RNA Expression Levels Research Articles

Knockdown of circ_0076305 decreases the paclitaxel resistance of non-small cell lung cancer cells by regulating TMPRSS4 via miR-936.

Paclitaxel (PTX) is a commonly used as a chemotherapeutic drug for non-small cell lung cancer (NSCLC). Exploring the underlying mechanism of PTX resistance is of great significance for NSCLC treatment. The expression levels of RNA and protein were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot assays. The targeted relationship was confirmed by dual-luciferase reporter assay and RNA-pull down assay. The PTX resistance and cell proliferation were assessed by cell counting kit-8 (CCK-8) assay and 5-Ethynyl-2'-deoxyuridine (EDU) assay, respectively. Cell migration and invasion were analyzed by transwell assays. Cell apoptosis was analyzed by flow cytometry, and cell glycolysis was analyzed using the commercial kits. The role of circular RNA_0076305 (circ_0076305) in regulating the PTX sensitivity in vivo was explored in xenograft tumor model. Circ_0076305 was up-regulated in PTX-resistant NSCLC tissues and cells. Mechanically, circ_0076305 bound to microRNA-936 (miR-936), and miR-936 targeted transmembrane serine protease 4 (TMPRSS4). Circ_0076305 could up-regulate TMPRSS4 expression by sponging miR-936 in NSCLC cells. miR-936 knockdown or TMPRSS4 overexpression reversed the anti-tumor effects of circ_0076305 knockdown in NSCLC cells with PTX treatment. Circ_0076305 silencing increased the PTX sensitivity of xenograft tumors in vivo. Circ_0076305 silencing promoted PTX sensitivity by targeting miR-936/TMPRSS4 axis in NSCLC cells.

MicroRNA-505-3p mediates cell motility of epithelial ovarian cancer via suppressing PEAK1 expression.

MicroRNAs (miRNAs) are a class of small RNA genes with important roles in cancer biology regulation. There are considerable studies regarding the roles of microRNA-505-3p (miR-505-3p) in cancer development and progression, but the function of miR-505-3p in epithelial ovarian cancer (EOC) has not been fully clarified. Comparative analysis of miRNA expression data set was used to select differentially expressed miRNAs. Quantitative real-time polymerase chain reaction was applied to detect expression levels of RNAs, while western blot and immunofluorescence staining were performed to detect expression levels of proteins of interest. The motility of EOC cells was assessed by wound healing and transwell assays. The binding and regulating relationship between miRNA and its direct target gene was investigated by dual-luciferase assay. Our results showthat miR-505-3p was upregulated in recurrent EOC, which significantly inhibits EOC cell motility via modulating cell epithelial-mesenchymal transition. Furthermore, our results indicated that PEAK1 expression was inhibited by direct binding of miR-505-3p into its 3'-URT in EOC cells. Importantly, knockdown of PEAK1 attenuated the effect of mi-505-3p inhibitor on EOC cell migration and invasion. In conclusion, our findings indicate that miRNA-505-3p inhibits EOC cell motility by targeting PEAK1.

Role of Native Probiotic Lactobacillus Species via TGF-β Signaling Pathway Modulation in CRC

Colon microbiome composition in colorectal cancer (CRC) patients undergoes remarkable changes. The present study was designed to assess the impact of Lactobacillus mixture on the regulating the CRC by influencing the transforming growth factor beta (TGF-β) signaling pathway in both in vitro (HT-29 cancer cells) and in vivo (BALB/c mice) models. In this study, the antiproliferative effect of a native potential probiotic Lactobacillus mixture on HT-29 cancer cells was evaluated using the MTT assay method. Also, qRT-PCR was performed to assess the RNA expression level of genes associated with the TGF-β signaling pathway at three levels: receptor, regulatory, and inhibitory SMADs. Finally, the in vivo assays were investigated by three groups of mice: a naive group (PBS), a disease group (azoxymethane [AOM]/ dextran sulfate sodium [DSS] + PBS), and a treatment group (AOM/DSS + Lactobacillus mixture in PBS). The MTT results showed a significant decrease in proliferation of HT-29 cancer cells after 120 h of treatment. Furthermore, qRT-PCR demonstrated the downregulation of the smad2/3 gene expression in HT-29-treated cells and also reduction in the level of smad4 gene expression. In addition, in the mouse model, the tgf-βR1 gene was downregulated in the group treated with AOM/DSS/Lactobacillus, but not the AOM/DSS group. A downregulation of smad4 gene expression was also observed in in vivo models. The obtained results suggest that our novel probiotic Lactobacillus mixture could have a positive impact on the inhibition of the CRC progression by downregulating the TGF-β signaling pathway.

RNA editing landscape of adipose tissue in polycystic ovary syndrome provides insight into the obesity-related immune responses.

Polycystic ovary syndrome (PCOS) is the most common reproductive-endocrine disorder with wide-ranging metabolic implications, including obesity. RNA editing, a post-transcriptional modification, can fine-tune protein function and introduce heterogeneity. However, the role of RNA editing and its impact on adipose tissue function in PCOS remain poorly understood. This study aimed to comprehensively analyze RNA-editing events in abdominal and subcutaneous adipose tissue of PCOS patients and healthy controls using high-throughput whole-genome sequencing (WGS) and RNA sequencing. Our results revealed that PCOS patients exhibited more RNA-editing sites, with adenosine-to-inosine (A-to-I) editing being prevalent. The expression of ADAR genes, responsible for A-to-I editing, was also higher in PCOS. Aberrant RNA-editing sites in PCOS adipose tissue was enriched in immune responses, and interleukin-12 biosynthetic process. Tumor necrosis factor (TNF) signaling, nuclear factor kappa B (NF-κB) signaling, Notch signaling, terminal uridylyl transferase 4 (TUT4), hook microtubule tethering protein 3 (HOOK3), and forkhead box O1 (FOXO1) were identified to be of significant differences. Differentially expressed genes (DEGs) in PCOS adipose tissue were enriched in immune responses compared with controls, and the DEGs between subcutaneous and abdominal adipose tissue were also enriched in immune responses suggesting the important role of subcutaneous adipose tissue. Furthermore, we identified the correlations between RNA editing levels and RNA expression levels of specific genes, such as ataxia-telangiectasia mutated (ATM) and mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) in inflammation pathways and ATM, TUT4, and YTH N6-methyladenosine RNA-binding protein C2 (YTHDC2) in oocyte development pathway. These findings suggest that RNA-editing dysregulation in PCOS adipose tissue may contribute to inflammatory dysregulations. Understanding the interplay between RNA editing and adipose tissue function may unveil potential therapeutic targets for PCOS management. However, further research and validation are required to fully elucidate the molecular mechanisms underlying these associations.

Effect of microRNA-141-3p, E2F3, CDK3, and KAT2B overexpression on histologic tumor grade and metastasis status in untreated breast cancer tissues

Introduction: Increasing evidence has reported gene expression alterations in breast cancer (BC) tissues, necessitating their investigating to highlight the molecular basis of the disease development or progression. This study investigated the expression of miR-141, E2F3, CDK3, TP53, and KAT2B, and their association with histologic grade and metastasis in BC tissues. Methods: The RNA expression level of miR-141, E2F3, CDK3, TP53, and KAT2B genes was analyzed in 23 BC and 23 normal tissue samples by RT-qPCR. The associations of the expression level of these genes with clinicopathological features of the BC tissue samples were evaluated. The study also explored the correlation between RNA levels of genes and miR-141. Results: Expression of miR-141, E2F3, CDK3, and KAT2B demonstrated significantly higher levels in BC tumor than normal tissues. TP53 expression showed an increase in tumor compared to normal tissues, although it was insignificant. Moreover, increased RNA expression of miR-141, E2F3, CDK3, and KAT2B corresponded to the advanced stage and regional metastasis of BC. Additionally, the results demonstrated a significant correlation between RNA expression levels of miR-141 with CDK3 and E2F3 with KAT2B. Conclusion: Our findings highlighted clinicopathologic indicators that were relevant to aggressive BC. Besides, Correlations between overexpression of miR-141, E2F3, CDK3, and KAT2B in BC tissues suggest regulatory effects. Taken together, it seems results of this study could provide evidence that dysregulation of gene expression contributes significantly to unveiling the underlying molecular basis of BC.

Characterization of the Expressions and m6A Methylation Modification Patterns of mRNAs and lncRNAs in a Spinal Cord Injury Rat Model.

Spinal cord injury (SCI) is a serious central nervous system disease with no effective treatment strategy presently due to its complex pathogenic mechanism. N6-methyladenosine (m6A) methylation modification plays an important role in diverse physiological and pathological processes. However, our understanding of the potential mechanisms of messenger RNA (mRNA) and long non-coding RNAs (lncRNA) m6A methylation in SCI is currently limited. Here, comprehensive m6A profiles and gene expression patterns of mRNAs and lncRNAs in spinal cord tissues after SCI were identified using microarray analysis of immunoprecipitated methylated RNAs. A total of 3745 mRNAs (2343 hypermethylated and 1402 hypomethylated) and 738 lncRNAs (488 hypermethylated and 250 hypomethylated) were differentially methylated with m6A modifications in the SCI and sham rats. Functional analysis revealed that differentially m6A-modified mRNAs were mainly involved in immune inflammatory response, nervous system development, and focal adhesion pathway. In contrast, differentially m6A-modified lncRNAs were mainly related to antigen processing and presentation, the apoptotic process, and the mitogen-activated protein kinases (MAPKs) signaling pathway. In addition, combined analysis of m6A methylation and RNA expression results revealed that 1636 hypermethylated mRNAs and 262 hypermethylated lncRNAs were up-regulated, and 1571 hypomethylated mRNAs and 204 lncRNAs were down-regulated. Furthermore, we validated the altered levels of m6A methylation and RNA expression of five mRNAs (CD68, Gpnmb, Lilrb4, Lamp5, and Snap25) and five lncRNAs (XR_360518, uc.393 + , NR_131064, uc.280 - , and XR_597251) using MeRIP-qPCR and qRT-PCR. This study expands our understanding of the molecular mechanisms underlying m6A modification in SCI and provides novel insights to promote functional recovery after SCI.

A Study on the Protective Impact of Resveratrol on Liver Damage in Rats with Obstructive Jaundice.

Obstructive Jaundice (OJ) is a common clinical condition with potential outcomes, including hepatocyte necrosis, bile duct hyperplasia, significant cholestatic liver fibrosis, and, in severe cases, liver failure. Resveratrol (RES), a polyphenol present in grapes and berries, has demonstrated efficacy in improving OJ. However, the precise mechanism of its action remains unclear. In this study, we employed network pharmacology to investigate the underlying molecular mechanism of RES in the treatment of OJ. The targets of RES were identified using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), SuperPred, and SwissTargetPrediction database. The targets related to OJ were gathered from the DisGeNET, GeneCards, DrugBank, and Online Mendelian Inheritance in Man (OMIM) databases, and the intersection of these targets was determined using Venny2.1.0. Subsequently, an active component-target network was constructed using Cytoscape software. The Protein-Protein Interaction (PPI) network was generated using the String database and Cytoscape software. Following this, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted using the Bioconductor platform. Finally, quantitative Real-Time PCR (qRT-PCR), Western Blotting (WB), and Enzyme-Linked Immunosorbent Assay (ELISA) were employed to assess RNA and protein expression levels in related pathways. The findings revealed a selection of 56 potential targets for RES, and a search through the online database identified 2,742 OJ-related targets with overlapping in 27 targets. In the PPI network, mTOR, CYP2C9, CYP1A1, CYP3A4, AHR, ESR1, and HSD17B1 emerged as core targets. KEGG analyses demonstrated that the primary pathways of RES in treating OJ, particularly those related to lipid metabolism, include linoleic acid metabolism, arachidonic acid metabolism, metabolism of xenobiotics by cytochrome P450, lipid and atherosclerosis, tyrosine metabolism, steroid hormone biosynthesis, and pentose and glucuronate interconversions signaling pathways. Furthermore, in vivo experiments indicated that RES significantly ameliorated liver injury induced by Common Bile Duct Ligation (CBDL) in rats with OJ. It lowered serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, reduced liver tissue MDA levels, increased glutathione (GSH) content, and enhanced activity of superoxide dismutase (SOD), alleviating liver damage. Metabolomics analysis revealed that the therapeutic effect of RES in OJ involved alterations in lipid metabolic pathways, hinting at the potential mechanism of RES in treating OJ. ELISA, qRTPCR, and WB analyses confirmed lower expression levels of mTOR, CYP1A1, and CYP2C9 in the RES group compared to the model group, validating their involvement in the lipid metabolism pathway. In conclusion, RES exhibited a protective effect on liver function in rats with OJ. The underlying mechanism appears to be linked to antioxidant activity and modulation of lipid metabolism pathways.

Needle-scalpel therapy inhibits the apoptosis of nucleus pulposus cells via the SDF-1/CXCR4 axis in a rat degenerative cervical intervertebral disc model.

As a common disease, cervical spondylosis (CS) results from the degeneration of the cervical intervertebral disc. However, there are still no effective clinical strategies for the treatment of this disease. Needle-scalpel (Ns), a therapy guided by traditional Chinese medicine theory, alleviates intervertebral disc degradation and is widely used in the clinic to treat CS. Stromal cell-derived factor-1 (SDF-1) and its receptor CXC receptor 4 (CXCR4) in nucleus pulposus cells play an important role in CS onset and development. This study aimed to explore whether Ns can relieve pain and regulate the SDF-1/CXCR4 axis in nucleus pulposus cells to inhibit apoptosis, thereby delaying cervical intervertebral disc degradation in a rat model of CS. It was found that the Ns-treated groups exhibited higher mechanical allodynia scores than the model group, and H&E staining, MRI, and scanning electron microscopy revealed that Ns therapy inhibited intervertebral disc degeneration. Additionally, Ns therapy significantly inhibited increases in the RNA and protein expression levels of SDF-1 and CXCR4. Furthermore, these treatments alleviated the apoptosis of nucleus pulposus cells, which manifested as a decline in the proportion of apoptotic nucleus pulposus cells and inhibition of the decrease in the levels of Bcl-2/Bax. These findings indicated that Ns mitigated CS-induced pain, inhibited the apoptosis of nucleus pulposus cells, and alleviated intervertebral disc degeneration in CS rats. These effects may be mediated by specifically regulating the SDF-1/CXCR4 signaling axis. Based on these findings, we conclude that Ns might serve as a promising therapy for the treatment of CS.

Identification of KRT80 as a Novel Prognostic and Predictive Biomarker of Human Lung Adenocarcinoma via Bioinformatics Approaches.

According to the 2022 Global Cancer Statistics, lung cancer is the leading cause of cancer-related mortality worldwide. Lung adenocarcinoma (LUAD), which is a histological subtype of Non- Small Cell Lung Cancer (NSCLC), accounts for 40% of primary lung cancer. Therefore, there is an urgent need to identify new prognostic markers as clinical predictive markers for LUAD. This study aimed to investigate the role of Keratin 80 (KRT80) in the prognosis of LUAD and its underlying mechanisms. Bioinformatics analysis was conducted using data retrieved from The Cancer Genome Atlas (TCGA) databases. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were employed to predict the involved biological processes and signaling pathways, respectively. The LinkedOmics database was utilized to identify differentially expressed genes (DEGs) correlated with KRT80. Nomograms and Kaplan-Meier plots were constructed to evaluate the survival outcomes of patients diagnosed with LUAD. Moreover, TIMER was employed to conduct correlation analyses between KRT80 expression and immune cell infiltration, shedding light on the intricate interplay between KRT80 and the tumor microenvironment in LUAD. To ascertain the RNA and protein expression levels of KRT80 in LUAD and adjacent normal tissues, Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR) and immunohistochemistry techniques were employed, respectively. Scrutiny of the TCGA dataset revealed KRT80 up-regulation across pan-cancer tissues, notably elevated in LUAD compared to healthy lung tissues. This finding was validated in our clinical samples, where Kaplan-Meier survival curves indicated poorer survival rates for high KRT80 expression in LUAD. A positive correlation was found between the transcription level of KRT80 in LUAD samples and clinical parameters, such as lymph node metastasis stage, distant metastasis, and pathological stage. Survival, logistic regression, and Cox regression analyses emphasized the clinical prognostic significance of high KRT80 expression in LUAD. Nomogram results underscored the robust predictive potential of KRT80 for the survival of LUAD patients. Gene functional enrichment analyses mainly associated KRT80 with cytokine-cytokine receptor interactions, cell cycle, apoptosis, and chemokine signaling pathways. Based on the results of the immune infiltration analysis, it can be found that the expression of KRT80 is related to the immune cell subsets and survival rate of patients with LUAD. Our research revealed a significant upregulation of KRT80 in LUAD, with heightened KRT80 expression correlating with unfavorable prognosis. This study represents a comprehensive and systematic evaluation of KRT80 expression in LUAD, encompassing its prognostic and diagnostic significance, as well as underlying mechanisms. Our findings suggest that KRT80 may emerge as a novel prognostic and predictive biomarker in LUAD.

β3-adREnoceptor Analysis in CORD Blood of Neonates (β3 RECORD): Study Protocol of a Pilot Clinical Investigation.

Background and Objective: The embryo and the fetus develop in a physiologically hypoxic environment, where vascularization is sustained by HIF-1, VEGF, and the β-adrenergic system. In animals, β3-adrenoceptors (β3-ARs), up-regulated by hypoxia, favor global fetal wellness to such an extent that most diseases related to prematurity are hypothesized to be induced or aggravated by a precocious β3-AR down-regulation, due to premature exposure to a relatively hyperoxic environment. In animals, β3-AR pharmacological agonism is currently investigated as a possible new therapeutic opportunity to counteract oxygen-induced damages. Our goal is to translate the knowledge acquired in animals to humans. Recently, we have demonstrated that fetuses become progressively more hypoxemic from mid-gestation to near-term, but starting from the 33rd-34th week, oxygenation progressively increases until birth. The present paper aims to describe a clinical research protocol, evaluating whether the expression level of HIF-1, β3-ARs, and VEGF is modulated by oxygen during intrauterine and postnatal life, in a similar way to animals. Materials and Methods: In a prospective, non-profit, single-center observational study we will enroll 100 preterm (group A) and 100 full-term newborns (group B). We will collect cord blood samples (T0) and measure the RNA expression level of HIF-1, β3-ARs, and VEGF by digital PCR. In preterms, we will also measure gene expression at 48-72h (T1), 14 days (T2), and 30 days (T3) of life and at 40 ± 3 weeks of post-menstrual age (T4), regardless of the day of life. We will compare group A (T0) vs. group B (T0) and identify any correlations between the values obtained from serial samples in group A and the clinical data of the patients. Our protocol has been approved by the Pediatric Ethical Committee for Clinical Research of the Tuscany region (number 291/2022). Expected Results: The observation that in infants, the HIF-1/β3-ARs/VEGF axis shows similar modulation to that of animals could suggest that β3-ARs also promote fetal well-being in humans.

Neuropeptide Y receptor Y8b (npy8br) regulates feeding and digestion in Japanese medaka (Oryzias latipes) larvae: evidence from gene knockout.

Neuropeptide Y receptor Y8 (NPY8R) is a fish-specific receptor with two subtypes, NPY8AR and NPY8BR. Changes in expression levels during physiological processes or in vivo regulation after ventricular injection suggest that NPY8BR plays an important role in feeding regulation; this has been found in only a few fish, at present. In order to better understand the physiological function of npy8br, especially in digestion, we used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology to generate npy8br-/- Japanese medaka (Oryzias latipes). We found that the deletion of npy8br in medaka larvae affected their feeding and digestion ability, ultimately affecting their growth. Specifically, npy8br deficiency in medaka larvae resulted in decreased feed intake and decreased expression levels of orexigenic genes (npy and agrp). npy8br-/- medaka larvae fed for 10 d (10th day of feeding) still had incompletely digested brine shrimp (Artemia nauplii) in the digestive tract 8 h after feeding, the messenger RNA (mRNA) expression levels of digestion-related genes (amy, lpl, ctra, and ctrb) were significantly decreased, and the activity of amylase, trypsin, and lipase also significantly decreased. The deletion of npy8br in medaka larvae inhibited the growth and significantly decreased the expression of growth-related genes (gh and igf1). Hematoxylin and eosin (H&E) sections of intestinal tissue showed that npy8br-/- medaka larvae had damaged intestine, thinned intestinal wall, and shortened intestinal villi. So far, this is the first npy8br gene knockout model established in fish and the first demonstration that npy8br plays an important role in digestion.

LncTIMP3 promotes osteogenic differentiation of bone marrow mesenchymal stem cells via miR-214/Smad4 axis to relieve postmenopausal osteoporosis.

Promoting the balance between bone formation and bone resorption is the main therapeutic goal for postmenopausal osteoporosis (PMOP), and bone marrow mesenchymal stem cells (BMSCs) osteogenic differentiation plays an important regulatory role in this process. Recently, several long non-coding RNAs (lncRNAs) have been reported to play an important regulatory role in the occurrence and development of OP and participates in a variety of physiological and pathological processes. However, the role of lncRNA tissue inhibitor of metalloproteinases 3 (lncTIMP3) remains to be investigated. The characteristics of BMSCs isolated from the PMOP rat model were verified by flow cytometry assay, alkaline phosphatase (ALP), alizarin red and Oil Red O staining assays. Micro-CT and HE staining assays were performed to examine histological changes of the vertebral trabeculae of the rats. RT-qPCR and western blotting assays were carried out to measure the RNA and protein expression levels. The subcellular location of lncTIMP3 was analyzed by FISH assay. The targeting relationships were verified by luciferase reporter assay and RNA pull-down assay. The trabecular spacing was increased in the PMOP rats, while ALP activity and the expression levels of Runx2, Col1a1 and Ocn were all markedly decreased. Among the RNA sequencing results of the clinical samples, lncTIMP3 was the most downregulated differentially expressed lncRNA, also its level was significantly reduced in the OVX rats. Knockdown of lncTIMP3 inhibited osteogenesis of BMSCs, whereas overexpression of lncTIMP3 exhibited the reverse results. Subsequently, lncTIMP3 was confirmed to be located in the cytoplasm of BMSCs, implying its potential as a competing endogenous RNA for miRNAs. Finally, the negative targeting correlations of miR-214 between lncTIMP3 and Smad4 were elucidated in vitro. lncTIMP3 may delay the progress of PMOP by promoting the activity of BMSC, the level of osteogenic differentiation marker gene and the formation of calcium nodules by acting on the miR-214/Smad4 axis. This finding may offer valuable insights into the possible management of PMOP.

MiR-25–5p in exosomes derived from UVB-induced fibroblasts regulates melanogenesis via TSC2-dominated cellular organelle dysfunction

BackgroundFew reports have confirmed whether exosomes derived from fibroblasts can regulate the process of melanogenesis. We wondered whether exosomes derived from fibroblasts could have a potent regulatory effect on melanogenesis and explored the underlying mechanisms. ObjectiveThis study aimed to find the role of fibroblasts in melanocytes and revealed the related mechanisms. MethodsRT-qPCR, Western blot analysis were conducted to measure the RNA and protein expression level of various related genes. miRNA sequencing, mass spectrum analysis and subsequent bioinformatics analysis were employed to find the underlying targets. Zebrafish were employed to measure the melanin synthesis related process in vivo. Furthermore, electron microscopy, ROS measurement and dual-luciferase reporter assay were adopted to investigate the relationship between these processes. ResultsWe found that exosomes derived from human primary dermal fibroblasts were internalized by human primary melanocytes and MNT1 cells and that the melanin content and the expression of melanin synthesis-related proteins TYR and MITF was inhibited by exosomes derived from UVB-induced human primary dermal fibroblasts. The miRNA expression profile in secreted exosomes changed significantly, with miR-25–5p identified as capable of regulating TSC2 expression via the CDS region. The miR-25–5p-TSC2 axis could affect the melanin content through subsequent cellular organelle dysfunction, such as mitochondrial dysfunction, endoplasmic reticulum stress and dysregulation of lysosomal cysteine proteases. ConclusionWe unveiled a novel regulatory role of fibroblasts in melanocytes, facilitated by the secretion of exosomes. miR-25–5p within exosomes plays a pivotal role in regulating melanogenesis via TSC2-induced cellular organelle dysfunction.

Diversity in B7-H3/CD276 expression across cancer types: Exploring a dynamic novel immune checkpoint.

3133 Background: The resistance encountered in immune checkpoint blockade has persistently impaired the achievement of robust, durable antitumor immune responses in most cancer patients, prompting the search for additional immune targets. B7 homolog 3 (B7-H3)/CD276 is an emerging immune biomarker whose contrasting immune functions challenge conventional immune marker classifications, making it a compelling target. The present study aimed to characterize the expression of B7-H3/CD276 in a pan-cancer setting, analyze its co-expression patterns with potentially co-targetable immune checkpoints, and discuss implications for ongoing clinical trials. Methods: We conducted a focused analysis of B7-H3/CD276 expression in 514 cancer patients with diverse tumor histologies evaluated under the PREDICT protocol at the University of California San Diego. RNA sequencing of tissue specimens was performed, and results were used to characterize the RNA expression levels of B7-H3/CD276 and its correlation with key cancer immunity markers including PD-1, PD-L1, PD-L2, CTLA4, LAG3, BTLA, TIM3, VISTA, ADORA2A and IDO1. Results: Elevated expression levels of B7-H3/CD276 were observed in 41.6% [214/514] of the 514 tumor samples analyzed. When stratified by cancer type, the highest rates of elevated expression were found in sarcomas (58.30%, 14/24), followed by pancreatic (58.20%, 32/55) and liver and bile duct cancers (52.60%, 10/19). The independent association with pancreatic cancers was statistically significant on multivariate analysis (odds ratio 2.20, 95% confidence interval 1.09 – 4.45, p = 0.027). Ninety-three unique patterns of immune co-expression were observed among the 214 patients with high B7-H3/CD276 expression levels. Among them, the most commonly observed pattern was high expression of B7-H3/CD276 alone (26.2%, 56/214) followed by co-expression with high VISTA (7.0%, [15/214]) and high ADORA2A (5.6%, [12/214]). Of note, there was significant heterogeneity observed in the patterns of co-expression in cancer immunity markers, with 72.04% (67/93) in the cohort being unique to one patient. Conclusions: We have observed immunomic diversity in our analysis of B7-H3/CD276 expression levels across varied tumor types as well as in its co-expression patterns with other immune markers. Our findings suggest that leveraging immune profiling to build customized therapeutic regimens may be pertinent to optimizing response rates and minimizing toxicity to B7-H3/CD276 targeting therapies.

Integrated analysis of ncRNA in hepatocellular carcinoma with CTNNB1 mutations reveals miR-205-5p and miR-3940-3p Axes

BackgroundCatenin beta 1 (CTNNB1) mutations are one of the most common mutations involved in hepatocellular carcinoma (HCC) progression. However, the association between CTNNB1 mutations and HCC remains controversial. MethodsFive tumor samples with wild-type CTNNB1 and three tumor samples with CTNNB1 mutations were collected from patients with HCC for whole transcriptome sequencing. Selected ncRNAs and mRNAs were validated by qPCR in 48 HCC tumors. Selected ncRNA regulatory axes were verified in HCC cells by transfecting mimics and inhibitors of miRNA. ResultsA network of differentially expressed (DE) lncRNA/circRNA–miRNA–mRNA was constructed to explore the effects of CTNNB1 mutations on ncRNA regulation. TXNRD1, CES1, MATN2, SERPINA5, lncRNA STAT4-210, hsa_circ_0007824, hsa_circ_0008234, hsa-miR-205-5p and hsa-miR-199a-5p were verified at the RNA expression level to validate the sequencing results. The down-up-down axes GLIS3-209/circ_0085440–miR-205-5p–GHRHR and WNK2-213–miR-3940-3p–LY6E were verified at the expression level, and proved to inhibit and promote cell proliferation, respectively. ConclusionThis study demonstrated CTNNB1 mutations associated ncRNA regulatory axes playing different roles in HCC cell proliferation, providing novel insights into the controversial role of CTNNB1 in HCC.

Asporin expression is associated with human atherosclerotic plaque integrity

Abstract Funding Acknowledgements Type of funding sources: Other. Main funding source(s): This work was supported by the Swedish Research Council, the Swedish Heart and Lung Foundation, Skåne University Hospital funds Background Calcification serves as a clinical marker of atherosclerotic plaque burden. Recent studies have also shed light on its intricate association with plaque vulnerability, likely depend on calcification structures and the surrounding environment. Asporin (ASPN) is a member of the small-leucine-rich proteoglycans that contains a unique and well-conserved stretch of aspartate (D) residues, which mimic the role of glycosaminoglycans. Via its D-repeat, ASPN can interact with calcium and several other extracellular matrix molecules, thus influencing collagen mineralization and TGF-β signaling. ASPN expression has been shown to be higher in asymptomatic compared to symptomatic atherosclerotic plaques, suggesting a potential protective role. However, the role of ASPN in maintaining plaque integrity remains unknown. Purpose Here we aimed to explore the role of ASPN in atherosclerosis, by investigating if ASPN was expressed in human carotid plaques and by determining if ASPN is associated with risk for postoperative cardiovascular events and death. Methods ASPN and TGF-β protein levels were measured by ELISA and Milliplex in 176 human plaque tissue homogenates obtained from the Carotid Plaque Imaging Project (CPIP) biobank. Plaque tissue sections were stained for ASPN, destabilizing (CD68, oil red O, glycophorin A) and stabilizing plaque components (Movat, α-actin). Plaque tissue RNA expression levels were assessed in 82 plaques using bulk RNA sequencing. Plaque levels of phosphate, elastin and collagen were measured using ELISA. Results ASPN protein levels were higher in plaques from asymptomatic patients compared to symptomatic patients (p=0.011). ASPN RNA levels correlated positively to RNA levels of the smooth muscle cell markers ACTA2, MYH11 and TAGLN. Moreover, protein levels correlated with plaque levels of phosphate (r=0.258, p=0.001), elastin (r=0.423, p<0.001), collagen (r=0.248, p=0.002) and stability factors like the TGF-β family members, TGF-β1 (r=0.176, p=0.035), TGF-β2 (r=0.581, p<0.001) and TGF-β3 (r=0.396, p<0.001). Furthermore, plaque ASPN protein concentration, correlated inversely with a histological plaque vulnerability index (r=-0.265, p=0.0029), which is ratio between the plaque area of destabilizing (macrophages, lipids, intra-plaque hemorrhage) and stabilizing plaque components (collagen and smooth muscle cells). Additionally, above median ASPN levels were associated with lower risk for postoperative cardiovascular events (p=0.008) and death (p=0.009). Conclusion Our findings suggest that elevated levels of ASPN might have a crucial role in maintaining plaque stability, affecting plaque calcification.

Changes in Daily Apparent Diffusion Coefficient on Fully Quantitative Magnetic Resonance Imaging Correlate With Established Genomic Pathways of Radiation Sensitivity and Reveal Novel Biologic Associations

Changes in quantitative magnetic resonance imaging (qMRI) are frequently observed during chemotherapy or radiation therapy (RT). It is hypothesized that qMRI features are reflective of underlying tissue responses. It's unknown what underlying genomic characteristics underly qMRI changes. We hypothesized that qMRI changes may correlate with DNA damage response (DDR) capacity within human tumors. Therefore, we designed the current study to correlate qMRI changes from daily RT treatment with underlying tumor transcriptomic profiles. Study participants were prospectively enrolled (National Clinical Trial 03500081). RNA expression levels for 757 genes from pretreatment biopsies were obtained using a custom panel that included signatures of radiation sensitivity and DDR. Daily qMRI data were obtained from a 1.5 Tesla MR linear accelerator. Using these images, d-slow, d-star, perfusion, and apparent diffusion coefficient-mean values in tumors were plotted per-fraction, over time, and associated with genomic pathways. A total of 1022 qMRIs were obtained from 39 patients and both genomic data and qMRI data from 27 total patients. For 20 of those patients, we also generated normal tissue transcriptomic data. Radio sensitivity index values most closely associated with tissue of origin. Multiple genomic pathways including DNA repair, peroxisome, late estrogen receptor responses, KRAS signaling, and UV response were significantly associated with qMRI feature changes (P < .001). Genomic pathway associations across metabolic, RT sensitivity, and DDR pathways indicate common tumor biology that may correlate with qMRI changes during a course of treatment. Such data provide hypothesis-generating novel mechanistic insight into the biologic meaning of qMRI changes during treatment and enable optimal selection of imaging biomarkers for biologically MR-guided RT.

The extracellular matrix proteoglycan serglycin is associated with human atherosclerotic plaque inflammation

Abstract Funding Acknowledgements Type of funding sources: Other. Main funding source(s): This work was supported by grants from the Swedish Research Council, the Swedish Heart and Lung Foundation, Skåne University Hospital funds, Royal Physiographic Society in Lund and O.E. and Edla Johanssons Foundation. Background Atherosclerotic cardiovascular disease is caused by the formation of plaques in the arterial wall which contain lipids, cells, cell debris and a unique extracellular matrix (ECM) signature. These plaques may ultimately rupture or erode away causing thrombosis, thereby leading to myocardial infarction or stroke. Rupture-prone plaques generally bear a greater inflammatory activity, with ECM proteins suggested to influence the inflammatory activity. Among many ECM proteins, serglycin (SRGN) is highly expressed by inflammatory cells, and SRGN has been linked to modulation of the inflammatory response in disease such as cancer. Yet, if SRGN contributes to the inflammatory circuitry in human atherosclerotic plaques remains to be explored. Purpose We aimed to investigate if SRGN was associated with inflammation in atherosclerotic plaques. Methods Plaque protein levels of SRGN were measured by ELISA in human carotid plaques obtained from the Carotid Plaque Imaging Project (CPIP) biobank. Plaque RNA expression levels of SRGN and cell markers were assessed by plaque bulk RNA sequencing. SRGN expression and distribution were further examined in tissue plaque sections through histology and multispectral imaging. In vitro models were used to investigate the functional consequences of SRGN expression changes, using SRGN CRISPR knockout THP1 monocytic cells. Immunoprecipitation of SRGN and immunoblotting against SRGN, heparan and chondroitin sulfate chains were employed to structurally characterize plaque protein SRGN. Results SRGN protein levels were significantly higher in plaques from asymptomatic patients compared to symptomatic. Multispectral imaging showed that SRGN protein was observed in plaque areas rich in macrophages and lipids. Using immunoprecipitation and immunoblotting, we identified a unique low glycosylated plaque SRGN, with both heparan sulfate and chondroitin sulfate glycosaminoglycans decorating its protein core. This exclusive configuration suggests that the plaque cell origin of SRGN may be macrophages or mast cells. In support of this, plaque SRGN RNA expression correlated with RNA levels of pro-inflammatory macrophage cells markers like CD68, CD163, MRC1, ITGAX. Plaque SRGN protein levels correlated with plaque levels of oxidised lipoproteins, proatherogenic (CCL1, IL7, MCP-4 and TNFα) and antiatherogenic cytokines (IL33, IL16 and CXCL13), as well as with growth factors, such as TGF-β. On the one hand, in vitro suppression of SRGN expression in monocytes led to a reduction in the anti-inflammatory M2-polarized phenotype, while M1 pro-inflammatory macrophages were unskewed. Furthermore, our in vitro studies showed that upon SRGN knockdown M2 macrophages retained more oxidized lipoproteins. Conclusions Our results suggest that SRGN may be a crucial modulator of inflammation in human atherosclerotic plaques, potentially by affecting the bioavailability of effector molecules such as growth factors and cytokines.

Inhibition of Adipocyte Necroptosis Alleviates Fat Necrosis and Fibrosis After Grafting in a Murine Model.

Because of the delicate structure of the adipose tissue, fat necrosis accounts for 43.7% of all complications after autologous fat grafting; however, its regulation remains unclear. The purpose of this study was to examine the role of necroptosis in fat graft remodeling after grafting. Clinical fat graft necrosis samples were collected, and the expression levels of the necroptosis marker phosphorylated(p)-MLKL were analyzed. Transcriptome analysis was performed on fat grafts before and 1 week after transplantation in C57BL/6 mouse fat grafting models. Additionally, the in vivo effects of RIPK1 inhibitor Nec-1s or RIPK3 inhibitor GSK'872 on the fat grafting complications, including fat necrosis and fibrosis, were investigated. Necroptosis markers were observed and associated with higher occurrence of fibrosis in clinical fat graft necrosis samples compared to normal fat tissue. Amplification and RNA-Seq were conducted on RNA isolated from fat grafts before and after grafting. MLKL, RIPK1, and RIPK3's expression levels were significantly upregulated in comparison to controls. Higher expression levels of necroptotic RNAs were associated with higher levels of DAMPs, including Cxcl2, HMGB1, S100a8, S100a9, Nlrp3, and IL33, and activated proinflammatory signaling pathways, including the TNF, NF-kappa B, and chemokine signaling pathways. Necroptotic inhibitor Nec-1s and GSK'872 robustly suppressed the p-MLKL expression level and significantly inhibited necroptotic cell death, especially in adipocytes. Moreover, administration of Nec-1s and GSK'872 significantly alleviated fat necrosis and subsequent fibrosis in fat grafts. Collectively, our study findings highlight the potential therapeutic applications of necroptosis inhibitors in preventing fat necrosis and fibrosis after grafting.

Investigation of lactotransferrin messenger RNA expression levels as an anti–type 2 asthma biomarker

Lactotransferrin (LTF) has an immunomodulatory function, and its expression levels are associated with asthma susceptibility. We sought to investigate LTF messenger RNA (mRNA) expression levels in human bronchial epithelial cells (BECs) as an anti-type 2 (T2) asthma biomarker. Association analyses between LTF mRNA expression levels in BECs and asthma-related phenotypes were performed in the Severe Asthma Research Program (SARP) cross-sectional (n= 155) and longitudinal (n= 156) cohorts using a generalized linear model. Correlation analyses of mRNA expression levels between LTF and all other genes were performed by Spearman correlation. Low LTF mRNA expression levels were associated with asthma susceptibility and severity (P< .025), retrospective andprospective asthma exacerbations, and low lung function (P< 8.3×10-3). Low LTF mRNA expression levels were associated with high airway T2 inflammation biomarkers (sputum eosinophils and fractional exhaled nitric oxide; P<8.3×10-3) but were not associated with blood eosinophils or total serum IgE. LTF mRNA expression levels were negatively correlated with expression levels of TH2 or asthma-associated genes (POSTN, NOS2, and MUC5AC) and eosinophil-related genes (IL1RL1, CCL26, and IKZF2) and positively correlated with expression levels of TH1 and inflammation genes (IL12A, MUC5B, and CC16) and TH17-driven cytokines or chemokines for neutrophils (CXCL1,CXCL6, and CSF3) (P< 3.5×10-6). Low LTF mRNA expression levels in BECs are associated with asthma susceptibility, severity, and exacerbations through upregulation of airway T2 inflammation. LTF is a potential anti-T2 biomarker, and its expression levels may help determine the balance of eosinophilic and neutrophilic asthma.