and secreted from macrophages were increased. H. pylori-infected macrophage culture medium and CCL3 disrupted gastric epithelial cells tight junctions, while CCL3 neutralizing antibody and receptor inhibitor of CCL3 improved the disruption of tight junctions between cells. In addition, H. pylori-infected macrophage culture medium and CCL3 recombinant proteins stimulated P38 phosphorylation, and P38 phosphorylation inhibitor improved the disruption of tight junctions between cells. Besides, it was identified that STAT1 was a transcription factor of CCL3 and H. pylori stimulated macrophage to secret CCL3 through the JAK1-STAT1 pathway. Finally, after mice were injected with murine CCL3 recombinant protein, the gastric mucosal injury and inflammation were aggravated, and the phosphorylation level of P38 was increased.ConclusionsIn summary, our findings demonstrate that H. pylori infection stimulates macrophages to secrete CCL3 via the JAK1-STAT1 pathway. Subsequently, CCL3 damages gastric epithelial tight junctions through the phosphorylation of P38. This may be a novel mechanism of gastric mucosal injury in H. pylori-associated gastritis.

LncRNA NUTM2A-AS1 silencing inhibits glioma via miR-376a-3p/YAP1 axis

The lncRNA NUTM2A-AS1 has been shown to be dysregulated in gastric cancer, while the roles in glioma is unclear. The aim of this study was to investigate the roles and potential mechanisms of lncRNA NUTM2A-AS1 in the proliferation and apoptosis of glioma cells. The StarBase software and dual luciferase reporter assay were used to identify the relationship between lncRNA NUTM2A-AS1 and miR-376a-3p, and miR-376a-3p and YAP1. The expression of lncRNA NUTM2A-AS1, miR-376a-3p, and YAP1 in human glioma cell lines was detected by qRT-PCR. MTT and flow cytometry were used to detect the effects of lncRNA NUTM2A-AS1 or miR-376a-3p on the proliferation and apoptosis of U251 and A172 cells, respectively. In addition, changes of Bax and Bcl-2 expression in glioma cells were further verified by western blotting and qRT-PCR. The results showed that the expression of lncRNA NUTM2A-AS1 was elevated in glioma cell lines, while miR-376a-3p was decreased. LncRNA NUTM2A-AS1 was negatively correlated with miR-376a-3p. Silencing of lncRNA NUTM2A-AS1 enhanced the levels of miR-376a-3p, leading to reduced cell proliferation and increased apoptosis in glioma cells. YAP1 was a direct target of miR-376a-3p, and it was negatively regulated by miR-376a-3p in U251 and A172 cells. Further mechanistic studies suggested that miR-376a-3p reduced glioma cell proliferation and increased apoptosis by inhibiting YAP1 expression. In addition, lncRNA NUTM2A-AS1 positively regulated of YAP1 expression in glioma cells. In conclusion, silencing of lncRNA NUTM2A-AS1 inhibited proliferation and induced apoptosis in human glioma cells via the miR-376a-3p/YAP1 axis.

Exosomes from hypoxic ADSCs ameliorate neuronal damage post spinal cord injury through circ-Wdfy3 delivery and inhibition of ferroptosis

BackgroundExosomes generated from adipose-derived mesenchymal stem cells (Exos), and in particular hypoxia-pretreated ADSCs (HExos), possess therapeutic properties that promote spinal cord repair following spinal cord injury (SCI). Nevertheless, the regulatory mechanisms through which HExos exert their effects remain unclear. MethodsHere, next-generation sequencing (NGS) was utilized to examine abnormal circRNA expression comparing HExos to Exos. Bioinformatics analysis and RNA pulldown assays together with luciferase reporter assays were applied to determine interactions among miRNAs, mRNAs and circRNAs. ELISA and immunofluorescence staining were used to examine inflammatory cytokine levels, apoptosis and ROS deposition in LPS-treated HT-22 cells, respectively. The therapeutic effects of Exos and HExos on a mouse model of SCI were analyzed by immunohistochemistry and immunofluorescence staining. ResultsOur findings confirmed that HExos have more significant therapeutic influences on decreasing ROS and inflammatory cytokine levels post-SCI than Exos. NGS revealed that circ-Wdfy3 expression levels were significantly higher in HExos than Exos. Downregulation of circ-Wdfy3 led to a decrease in HExo-induced therapeutic effects on spinal cord repair post-SCI, indicating that circ-Wdfy3 has a critical role in the regulation of HExo-mediated protection against SCI. Our bioinformatics, RNA pulldown and luciferase reporter data demonstrated that GPX4 and miR-423-3p were downstream targets of circ-Wdfy3. GPX4 downregulation or miR-423-3p overexpression reversed the protective effects of circ-Wdfy3 on LPS-treated HT-22 cells. Furthermore, overexpression of circ-Wdfy3 led to an in increase in the Exo-induced therapeutic effects on spinal cord repair post-SCI through the inhibition of ferroptosis. Conclusionscirc-WDfy3-overexpressing Exos promote spinal cord repair post-SCI through mediation of ferroptosis via the miR-138-5p/GPX4 pathway.

Blocking the MIR155HG/miR-155 axis reduces CTGF-induced inflammatory cytokine production and α-SMA expression via upregulating AZGP1 in hypertrophic scar fibroblasts

Hypertrophic scarring (HS) is a pathological condition characterized by excessive fibrosis and inflammation, resulting in excessive extracellular matrix formation in the skin. MIR155HG, a long non-coding RNA, is abnormally upregulated in fibrotic tissues; however, its underlying mechanism is poorly understood. Using single-cell sequencing data, we analyzed connective tissue growth factor (CTGF) expression in various cell types in HS and normal skin tissues and MIR155HG expression in clinical samples. To investigate the mechanism of fibrosis, an in vitro model using CTGF-treated hypertrophic scar fibroblasts (HSFBs) was established and qRT-PCR, western blotting and ELISA assays were performed to investigate the expression of interleukin (IL)-1β, IL-6, and mesenchymal markers α-smooth muscle actin (α-SMA). CTGF stimulates MIR155HG level through phosphorylated STAT3 binding to the MIR155HG promoter. We analyzed the methylation of MIR155HG, assessed the levels of miR-155-5p/−3p in CTGF-treated HSFBs and identified differentially expressed genes among HS and NS samples using the Gene Expression Omnibus RNA sequencing data. The binding between miR-155-5p/−3p and AZGP1 was confirmed using a dual-luciferase assay and inflammatory cytokine production and α-SMA expression were investigated in rescue experiments. The findings revealed that CTGF elevated inflammatory cytokine production, α-SMA and MIR155HG expression in HSFBs. MIR155HG is upregulated in HS tissues due to low DNA methylation. Mechanistically, miR-155-5p/−3p was directly bound to MIR155HG 3’UTR. MIR155HG silencing inhibited cytokine production and α-SMA expression by repressing the generation of miR-155-5p/−3p in CTGF-treated HSFBs. Bioinformatics analysis and luciferase reporter assays revealed that miR-155-5p/−3p targets AZGP1. In addition, transfection with plasmids carrying AZGP1 cDNA significantly inhibited the signaling activity of miR-155-5p/−3 p-overexpressing HSFBs. Our findings highlight the importance of the MIR155HG/miR-155/AZGP1 axis in regulating cytokine production and α-SMA in HS.

Multiplex profiling of developmental cis-regulatory elements with quantitative single-cell expression reporters.

The inability to scalably and precisely measure the activity of developmental cis-regulatory elements (CREs) in multicellular systems is a bottleneck in genomics. Here we develop a dual RNA cassette that decouples the detection and quantification tasks inherent to multiplex single-cell reporter assays. The resulting measurement of reporter expression is accurate over multiple orders of magnitude, with a precision approaching the limit set by Poisson counting noise. Together with RNA barcode stabilization via circularization, these scalable single-cell quantitative expression reporters provide high-contrast readouts, analogous to classic in situ assays but entirely from sequencing. Screening >200 regions of accessible chromatin in a multicellular in vitro model of early mammalian development, we identify 13 (8 previously uncharacterized) autonomous and cell-type-specific developmental CREs. We further demonstrate that chimeric CRE pairs generate cognate two-cell-type activity profiles and assess gain- and loss-of-function multicellular expression phenotypes from CRE variants with perturbed transcription factor binding sites. Single-cell quantitative expression reporters can be applied in developmental and multicellular systems to quantitatively characterize native, perturbed and synthetic CREs at scale, with high sensitivity and at single-cell resolution.

MiR-26b-5p/TET3 regulates the osteogenic differentiation of human bone mesenchymal stem cells and bone reconstruction in female rats with calvarial defects.

MicroRNAs (miRNAs) play critical roles in the osteogenic differentiation of human bone mesenchymal stem cells (hBMSCs), but the mechanism by which miRNAs indirectly modulate osteogenesis remains unclear. Here, we explored the mechanism by which miRNAs indirectly modulate gene expression through histone demethylases to promote bone regeneration. Bioinformatics analysis was performed on hBMSCs after 7 days of osteogenic induction. The differentially expressed miRNAs were screened, and potential target mRNAs were identified. To determine the bioactivity and stemness of hBMSCs and their potential for bone repair, we performed wound healing, Cell Counting Kit-8 (CCK-8), real-time reverse transcription quantitative polymerase chain reaction (RT‒qPCR), alkaline phosphatase activity, alizarin red S (ARS) staining and radiological and histological analyses on SD rats with calvarial bone defects. Additionally, a dual-luciferase reporter assay was utilized to investigate the interaction between miR-26b-5p and ten-eleven translocation3 (TET3) in human embryonic kidney 293T cells. The in vitro and in vivo results suggested that miR-26b-5p effectively promoted the migration, proliferation and osteogenic differentiation of hBMSCs, as well as the bone reconstruction of calvarial defects in SD rats. Mechanistically, miR-26b-5p bound to the 3' untranslated region of TET3 mRNA to mediate gene silencing. MiR-26b-5p downregulated the expression of TET3 to increase the osteogenic differentiation of hBMSCs and bone repair in rat calvarial defects. MiR-26b-5p/TET3 crosstalk might be useful in large-scale critical bone defects.

Genome-wide identification and transcription factor regulation of monolignol biosynthetic genes in Ginkgo biloba L.

BackgroundGinkgo biloba L. is a widely cultivated Mesozoic relict gymnosperm, and the wood is resource-rich. As a main component of plant cell walls, lignin serves for plant water transport and mechanical support, as well as directly defining the properties of wood-based products. The lignin biosynthesis pathway in gymnosperms is simpler than that in angiosperms, but has been poorly studied due to the lack of model plants. ResultsIn the present study, we systematically identified a total of 157 monolignol biosynthetic genes from 10 families in G. biloba, including 12 Gb4CLs, 34 GbC3Hs, 7 GbC4Hs, 34 GbCADs, 8 GbCCoAOMTs, 19 GbCCRs, 13 GbCOMTs, 14 GbCSEs, 11 GbHCTs and 5 GbPALs. The chromosome localization, gene structure, gene promoter and other analyses were also conducted. We established a regulatory network between the identified monolignol biosynthetic genes and TFs generated by TGMI algorithm using 133 public RNA-seq datasets of various G. biloba tissues. We then examined 2 gene pairs from the network of their regulatory relationship via yeast one-hybrid and dual-luciferase reporter assay, and the results were positive confirming interactions existing between the GbMYB-025 protein and GbCAD31 promoter, GbERF-174 protein and GbCSE13 promoter in vivo. ConclusionsOur results provided a new effective approach to find transcriptional regulatory relationships and established an important foundation for molecular mechanisms and genetic regulation of monolignol biosynthesis in G. biloba.

Mechanism study of lncRNA RMRP regulating esophageal squamous cell carcinoma through miR-580-3p/ATP13A3 axis

ObjectiveIt is well-known that lncRNAs regulate energy metabolism in tumors. This study focused on the action of RMRP on esophageal squamous cell carcinoma (ESCC) cell proliferation, apoptosis, and glycolysis.MethodsIn the resected ESCC tissues and adjacent tissues from patients, RMRP/miR-580-3p/ATP13A3 expressions were evaluated. ESCC cell proliferation rates and apoptotic rates were measured by CCK-8 and flow cytometry, respectively. Apoptosis related markers were examined by Western blot. Moreover, glucose uptake, lactic acid, and ATP were measured by commercial kits, whereas HK2 and PKM2 were evaluated by Western blot to study ESCC cell glycolysis. Finally, the editing program of RMRP/miR-580-3p/ATP13A3 was translated by luciferase reporter assay and RIP analysis.ResultsRMRP and ATP13A3 were induced, while miR-580-3p was reduced in their expression in ESCC tissues. Silencing RMRP reduced proliferation, glycolysis, and anti-apoptosis ability of ESCC cells. RMRP sequestered miR-580-3p to target ATP13A3. Silenced ATP13A3 or overexpressed miR-580-3p rescued overexpressed RMRP-mediated promotion of proliferation, glycolysis, and anti-apoptosis of ESCC cells.ConclusionRMRP accelerates ESCC progression through the miR-580-3p/ATP13A3 axis, renewing a reference for lncRNA-based therapies for tumors.

SLC15A3 is transcriptionally regulated by HIF1α and p65 to worsen neuroinflammation in experimental ischemic stroke.

Systemic inflammatory stimulus is a risk factor for the incidence of ischemic stroke and contributes to poorer clinical outcomes. Solute carrier 15A3 (SLC15A3) is a peptide/histidine transporter that is implicated in regulating inflammatory responses. However, whether SLC15A3 affects the progression of ischemic stroke associated with systemic inflammation is unclear. The transient middle cerebral artery occlusion (tMCAO) mice with LPS administration (LPS/tMCAO) were prepared as an in vivo model, and LPS-induced BV2 cells under oxygen-glucose deprivation (OGD) exposure were utilized as an in vitro model. We found that SLC15A3 was highly expressed in the ischemic penumbra of LPS/tMCAO mice, and its inhibition reduced infarct area, attenuated neurological deficit, recovered motor function, and mitigated apoptotic neurons. Knockdown of SLC15A3 suppressed the proinflammatory M1-type markers and promoted the levels of M2-associated genes. The in vitro results confirmed that SLC15A3 overexpression promoted microglia polarizing towards M1 subtypes, while SLC15A3 inhibition exerted an opposite effect. In addition, we demonstrated that the p65 signaling pathway and HIF1α were activated by LPS/OGD. Luciferase reporter assay showed that inhibiting p65 using its specific inhibitor BAY 11-7082 or silencing HIF1α using siRNAs reduced the transcriptional activity of SLC15A3in LPS/OGD-induced BV2 cells. Results in NIH 3T3 cells also confirmed that p65 and HIF1α directly bound to the SLC15A3 promoter to activate SLC15A3 transcription. In conclusion, this work shows that SLC15A3, transcriptionally activated by p65 and HIF1α, contributes to poor outcomes in ischemic stroke associated with systemic inflammation by promoting microglial cells polarizing towards M1 types.

MiR-143-3p regulates chondrogenic differentiation of synovium derived mesenchymal stem cells under mechanical stress through the BMPR2-Smad signalling pathway by targeting BMPR2.

Mesenchymal stem cells (MSCs) derived from the synovium, known as synovium mesenchymal stem cells (SMSCs), exhibit significant potential for articular cartilage regeneration owing to their capacity for chondrogenic differentiation. However, the microRNAs (miRNAs) governing this process and the associated mechanisms remain unclear. While mechanical stress positively influences chondrogenesis in MSCs, the miRNA-mediated response of SMSCs to mechanical stimuli is not well understood. This study explores the miRNA-driven mechano-transduction in SMSCs chondrogenesis under mechanical stress. The surface phenotype of SMSCs was analysed by flow cytometry. Chondrogenesis capacities of SMSCs were examined by Alcian blue staining. High throughput sequencing was used to screen mechano-sensitive miRNAs of SMSCs. The RNA expression level of COL2A1, ACAN, SOX9, BMPR2 and miR-143-3p of SMSCs were tested by quantitative real-time polymerase chain reaction (qRT-PCR). The interaction between miR-143-3p and TLR4 was confirmed by luciferase reporter assays. The protein expression levels of related genes were assessed by western blot. High-throughput sequencing revealed a notable reduction in miR-143-3p levels in mechanically stressed SMSCs. Gain- or loss-of-function strategies introduced by lentivirus demonstrated that miR-143-3p overexpression hindered chondrogenic differentiation, whereas its knockdown promoted this process. Bioinformatics scrutiny and luciferase reporter assays pinpointed a potential binding site for miR-143-3p within the 3'-UTR of bone morphogenetic protein receptor type 2 (BMPR2). MiR-143-3p overexpression decreased BMPR2 expression and phosphorylated Smad1, 5 and 8 levels, while its inhibition activated BMPR2-Smad pathway. This study elucidated that miR-143-3p negatively regulates SMSCs chondrogenic differentiation through the BMPR2-Smad pathway under mechanical tensile stress. The direct targeting of BMPR2 by miR-143-3p established a novel dimension to our understanding of mechano-transduction mechanism during SMSC chondrogenesis. This understanding is crucial for advancing strategies in articular cartilage regeneration.

MiRNA-200c-3p deficiency promotes epithelial-mesenchymal transition in triple-negative breast cancer by activating CRKL expression

Epithelial-mesenchymal transition (EMT) plays an important role in malignant progression of Triple-negative breast cancer (TNBC). Many studies have confirmed that miRNA-200c-3p is related to EMT. And we found that it is involved in the regulation of EMT, but the exact mechanism is unclear. CRKL is highly expressed in a variety of tumors and plays a role in EMT. In this study, the potential targets of miRNA-200c-3p were searched in miRPathDB, Targetscan and PicTar. And there are 68 potential targets at the intersection of the three databases. Then, bioinformatics and text mining performed by Coremine Medica, and found that among 68 potential targets, CRKL has the strongest correlation with EMT in TNBC. Therefore, we speculated that miRNA-200c-3p involvement in EMT might be related to CRKL. To verify miRNA-200c-3p inhibits the malignant phenotype of TNBC by regulating CRKL, RT‒PCR, western blotting, Clonal formation assays,CCK-8 proliferation assays, transwell invasion assays, Luciferase reporter assay and nude mouse transplantation tumor assay were performed. In this study, we found that miRNA-200c-3p is under-expressed and EMT-related genes are up-regulated in TNBC, and miRNA-200c-3p can inhibit cancer cell proliferation, invasion and the expression of EMT-related genes and proteins in TNBC. Further research confirmed that miRNA-200c-3p could inhibit EMT by inhibiting the expression of CRKL that directly combining CRKL gene.

CircRAB11A act as miR-24-5p sponge promotes proliferation and resists apoptosis of chicken granulosa cell via EGFR/ERK1/2 and RAB11A/ PI3K/AKT pathways

Circular RNAs (circRNAs) are a class of endogenous non-coding RNAs that have been implicated in mediating granulosa cell (GC) proliferation and apoptosis. CircRAB11A was found to have a significantly higher expression in normal follicles compared to atrophic follicles. In this study, we determined that the knockdown of circRAB11A resulted in the inhibition of proliferation and promotion of apoptosis in GCs of chicken. Moreover, circRAB11A was found to act as a sponge for miR-24-5p, both member RAS oncogene family (RAB11A) and epidermal growth factor receptor (EGFR) were revealed to be targets of miR-24-5p through a dual-luciferase reporter assay. RAB11A or EGFR promoted proliferation and suppressed apoptosis in GCs through the phosphatidylinositol-kinase (PI3K)/AKT or extracellular signal-regulated kinase (ERK)1/2 pathway. These findings suggest that circRAB11A may function as a competing endogenous RNA (ceRNA) by targeting the miR-24-5p/RAB11A and miR-24-5p/EGFR axes and activating the ERK1/2 and PI3K/AKT pathways, offering a potential avenue for exploring the mechanism of follicle development.

Functional Analysis of the Zinc Finger Modules of the S. cerevisiae Splicing Factor Luc7.

Identification of splice sites is a critical step in pre-mRNA splicing since definition of the exon/intron boundaries controls what nucleotides are incorporated into mature mRNAs. The intron boundary with the upstream exon is initially identified through interactions with the U1 snRNP. This involves both base pairing between the U1 snRNA and the pre-mRNA as well as snRNP proteins interacting with the 5' splice site/snRNA duplex. In yeast, this duplex is buttressed by two conserved protein factors, Yhc1 and Luc7. Luc7 has three human paralogs (LUC7L, LUC7L2, and LUC7L3) which play roles in alternative splicing. What domains of these paralogs promote splicing at particular sites is not yet clear. Here, we humanized the zinc finger domains of the yeast Luc7 protein in order to understand their roles in splice site selection using reporter assays, transcriptome analysis, and genetic interactions. While we were unable to determine a function for the first zinc finger domain, humanization of the second zinc finger domain to mirror that found in LUC7L or LUC7L2 resulted in altered usage of nonconsensus 5' splice sites. In contrast, the corresponding zinc finger domain of LUC7L3 could not support yeast viability. Further, humanization of Luc7 can suppress mutation of the ATPase Prp28, which is involved in U1 release and exchange for U6 at the 5' splice site. Our work reveals a role for the second zinc finger of Luc7 in splice site selection and suggests that different zinc finger domains may have different ATPase requirements for release by Prp28.

The miR184-3p targets neuron-specific ecdysone inducible protein 78 to promote rice black streaked dwarf virus propagation in its planthopper vector.

MicroRNAs (miRNAs) are non-coding RNAs that play a pivotal role in antiviral infection. The miR184-3p has been identified to promote rice black streaked dwarf virus (RBSDV) infection in vector Laodelphax striatellus, whether it targets other genes of L. striatellus to modulate RBSDV propagation remains unknown. We first analyzed the expression profiles of miR184-3p and its role in regulating RBSDV infection in L. striatellus. Then the candidate genes expression of miR184-3p were systemically analyzed with gain and loss function of miR184-3p, and the interaction of candidate gene, ecdysone inducible protein 78 (Eip78) with miR184-3p was verified by dual luciferase reporter assay. We found Eip78 is evolutionary conserved among agricultural pests and predominantly expressed in the central nervous system (CNS) of L. striatellus. Knockdown of Eip78 effectively increased RBSDV propagation and transmission. Blockade with Eip78 antibody or injection with Eip78 protein could significantly regulate RBSDV infection. Further analysis revealed that knockdown of Eip78 specifically suppresses RBSDV infection in the head part but not in the body part of L. striatellus. Besides, knockdown of ecdysone receptor (EcR) notably restricted Eip78 expression and increased RBSDV accumulation in L. striatellus. Taken together, we identified a novel target gene of miR184-3p, Eip78, a member of the ecdysone signaling pathway, and revealed the anti-RBSDV role of Eip78 in the CNS of L. striatellus. These results shed light on the interaction mechanisms of miRNAs, virus and ecdysone signaling pathway in insect vector. © 2024 Society of Chemical Industry.

Codon Optimization-based Whole-gene Scanning Identifies Hidden Nucleotides Essential for Bombyx mori Nucleopolyhedrovirus polyhedrin Hyperexpression

During the late stage of infection, alphabaculoviruses produce many occlusion bodies (OBs) in the nuclei of the insect host’s cells through the hyperexpression of polyhedrin (POLH), a major OB component encoded by polh. The strong polh promoter has been used to develop a baculovirus expression vector system for recombinant protein expression in cultured insect cells and larvae. However, the relationship between POLH accumulation and the polh coding sequence remains largely unelucidated. This study aimed to assess the importance of polh codon usage and/or nucleotide sequences in POLH accumulation by generating a baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV) expressing mutant polh (co-polh) optimized according to the codon preference of its host insect. Although the deduced amino acid sequence of CO-POLH was the same as that of wild-type POLH, POLH accumulation was significantly lower in cells infected with the co-polh mutant. This reduction was due to decreased polh mRNA levels rather than translational repression. Analysis of mutant viruses with chimeric polh revealed that a 30 base-pair (bp) 5ʹ proximal polh coding region was necessary for maintaining high polh mRNA levels. Sequence comparison of wild-type polh and co-polh identified five nucleotide differences in this region, indicating that these nucleotides were critical for polh hyperexpression. Furthermore, luciferase reporter assays showed that the 30 bp 5ʹ coding region was sufficient for maintaining the polh promoter-driven high level of polh mRNA. Thus, our whole-gene scanning by codon optimization identified important hidden nucleotides for polh hyperexpression in alphabaculoviruses.

TRIM58 downregulation maintains stemness via MYH9-GRK3-YAP axis activation in triple-negative breast cancer stem cells.

TRIM58 is a member of the TRIM protein family, which possess with E3 ubiquitin ligase activities. Studies have revealed that low expression of TRIM58 plays key roles, has been implicated in the tumor progression of tumor formation due to its reduced expression. However, its role in regulating the stemness of breast cancer stem cells (CSCs) remains unexplored. Here, we found that TRIM58 was underexpressed in TNBC tissues and cells compared to adjacent mucosa tissue, and its downregulation was significantly associated with shorter survival. Overexpression of TRIM58 reduced the proportion of CD44 + /CD24- cells, upregulated differentiation genes, and inhibited stemness-related gene expression in TNBC CSCs. In vitro and in vivo experiments revealed that TRIM58 overexpression in CSCs suppressed tumor sphere formation and tumorigenic capacity. Co-IP results indicated direct interaction between TRIM58 and MYH9, with TRIM58 inducing MYH9 degradation via ubiquitination in differentiated cells. Label-free quantitative proteomics identified GRK3 and Hippo-YAP as downstream targets and signaling pathways of MYH9. TIMER database analysis, immunohistochemistry, western blotting, DNA-protein pulldown experiments, and dual luciferase reporter assays demonstrated that MYH9 regulated GRK3 transcriptional activation in CSCs. In conclusion, elevated TRIM58 expression in CSCs downregulates MYH9 protein levels by promoting ubiquitin-mediated degradation, thereby inhibiting downstream GRK3 transcription, inactivating the YAP stemness pathway, and ultimately promoting CSC differentiation.

KIF22 promotes multiple myeloma progression by regulating the CDC25C/CDK1/cyclinB1 pathway

PurposeMultiple myeloma (MM) is an incurable hematological malignancy characterized by clonal proliferation of malignant plasma B cells in bone marrow, and its pathogenesis remains unknown. The aim of this study was to determine the role of kinesin family member 22 (KIF22) in MM and elucidate its molecular mechanism.MethodsThe expression of KIF22 was detected in MM patients based upon the public datasets and clinical samples. Then, in vitro assays were performed to investigate the biological function of KIF22 in MM cell lines, and subcutaneous xenograft models in nude mice were conducted in vivo. Chromatin immunoprecipitation (ChIP) and luciferase reporter assay were used to determine the mechanism of KIF22-mediated regulation.ResultsThe results demonstrated that the expression of KIF22 in MM patients was associated with several clinical features, including gender (P = 0.016), LDH (P < 0.001), β2-MG (P = 0.003), percentage of tumor cells (BM) (P = 0.002) and poor prognosis (P < 0.0001). Furthermore, changing the expression of KIF22 mainly influenced the cell proliferation in vitro and tumor growth in vivo, and caused G2/M phase cell cycle dysfunction. Mechanically, KIF22 directly transcriptionally regulated cell division cycle 25C (CDC25C) by binding its promoter and indirectly influenced CDC25C expression by regulating the ERK pathway. KIF22 also regulated CDC25C/CDK1/cyclinB1 pathway.ConclusionKIF22 could promote cell proliferation and cell cycle progression by transcriptionally regulating CDC25C and its downstream CDC25C/CDK1/cyclinB1 pathway to facilitate MM progression, which might be a potential therapeutic target in MM.

Circ_PPAPDC1A promotes Osimertinib resistance by sponging the miR-30a-3p/ IGF1R pathway in non-small cell lung cancer (NSCLC)

BackgroundRecent evidence has demonstrated that abnormal expression and regulation of circular RNA (circRNAs) are involved in the occurrence and development of a variety of tumors. The aim of this study was to investigate the effects of circ_PPAPDC1A in Osimertinib resistance in NSCLC.MethodsHuman circRNAs microarray analysis was conducted to identify differentially expressed (DE) circRNAs in Osimertinib-acquired resistance tissues of NSCLC. The effect of circ_PPAPDC1A on cell proliferation, invasion, migration, and apoptosis was assessed in both in vitro and in vivo. Dual-luciferase reporter assay, RT-qPCR, Western-blot, and rescue assay were employed to confirm the interaction between circ_PPAPDC1A/miR-30a-3p/IGF1R axis.ResultsThe results revealed that circ_PPAPDC1A was significantly upregulated in Osimertinib acquired resistance tissues of NSCLC. circ_PPAPDC1A reduced the sensitivity of PC9 and HCC827 cells to Osimertinib and promoted cell proliferation, invasion, migration, while inhibiting apoptosis in Osimertinib-resistant PC9/OR and HCC829/OR cells, both in vitro and in vivo. Silencing circ_PPAPDC1A partially reversed Osimertinib resistance. Additionally, circ_PPAPDC1A acted as a competing endogenous RNA (ceRNA) by targeting miR-30a-3p, and Insulin-like Growth Factor 1 Receptor (IGF1R) was identified as a functional gene for miR-30a-3p in NSCLC. Furthermore, the results confirmed that circ_PPAPDC1A/miR-30a-3p/IGF1R axis plays a role in activating the PI3K/AKT/mTOR signaling pathway in NSCLC with Osimertinib resistance.ConclusionsTherefore, for the first time we identified that circ_PPAPDC1A was significantly upregulated and exerts an oncogenic role in NSCLC with Osimertinib resistance by sponging miR-30a-3p to active IGF1R/PI3K/AKT/mTOR pathway. circ_PPAPDC1A may serve as a novel diagnostic biomarker and therapeutic target for NSCLC patients with Osimertinib resistance.Graphical

Polymorphism rs2327430 in TCF21 predicts the risk and prognosis of gastric cancer by affecting the binding between TFAP2A and TCF21

BackgroundSingle nuclear polymorphisms (SNPs) have been published to be correlated with multiple diseases. Transcription Factor 21 (TCF21) is a critical transcription factor involved in various types of cancers. However, the association of TCF21 genetic polymorphisms with gastric cancer (GC) susceptibility and prognosis remains unclear.MethodsA case-control study comprising 890 patients diagnosed with GC and an equal number of cancer-free controls was conducted. After rigorous statistical analysis, molecular experiments were carried out to elucidate the functional significance of the SNPs in the context of GC.ResultsTCF21 rs2327430 (OR = 0.78, P = 0.026) provides protection against GC, while rs4896011 (OR = 1.39, P = 0.005) exhibit significant associations with GC risk. Furthermore, patients with the (TC + CC) genotype of rs2327430 demonstrate a relatively favorable prognosis (OR = 0.47, P = 0.012). Mechanistically, chromatin immunoprecipitation assay and luciferase reporter assay revealed that the C allele of rs2327430 disrupts the binding of Transcription Factor AP-2 Alpha (TFAP2A) to the promoter region of TCF21, resulting in increased expression of TCF21 and inhibition of malignant behaviors in GC cells.ConclusionOur findings highlight the significant role of TCF21 SNPs in both the risk and prognosis of GC and provide valuable insights into the underlying molecular mechanisms. Specifically, the disruptive effect of rs2327430 on TCF21 expression and its ability to modulate malignant cell behaviors suggest that rs2327430 may serve as a potential predictive marker for GC risk and prognosis.