Real-time Quantitative Reverse Transcription-polymerase Research Articles

Identifying the stability of housekeeping genes to be used for the quantitative real-time PCR normalization in retinal tissue of streptozotocin-induced diabetic rats.

To investigate the stability of the seven housekeeping genes: beta-actin (ActB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18s ribosomal unit 5 (18s), cyclophilin A (CycA), hypoxanthine-guanine phosphoribosyl transferase (HPRT), ribosomal protein large P0 (36B4) and terminal uridylyl transferase 1 (U6) in the diabetic retinal tissue of rat model. The expression of these seven genes in rat retinal tissues was determined using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) in two groups; normal control rats and streptozotocin-induced diabetic rats. The stability analysis of gene expression was investigated using geNorm, NormFinder, BestKeeper, and comparative delta-Ct (ΔCt) algorithms. The 36B4 gene was stably expressed in the retinal tissues of normal control animals; however, it was less stable in diabetic retinas. The 18s gene was expressed consistently in both normal control and diabetic rats' retinal tissue. That this gene was the best reference for data normalisation in RT-qPCR studies that used the retinal tissue of streptozotocin-induced diabetic rats. Furthermore, there was no ideal gene stably expressed for use in all experimental settings. Identifying relevant genes is a need for achieving RT-qPCR validity and reliability and must be appropriately achieved based on a specific experimental setting.

Identification and validation of stable reference genes for RT-qPCR analyses of Kobresia littledalei seedlings

BackgroundKobreisa littledalei, belonging to the Cyperaceae family is the first Kobresia species with a reference genome and the most dominant species in Qinghai-Tibet Plateau alpine meadows. It has several resistance genes which could be used to breed improved crop varieties. Reverse Transcription Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR) is a popular and accurate gene expression analysis method. Its reliability depends on the expression levels of reference genes, which vary by species, tissues and environments. However, K.littledalei lacks a stable and normalized reference gene for RT-qPCR analysis.ResultsThe stability of 13 potential reference genes was tested and the stable reference genes were selected for RT-qPCR normalization for the expression analysis in the different tissues of K. littledalei under two abiotic stresses (salt and drought) and two hormonal treatments (abscisic acid (ABA) and gibberellin (GA)). Five algorithms were used to assess the stability of putative reference genes. The results showed a variation amongst the methods, and the same reference genes showed tissue expression differences under the same conditions. The stability of combining two reference genes was better than a single one. The expression levels of ACTIN were stable in leaves and stems under normal conditions, in leaves under drought stress and in roots under ABA treatment. The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was stable in the roots under the control conditions and salt stress and in stems exposed to drought stress. Expression levels of superoxide dismutase (SOD) were stable in stems of ABA-treated plants and in the roots under drought stress. Moreover, RPL6 expression was stable in the leaves and stems under salt stress and in the stems of the GA-treated plants. EF1-alpha expression was stable in leaves under ABA and GA treatments. The expression levels of 28 S were stable in the roots under GA treatment. In general, ACTIN and GAPDH could be employed as housekeeping genes for K. littledalei under different treatments.ConclusionThis study identified the best RT-qPCR reference genes for different K. littledalei tissues under five experimental conditions. ACTIN and GAPDH genes can be employed as the ideal housekeeping genes for expression analysis under different conditions. This is the first study to investigate the stable reference genes for normalized gene expression analysis of K. littledalei under different conditions. The results could aid molecular biology and gene function research on Kobresia and other related species.

MicroRNA-671-5p regulates the inflammatory response of periodontal ligament stem cells via the DUSP8/p38 MAPK pathway.

MicroRNAs are differentially expressed in periodontitis tissues. They are involved in cellular responses to inflammation and can be used as markers for diagnosing periodontitis. Microarray analysis showed that the expression level of microRNA-671-5p in periodontal tissues of patients with periodontitis was increased. In this study, we investigated the mechanism of action of microRNA-671-5p in human periodontal ligament stem cells (hPDLSCs) under inflammatory conditions. HPDLSCs were treated with lipopolysaccharide (LPS) to establish an inflammation model. The cell survival rate was determined using the cell counting kit-8 (CCK8). Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot analyses were used to detect the expression of microRNA-671-5p and dual-specificity phosphatase (DUSP) 8 proteins, respectively, Interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α were detected using qRT-PCR and Enzyme-linked immunosorbent assay (ELISA). A dual-luciferase reporter system was employed to determine the relationship between micoRNA-671-5p and DUSP8 expression. Activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway was confirmed using western blot analysis. Following the treatment of hPDLSCs with LPS, the expression levels of microRNA-671-5p in hPDLSCs were increased, cell viability decreased, and the expression of inflammatory factors displayed an increasing trend. MicroRNA-671-5p targets and binds to DUSP8. Silencing microRNA-671-5p or overexpressing DUSP8 can improve cell survival rate and reduce inflammatory responses. When DUSP8 was overexpressed, the expression of p-p38 was reduced. microRNA-671-5p targets DUSP8/p38 MAPK pathway to regulate LPS-induced proliferation and inflammation in hPDLSCs.

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

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

Risk factors and circulation pattern of respiratory syncytial virus in children under 2years in Maputo, Mozambique.

Evidence on risk factors for respiratory syncytial virus (RSV) in low-resource settings is limited. In Mozambican children <2y of age with severe acute respiratory infection (SARI), we explored risk factors for RSV, described its seasonal variation and assessed associations between RSV and a life-threatening condition. We retrospectively included participants presenting in 2017-2018 in two hospitals in Maputo. RSV was detected and subtyped using real-time quantitative reverse transcription polymerase chain reaction on nasopharyngeal swabs. We used logistic regression and χ2 tests to assess associations and Spearman's correlation coefficient to assess the correlation between weather measurement and RSV positivity. RSV was detected in 23.1% (n=109) of 472 included children and in 50.0% (20/40) of those <3months old. Being <3months (vs >1y) was associated with RSV (adjusted odds ratio 4.3 [95% confidence interval 2.1-8.5]). RSV status was not associated with experiencing a life-threatening condition. RSV A and B co-circulated during the study period, but one type predominated in each year. In 2017, the RSV positivity rate was correlated with monthly average temperature (r=0.793, p=0.002) and precipitation (r=0.596, p=0.041). In Mozambican children with SARI, RSV was prevalent, especially in neonates. However, RSV was not associated with a life-threatening condition.

An Evaluation of High-Risk HPV in Squamous Cell Carcinomas of the Lip in a South African Cohort

BackgroundTo determine the prevalence of HR-HPV in a series of lip SCC from South African patients, using currently accepted HPV-testing methodologies and to define the clinical and histomorphologic features of HPV-associated lip SCC.MethodsFifty SCC of lip and 50 control cases were tested for HR-HPV using p16 and HR-HPV DNA PCR. p16-equivocal/positive and HPV DNA PCR-positive SCC were further evaluated for the expression of HPV-16 and HPV-18 mRNA transcripts using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) to confirm transcriptionally active HPV.Resultsp16 was positive in 22% (n = 11) and equivocal in 4% (n = 2) of the SCC. One p16-positive case showed positivity for both HPV-16 DNA and HPV-16 E6/E7 mRNA transcripts (HPV prevalence rate of 2%). The HPV-positive case was non-keratinizing and occurred in an 80-year-old female. The two p16-equivocal cases were HR-HPV DNA positive and mRNA PCR negative. p16 was found to have a positive predictive value of 9%.ConclusionFindings from our cohort of lip SCC suggest that HR-HPV may have an insignificant role in the pathogenesis of SCC at this site. Due to its low ppv, p16 is insufficient to establish HR-HPV infection in SCC of the lip. The combination of p16 and DNA PCR appears to correlate with the presence of transcriptionally active virus. HPV E6/E7 mRNA detection is the gold standard for identifying HR-HPV. mRNA testing is not widely available in sub-Saharan Africa due to technical and financial constraints; however, the test appears to be of great value in p16-equivocal lip SCC.

Abstract PO1-02-08: Comprehensive Molecular Profiling of Breast Cancer: A real-time PCR assay for identifying ESR1, PGR, ERBB2, MKI67 and a novel proliferative signature in core needle biopsies and resected invasive breast cancer tissues

Abstract Background: Accurate status determination of breast cancer (BC) biomarkers, including estrogen receptor (ER/ESR1), progesterone receptor (PR/PGR), human epidermal growth factor receptor 2 (HER2/ERBB2), and marker of proliferation Ki67 (MKI67), is crucial for guiding therapeutic decisions. However, current “gold standard” methods such as immunohistochemistry (IHC) for assessing these biomarkers in formalin-fixed paraffin-embedded (FFPE) tissues face challenges in standardization and exhibit substantial inter- and intra-laboratory variability, particularly for Ki67. To address these limitations, APIS Breast Cancer Subtyping Kit has been developed, utilizing reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) to evaluate the status of ER, PR, HER2, KI67, and a novel proliferative signature in breast cancer core needle biopsy (CNB) and resected specimens. The APIS Breast Cancer Subtyping Kit offers enhanced accuracy, improved reproducibility, and the ability to provide results in a timely manner. Herein we report the development and validation of the APIS Breast Cancer Subtyping Kit, assessing clinical performance using retrospective and prospective sample collection. Furthermore, reproducibility and reliability across three testing sites were evaluated. By comparing the results of the kit with those obtained through standard IHC, we demonstrate the reliability and utility of the APIS Breast Cancer Subtyping Kit for precise breast cancer subtyping and guiding personalized patient management decisions. Methods: FFPE tissue sections (n=652) taken by CNB or resection underwent histological analysis in accordance with laboratory’s standard of care methods. Samples with a HER2 score of 2+ were referred to fluorescence in situ hybridization (FISH) to determine ERBB2 amplification, with the FISH result replacing the IHC result in determining the HER2 status (positive/negative) of the sample. Tumors with greater than 1% positive stained nuclei for ER/PR were scored positive. A cut-off of 20% Ki67 staining was used for positive/negative status. The performance was assessed as the agreement with IHC/ISH status and reported as OPA (overall percent agreement) with corresponding 95% confidence intervals (95% CI). To assess the precision of the assay, a panel of RNA pools (samples) was repeatedly measured on different days across three independent laboratories. Reproducibility was evaluated using variance components analysis. Results: The APIS Breast Cancer Subtyping Kit showed high concordance (OPA) with IHC: 93.1% (95% CI: 90.9-94.8) for ER, 86.8% (95% CI: 84.0-89.2) for PR, 94.2% (95% CI: 92.2-95.8) for HER2 (IHC/FISH), 78.3% (95% CI: 75.0-81.3) for Ki67, and 80.1% (95% CI: 76.8-83.1) for proliferative signature (assessed against MKI67 IHC status). Inter-site reproducibility demonstrated excellent agreement in quantitative measurements, with a total SD ranging from 0.00 to 0.22. Binary single-marker status (positive/negative) calling achieved 100% reproducibility for ER, PR, and HER2 for all tested samples (negative/low positive/medium positive), as well as for most Ki67 samples (87% agreement for negative samples). Conclusions: APIS Breast Cancer Subtyping Kit demonstrated a high level of concordance with standard of care IHC/FISH in assessing breast cancer biomarker status. These findings suggest that the APIS Breast Cancer Subtyping Kit provides highly precise and reproducible quantitative assessment of BC biomarkers and molecular subtypes. Inclusion and exclusion criteria Citation Format: Anna Gasior, Joanna Gorniak, Sara Rollinson, Leanne Gough, Anne-Sophie Wegscheider, Axel Niendorf. Comprehensive Molecular Profiling of Breast Cancer: A real-time PCR assay for identifying ESR1, PGR, ERBB2, MKI67 and a novel proliferative signature in core needle biopsies and resected invasive breast cancer tissues [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-02-08.

Nanoscale dihydroartemisinin@zeolitic imidazolate frameworks for enhanced antigiardial activity and mechanism analysis.

An artificial semisynthetic material can be derived from artemisinin (ART) called dihydroartemisinin (DHA). Although DHA has enhanced antigiardial potential, its clinical application is limited because of its poor selectivity and low solubility. The drug's absorption has a direct impact on the cell, and mechanism research is limited to its destruction of the cytoskeleton. In this study, we used the zeolitic imidazolate framework-8 and loaded it with DHA (DHA@Zif-8) to improve its antigiardial potential. DHA@Zif-8 can enhance cellular uptake, increase antigiardial proliferation and encystation, and expand the endoplasmic reticulum compared with the DHA-treated group. We used RNA sequencing (RNA-seq) to investigate the antigiardial mechanism. We found that 126 genes were downregulated and 123 genes were upregulated. According to the KEGG and GO pathway analysis, the metabolic functions in G. lamblia are affected by DHA@Zif-8 NPs. We used real-time quantitative reverse transcription polymerase chain reaction to verify our results using the RNA-seq data. DHA@Zif-8 NPs significantly enhanced the eradication of the parasite from the stool in vivo. In addition, the intestinal mucosal injury caused by G. lamblia trophozoites markedly improved in the intestine. This research provided the potential of utilizing DHA@Zif-8 to develop an antiprotozoan drug for clinical applications.

Transcriptional regulation of the yersiniabactin receptor fyuA gene by the ferric uptake regulator in Klebsiella pneumoniae NTUH-K2044.

The ferric uptake regulator (Fur) is a global regulator that influences the expression of virulence genes in Klebsiella pneumoniae. Bioinformatics analysis suggests Fur may involve in iron acquisition via the identified regulatory box upstream of the yersiniabactin receptor gene fyuA. To observe the impact of the gene fyuA on the virulence of K. pneumoniae, the gene fyuA knockout strain and complementation strain were constructed and then conducted a series of phenotypic experiments including chrome azurol S(CAS) detection, crystal violet staining, and wax moth virulence experiment. To examine the regulatory relationship between Fur and the gene fyuA, green fluorescent protein (GFP) reporter gene fusion assay, real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR), gel migration assay (EMSA), and DNase I footprinting assay were used to clarify the regulatory mechanism of Fur on fyuA. CAS detection revealed that the gene fyuA could affect the generation of iron carriers in K. pneumoniae. Crystal violet staining experiment showed that fyuA could positively influence biofilm formation. Wax moth virulence experiment indicated that the deletion of the fyuA could weaken bacterial virulence. GFP reporter gene fusion experiment and RT-qPCRanalysis revealed that Fur negatively regulated the expression of fyuA in iron-sufficient environment. EMSA experiment demonstrated that Fur could directly bind to the promoter region of fyuA, and DNase I footprinting assay further identified the specific binding site sequences. The study showed that Fur negatively regulated the transcriptional expression of fyuA by binding to upstream of the gene promoter region, and then affected the virulence of K. pneumoniae.

Noninvasive PCA3 detection for screening prostate diseases using a graphene oxide-based method

Prostate Cancer Associated 3 (PCA3) as a promising biomarker to replace serum prostate-specific antigen (PSA) for differential diagnosis of benign and malignant prostate diseases. However, the detection of PCA3 in urine requires the collection of digital rectal examination (DRE) urine, which limits the application of PCA3 in screening prostate diseases. In the present study, a new reverse transcription quantitative real-time polymerase chain reaction (qRT-PCR) method was developed for PCA3 detection in non-DRE urine. The method uses the nanomaterial Graphene oxide (GO) to effectively prevent nonspecific amplification in qRT-PCR. GO-based qRT-PCR produced no nonspecific amplification. Receiver operating characteristic curve (ROC) results showed that the area under curve (AUC) of PCA3 to discriminate normal control subjects from prostate cancer (PCa) was 0.869 (95% confidence interval [CI], 0.689–1.000), and using 2.711 as the cutoff value of PCA3, the sensitivity was 81.8% and the specificity was as high as 88.9%. For prostatitis (PTIS) and PCa, the AUC value of PCA3 to distinguish them was 0.636 (95% CI, 0.396–0.877), with 81.8% sensitivity and 58.3% specificity. Compared with the 8.3% specificity of PAS, the specificity of PCA3 was improved by 50%, which suggested that PCA3, in non-DRE urine, has a significant potential application in the differential diagnosis of prostate diseases.

Mogroside V Promotes Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells from Diabetic Mice by Altering MicroRNA Profiles.

Mogroside V (MV), a triterpene glycoside, exhibits diverse biological functions. However, its ability to promote the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) under diabetic conditions is yet to be elucidated. To study the regulation of osteogenic differentiation of BMSCs in diabetic mice by MV and determine the potential mechanism. BMSCs were isolated from both normal (referred to as N-BMSCs) and diabetic (referred to as DM-BMSCs) C57BL/6 mice. DM-BMSCs were treated with different concentrations of MV for varying durations, and cell viability was detected using the cell counting kit-8 assay. Following 2 weeks of osteogenic induction, osteogenic differentiation capability was evaluated using alizarin red S staining, alkaline phosphatase (ALP) activity analysis, and quantitative real-time reverse transcription polymerase chain reaction. Furthermore, the microRNA (miRNA) expression profiles of N-BMSCs, DM-BMSCs, and DM-BMSCs treated with MV were tested using high-throughput sequencing. Treatment with MV enhanced the viability of DM-BMSCs and mitigated the reduction of calcium nodule deposition, ALP activity, and mRNA expression of ALP, osteocalcin, and runt-related transcription factor 2. Of the analyzed miRNAs, miR-10b-5p was the only one that exhibited differential expression in N-BMSCs, DM-BMSCs, and DM-BMSCs treated with MV. An analysis of the top four protein clusters based on KEGG suggested that the target genes of differentially expressed miRNAs were closely linked to the PI3K/AKT pathway. MV significantly enhances the viability and osteogenic differentiation of BMSCs under diabetic conditions. The alteration of miRNA profiles provides a foundation for further research into the regulatory role of miRNAs and MV in this process.

SIRT1/P53 in retinal pigment epithelial cells in diabetic retinopathy: a gene co-expression analysis and He-Ying-Qing-Re formula treatment.

Diabetic retinopathy (DR) is a severe diabetic complication that leads to severe visual impairment or blindness. He-Ying-Qing-Re formula (HF), a traditional Chinese medicinal concoction, has been identified as an efficient therapy for DR with retinal vascular dysfunction for decades and has been experimentally reported to ameliorate retinal conditions in diabetic mice. This study endeavors to explore the therapeutic potential of HF with key ingredients in DR and its underlying novel mechanisms. Co-expression gene modules and hub genes were calculated by weighted gene co-expression network analysis (WGCNA) based on transcriptome sequencing data from high-glucose-treated adult retinal pigment epithelial cell line-19 (ARPE-19). The chromatographic fingerprint of HF was established by ultra-performance liquid chromatography coupled with high-resolution mass spectrometry (UPLC-Q-TOF-MS). The molecular affinity of the herbal compound was measured by molecular docking. Reactive oxygen species (ROS) was measured by a DCFDA/H2DCFDA assay. Apoptosis was detected using the TUNEL Assay Kit, while ELISA, Western blot, and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used for detecting the cytokine, protein, and mRNA expressions, respectively. Key compounds in HF were identified as luteolin, paeoniflorin, and nobiletin. For WGCNA, ME-salmon ("protein deacetylation") was negatively correlated with ME-purple ("oxidative impairment") in high-glucose-treated ARPE-19. Luteolin has a high affinity for SIRT1 and P53, as indicated by molecular docking. Luteolin has a hypoglycemic effect on type I diabetic mice. Moreover, HF and luteolin suppress oxidative stress production (ROS and MDA), inflammatory factor expression (IL-6, TNF-α, IL1-β, and MCP-1), and apoptosis, as shown in the in vivo and in vitro experiments. Concurrently, treatment with HF and luteolin led to an upregulation of SIRT1 and a corresponding downregulation of P53. Using HF and its active compound luteolin as therapeutic agents offers a promising approach to diabetic retinopathy treatment. It primarily suppressed protein acetylation and oxidative stress via the SIRT1/P53 pathway in retinal pigment epithelial cells.

Turmeric Inhibits MDA-MB-231 Cancer Cell Proliferation, Altering miR-638-5p and Its Potential Targets.

Recent research suggests curcumin extracted from the turmeric plant may inhibit the proliferation of cancer cells by controlling the expression of microRNAs (miRNAs). The effect of phenolic curcumin on miR-638-5p and potential target gene expressions in the triple negative breast cancer (TNBC) cell line MDA-MB-231 was investigated in this study. GSE154255 and GSE40525 datasets were downloaded and analyzed using GEO2R to identify dysregulated miRNAs in TNBC. To find differently expressed genes in breast cancer (BRCA), The Cancer Genome Atlas Program data was examined. Utilizing in silico tools, KEGG, GO, and other enrichment analyses were performed. The databases miRNet, miRTarBase v8.0, and TarBase v.8 were used for miRNA and mRNA matching. Real-time quantitative reverse transcription polymerase chain reaction was used to examine the levels of miRNA and its targets in miRNA mimic transfected/curcumin-treated MDA-MB-231 cultures and controls. The cell viability detection kit-8 method was used to assess cell viability, and the scratch assay was used to conduct migration assessment. Bioinformatics analysis showed that miR-638-5p was significantly reduced in TNBC patients. Experimental results showed that miR-638-5p was upregulated in MDA-MB-231 treated with curcumin, while the potential target genes of miR-638-5p, CFL1, SIX4, MAZ, and CDH1 were downregulated. Mimic miR-638-5p transfection inhibited MDA-MB-231 cell proliferation and reduced migration and expression of CFL1, SIX4, and MAZ genes was decreased in mimic miR-638-5p transfected cells. These findings suggest that curcumin exerts its anticancer effects on MDA-MB-231 cells by modulating the expression of miR-638-5p and its possible target genes.

Investigating diagnostic potential of long non-coding RNAs in head and neck squamous cell carcinoma using TCGA database and clinical specimens

Head and neck squamous cell carcinoma (HNSCC) is a prevalent and prognostically challenging cancer worldwide. The role of long non-coding RNAs (lncRNAs) in cancer regulation is progressively being understood. This study aims to identify lncRNAs with diagnostic potential as biomarkers for HNSCC. Statistical analysis was performed on expression data from the Cancer Genome Atlas (TCGA) database to identify potential lncRNAs associated with HNSCC. Four selected lncRNAs were validated using real-time quantitative reverse transcription polymerase chain reaction and correlated with clinical factors. Functional roles were further investigated. A total of 488 differentially expressed lncRNAs were identified in TCGA-HNSC. After rigorous evaluation based on p-values, survival analysis, and ROC analysis, 24 lncRNAs were prioritized for additional investigation. LINC00460, LINC00941, CTC-241F20.4, and RP11-357H14.17 were established as candidate diagnostic biomarkers. These lncRNAs exhibited elevated expression in HNSCC tissues and were associated with poor prognosis. Combining them showed high diagnostic accuracy. Notably, LINC00460 and CTC-241F20.4 demonstrated a significant elevation in the advanced stages of HNSCC. We constructed an lncRNA-mRNA regulatory network, and the array of significant regulatory pathways identified included focal adhesion, regulation of epithelial cell migration, and others. Additionally, these lncRNAs were found to influence immune responses by modulating immune cell infiltration in the HNSCC microenvironment. Our research indicates that LINC00460, LINC00941, RP11-357H14.17, and CTC-241F20.4 may have diagnostic and prognostic importance in HNSCC. Furthermore, we have gained insights into their potential functional roles, particularly about immune responses and interactions in the microenvironment.

Hsa_circTCF25 Facilitates Oncogenesis in Gastric Cancer Cells by Modulating miR-149 Expression.

Circular RNA (circRNA) is related to gastric carcinogenesis and progression. This study explored the effects of circTCF25 on gastric cancer cell proliferation, migration, invasion, and cancer stem cell markers, as well as the potential network of circTCF25-miR and miR-149. circTCF25 expression was detected in tissue specimens and cells by real-time quantitative reverse transcription polymerase chain reaction. Cell Counting Kit-8 and transwell assays were used to measure the effects of circTCF25 knockdown on proliferation, migration and invasion. The potential network of circTCF25 was analyzed using bioinformatic analysis. circTCF25 was overexpressed in human gastric cancer tissues, and a series of cancer cell lines, and was associated with shorter overall survival. Interfering with circTCF25 reduced gastric cancer cell proliferation, migration, invasion and expression of cancer stem cell markers. CircTCF25 reduced expression of miR-149, apparently by acting as a miR-149 sponge. A new circTCF25-miR-149 competitive endogenous RNA network in gastric cancer was constructed, and most core genes were associated with the malignant growth and metastatic behavior of gastric cancer. circTCF25 may have prognostic value and an oncogenic role in gastric cancer. A circTCF25-miR-149 RNA regulatory network was established which may provide novel biomarkers or potential therapeutic targets for treating gastric cancer.

Abstract 4728: Circ-EGFR enhances cetuximab efficacy by regulating miR-942-3p/GAS1 signaling axis in colorectal cancer

Abstract Background: Cetuximab, an epidermal growth factor receptor (EGFR)-targeting IgG1 monoclonal antibody (mAb), results in beneficial, yet limited, clinical improvement in metastatic colorectal cancer (mCRC) patients with wild-type (WT) KRAS gene. Numerous studies have shown that circRNAs are aberrantly expressed in tumors and play crucial roles in tumor growth and metastasis in various human malignancies, including colorectal cancer (CRC). One such circRNA, circ-EGFR, has been identified as having associations with tumor progression and chemotherapy response in diverse cancer types. However, exploring the functional role and underlying mechanisms of circ-EGFR in the context of the response to EGFR-targeted therapy may offer new therapeutic avenues for mCRC. Methods: The expression level of circ-EGFR (hsa_circ_0080229) was evaluated in cetuximab-sensitive and resistant RAS and BRAF WT CRC cell lines by using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). The biological function of circ-EGFR was investigated by loss- and gain-of-function assays in cultured cells and an animal model. The potential targets of circ-EGFR were identified through in-silico analysis, and the circ-EGFR/miR-942-3p/GAS1 axis pathway was verified using the luciferase reporter assay and RT-qPCR. The association of circ-EGFR expression with clinical prognosis in mCRC patients with different cetuximab responses was analyzed by Kaplan-Meier survival analysis. Results: The expression of circ-EGFR was remarkedly higher in sensitive (DiFi and SW48) compared with resistant cell lines (CaCO2 and SNU-C1) after exposure of cells to cetuximab (sensitive vs. resistant: p &amp;lt; 0.001). Biologically, genetic depletion of circ-EGFR significantly enhanced cell proliferation, migration [DiFi: sh-circ-EGFR vs. shNC: Foldchange (FC) = 1.97, p &amp;lt; 0.001; SW48: FC = 1.96, p &amp;lt; 0.001], and invasion (DiFi: sh-circ-EGFR vs. shNC: FC = 2.23, p &amp;lt; 0.001; SW48: FC = 1.78, p &amp;lt; 0.001) after 24-48 hours of cetuximab treatment in DiFi and SW48 cell lines, and its overexpression suppressed tumor growth in vivo under cetuximab treatment. Mechanically, we identified circ-EGFR as a sponge for miR-942-3p to regulate the expression of Growth Arrest Specific 1 (GAS1), which promoted sensitivity to cetuximab treatment in CRC. Notably, our findings identified circ-EGFR as a promising therapeutic indicator in clinical applications. Conclusion: Our study illustrated that circ-EGFR plays a vital role in affecting cetuximab efficacy by regulating the miR-942-3p/GAS1 axis in CRC. This holds promise for improving patient selection and management in this malignancy. Citation Format: Silei Sui, Caiming Xu, Yuan Li, Wenyi Wei, Joan Maurel, Man Li, Ajay Goel. Circ-EGFR enhances cetuximab efficacy by regulating miR-942-3p/GAS1 signaling axis in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4728.

A first complete catalog of highly expressed genes in eight chicken tissues reveals uncharacterized gene families specific for the chicken testis.

Based on next-generation sequencing, we established a repertoire of differentially overexpressed genes (DoEG) in eight adult chicken tissues: testis, brain, lung, liver, kidney, muscle, heart, and intestine. With 4499 DoEG, the testis had the highest number and proportion of DoEG compared with the seven somatic tissues. The testis DoEG set included the highest proportion of long noncoding RNAs (lncRNAs; 1851, representing 32% of the lncRNA genes in the whole genome) and the highest proportion of protein-coding genes (2648, representing 14.7% of the protein-coding genes in the whole genome). The main significantly enriched Gene Ontology terms related to the protein-coding genes are "reproductive process," "tubulin binding," "microtubule cytoskeleton." By using real-time quantitative reverse transcription polymerase chain reaction, we confirmed the overexpression of genes that encode proteins already described in chicken sperm (such as CABYR, SPAT18 and CDK5RAP2) but whose testis origin had not been confirmed previously. Moreover, we demonstrated the overexpression of vertebrate orthologs of testis genes not yet described in the adult chicken testis (such as NEK2, AK7 and CCNE2). Using clustering according to primary sequence homology, we found that 67% (1737) of the 2648 testis protein-coding genes were unique genes. This proportion was significantly higher than the somatic tissues except muscle. We clustered the other 911 testis protein-coding genes into 495 families, from which 47 had all paralogs overexpressed in the testis. Among these 47 testis-specific families, eight contain uncharacterized members without orthologs in other metazoans except birds: these families are then specific for chickens (probably more broadly birds).

Age-related dynamics in acute myeloid leukemia: Implications for prognosis, risk stratification, and treatment response

Abstract BACKGROUND: Acute myeloid leukemia (AML) is a complex, heterogeneous disease driven by acquired somatic mutations. The presence of specific mutations advances stratification, treatment, and prognosis. Linear accumulation of mutations over time is a crucial factor in cancer development, particularly among elderly patients. Our recent study on gene rearrangement in AML revealed a significant association between age and adverse risk cases. AIM: The aim of this study was to examine the distribution of age, molecular characteristics, risk stratification, and treatment response based on age among patients with de novo AML in Iraq. PATIENTS AND METHODS: A prospective cohort study enrolled 115 Iraqi adult patients diagnosed with de novo AML using morphology and flow cytometry from December 2020 to May 2022. The Leukemia Q-Fusion Screening Kit, employing multiplex reverse transcription–real-time quantitative polymerase chain reaction with 30 gene rearrangements, was employed for the identification of gene rearrangement. The patients received care and follow-up at the Hematology Unit of Baghdad Teaching Hospital in Medical City. Ethical approval from the College of Medicine’s Ethical Committee at the University of Baghdad was secured before commencing the research, ensuring adherence to ethical standards throughdout the study. RESULTS: The age distribution exhibited a bimodal pattern, with a mean of 45.1 ± 17.5 years, ranging from 18 to 84 years, and a median of 46 years. A total of 39.1% of patients were diagnosed with AML before the age of 35 years, while 43% were diagnosed after the age of 51 years. AML patients with RARA mutations, RUNX1:: RUNX1T1 alterations, and NPM1 mutations were predominantly observed in younger individuals, as well as those diagnosed with AML defined by differentiation. Conversely, KMT2A rearrangements were more prevalent among older age groups, with a statistically significant difference in the distribution of AML classifications according to the World Health Organization (WHO) by age categories (P = 0.001). The risk stratification based on age and response assessment showed a notable higher risk profile observed among elderly patients that was associated with adverse risk and poorer response and mortality (P &lt; 0.05). The prediction of treatment response accuracy rate was improved by adding age to the WHO classification and ELN 2022 risk stratification (73.5%–87.9%). CONCLUSION: Age significantly influences AML prognosis and treatment response. Incorporating age into risk stratification improves accuracy. Tailored approaches considering age are vital for optimizing AML management and outcomes.

3D printed high-precision porous scaffolds prepared by fused deposition modeling induce macrophage polarization to promote bone regeneration

Macrophage-mediated bone immune responses significantly influence the repair of bone defects when utilizing tissue-engineered scaffolds. Notably, the scaffolds’ physical structure critically impacts macrophage polarization. The optimal pore size for facilitating bone repair remains a topic of debate due to the imprecision of traditional methods in controlling scaffold pore dimensions and spatial architecture. In this investigation, we utilized fused deposition modeling (FDM) technology to fabricate high-precision porous polycaprolactone (PCL) scaffolds, aiming to elucidate the impact of pore size on macrophage polarization. We assessed the scaffolds’ mechanical attributes and biocompatibility. Real-time quantitative reverse transcription polymerase chain reaction was used to detect the expression levels of macrophage-related genes, and enzyme linked immunosorbent assay for cytokine secretion levels. In vitro osteogenic capacity was determined through alkaline phosphatase and alizarin red staining. Our findings indicated that macroporous scaffolds enhanced macrophage adhesion and drove their differentiation towards the M2 phenotype. This led to the increased production of anti-inflammatory factors and a reduction in pro-inflammatory agents, highlighting the scaffolds’ immunomodulatory capabilities. Moreover, conditioned media from macrophages cultured on these macroporous scaffolds bolstered the osteogenic differentiation of bone marrow mesenchymal stem cells, exhibiting superior osteogenic differentiation potential. Consequently, FDM-fabricated PCL scaffolds, with precision-controlled pore sizes, present promising prospects as superior materials for bone tissue engineering, leveraging the regulation of macrophage polarization.

Acute and Chronic Exposure to Linagliptin, a Selective Inhibitor of Dipeptidyl Peptidase-4 (DPP-4), Has an Effect on Dopamine, Serotonin and Noradrenaline Level in the Striatum and Hippocampus of Rats.

Linagliptin is a selective dipeptidyl peptidase-4 (DPP-4) inhibitor that indirectly elevates the glucagon-like peptide-1 (GLP-1) level. The aim of the present study was to check whether linagliptin has an influence on neurotransmission in rat brain. Rats were acutely and chronically exposed to linagliptin (10 and 20 mg/kg, intraperitoneally (i.p.)). Twenty-four hours later, the striatum and hippocampus were selected for further studies. In neurochemical experiments, using high-performance liquid chromatography with electrochemical detection (HPLC-ED), the concentrations of three major neurotransmitters-dopamine, serotonin and noradrenaline-and their metabolites were measured. The analysis of mRNA expression of dopamine (D1 and D2), serotonin (5-HT-1 and 5-HT-2) and noradrenaline (α1 and α2a) receptors was also investigated using real-time quantitative reverse transcription polymerase chain reaction (RQ-PCR) in the same brain areas. Linagliptin has the ability to influence the dopaminergic system. In the striatum, the elevation of dopamine and its metabolites was observed after repeated administration of that linagliptin, and in the hippocampus, a reduction in dopamine metabolism was demonstrated. Acute linagliptin exposure increases the serotonin level in both areas, while after chronic linagliptin administration a tendency for the mRNA expression of serotoninergic receptors (5-HT1A and 5-HT2A) to increase was observed. A single instance of exposure to linagliptin significantly modified the noradrenaline level in the striatum and intensified noradrenaline turnover in the hippocampus. The recognition of the interactions in the brain between DPP-4 inhibitors and neurotransmitters and/or receptors is a crucial step for finding novel discoveries in the pharmacology of DPP-4 inhibitors and raises hope for further applications of DPP-4 inhibitors in clinical practices.