RNA Expression Levels Research Articles

Predictive methylation signature for gemcitabine sensitivity in pancreatic ductal adenocarcinoma: A pathway to personalized treatment.

768 Background: Gemcitabine (Gem) is a key drug used in chemotherapy regimens for treating pancreatic ductal adenocarcinoma (PDA). Identifying a biomarker to predict patient response to Gem could be crucial in personalizing treatment selection between Gemcitabine and fluorouracil (5-FU)-based therapies, thereby improving patient outcomes. We hypothesize that DNA methylation changes in genes linked to Gem resistance may serve as surrogates for the expression of corresponding proteins, thereby predicting treatment outcomes in patients with PDA receiving this therapy. Methods: We curated a 93-gene panel through a literature review spanning from January 1970 to April 2024. The proteins encoded by these genes have demonstrated either preclinical evidence (from cell line or mouse models) or clinical evidence (from tissue-based studies) of affecting the response to Gem in PDA. We evaluated the prognostic significance of methylation at specific CpG (cytosine-phosphate-guanine) sites within these genes using data from The Cancer Genome Atlas (TCGA) database, focusing on patients who had received Gem (106 /184 patients). We utilized elastic net multivariate analysis to analyze survival and created Kaplan-Meier plots based on our gene panel to estimate overall survival (OS). We compared RNA and gene expression levels for the clinically significant gene signatures between normal (NT) and PDA tissues using TNMplot.com, a web-based tool developed from data in the Gene Expression Omnibus of the National Center for Biotechnology Information (NCBI-GEO), TCGA, Therapeutically Applicable Research to Generate Effective Treatments (TARGET), and The Genotype-Tissue Expression (GTEx) repositories. This tool employs Mann-Whitney or Kruskal-Wallis tests to determine statistical significance. Results: We identified an 8-CpG site methylation signature across eight genes, distinguishing a high-risk subgroup within the Gem-treated cohort in the TCGA database. Patients exhibiting methylation at specific CpG sites in this signature experienced significantly worse OS, with median survival times of 20.35 months compared to 49.38 months (p=0.0068). Additionally, the RNA expression (fold change: 2.11, p=2.12e-55) and gene expression (fold change: 1.42, p=3.14e-18) of these genes were markedly higher in PDA tissues compared to normal tissues. Notably, the candidate genes in our signature do not overlap with those used to classify PDA into classical versus basal subtypes. In an ongoing study, we are also investigating methylation changes of these candidate genes in the blood (cell-free DNA) of PDA patients. Conclusions: We present a signature that can predict Gem sensitivity or resistance in patients with PDA. This signature serves as a critical first step toward personalizing treatment strategies.

Leveraging large-scale PDO-based assays to optimize antibody-drug conjugate efficacy in CRC.

261 Background: Clinical trials are one of the major barriers in contemporary drug development. Functional assays based on patient-derived organoids (PDO) are a promising new tool for derisking clinical development of new therapies, but their use has been limited due to small cohort sizes and the absence of systematic validation studies. Using the largest cohort of matched PDOs, longitudinal clinical data, transcriptomic and WES data in CRC, we explore the use of large PDOs collections to characterize ADC efficacy and affinity through systematic mapping of the relationship between target antigen affinity and payload efficacy. Methods: We have assembled a collection of 85 PDOs from colorectal cancer (CRC), generated from primary tumour samples and metastatic biopsies. Patients ranged from 32 to 89 years old and had experienced an average of 1.54 lines of treatment prior to the PDO-generating tissue sampling. Using gene expression levels of well known target antigens such as CEACAM5, ERBB2, MSLN and TROP2 as a proxy for protein concentration, we explore the relationship between the target antigen affinity and the efficacy of a subset of common payloads including topoisomerase and microtubule inhibitors. This allows us to identify the relationship between two of the three main vectors of ADC efficacy in CRC. Results: We show that topoismerase inhibitor and microtubule inhibitor efficacy relates to the RNA expression level of multiple target antigens commonly used in active clinical trials in CRC. These data match the target antigen and payload couples currently under development or approved in the clinic. We are developing a tool to optimize clinical trial design. This approach will allow us to match patients more accurately with the appropriate payload, ensuring a higher likelihood of response. Conclusions: We report that large-scale PDO-based assays can be a useful tool to anticipate clinical readouts. This work suggests that well-characterized PDO collections could also help redefine patient populations, increasing the likelihood of identifying those more likely to respond to a given ADC, and thus improving the success rates of clinical studies for new treatments in CRC.

Photoperiod and Light Spectrum Modulate Daily Rhythms and Expression of Genes Involved in Cell Proliferation, DNA Repair, Apoptosis and Oxidative Stress in a Seabream Embryonic Stem Cell Line

The use of cell lines as alternative models for environmental physiology studies opens a new window of possibilities and is becoming an increasingly used tool in marine research to fulfil the 3R’s rule. In this study, an embryonic monoclonal stem cell line obtained from a marine teleost (gilthead seabream, Sparus aurata) was employed to assess the effects of photoperiod (light/dark cycles vs constant dark) and light spectrum (white, blue, green, blue/green and red lights) on gene expression and rhythms of cellular markers of proliferation, DNA repair, apoptosis and cellular/oxidative stress by RT-qPCR and cosinor analyses. The results obtained revealed the optimal performance of cells under blue light (LDB), with all the genes analysed showing their highest RNA expression levels and most robust daily variations/rhythms in this condition. Under LDB, the mRNA levels of cell proliferation (pcna), DNA repair (cry5), anti-apoptotic (bcl2) and oxidative stress (prdx2) markers peaked at the day-night transition, whereas pro-apoptotic (bax) and cell stress (hsp70) markers showed their highest expression at the night-day transition, evidencing the strong synchronisation of the transcription of key genes involved in the cell cycle in this photoregime. The persistence of significant pcna, cry5, hsp70 and prdx2 rhythms after 3 days in constant darkness reveals the endogenous and circadian nature of these rhythms. Our results highlight the importance of implementing photoperiods with light–dark cycles of blue wavelengths when performing fish cell culture research. These results reinforce and extend our previous studies, confirming the importance of lighting conditions that mimic the natural environment for the proper development of fish embryos and larvae in aquaculture.

Gene expression of psychiatric disorder-related kinesin superfamily proteins (Kifs) is potentiated in alternatively activated primary cultured microglia.

Reactivity of microglia, the resident cells of the brain, underlies innate immune mechanisms (e.g., injury repair), and disruption of microglial reactivity has been shown to facilitate psychiatric disorder dysfunctions. Although cellular analyses based on cultured microglia have been conducted, the molecular mechanism regulating microglial polarization remains elusive. We established a primary microglia culture that enabled manipulation of the substate of cells. This allowed us to investigate the expression levels of psychiatric disorder-related Kifs messenger RNA (mRNA) in each condition. Kifs encode molecular motor proteins that transport cargo along microtubules, which are thought to dynamically reorganize during a substate change. As a candidate for a crucial Kifs gene that is associated with microglia polarization, we selected psychiatric disorder-related Kifs including Kif17. We found that the relative amounts of Kif3a, Kif17, and Kif13a mRNA were potentiated in alternatively activated microglia, whereas there were no significant changes in activated microglia. Furthermore, the microglia derived from a mouse line which possesses a mutation inducing truncated KIF17 indicated disrupted morphological phenotype of alternatively activated microglia. These results suggest that the potentiation of specific molecular motor expression is required to maintain the function of alternatively activated microglia.

High Expression Levels of the Long Non-Coding RNAs Lnc-IRF2-3 and Lnc-KIAA1755-4 Are Markers of Poor Prognosis in Chronic Lymphocytic Leukemia

Long non-coding RNAs (lncRNAs) play complex roles at multiple levels of gene regulation, thus modulating key cellular processes involved in the pathogenesis and progression of cancer. Aberrant expression of lncRNAs has been reported in various malignancies, including chronic lymphocytic leukemia (CLL). We investigated the expression of lnc-IRF2-3 and lnc-KIAA1755-4 in peripheral blood mononuclear cells of 112 previously untreated CLL patients by quantitative reverse-transcriptase polymerase chain reaction. Both lncRNAs were found to be overexpressed in CLL samples in comparison to healthy controls, and their high levels were associated with adverse clinico-biological characteristics of patients at diagnosis. High lnc-IRF2-3 expression was associated with high leukocyte and lymphocyte counts, high β2-microglobulin, advanced Binet stage, unfavorable cytogenetics, CD38-positivity and IGHV-unmutated status. Regarding lnc-KIAA1755-4, its high expression was associated with high leukocyte count, lymphocyte count, β2-microglobulin, lactate dehydrogenase and low hemoglobin, as well as with IGHV-unmutated status. In addition, we observed shorter time to first treatment and overall survival of patients expressing high levels of both lncRNAs in comparison to low-expressing patients. In summary, our study showed that high lnc-IRF2-3 and lnc-KIAA1755-4 expression at diagnosis predicts poor survival in CLL. The mechanisms of their upregulation, as well as their specific targets in CLL cells, remain to be elucidated.

Intratumoral or Subcutaneous MK-2118, a Non-Cyclic Dinucleotide STING Agonist, With or Without Pembrolizumab for Advanced or Metastatic Solid Tumors or Lymphomas.

We evaluated the non-cyclic dinucleotide stimulator of interferon genes agonist MK-2118 ± pembrolizumab in patients with advanced solid tumors or lymphomas. This first-in-human study (NCT03249792) enrolled patients with refractory, advanced solid tumors or lymphomas. Patients received intratumoral (IT) MK-2118 100-20,000 µg (arm 1), IT MK-2118 900-15,000 µg plus intravenous (IV) pembrolizumab 200 mg every 3 weeks (Q3W; arm 2), or subcutaneous (SC) MK-2118 5000-150,000 µg plus IV pembrolizumab 200 mg Q3W (arm 4); arm 3 (visceral injection of MK-2118) was not pursued. IT dosing used an accelerated titration design and modified toxicity probability interval method; SC dosing (arm 4) was started subsequent to arms 1 and 2. Primary objectives were safety/tolerability. MK-2118 pharmacokinetics were a secondary endpoint; objective responses and biomarkers were exploratory endpoints. 140 patients were enrolled (arm 1, n=27; arm 2, n=57; arm 4, n=56). Grade 3/4 treatment-related adverse events occurred in 22%, 23%, and 11% of patients, respectively, but no maximum tolerated dose was identified up to MK-2118 20,000, 15,000, and 150,000 µg across the 3 arms. Dose-dependent increases in MK-2118 systemic exposure were observed following IT and SC administration. Objective responses were seen in 0%, 6%, and 4% of patients, respectively. IT MK-2118 led to dose-dependent changes in STING-based blood RNA expression levels, interferon-gamma, interferon-gamma‒induced protein 10, and interleukin-6; SC MK-2118 did not generate dose-related immune responses. IT MK-2118 ± pembrolizumab and SC MK-2118 plus pembrolizumab had manageable toxicity and limited antitumor activity. IT but not SC administration demonstrated systemic immune effects.

PET Quantification in Healthy Humans of Cyclooxygenase-2, a Potential Biomarker of Neuroinflammation.

Cyclooxygenase-2 (COX-2) is present in a healthy brain at low densities but can be markedly upregulated by excitatory input and by inflammogens. This study evaluated the sensitivity of the PET radioligand [11C]-6-methoxy-2-(4-(methylsulfonyl)phenyl)-N-(thiophen-2-ylmethyl)pyrimidin-4-amine ([11C]MC1) to detect COX-2 density in a healthy human brain. Methods: The specificity of [11C]MC1 was confirmed using lipopolysaccharide-injected rats and transgenic mice expressing the human COX-2 gene, with 120-min baseline and blocked scans using COX-1 and COX-2 selective agents. Twenty-seven healthy participants were injected with [11C]MC1. Ten of these participants received 2 PET scans: a baseline study followed by blockade with celecoxib (600 mg orally), a preferential COX-2 inhibitor. Seventeen participants underwent test-retest imaging. All scans included concurrent arterial sampling. The tissue-to-plasma ratio at equilibrium (i.e., total distribution volume) was determined using a 2-tissue compartment model (2TCM). Results: In humanized transgenic COX-2 mice, 70%-90% of [11C]MC1 brain uptake was blocked by nonradioactive MC1 and celecoxib (a COX-2 selective inhibitor) but not by PS13 (a COX-1 selective inhibitor), thereby confirming specific binding to human COX-2. Radioactivity in the human brain peaked at a concentration of about 4.0 SUV, indicating good passage through the blood-brain barrier. Values for the total distribution volume achieved stability after 80 min, indicating no radiometabolite contamination. Celecoxib reduced radioligand binding in neocortical areas by 25% but had little or no effect in subcortical regions and the cerebellum, which correlated with COX-2 messenger RNA expression levels. Binding site occupancy by celecoxib was virtually complete, as determined by the Lassen plots. Test-retest reliability was moderate (intraclass correlation coefficient, 0.65) but had relatively large variability (absolute retest variability, 20%). Reference tissue methods yielded results comparable to those of 2TCM but reduced retest variability by up to 75% and reduced intersubject variability (coefficient of variation) by about half. Thus, compared with 2TCM, which requires arterial blood, the reference tissue method is expected to significantly reduce the sample sizes required to detect statistically significant differences between groups. Conclusion: [11C]MC1 has adequate sensitivity to measure the low density of COX-2 in a healthy human brain, suggesting it can also quantify the COX-2 elevations expected in human disorders associated with neuroinflammation.

SNHG17 Reprograms Energy Metabolism of Breast Cancer by Activating Mitochondrial DNAs Transcription.

In most solid tumors, cellular energy metabolism is primarily dominated by aerobic glycolysis, which fulfills the high demand for biomacromolecules at the expense of reduced ATP production efficiency. Elucidation of the mechanisms by which rapidly proliferating malignant cells acquire sufficient energy in this state of inefficient ATP production from glycolysis could enable development of metabolism targeted therapeutic strategies. In this study, we observed a significant association between elevated expression levels of the long non-coding RNA (lncRNA) SNHG17 and unfavorable prognosis in breast cancer (BCa). SNHG17 promoted BCa cell proliferation by augmenting mitochondrial ATP production. Mechanistically, SNHG17 directly interacted with the p65 subunit of NF-κB and phosphorylated p65 at the threonine 505 site. SNHG17 bound to p65 at its truncated loop2 site, recruited p65 to mitochondria, and co-regulated the transcriptional activation of mitochondrial DNA to promote ATP production. Accordingly, targeting SNHG17 with an anti-sense oligonucleotide (ASO) significantly reduced BCa tumor growth both in vitro and in vivo. Overall, these results established a role for SNHG17 in promoting BCa progression by increasing ATP production and provided insight into the reprogramming of energy metabolism in solid tumors.

Lidocaine could promote the cuproptosis through up-regulating the long noncoding RNA DNMBP-AS1 in Hep-2 cells

BackgroundLidocaine is a traditional local anesthetic, which has been reported to trigger apoptosis through the mitochondrial pathway, independent of death receptor signaling. Cuproptosis is a copper triggered mitochondrial cell death mode. In this study, we explored the biological effects of lidocaine on cuproptosis in Hep-2 cells and studied the relevant mechanisms.Methodsquantitative RT-PCR was used to measure the expression level of long noncoding RNA (IncRNA) DNMBP-AS1. DNMBP-AS1 siRNA (si-DNMBP-AS1) were transfected into Hep-2 cells to verify the roles of DNMBP-AS1 in cuproptosis. 24 h treatment with 20 nM elesclomol and 2 µM CuCl2 was performed to promote the occurrence of Cuproptosis. Cell proliferation, migration and apoptosis assays ware utilized to analyze biological effect of lidocaine and DNMBP-AS1 on Hep-2 cells. Active caspase-3 were also determined after treatment.ResultsDNMBP-AS1 was significantly upregulated during cuproptosis in Hep-2 cells. The si-DNMBP-AS1 significantly increased the cell viability with nonactivated caspase-3, promoted the cell migration and suppress the cuproptosis. Lidocaine was cytotoxic to the Hep-2 cells in a dose- and time-dependent manner. Exposure to 10 µM of lidocaine for 24 h did not reduce the viability or activated the caspase-3, but significantly increased the expression of DNMBP-AS1, and promote the cuproptosis. Anymore, si-DNMBP-AS1 reversed the pro-cuproptosis function of lidocaine.Conclusionslidocaine was cytotoxic to Hep-2 cells in a time- and dose-dependent manner, promoted the cuproptosis through up-regulating DNMBP-AS1. The results of this study offered initial optimism that lidocaine could be used in an adjuvant or neoadjuvant fashion in cancer treatment.

APOBEC3A deaminates CTG hairpin loops to promote fragility and instability of expanded CAG/CTG repeats

CAG/CTG repeats are prone to expansion, causing several inherited human diseases. The initiating sources of DNA damage which lead to inaccurate repair of the repeat tract to cause expansions are not fully understood. Expansion-prone CAG/CTG repeats are actively transcribed and prone to forming stable R-loops with hairpin structures forming on the displaced single-stranded DNA (S-loops). We previously determined that damage by the Saccharomyces cerevisiae cytosine deaminase, Fcy1, was required for both fragility and instability of CAG/CTG tracts engaged in R-loops. To determine whether this mechanism is more universal, we expressed human cytidine deaminases APOBEC3A (A3A), APOBEC3B (A3B), or activation-induced cytidine deaminase (AID) in our yeast system. We show that mutagenic activity of Apolipoprotein B messenger RNA-editing enzyme, catalytic polypeptides causes CAG/CTG fragility and instability, with A3A having the greatest effect followed by A3B and least from AID. A3A-induced repeat fragility was exacerbated by enrichment of R-loops at the repeat site. A3A and A3B-induced instability was dependent on the MutLγ nuclease and to a lesser extent, base excision repair factors. Deaminase activity assays on hairpin substrates containing CTG and GTC triplet sequences revealed that A3A prefers cytidines within the hairpin loop, and bulges in the hairpin stem alter preferred locations. Analysis of RNA expression levels in human cortex samples revealed that A3A is expressed in brain tissue that exhibits CAG/CTG repeat expansions and its expression is elevated in Huntington's disease (HD) patient samples. These results implicate cytidine deamination by A3A as a potential source of repeat expansions in HD and other CAG/CTG repeat expansion disorders.

Protective effects of small RNAs encapsulated in Artemisia Capillaris-derived exosomes against non-alcoholic fatty liver disease.

Artemisia capillaris, a traditional medicinal plant, is renowned for its therapeutic properties, including the promotion of anti-inflammatory and bile secretion. Notably, it has demonstrated efficacy in the treatment of jaundice. This study aimed to evaluate the potential of Artemisia capillaris-derived exosomes (ACDEs) as a novel therapeutic approach in non-alcoholic fatty liver disease (NAFLD). The physicochemical properties of ACDEs were isolated and characterized using differential centrifugation, and the therapeutic efficacy was evaluated in an in vivo methionine-choline-deficient (MCD) diet induced NAFLD mouse model. In vitro, mouse hepatocytes were treated with palmitic acid (PA) to simulate a high fat environment. Intracellular triglycerides (TG) and total cholesterol (TC) levels were quantified, and Oil Red O staining was assessed. Additionally, the expression levels of proteins and RNAs associated with lipogenesis and inflammation were analyzed. The NAFLD mouse model exhibited notable liver damage, including lipid deposition and inflammatory responses. However, treatment with ACDEs exhibited broad pharmacological activities, effectively reversing hepatic lipid accumulation and inflammatory damage. In vitro experiments revealed that ACDEs were internalized by AML12 cells via macropinocytosis and caveolin-mediated endocytosis. This treatment ameliorated dysregulated lipid metabolism and inhibited inflammatory responses. High throughput sequencing further identified a distinct small RNA profile in ACDEs, indicating the potential involvement in interspecies physiological regulation. In conclusion, this study provides evidence for the therapeutic potential of ACDEs in NAFLD and offers a novel perspective for the development of Artemisia capillaris-based therapies for NAFLD, related metabolic disorders, and hepatitis.

The levels of the long non-coding RNA MALAT1 affect cell viability and modulate TDP-43 binding to mRNA in the nucleus.

TAR DNA-binding protein (TDP-43) and Metastasis Associated Lung Adenocarcinoma Transcript (MALAT1) RNA are both abundantly expressed in the human cell nucleus. Increased interaction of TDP-43 and MALAT1, as well as dysregulation of TDP-43 function, was previously identified in brain samples from patients with neurodegenerative disease compared to healthy brain tissues. We hypothesized that TDP-43 function may depend in part on MALAT1 expression levels. Here, we find that alterations in MALAT1 expression affect cell viability and can modulate TDP-43 binding to other mRNAs in HEK293 and SH-SH5Y human cell lines. Disruption of either MALAT1 or TDP-43 expression induces cell death, indicating that both macromolecules contribute positively to survival. Depletion of MALAT1 RNA results in increased binding of TDP-43 to other mRNA transcripts at the 3' UTR. Finally, we examined the contribution of MALAT1 expression to survival in a cell culture model of neurodegeneration using MPP+ treatment in SH-SY5Y cells. Depletion of MALAT1 RNA protects against toxicity in a cellular model of neurodegeneration and modulates TDP-43 binding to mRNA transcripts involved in apoptotic cell death. Taken together, we find that MALAT1 RNA and TDP-43 interactions can affect mRNA levels and cell viability. A tightly regulated network of non-coding RNA, messenger RNA, and protein interactions could provide a mechanism to maintain appropriate RNA expression levels and contribute to neuronal function.

Therapeutic Potential of Brassica carinata Microgreens Extract in Alleviating Symptoms of Type 2 Diabetes in Wistar Rats.

Microgreens of Brassica plants have attracted increasing research interest in the management of the prevailing epidemic of Type 2 diabetes mellitus (T2DM) because of their high nutritional value. This study evaluated the antidiabetic effects of Brassica carinata Microgreens Ethanolic Extract (BMEE) in type-2 diabetic rats. For the normoglycemic assay, rats were divided into five groups and received a single oral dose of 100, 250, and 500 mg/kg of BMEE while the control groups received distilled water and Glibenclamide. Fasting blood glucose (FBG) levels were determined on a weekly basis for 28 days in diabetic rats after treatment with BMEE at 250 and 500 mg/kg dosage levels. Oral glucose tolerance test (OGTT), serum insulin levels, lipid profile and messenger RNA expression levels of Insulin receptor substrate 1 (IRS-1), Glucose transporter 2 (GLUT2), and Nuclear factor kappa light-chain enhancer of activated B cells (NFKβ) genes were determined. BMEE did not induce hypoglycemic effects in rats with normal blood glucose levels, but induced antidiabetic activities in the experimental type-2 diabetic rats. BMEE lowered FBG levels, increased oral glucose tolerance, increased insulin sensitization, and reduced insulin resistance. Treatment of diabetic rats with BMEE increased lipid metabolism and relatively higher expression levels of IRS-1 and GLUT2 genes, and led to reduced expression levels of NFKβ in the liver. Overall, this study reports that BMEE has potential as a nutraceutical to be utilized in the management of T2DM.

Interleukin-6 promotes the epithelial mesenchymal transition in canine tonsillar squamous cell carcinoma cells

Canine oral squamous cell carcinoma (CoSCC) is often associated with suppurative inflammation. Metastasis of malignant tumors is one of the signs of major interest in oncology because it speaks of disease progression, where the involvement of interleukin-6 (IL-6) in cancer progression is so far unknown. Therefore, the aim of this study was the determination of the role of IL-6 in metastasis in CoSCC cells model through expression analysis of mRNA and protein using real-time PCR and western blotting and assessment of cell migration and invasion. The messenger RNA (mRNA) expression level of IL-6 was elevated in CoSCC tissues, and the IL-6 receptor protein was expressed in CoSCC cell lines. Furthermore, IL-6 levels were associated and showed negative correlation with survival time (rs = −0.92857) in dogs with tonsillar SCC. Recombinant canine IL-6 (rcIL-6) treatment promoted migration and invasion, in addition to increasing the viable cell number of the tonsillar SCC cell line (TSCCLN#6). Consistently, the protein expression of phosphorylated ERK1/2 and STAT3 and Fascin1 (FSCN1) was upregulated by treatment with rcIL-6 in a dose-dependent manner. Treatment with ERK or STAT3 inhibitors abolished the effects of rcIL-6, and the ERK inhibitor successfully downregulated the expression of FSCN1. In conclusion, IL-6 may be involved in tonsillar CoSCC invasion and metastasis through the activation of the mitogen-activated protein kinase and Janus tyrosine kinase/signal transducer and activator of transcription signaling.

Exploring the role of ATF3 and ferroptosis-related RNA expression in osteoarthritis: An RNA analysis approach to immune infiltration.

Osteoarthritis (OA) is a widespread joint disorder that is primarily noted for the progressive degeneration of joint cartilage, accompanied by a significant inflammatory response. Recently, there has been a growing interest in understanding the roles of ATF3 and ferritin-related RNAs in the context of immune responses and inflammatory processes. However, their specific functions and mechanisms in the progression of osteoarthritis have remained largely ambiguous and underexplored. The primary objective of this study was to thoroughly investigate the changes in expression levels of ATF3 and ferritin-related RNAs within osteoarthritic tissues, as well as to examine their potential effects on immune cell infiltration. To achieve this, advanced RNA sequencing technology was employed to meticulously analyze the expression levels of ATF3 and the ferritin-related RNAs. Furthermore, bioinformatics methods were utilized to assess the infiltration patterns of various immune cells and to explore the correlation between these infiltration patterns and the expression levels of RNA. The findings from this study revealed that both ATF3 and ferritin-related RNAs exhibited significantly elevated expression levels in tissues affected by osteoarthritis. Additionally, the immunoinfiltration analysis highlighted a positive correlation between the degree of infiltration of T cells and macrophages and the levels of ferritin-related RNAs. Such findings suggest that the presence of these immune cells is intricately linked to the expression of ferritin-associated RNAs. Further investigations indicated that ferritin-associated RNAs play a critical role in the progression of osteoarthritis by modulating inflammatory responses and influencing the activity of various immune cells. Consequently, both ATF3 and ferritin-related RNAs demonstrate abnormal expression patterns in osteoarthritis, which are closely associated with the infiltration of immune cells.

Biotin-Based Northern Blotting (BiNoB): A Cost-Efficient Alternative for Detection of Small RNAs.

Advances in sequencing technology have led to the discovery of diverse types of regulatory RNAs. Differential transcript levels regulate cellular processes and influence disease severity. Identifying these variations through reliable methods is crucial for understanding the regulatory roles and disease mechanisms of regulatory RNAs. Northern blotting, which is considered the gold standard for differential expression analysis, poses challenges due to various limitations associated with RNA quality and integrity, radioactivity exposure, and associated reagents and expenses. In this protocol, we employ a biotin-based northern blotting (BiNoB) approach that is both convenient and inexpensive, eliminating the need for specialized settings as required with radioactivity-based northern blotting. We comprehensively target various RNA types, making this technique a versatile tool for RNA detection. Additionally, we conduct a comparison between 3'-end labeled probes that were labeled in-house and 5'-end labeled probes that were obtained commercially. Remarkably, our results reveal relatively higher sensitivity with 3'-end labeled probes. Furthermore, we demonstrated that the use of an in-house buffer offered comparable sensitivity to a commercially available buffer, providing another cost-effective alternative. We also aimed to determine the minimum quantity of total RNA required to detect small non-coding RNAs such as tRNA fragments. Whereas previous studies reported the use of 5-10 µg total RNA for tRNA fragment detection, our findings revealed that as little as 1 µg total RNA is sufficient to detect small RNAs like tRNAs and their fragments. This concentration may vary depending on the expression levels of the specific RNAs being detected. © 2024 Wiley Periodicals LLC. Basic Protocol: Biotin-based northern blotting.

Identification of Serum-Derived CricRNA Diagnostic Panel and Revealing Their Regulatory Mechanisms in HCV-HCC: A Cross-Sectional Study.

Hepatitis C virus (HCV) infection is a significant risk factor for the development of hepatocellular carcinoma (HCC). Serum-derived circular RNAs (circRNAs) play several crucial roles in HCV and HCC. They represent a promising area of research for improving the diagnosis and understanding the mechanisms of HCV-HCC. This study aims to identify a serum-derived circular RNA (circRNA) diagnostic panel for HCV-HCC and to elucidate the regulatory mechanisms underlying their role in cancer progression. In this study, data mining and in silico analysis were conducted to identify the role of circular RNAs (hsa_circ_0003288, circ-RNF13, hsa_circ_0004277, circANRIL, circUHRF1, hsa_circ_103047) and their associated biomarkers (IL-6 and NF-κB) in HCV-HCC pathogenesis. Additionally, RT-PCR was performed to assess their expression levels across different study groups (G0 = control, G1 = HCV, G2 = HCC, and G3 = HCV-induced HCC). The expression levels of circular RNAs, including hsa_circ_0003288, circ-RNF13, hsa_circ_0004277, circANRIL, circUHRF1, and hsa_circ_103047, as well as the biomarkers IL-6 and NF-κB, were significantly elevated in the G3 group compared to the G0 group. ROC analysis also revealed significantly different expression rates for G3 group and G0 group. The data revealed that cricRNAs panel (hsa_circ_0003288, circ-RNF13, circANRIL, circUHRF1, and hsa_circ_103047) could serve as a diagnostic biomarker and therapeutic target for HCV-induced HCC.

Targeting LncRNA ADAMTS9-AS1 is a Promising Therapeutic Strategy to Inhibit the Progression of Bladder Cancer.

Bladder cancer (BC) is a malignant tumor that begins in the cells of the bladder, characterized by poor cell differentiation and strong invasion capacity, with a high incidence rate. Identifying key molecules that enhance BC cells' cisplatin sensitivity can help improve the clinical efficacy of BC treatment. Hence, this study aimed to determine the expression level of long non-coding RNA (lncRNA) ADAM Metallopeptidase with Thrombospondin Type 1 Motif 9 Antisense RNA 1 (ADAMTS9-AS1) in BC and explore its related mechanism underlying the amplification of cisplatin sensitivity. Cancer tissues and para-cancerous tissues of 10 BC patients treated in The 908th Hospital of Joint Logistic Support Force of PLA were collected retrospectively and analyzed for the expression of the lncRNA ADAMTS9-AS1 and fused in sarcoma (FUS) in this tissue. Normal bladder epithelial cell line SV-HUC1, and BC cell lines such as T24, J82, 5637, KU-19-19, and EJ were cultured for in vitro experimentation. Then, the expression levels of ADAMTS9-AS1, FUS mRNA, and FUS protein were detected by means of reverse-transcription quantitative polymerase chain reaction (RT-qPCR), Western blotting, and immunohistochemistry. pcDNA3.1 vector, pcDNA3.1-ADAMTS9-AS1, or pcDNA3.1-ADAMTS9-AS1 and FUS overexpression plasmid was transfected into the cultured T24 and 5637 cells. A series of tests were performed to detect cell proliferation, migration capacity, apoptosis, and cisplatin half-effective concentration (IC50) values of BC cells using Cell Counting Kit-8 (CCK-8) assay, colony formation assay, wound healing assay, flow cytometry, and gradient cisplatin culturation. Compared with SV-HUC1 cell line and adjacent normal tissues, ADAMTS9-AS1 levels were significantly decreased in T24, J82, 5637, KU-19-19, EJ cell lines, and BC tissues, while FUS mRNA and protein expression levels were up-regulated (p < 0.05). After transfection with pcDNA3.1-ADAMTS9-AS1, the colony number, cell viability, wound healing ratio, and cisplatin IC50 value, were remarkably reduced (p < 0.05), but apoptosis ratio, cleaved-caspase3 and cleaved-poly-ADP-ribose polymerases (PARP) expressions were increased (p < 0.05). ADAMTS9-AS1 was found to directly target FUS, and overexpression of FUS reversed ADAMTS9-AS1 effects on BC cells. ASAMTS9-AS1 can inhibit the proliferation and migration, and promote apoptosis and cisplatin sensitivity of BC cells through regulating FUS, thus providing a theoretical basis for ADAMTS9-AS1 as a potential therapeutic target in BC treatment.

Elevated MCL1 expression drives esophageal squamous cell carcinoma stemness and induces resistance to radiotherapy.

Esophageal squamous cell carcinoma (ESCC) stands as the sixth most common cause of cancer-related mortality on a global scale, with a strikingly high proportion-over half-of these fatalities occurring within China. The emergence of radiation resistance in ESCC patients significantly diminishes overall survival rates, complicating treatment regimens and reducing clinical outcomes. There is an urgent need to explore the molecular mechanisms that underpin radiation resistance in ESCC, which could lead to the identification of new therapeutic targets aimed at overcoming this resistance. This study aims to elucidate the role of myeloid cell leukemia-1 (MCL1) in ESCC and its association with radioresistance, thereby providing a novel strategy for enhancing the efficacy of radiotherapy. We used The Cancer Genome Atlas (TCGA) database, Genotype-Tissue Expression (GTEx) project and real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) of 10 pairs of fresh endoscopic biopsy samples from patients with ESCC to analyze the messenger RNA (mRNA) expression levels of MCL1 in esophageal cancer tissues as compared to normal tissues. Immunohistochemistry (IHC) staining and Western blotting were performed using an anti-MCL1 antibody to visualize protein expression. The mechanism of radioresistance of ESCC was examined by combining bioinformatics analysis, Western blotting, and clone formation and stemness sphere formation assays. The analysis of TCGA database and the results of RT-qPCR indicated that the mRNA level of MCL1 was overexpressed in esophageal cancer tissues. Subsequently, the results of IHC and Western blotting showed that the protein level of MCL1 expression in cancer tissues was significantly higher than that in adjacent normal tissues. Moreover, there was a significant upregulation of MCL1 in ESCC tissues and in radioresistant tissues and cells, with its overexpression correlating with the acquisition of stemness properties in ESCC. In terms of mechanism, MCL1 induced cell cycle arrest by regulating the expression of cyclin D3 and p21 through the JAK-STAT signaling pathway. G0/G1 phase arrest contributed to the stem cell-like phenotype. Blocking JAK-STAT signaling significantly improved the efficacy of radiotherapy for ESCC. These findings indicate that MCL1 is a critical cell cycle regulator that drives the stemness and radioresistance of ESCC and may thus be a potential target in a combined therapeutic strategy aimed at overcoming radioresistance.

CSE1L in enhancing the effect of defactinib on gastric cancer cells via the inhibition of FAK phosphorylation.

Chromosome segregation 1 like (CSE1L) overexpression can promote proliferation and migration in cancer. In previous study, we found that CSE1L expression was higher in gastric cancer (GC) tissues compared to normal tissues. However, the biological function and molecular mechanism of CSE1L in GC remains unclear. In this study, we investigate the function of CSE1L in GC biology and its related molecular mechanisms and therapeutic potentials. Transcriptome data from public databases were used to assess CSE1L messenger RNA (mRNA) expression levels in GC. A total of 83 pairs of GC surgical samples were evaluated to determine CSE1L protein expression levels. CSE1L was knocked out in MGC-803 and overexpressed in BGC-823 to evaluate its biological function in GC cells. RNA sequencing (RNA-seq) was used to identify the signaling pathways regulated by CSE1L and the underlying molecular mechanisms, and the transcriptome data were validated. Western blotting and immunofluorescence were used to clarify the regulatory effect of CSE1L on phosphorylated focal adhesion kinase (p-FAK). CSE1L mRNA was increased in patients with GC, and the high expression of CSE1L was associated with poor prognosis in these patients. Protein levels of CSE1L were also increased in GC surgical samples. CSE1L promoted cell proliferation, cell migration, cell invasion, clone formation, and adhesion ability in GC cells. RNA-seq results suggested that CSE1L upregulated the focal adhesion pathway, which was verified at the mRNA level and protein level. Moreover, CSE1L upregulated p-FAK tyrosine 397 [p-FAK (Y397)] and enhanced the efficacy of defactinib. After the knockout of CSE1L, the killing effect of defactinib on GC cells was intensified. CSE1L is a potential biomarker for evaluating the prognosis of patients with GC. Knockdown of CSE1L can enhance the efficacy of defactinib by inhibiting p-FAK (Y397), which may be a synergistic target of defactinib.