Dual RNA Research Articles

The orchestration of coding and non-coding genes at the CDH13 locus in coronary artery disease

Abstract Background The CDH13 locus was associated with coronary artery disease (CAD) by genome-wide association studies (GWAS). However, the causal gene(s) and underlying disease mechanisms at this locus remain undetermined. Purpose We intended to pinpoint and functionally validate the causal coding and non-coding genes at the CDH13 locus and delineate the underlying mechanisms for CAD. Methods and results We conducted the transcriptome-wide association analysis (TWAS) using nine types of CAD-relevant tissues from Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task (STARNET) and Genotyped Tissue Expression (GTEx) projects and identified CDH13 (T-cadherin) and four long non-coding RNA (lncRNA) genes as candidate causal genes at The CDH13 locus. One of the antisense(AS) lncRNAs, CDH13-AS2, was predicted to bind on the 3’ untranslated region (UTR) of CDH13 using LncRRIsearch and RNAup servers. The binding interaction was validated by dCas13-medicated RNA immunoprecipitation. Weighted correlation network analysis (WGCNA) using transcriptomic data from CAD-relevant tissues of ~600 human subjects suggested a positive expression correlation between CDH13 and CDH13-AS2 in artery tissues. CRISPR/Cas9-based knockout (KO) of CDH13 or CDH13-AS2 in human umbilical vein endothelial cells (HUVECs) indicated synergistically atherogenic phenotypes, including reduced proliferation and migration, and increased apoptosis and monocyte adhesion. The data matched with the downregulation of CDH13 in artery samples of atherosclerosis patients. The cellular phenotypes were also in line with our in vivo data of Cdh13 and Apoe double-KO (Cdh13-/-/Apoe-/-) mice, which showed increased aortic lesion areas when fed on a high-fat diet compared to Apoe-/- mice. Furthermore, prediction using TargetScan, miRWalk, and scanMiRApp databases identified the potential binding of several EC-expression microRNAs (mir) on 3’UTR of CDH13. Several disease-associated variants were mapped to the predicted binding sites of a few microRNAs, including mir-let7, mir-30, and mir-125. The dual luciferase-based RNA interference (RNAi) assay confirmed the binding of mir-let7 on 3’UTR of CDH13. Transcriptional activation of CDH13-AS2 by the enzymatically inactivated Cas9 (dCas9) system diminished the binding of mir-let7 and increased CDH13 expression. Conclusions At the CDH13 locus, CDH13 and CDH13-AS2 are two causal genes, whose downregulation likely contributes to CAD. CDH13-AS2 bound and stabilized CDH13 partially by preventing mir-let7 mediated degradation of CDH13 mRNA, suggesting therapeutic potentials.

Dual RNA-seq reveals distinct families of co-regulated and structurally conserved effectors in Botrytis cinerea infection of Arabidopsis thaliana

BackgroundBotrytis cinerea is a broad-host-range pathogen causing gray mold disease and significant yield losses of numerous crops. However, the mechanisms underlying its rapid invasion and efficient killing of plant cells remain unclear.ResultsIn this study, we elucidated the dynamics of B. cinerea infection in Arabidopsis thaliana by live cell imaging and dual RNA sequencing. We found extensive transcriptional reprogramming events in both the pathogen and the host, which involved metabolic pathways, signaling cascades, and transcriptional regulation. For the pathogen, we identified 591 candidate effector proteins (CEPs) and comprehensively analyzed their co-expression, sequence similarity, and structural conservation. The results revealed temporal co-regulation patterns of these CEPs, indicating coordinated deployment of effectors during B. cinerea infection. Through functional screening of 48 selected CEPs in Nicotiana benthamiana, we identified 11 cell death-inducing proteins (CDIPs) in B. cinerea.ConclusionsThe findings provide important insights into the transcriptional dynamics and effector biology driving B. cinerea pathogenesis. The rapid infection of this pathogen involves the temporal co-regulation of CEPs and the prominent role of CDIPs in host cell death. This work highlights significant changes in gene expression associated with gray mold disease, underscoring the importance of a diverse repertoire of effectors crucial for successful infection.

The chronic wound characterisation study and biobank: a study protocol for a prospective observational cohort investigation of bacterial community composition, inflammatory responses and wound-healing trajectories in non-healing wounds

IntroductionChronic wounds affect 1%–2% of the global population, with rising incidence due to ageing and lifestyle-related diseases. Bacterial biofilms, found in 80% of chronic wounds, and scattered single-cell bacteria may...

Circ_0006988 promotes gastric cancer cell proliferation, migration and invasion through miRNA-92a-2-5p/TFAP4 axis.

Aim: To explore precise function and underlying mechanism of circ_0006988 in gastric cancer (GC).Materials & methods: GC tissues were collected clinically, and GC cells were purchased from the company. Quantitative real-time polymerase chain reaction and western blot were used to detect mRNA and protein expression. Functional analysis was performed through CCK-8, Transwell and scratch experiment. Binding relationship was validated through dual luciferase reporter and RNA immunoprecipitation assays. HGC-27 cells were subcutaneously injected into mice to construct a xenograft tumor model.Results: In GC tissues and cells, circ_0006988 overexpressed, promoting proliferation, migrationand invasion. MiRNA-92a-2-5p downregulation or TFAP4 overexpression weakened effects of circ_0006988 silencing on GC progression.Conclusion: circ_0006988 facilitates GC development through miRNA-92a-2-5p/TFAP4 axis.

DMRT3-mediated lncRNA OIP5-AS1 promotes the pyroptosis of bronchial epithelial cells by binding with EIF4A3 to enhance YAP mRNA stability.

Asthma is featured by persistent airway inflammation. Long noncoding RNAs (lncRNAs) are reported to play critical roles in asthma. However, the function of Opa interacting protein 5-antisense 1 (OIP5-AS1) in pyroptosis during the development of asthma remains unexplored. The blood samples of asthma patients (n = 32) as well as the baseline characteristics of asthma patients or healthy people were collected. An in vivo model of asthma was established using house dust mites (HDM). To mimic asthma in vitro, BEAS-2B cells were treated with HDM. Cell pyroptosis and apoptosis were examined by flow cytometry. The levels of interleukin-1 beta (IL-1β) and interleukin-18 (IL-18) were detected by enzyme-linked immunosorbent assay (ELISA). The binding among messenger RNAs (mRNAs) was assessed by chromatin immunoprecipitation (ChIP), dual luciferase report assay, RNA immunoprecipitation (RIP), co-immunoprecipitation (Co-IP), and RNA pull-down assay, respectively. The cellular localization was observed by fluorescence in situ hybridization (FISH) staining. The level of OIP5-AS1 was upregulated in asthma patients. HDM induced pyroptosis and increased the levels of IL-18, IL-1β, and lactate dehydrogenase (LDH) in BEAS-2B cells, which was obviously reversed by OIP5-AS1 knockdown. Consistently, the expressions of NOD-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), c-caspase 1, and pyroptosis-related gasdermin D-1 (GSDMD-1) in BEAS-2B cells were upregulated by HDM treatment, while these phenomena were partially abolished by silencing of OIP5-AS1. Moreover, HDM promoted the progression of asthma in vivo, which was rescued by the downregulation of OIP5-AS1. OIP5-AS1 silencing decreased HDM-induced cell pyroptosis by inactivation of NLRP3. More importantly, OIP5-AS1 promoted the mRNA stability of yes-associated protein (YAP) via binding with eukaryotic translation initiation factor 4A3 (EIF4A3), and OIP5-AS1 was transcriptionally upregulated by doublesex and mab-3 related transcription factor 3 (DMRT3). DMRT3-mediated OIP5-AS1 aggravated the progression of asthma by mediation of the EIF4A3/YAP axis, which might provide a new therapeutic strategy against asthma.

Bone marrow-derived mesenchymal stem cell-derived exosome-loaded miR-129-5p targets high-mobility group box 1 attenuates neurological-impairment after diabetic cerebral hemorrhage

BACKGROUND Diabetic intracerebral hemorrhage (ICH) is a serious complication of diabetes. The role and mechanism of bone marrow mesenchymal stem cell (BMSC)-derived exosomes (BMSC-exo) in neuroinflammation post-ICH in patients with diabetes are unknown. In this study, we investigated the regulation of BMSC-exo on hyperglycemia-induced neuroinflammation. AIM To study the mechanism of BMSC-exo on nerve function damage after diabetes complicated with cerebral hemorrhage. METHODS BMSC-exo were isolated from mouse BMSC media. This was followed by transfection with microRNA-129-5p (miR-129-5p). BMSC-exo or miR-129-5p-overexpressing BMSC-exo were intravitreally injected into a diabetes mouse model with ICH for in vivo analyses and were cocultured with high glucose-affected BV2 cells for in vitro analyses. The dual luciferase test and RNA immunoprecipitation test verified the targeted binding relationship between miR-129-5p and high-mobility group box 1 (HMGB1). Quantitative polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay were conducted to assess the levels of some inflammation factors, such as HMGB1, interleukin 6, interleukin 1β, toll-like receptor 4, and tumor necrosis factor α. Brain water content, neural function deficit score, and Evans blue were used to measure the neural function of mice. RESULTS Our findings indicated that BMSC-exo can promote neuroinflammation and functional recovery. MicroRNA chip analysis of BMSC-exo identified miR-129-5p as the specific microRNA with a protective role in neuroinflammation. Overexpression of miR-129-5p in BMSC-exo reduced the inflammatory response and neurological impairment in comorbid diabetes and ICH cases. Furthermore, we found that miR-129-5p had a targeted binding relationship with HMGB1 mRNA. CONCLUSION We demonstrated that BMSC-exo can reduce the inflammatory response after ICH with diabetes, thereby improving the neurological function of the brain.

Bone marrow-derived mesenchymal stem cell-derived exosome-loaded miR-129-5p targets high-mobility group box 1 attenuates neurological-impairment after diabetic cerebral hemorrhage.

Diabetic intracerebral hemorrhage (ICH) is a serious complication of diabetes. The role and mechanism of bone marrow mesenchymal stem cell (BMSC)-derived exosomes (BMSC-exo) in neuroinflammation post-ICH in patients with diabetes are unknown. In this study, we investigated the regulation of BMSC-exo on hyperglycemia-induced neuroinflammation. To study the mechanism of BMSC-exo on nerve function damage after diabetes complicated with cerebral hemorrhage. BMSC-exo were isolated from mouse BMSC media. This was followed by transfection with microRNA-129-5p (miR-129-5p). BMSC-exo or miR-129-5p-overexpressing BMSC-exo were intravitreally injected into a diabetes mouse model with ICH for in vivo analyses and were cocultured with high glucose-affected BV2 cells for in vitro analyses. The dual luciferase test and RNA immunoprecipitation test verified the targeted binding relationship between miR-129-5p and high-mobility group box 1 (HMGB1). Quantitative polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay were conducted to assess the levels of some inflammation factors, such as HMGB1, interleukin 6, interleukin 1β, toll-like receptor 4, and tumor necrosis factor α. Brain water content, neural function deficit score, and Evans blue were used to measure the neural function of mice. Our findings indicated that BMSC-exo can promote neuroinflammation and functional recovery. MicroRNA chip analysis of BMSC-exo identified miR-129-5p as the specific microRNA with a protective role in neuroinflammation. Overexpression of miR-129-5p in BMSC-exo reduced the inflammatory response and neurological impairment in comorbid diabetes and ICH cases. Furthermore, we found that miR-129-5p had a targeted binding relationship with HMGB1 mRNA. We demonstrated that BMSC-exo can reduce the inflammatory response after ICH with diabetes, thereby improving the neurological function of the brain.

Long non-coding RNA LNC-POTEM-4 promotes HCC progression via the LNC-POTEM-4/miR-149-5p/Wnt4 signaling axis

Information on the potential role of the long non-coding RNA LNC-POTEM-4 in cancer progression is limited. Our preliminary study found that LNC-POTEM-4 was overexpressed in hepatocellular carcinoma (HCC) tissues, which led us to further investigate the biological function and molecular mechanism of LNC-POTEM-4 in HCC development. LNC-POTEM-4 expression in HCC tissues was examined using transcriptome sequencing and quantitative reverse transcription PCR. The relationships between LNC-POTEM-4 and the stage and prognosis of HCC in patient data from the TCGA database were analyzed. The effects of LNC-POTEM-4 on proliferation, invasion/migration, and epithelial-mesenchymal transition marker expression in HCC cells were evaluated in vitro using gain- and loss-of-function assays, while its effects on tumor growth and metastasis were explored through animal experiments. A LNC-POTEM-4/microRNA (miR)-149-5p/Wnt4 regulatory signaling axis was identified using bioinformatics analysis, and dual luciferase reporter, RNA immunoprecipitation, and RNA pull-down assays. Co-transfection of LNC-POTEM-4 and Wnt4 expression plasmids was employed to confirm the new signaling pathway. We found that LNC-POTEM-4 was overexpressed in HCC tissues and was linked to poor staging and prognosis. LNC-POTEM-4 promoted proliferation, invasion, migration, and the epithelial-mesenchymal transition of HCC cells in vitro. Silencing of LNC-POTEM-4 inhibited HCC growth and distant metastasis in vivo. Mechanically, LNC-POTEM-4 was found to function as a competitive endogenous RNA, upregulating Wnt4 by sponging miR-149-5p to promote HCC progression. Wnt4 overexpression may have counteracted the tumor-inhibition effect of LNC-POTEM-4 silencing. In conclusion, LNC-POTEM-4 upregulated Wnt4 to activate the Wnt signaling pathway and stimulate the malignancy tendency of HCC by sponging miR-149-5p, providing a prospective target for the detection and therapy of HCC. However, the effects of LNC-POTEM-4 on the miR-149-5p/Wnt4 signaling axis should be further studied in animal experiments.

Dual RNA sequencing during Trichoderma harzianum-Phytophthora capsici interaction reveals multiple biological processes involved in the inhibition and highlights the cell wall as a potential target.

Phytophthora capsici is a destructive oomycete pathogen, causing huge economic losses for agricultural production. The genus Trichoderma represents one of the most extensively researched categories of biocontrol agents, encompassing a diverse array of effective strains. The commercial biocontrol agent Trichoderma harzianum strain T-22 exhibits pronounced biocontrol effects against many plant pathogens, but its activity against P. capsici is not known. T. harzianum T-22 significantly inhibited the growth of P. capsici mycelia and the culture filtrate of T-22 induced lysis of P. capsici zoospores. Electron microscopic analyses indicated that T-22 significantly modulated the ultrastructural composition of P. capsici, with a severe impact on the cell wall integrity. Dual RNA sequencing revealed multiple biological processes involved in the inhibition during the interaction between these two microorganisms. In particular, a marked upregulation of genes was identified in T. harzianum that are implicated in cell wall degradation or disruption. Concurrently, the presence of T. harzianum appeared to potentiate the susceptibility of P. capsici to cell wall biosynthesis inhibitors such as mandipropamid and dimethomorph. Further investigations showed that mandipropamid and dimethomorph could strongly inhibit the growth and development of P. capsici but had no impact on T. harzianum even at high concentrations, demonstrating the feasibility of combining T. harzianum and these cell wall synthesis inhibitors to combat P. capsici. These findings provided enhanced insights into the biocontrol mechanisms against P. capsici with T. harzianum and evidenced compatibility between specific biological and chemical control strategies. © 2024 Society of Chemical Industry.

Dual transcriptomic analysis reveals early induced Castanea defense-related genes and Phytophthora cinnamomi effectors.

Phytophthora cinnamomi Rands devastates forest species worldwide, causing significant ecological and economic impacts. The European chestnut (Castanea sativa) is susceptible to this hemibiotrophic oomycete, whereas the Asian chestnuts (Castanea crenata and Castanea mollissima) are resistant and have been successfully used as resistance donors in breeding programs. The molecular mechanisms underlying the different disease outcomes among chestnut species are a key foundation for developing science-based control strategies. However, these are still poorly understood. Dual RNA sequencing was performed in C. sativa and C. crenata roots inoculated with P. cinnamomi. The studied time points represent the pathogen's hemibiotrophic lifestyle previously described at the cellular level. Phytophthora cinnamomi expressed several genes related to pathogenicity in both chestnut species, such as cell wall-degrading enzymes, host nutrient uptake transporters, and effectors. However, the expression of effectors related to the modulation of host programmed cell death (elicitins and NLPs) and sporulation-related genes was higher in the susceptible chestnut. After pathogen inoculation, 1,556 and 488 genes were differentially expressed by C. crenata and C. sativa, respectively. The most significant transcriptional changes occur at 2 h after inoculation (hai) in C. sativa and 48 hai in C. crenata. Nevertheless, C. crenata induced more defense-related genes, indicating that the resistant response to P. cinnamomi is controlled by multiple loci, including several pattern recognition receptors, genes involved in the phenylpropanoid, salicylic acid and ethylene/jasmonic acid pathways, and antifungal genes. Importantly, these results validate previously observed cellular responses for C. crenata. Collectively, this study provides a comprehensive time-resolved description of the chestnut-P. cinnamomi dynamic, revealing new insights into susceptible and resistant host responses and important pathogen strategies involved in disease development.

CircCHSY1 protects hearts against ischemia/reperfusion injury by enhancing heme oxygenase 1 expression via miR-24-3p.

Circular RNAs are important players involving in a variety of physiological and pathological processes. However, their functions and mechanisms during myocardial ischemic injury and protection remain largely unknown. We recently found significant alterations of many circRNAs including circCHSY1 following myocardial ischemia/reperfusion (I/R) injury, whereas their exact functions are unclear. Here, we investigated roles of circCHSY1 in the acute myocardial I/R injury and the potential mechanisms involved. The expression of circCHSY1 was detected in cardiomyocytes from mouse, rat and human embryonic stem cells (hESC-CMs). It was further upregulated in mouse I/R (30 min/24 h) hearts, oxygen glucose deprivation/reperfused (OGD/R, 6 h/2 h) primary neonatal rat ventricular cardiomyocytes (NRCMs) and OGD/R (48 h/2 h) hESC-CMs. Adenovirus-mediated circCHSY1-overexpression significantly decreased infarct size and lactate dehydrogenase (LDH) release in mouse I/R hearts. Consistently, circCHSY1 overexpression reduced the LDH release in the OGD/R NRCMs and hESC-CMs, improved cell viability, and preserved mitochondrial function in the OGD/R NRCMs, whereas there were no significant differences in cell viability and LDH release between the OGD/R NRCMs with and without siRNA-mediated circCHSY1 knockdown. Mechanistically, circCHSY1 was detected to bind with miR-24-3p analyzed by dual luciferase assay and RNA pull-down assays. CircCHSY1 overexpression-mediated protective effects on cells and mitochondria in OGD/R NRCMs were reversed by the miR-24-3p mimic. Further, dual luciferase assay showed that miR-24-3p directly bound to heme oxygenase 1 (HO1) via its 3'UTR. The protein level of HO1 was downregulated by miR-24-3p mimic in OGD/R NRCMs but enhanced by the circCHSY1 overexpression in vitro and in vivo. Functionally, the HO1 knockdown by adenovirus in vivo and by siRNA in vitro eliminated cardioprotective effects of circCHSY1 overexpression. CircCHSY1 is upregulated following myocardial I/R injury. The higher level of circCHSY1 protects I/R hearts and cardiomyocytes. The protection of circCHSY1 is mediated through enhancement of the HO1 level, resulting in preserving mitochondrial homeostasis via targeting miR-24-3p in cardiomyocytes. These findings suggest circCHSY1 as a protective factor.

Exosomes derived from mir-337-3p over-expressing tendon stem cells protect against apoptosis of tenocytes via targeting caspase3

BackgroundTendons are important dense fibrous structures connecting muscle to bone, and tendon stem cells (TDSCs) affect their repair and regeneration. The role of TDSC-derived exosomes (TDSC-Exos) is still being unexplored; therefore, this study aimed to investigate the protective effect of TDSC-Exos on tenocytes.MethodsThe TDSCs and tenocytes were all derived from Sprague Dawley (SD) rats. The expression of positive and negative markers of TDSCs were detected by flow cytometry, and the multi-differentiation ability was also detected to identify TDSCs. Exos were derived from TDSCs using ultracentrifugation; furthermore, Exos enriched with microRNA(miR)-377-3p were generated from TDSCs stably overexpressing miR-377-3p after transfection, identified with transmission electron microscopy (TEM), western blot and PKH26 staining assay. Moreover, the cell functions of tenocytes were evaluated by MTT, EdU, transwell, and flow cytometry. Dual luciferase reporter and RNA pull-down assays were used to verify the binding sites of miR-337-3p and caspase3 (CASP3) predicted by Targetscan.ResultsExos (miR-337-3p) were taken up by tenocytes, and promoted the proliferation, migration, and invasion and suppressed the apoptosis of tenocytes in a dose-dependent manner. Bioinformatics analysis showed that CASP3 was a target of miR-377-3p, which was further verified by luciferase and RNA pull-down assays. Moreover, over-expressed CASP3 reversed the effects of Exos (miR-337-3p) on cell functions of tenocytes.ConclusionsOur findings suggest that Exos derived from miR-337-3p over-expressing TDSCs could potentially protect against tenocyte apoptosis by regulating CASP3. This novel therapeutic approach holds promise for the treatment of tendon injury, offering a glimmer of hope for improved patient outcomes.

Phage anti-CRISPR control by an RNA- and DNA-binding helix-turn-helix protein.

In all organisms, regulation of gene expression must be adjusted to meet cellular requirements and frequently involves helix-turn-helix (HTH) domain proteins1. For instance, in the arms race between bacteria and bacteriophages, rapid expression of phage anti-CRISPR (acr) genes upon infection enables evasion from CRISPR-Cas defence; transcription is then repressed by an HTH-domain-containing anti-CRISPR-associated (Aca) protein, probably to reduce fitness costs from excessive expression2-5. However, how a single HTH regulator adjusts anti-CRISPR production to cope with increasing phage genome copies and accumulating acr mRNA is unknown. Here we show that the HTH domain of the regulator Aca2, in addition to repressing Acr synthesis transcriptionally through DNA binding, inhibits translation of mRNAs by binding conserved RNA stem-loops and blocking ribosome access. The cryo-electron microscopy structure of the approximately 40 kDa Aca2-RNA complex demonstrates how the versatile HTH domain specifically discriminates RNA from DNA binding sites. These combined regulatory modes are widespread in the Aca2 family and facilitate CRISPR-Cas inhibition in the face of rapid phage DNA replication without toxic acr overexpression. Given the ubiquity of HTH-domain-containing proteins, it is anticipated that many more of them elicit regulatory control by dual DNA and RNA binding.

Exosomal lncRNA HCP5 derived from human bone marrow mesenchymal stem cells improves chronic periodontitis by miR-24-3p/HO1/P38/ELK1 pathway

ObjectiveThe present study aimed to explore the function of human bone marrow mesenchymal stem cells (hBMMSCs)-derived exosomal long noncoding RNA histocompatibility leukocyte antigen complex P5 (HCP5) in the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) to improve chronic periodontitis (CP). MethodsExosomes were extracted from hBMMSCs. Alizarin red S staining was used to detect mineralised nodules. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to measure HCP5 and miR-24-3p expression. The mRNA and protein levels of alkaline phosphatase (ALP), osteocalcin, osterix, runt-related transcription factor 2, bone morphogenetic protein 2, osteopontin, fibronectin, collagen 1, heme oxygenase 1 (HO1), P38, and ETS transcription factor ELK1 (ELK1) were detected using RT-qPCR and Western blot. Enzyme-linked immunosorbent assay (ELISA) kits were used to determine the HO1 and carbon monoxide concentrations. Heme, biliverdin, and Fe2+ levels were determined using detection kits. Micro-computed tomography, hematoxylin and eosin staining, ALP staining, tartrate-resistant acid phosphatase staining, ELISA, and RT-qPCR were conducted to evaluate the effect of HCP5 on CP mice. Dual luciferase, RNA immunoprecipitation, and RNA pulldown experiments were performed to identify the interactions among HCP5, miR-24-3p, and HO1. ResultsThe osteogenic ability of hPDLSCs significantly increased when co-cultured with hBMMSCs or hBMMSCs exosomes. Overexpression of HCP5 and HO1 in hBMMSCs exosomes promoted the osteogenic differentiation of hPDLSCs, and knockdown of HCP5 repressed the osteogenic differentiation of hPDLSCs. HCP5 knockdown enhanced the inflammatory response and repressed osteogenesis in CP mice. MiR-24-3p overexpression diminished the stimulatory effect of HCP5 on the osteogenic ability of hPDLSCs. Mechanistically, HCP5 acted as a sponge for miR-24-3p and regulated HO1 expression, and HO1 activated the P38/ELK1 pathway. ConclusionHBMMSCs-derived exosomal HCP5 promotes the osteogenic differentiation of hPDLSCs and alleviates CP by regulating the miR-24-3p/HO1/P38/ELK1 signalling pathway.

The role of SPI1/VSIG4/THBS1 on glioblastoma progression through modulation of the PI3K/AKT pathway

IntroductionGlioblastoma multiforme (GBM) poses a significant challenge in terms of treatment due to its high malignancy, necessitating the identification of additional molecular targets. VSIG4, an oncogenic gene participates in tumor growth and migration in various cancer types. Nevertheless, the precise process through which VSIG4 facilitates the malignant progression of glioma remains to be elucidated. ObjectivesThis research aims to explore the function and molecular mechanism involving VSIG4 in the malignant progression of glioma. MethodsThe amount of VSIG4 was measured using qPCR, western blotting, and immunohistochemistry. Lentivirus infections were applied for upregulating or downregulating molecules within glioma cells. The incorporation of 5-ethynyl-20-deoxyuridine, Transwell, cell counting kit-8, and clone formation experiments, were applied to assess the biological functions of molecules on glioma cells. Dual luciferase reporter gene, RNA immunoprecipitation, and chromatin immunoprecipitation assays were used to explore the functional relationship among relevant molecules. ResultsThe upregulation of VSIG4 was observed in GBM tissues, indicating an adverse prognosis. Silencing VSIG4 in glioma cells resulted in a decrease in cell viability, invasion, proliferation, and tumorigenesis, an increase in cell apoptosis, and a stagnation in the cell cycle progression at the G0/G1 phase. Mechanistically, SPI1-mediated upregulation of VSIG4 expression led to binding between VSIG4 and THBS1 protein, ultimately facilitating the malignant progression of glioma cells through the activation of the PI3K/AKT pathway. The inhibited proliferative and invasive capabilities of glioma cells were reversed by overexpressing THBS1 following the knockdown of VSIG4. ConclusionOur findings provide evidence for the role of VSIG4 as an oncogene and reveal the previously unidentified contribution of the SPI1/VSIG4/THBS1 axis in the malignant progression of glioma. This signaling cascade enhances tumor growth and invasion by modulating the PI3K/AKT pathway. VSIG4 as a potential biomarker may be a viable strategy in the development of tailored molecular therapies for GBM.

An improved bacterial mRNA enrichment strategy in dual RNA sequencing to unveil the dynamics of plant-bacterial interactions

BackgroundDual RNA sequencing is a powerful tool that enables a comprehensive understanding of the molecular dynamics underlying plant-microbe interactions. RNA sequencing (RNA-seq) poses technical hurdles in the transcriptional analysis of plant-bacterial interactions, especially in bacterial transcriptomics, owing to the presence of abundant ribosomal RNA (rRNA), which potentially limits the coverage of essential transcripts. Therefore, to achieve cost-effective and comprehensive sequencing of the bacterial transcriptome, it is imperative to devise efficient methods for eliminating rRNA and enhancing the proportion of bacterial mRNA. In this study, we modified a strand-specific dual RNA-seq method with the goal of enriching the proportion of bacterial mRNA in the bacteria-infected plant samples. The enriched method involved the sequential separation of plant mRNA by poly A selection and rRNA removal for bacterial mRNA enrichment followed by strand specific RNA-seq library preparation steps. We assessed the efficiency of the enriched method in comparison to the conventional method by employing various plant-bacterial interactions, including both host and non-host resistance interactions with pathogenic bacteria, as well as an interaction with a beneficial rhizosphere associated bacteria using pepper and tomato plants respectively.ResultsIn all cases of plant-bacterial interactions examined, an increase in mapping efficiency was observed with the enriched method although it produced a lower read count. Especially in the compatible interaction with Xanthmonas campestris pv. Vesicatoria race 3 (Xcv3), the enriched method enhanced the mapping ratio of Xcv3-infected pepper samples to its own genome (15.09%; 1.45-fold increase) and the CDS (8.92%; 1.49-fold increase). The enriched method consistently displayed a greater number of differentially expressed genes (DEGs) than the conventional RNA-seq method at all fold change threshold levels investigated, notably during the early stages of Xcv3 infection in peppers. The Gene Ontology (GO) enrichment analysis revealed that the DEGs were predominantly enriched in proteolysis, kinase, serine type endopeptidase and heme binding activities.ConclusionThe enriched method demonstrated in this study will serve as a suitable alternative to the existing RNA-seq method to enrich bacterial mRNA and provide novel insights into the intricate transcriptomic alterations within the plant-bacterial interplay.

Circ_0000099/miR-223-3p/CTGF Regulates the Growth, Metastasis, and EMT Processes in TGF-β2-Stimulated Human Lens Epithelial Cells

Purpose Posterior capsule opacification (PCO) is the major complication of visual impairment after cataract surgery. Circular RNAs (circRNAs) are involved in the development of many diseases. The purpose of this study was to explore the role and molecular mechanism of circ_0000099 in PCO. Methods SRA01/04 cells were treated with TGF-β2 to establish a PCO cell model. The expression of circ_0000099, miR-223-3p, and connective tissue growth factor (CTGF) mRNA was determined by real-time quantitative polymerase chain reaction (qRT-PCR). Western blot assay was used to analyze the protein expression. Cell proliferation, migration, and invasion were analyzed by (4-5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), 5-ethynyl-2 ‘-Deoxyuridine (EdU), transwell, and wound healing tests. The circ_0000099/miR-223-3p/CTGF relationship was verified by dual luciferase reporter gene and RNA binding protein immunoprecipitation (RIP) assays. Results TGF-β2 treatment promoted SRA01/04 cell proliferation invasion, migration, and EMT. Circ_0000099 expression was increased in POC patients and TGF-β2-treated SRA01/04 cells. Knockdown of circ_0000099 suppressed TGF-β2-induced proliferation, invasion, migration, and EMT in SRA01/04 cells. miR-223-3p was identified as the target of circ_0000099, and miR-223-3p inhibitor might partly abolish the repression of circ_0000099 silencing on TGF-β2-triggered SRA01/04 cell disorders. MiR-223-3p directly targeted CTGF. Knockdown of CTGF suppressed TGF-β2-induced SRA01/04 cell injury. Circ_0000099 can regulate CTGF expression by targeting miR-223-3p. Conclusions Circ_0000099 silencing might relieve TGF-2-induced SRA01/04 cell injury by the miR-223-3p/CTGF axis, providing new avenues for the prevention and treatment of PCO.

LncRNA PGM5-AS1 inhibits non-small cell lung cancer progression by targeting miRNA-423-5p/SLIT2 axis

Non-small cell lung cancer (NSCLC) is a common and aggressive primary malignancy worldwide. Dysregulation of long non-coding RNAs (lncRNAs) has been shown to play an essential regulatory role in multiple cancers. However, the role of PGM5-AS1 in NSCLC remains unclear. Here, we found that PGM5-AS1 was down-regulated in NSCLC tissues and cells. Furthermore, reduced PGM5-AS1 expression levels were associated with larger tumor size, positive lymph node metastasis, advanced TNM stage and worse prognosis. We found that overexpression of PGM5-AS1 inhibited cell proliferation and metastasis, and induced apoptosis and G0/G1 cell cycle arrest in NSCLC cell lines. Using dual luciferase gene reporter and RNA immunoprecipitation assays, we confirmed that miR-423-5p interacted with PGM5-AS1, and that their expression levels were negatively correlated in NSCLC tissues. miR-423-5p was also found to reverse PGM5-AS1-induced malignant biological behavior. Moreover, we identified slit guidance ligand 2 (SLIT2) as a target gene of miR-423-5p. Using a dual luciferase gene reporter assay, we confirmed the regulatory relationship between SLIT2 and miR-423-5p and demonstrated that their expression levels were negatively correlated. Our rescue experiments showed that SLIT2 knockdown reversed miR-423-5p-mediated effects. Overall, this study identifies PGM5-AS1 as a potential prognostic biomarker for NSCLC and shows that PGM5-AS1 suppresses NSCLC development by regulating the miR-423-5p/SLIT2 axis.

Vaccination with a Protective Ipa Protein-Containing Nanoemulsion Differentially Alters the Transcriptomic Profiles of Young and Elderly Mice following Shigella Infection.

Shigella spp. are responsible for bacillary dysentery or shigellosis transmitted via the fecal-oral route, causing significant morbidity and mortality, especially among vulnerable populations. There are currently no licensed Shigella vaccines. Shigella spp. use a type III secretion system (T3SS) to invade host cells. We have shown that L-DBF, a recombinant fusion of the T3SS needle tip (IpaD) and translocator (IpaB) proteins with the LTA1 subunit of enterotoxigenic E. coli labile toxin, is broadly protective against Shigella spp. challenge in a mouse lethal pulmonary model. Here, we assessed the effect of LDBF, formulated with a unique TLR4 agonist called BECC470 in an oil-in-water emulsion (ME), on the murine immune response in a high-risk population (young and elderly) in response to Shigella challenge. Dual RNA Sequencing captured the transcriptome during Shigella infection in vaccinated and unvaccinated mice. Both age groups were protected by the L-DBF formulation, while younger vaccinated mice exhibited more adaptive immune response gene patterns. This preliminary study provides a step toward identifying the gene expression patterns and regulatory pathways responsible for a protective immune response against Shigella. Furthermore, this study provides a measure of the challenges that need to be addressed when immunizing an aging population.

CircMTO1/miR-30c-5p/SOCS3 axis alleviates oral submucous fibrosis through inhibiting fibroblast-myofibroblast transition.

circRNAs have been shown to participate in diverse diseases; however, their role in oral submucous fibrosis (OSF), a potentially malignant disorder, remains obscure. Our preliminary experiments detected the expression of circRNA mitochondrial translation optimization 1 homologue (circMTO1) in OSF tissues (n = 20) and normal mucosa tissues (n = 20) collected from Hunan Xiangya Stomatological Hospital, and a significant decrease of circMTO1 expression was showed in OSF tissues. Therefore, we further explored circMTO1 expression in OSF. Target molecule expression was detected using RT-qPCR and western blotting. The migration and invasion of buccal mucosal fibroblasts (BMFs) were assessed using wound healing and Transwell assays. The interaction between miR-30c-5p, circMTO1, and SOCS3 was evaluated using dual luciferase, RNA immunoprecipitation (RIP), and RNA pull-down assays. The colocalisation of circMTO1 and miR-30c-5p was observed using fluorescence in situ hybridisation (FISH). circMTO1 and SOCS3 expression decreased, whereas miR-30c-5p expression increased in patients with OSF and arecoline-stimulated BMFs. Overexpression of circMTO1 effectively restrained the fibroblast-myofibroblast transition (FMT), as evidenced by the increase in expression of Coll I, α-SMA, Vimentin, and the weakened migration and invasion functions in BMFs. Mechanistic studies have shown that circMTO1 suppresses FMT by enhancing SOCS3 expression by sponging miR-30c-5p and subsequently inactivating the FAK/PI3K/AKT pathway. FMT induced by SOCS3 silencing was reversed by the FAK inhibitor TAE226 or the PI3K inhibitor LY294002. circMTO1/miR-30c-5p/SOCS3 axis regulates FMT in arecoline-treated BMFs via the FAK/PI3K/AKT pathway. Expanding the sample size and in vivo validation could further elucidate their potential as therapeutic targets for OSF.