Differentially Expressed miRNAs Research Articles

Non-invasive detection of orthotopic human lung tumors by microRNA expression profiling of mouse exhaled breath condensates and exhaled extracellular vesicles.

The lung is the second most frequent site of metastatic dissemination. Early detection is key to improving survival. Given that the lung interfaces with the external environment, the collection of exhaled breath condensate (EBC) provides the opportunity to obtain biological material including exhaled miRNAs that originate from the lung. In this proof-of-principal study, we used the highly metastatic MDA-MB-231 subline 3475 breast cancer cell line (LM-3475) to establish an orthotopic lung tumor-bearing mouse model and investigate non-invasive detection of lung tumors by analysis of exhaled miRNAs. We initially conducted miRNA NGS and qPCR validation analyses on condensates collected from unrestrained animals and identified significant miRNA expression differences between the condensates of lung tumor-bearing and control mice. To focus our purification of EBC and evaluate the origin of these differentially expressed miRNAs, we developed a system to collect EBC directly from the nose and mouth of our mice. Using nanoparticle distribution analyses, TEM, and ONi super-resolution nanoimaging, we determined that human tumor EVs could be increasingly detected in mouse EBC during the progression of secondary lung tumors. Using our customizable EV-CATCHER assay, we purified human tumor EVs from mouse EBC and demonstrated that the bulk of differentially expressed exhaled miRNAs originate from lung tumors, which could be detected by qPCR within 1 to 2 weeks after tail vein injection of the metastatic cells. This study is the first of its kind and demonstrates that lung tumor EVs are exhaled in mice and provide non-invasive biomarkers for detection of lung tumors.

Study on miRNA expression and regulatory network of Amygdalus mongolica oil and amygdalin in the treatment of pulmonary fibrosis

IntroductionAmygdalus mongolica (Maxim.) Ricker is a traditional Chinese medicine known for its ability to relieve cough and expectorate phlegm and is commonly used to treat dry throat, dry cough, and bronchitis. A. mongolica oil and amygdalin were the active components isolated from the seed kernel seeds of A. mongolica. The main objective of this study was to investigate the effects of A. mongolica oil and amygdalin on bleomycin-induced pulmonary fibrosis (PF) in rats by integrating pharmacodynamic, transcriptomics, and bioinformatics, to determine the changes of miRNAs in both against PF, and to explore the underlying mechanisms. MethodsA rat model of PF was induced by administering bleomycin through the trachea. The rats were randomized into several groups: a control group (CON), an untreated model group (MOD), an active control group treated with pirfenidone (PFD), and groups receiving different doses of A. mongolica oil (5 g/kg [OIL-L], 10 g/kg [OIL-M], and 15 g/kg [OIL-H]), or amygdalin (20 mg/kg [AMY-L], 40 mg/kg [AMY-M], and 80 mg/kg [AMY-H]). After 28 days, samples were collected for subsequent pharmacodynamic and transcriptomic analyses. ResultsThe results demonstrated that A. mongolica oil and amygdalin effectively reduced the abnormally elevated levels of IL-1β, IL-6, HA, LN and COL-IV in PF rats and alleviated bleomycin-induced pulmonary fibrotic changes. Furthermore, compared to the MOD group, the expression levels of 27 miRNAs were up-regulated and 23 miRNAs were down-regulated in the OIL 10 g/kg group. Among the up-regulated miRNAs, two novel differentially expressed miRNAs (DE-miRNAs) were identified. In the AMY 20 mg/kg group, 12 miRNAs were up-regulated and 10 were down-regulated when compared to the MOD group. These DE-miRNAs were found to be enriched in pathways such as the PI3K-Akt signalling pathway, MAPK signalling pathway, and calcium signalling pathway. The microRNAs miR-301b-3p, miR-667-5p, miR-363-3p, and miR-540-3p may be associated with both the anti-PF mechanism of A. mongolica oil and the anti-PF mechanism of amygdalin. DiscussionNumerous studies have demonstrated the significance of various pathways, including PI3K-Akt, MAPK, and calcium signalling pathways, in the mechanism of PF and the development of anti-PF medications. Meanwhile, multiple miRNAs, including miR-301b-3p, miR-667-5p, miR-363-3p, and miR-540-3p, have been found to be associated with lung disease and fibrosis. ConclusionsThe present experiments confirmed the effectiveness of A. mongolica oil and amygdalin in reducing inflammation and fibrosis in bleomycin-induced PF rats, with particularly favorable outcomes observed in the A. mongolica oil 10 g/kg versus amygdalin 20 mg/kg. Meanwhile, miRNAs miR-301b-3p, miR-667-5p, miR-363-3p and miR-540-3p were found to be potentially associated with both A. mongolica oil and amygdalin anti-PF mechanisms. Based on our findings, we suggest that these miRNAs might play key roles in the anti-PF mechanism of A. mongolica. The individual miRNAs require further investigation.

Identification of cuproptosis-related miRNAs in triple-negative breast cancer and analysis of the miRNA–mRNA regulatory network

IntroductionThe close association between cuproptosis and tumor immunity in triple-negative breast cancer (TNBC) allows its monitoring for predicting the prognosis of patients with TNBC. Nevertheless, the biological function and prognostic value of cuproptosis-related miRNAs and their target genes have not been reported. PurposeTo construct the miRNA and mRNA-based risk models associated with cuproptosis for patients with TNBC. MethodsComparison of expression levels for genes associated with cuproptosis was executed between patients in the normal individuals and the TCGA-TNBC cohort. Conducting differential analysis resulted in the identification of differentially expressed miRNA (DE-miRNAs) and differentially expressed genes (DEGs) between the TNBC and Control samples. Screening for prognostic miRNAs and biomarkers involved employing univariate Cox analysis and least absolute shrinkage and selection operator regression analyses. These methods were utilized to construct risk models aimed at predicting the survival of patients with TNBC. Based on the median value of risk scores, patients were then stratified into low- and high-risk groups. Functional enrichment analysis was employed to explore the potential function and pathways of prognostic genes. Additionally, independent prognostic analysis was performed through univariate and multivariate Cox regression. Immune infiltration analysis was performed to examine disparities in the infiltration of immune cells between the two risk groups. Finally, the prognostic gene expression was mined in key cell types of TNBC. ResultsWe obtained 5213 DEGs and 204 DE-miRNAs related to cuproptosis between TNBC and Control samples. Five prognostic miRNAs (miR-203a-3p, miR-1277–3p, miR-135b-5p, miR-200c-3p, and miR-592) and three biomarkers (DENND5B, IGF1R, and MEF2C) were closely associated with TNBC. Significant differences in the functions of prognostic genes between the two risk groups were observed, encompassing adipogenesis, inflammatory response, and hormone metabolic process. The prognostic gene regulatory network revealed that miR200C-3p regulated ZFPM2 and CFL2, and miR-1277–3p regulated BMP2 and RORA. A nomogram was created based on riskScore, cancer status, and pathologic stage to predict 1/3/5-year survival of patients with TNBC. Immune infiltration analysis indicated that the immune microenvironment may be associated with the progression of TNBC. Interestingly, prognostic genes exhibited higher expression levels in T cells, fibroblasts, endothelial cells, and monocytes compared to other cells. ConclusionsFive prognostic miRNA (miR-203a-3p, miR-1277–3p, miR-135b-5p, miR-200c-3p, and miR-592) and three biomarkers (DENND5B, IGF1R, and MEF2C) were significantly associated with TNBC, it provides new therapeutic targets for the treatment and prognosis of TNBC.

Withaferin A alters the expression of microRNAs 146a-5p and 34a-5p and associated hub genes in MDA-MB-231 cells.

Triple-negative breast cancer (TNBC) is a highly metastatic subtype of breast cancer. Due to the absence of obvious therapeutic targets, microRNAs (miRNAs) provide possible hope to treat TNBC. Withaferin A (WA), a steroidal lactone, possesses potential anticancer activity with lesser side effects. The present study identifies hub genes (CDKN3, TRAF6, CCND1, JAK1, MET, AXIN2, JAG1, VEGFA, BRCA1, E2F3, WNT1, CDK6, KRAS, MYB, MYCN, TGFβR2, NOTCH1, SIRT1, MYCN, NOTCH2, WNT3A) from the list of predicted targets of the differentially expressed miRNAs (DEMs) in WA-treated MDA-MB-231 cells using in silico protein-protein interaction network analysis. CCND1, CDK6, and TRAF6 hub genes were predicted as targets of miR-34a-5p and miR-146a-5p, respectively. The study found the lower expression of miR-34a-5p and miR-146a-5p in MDA-MB-231 cells, and further, it was observed that WA treatment effectively restored the lost expression of miR-34a-5p and miR-146a-5p in MDA-MB-231 cells. An anti-correlation expression pattern was found among the miR-34a-5p and miR-146a-5p and the respective target hub genes in WA-treated TNBC cells. In conclusion, WA might exert anti-cancer effect in TNBC cells by inducing miR-34a-5p and miR-146a-5p expressions and decreasing CCND1, CDK6, and TARF6 target hub genes in TNBC cells.

Let-7 family regulates HaCaT cell proliferation and apoptosis via the ΔNp63/PI3K/AKT pathway.

We evaluated the expression profiles of differentially expressed miRNAs (DEmiRNAs) involved in human fetal skin development via high-throughput sequencing to explore the expression difference and the regulatory role of miRNA in different stages of fetal skin development. Analysis of expression profiles of miRNAs involved collecting embryo samples via high-throughput sequencing, then bioinformatics analyses were performed to validate DEmiRNAs. A total of 363 miRNAs were differentially expressed during the early and mid-pregnancy of development, and upregulated DEmiRNAs were mainly concentrated in the let-7 family. The transfection of let-7b-5p slowed down HaCaT cell proliferation and promoted apoptosis, as evidenced by the cell counting kit-8 assay, quantitative real-time polymerase chain reaction, and flow cytometry. The double luciferin reporter assay also confirmed let-7b-5p and ΔNp63 downregulation through the combination with the 3'-untranslated region of ΔNp63. Moreover, treatment with a let-7b-5p inhibitor upregulated ΔNp63 and activated the phosphoinositide 3-kinase (PI3K)-protein kinase B (AKT) signaling pathway. The let-7b-5p caused a converse effect on HaCaT cells because of Np63 upregulation. Let-7b-5p regulates skin development by targeting ΔNp63 via PI3K-AKT signaling, contributing to future studies on skin development and clinical scar-free healing.

Human and Viral microRNA Expression in Acute and Chronic HIV Infections.

Human and viral microRNAs (miRNAs) are involved in the regulation of gene transcription, and the establishment of their profiles in acute (AHI) and chronic (CHI) HIV infections may shed light on the pathogenetic events related to different phases of HIV disease. Next-generation sequencing (NGS) of miRNA libraries was performed, and the reads were used to analyze miRNA differential expression in the plasma with AHI and CHI. Functional analysis was then undertaken to investigate the biological processes characterizing the two phases of HIV infection. Except for hsa-miR-122-5p, which was found in 3.39% AHI vs. 0.18% CHI, the most represented human miRNAs were similarly represented in AHI and CHI. However, when considering the overall detected miRNAs in AHI and CHI, 15 displayed differential expression (FDR p < 0.05). Functional analysis identified 163 target mRNAs involved in promoting angiogenesis activation in AHI versus CHI through the action of hsa-miR10b-5p, hsa-miR1290, hsa-miR1-3p, and hsa-miR296-5p. The viral miRNAs detected, all belonging to herpesviruses, accounted for only 0.014% of total reads. The present data suggest that AHI patients exhibit strong innate immune activation through the upregulation of hsa-miR-122-5p and early activation of angiogenesis. More specific investigations are needed to study the role of viral miRNAs in HIV pathogenesis.

Abstract 5690: Urinary microRNA expression in pancreatic cancer with cachexia

Abstract Background: Cancer cachexia (CC) is a multifactorial syndrome characterized by appetite loss, weight loss and skeletal muscle wasting. It increases functional impairment, treatment-related toxicity, and a lower quality of life. Importantly, CC is one of the leading causes of cancer-related deaths, and its occurrence is particularly prevalent in advanced stages of pancreatic cancer (PC). The syndrome is thought to be associated with systemic inflammation and increased energy expenditure, yet the specific mechanisms remain to be fully understood. Although timely detection and intervention might be crucial in preventing CC’s progression, early diagnosis of this syndrome remains challenging due to the absence of suitable biomarkers. Here, we analyzed urinary microRNAs (miRNAs) as a potential noninvasive biomarkers for detecting CC in patients (pts) with PC. Methods: Clinical information including presence or absence of CC and urine samples were collected from pts with PC treated at National Cancer Center Hospital, Tokyo, Japan from March to October 2022. The diagnosis of CC was made according to the following definitions by Fearon. K et al. (1) weight loss of ≥5% within 6 months; (2) weight loss of ≥2% in pts with a BMI of &amp;lt;20 kg/m2; or (3) weight loss of ≥2% in pts with sarcopenia. In this study, pts who met the definition of CC within 6 months of enrollment were considered to have CC.Urinary miRNA profiles from pts with PC were sequenced by NGS and analyzed using DESeq2 to identify differentially expressed miRNAs (DEMs) between pts with CC and those without. DEMs were further explored for associated pathways and target genes using miRPathDB v.2.0. Results: Of the 64 subjects, 52 pts were included in the analysis after excluding three pts with low urine sample read coverage and nine pts with no information on CC status. Among those, 38 pts were classified as having CC. Patients’ background was as follows (CC/no CC); median age 70 [range: 44-80]/67 [57-74], male and female 19/7 pts, respectively, cStage {I (6/0), II (1/0), III (17/4), IV (14/10)} pts, median BMI (male 22.1 [17.7-27.2]/21.7 [18.2-28.2], female 21.7 [14.6-25.2]/21.9 [16.6-27.4]) kg/m2. Among 307 miRNAs detected in the urine of pts with PC, 49 miRNAs had previously been reported in CC research. The miR-141-5p was differentially expressed between pts with CC and those without CC (Wald test pBH = 0.079). It was down-regulated in pts with CC (Mann-Whitney U test p = 6.4e-4) and most significantly associated with the FoxO signaling pathway (ORA pBH = 1.84e-4), followed by the mTOR signaling pathway (ORA pBH = 0.008). Conclusions: Our study indicates that miR-141-5p holds potential as a noninvasive biomarker for the detection of CC in pts with PC. Although miR-141-5p has not been previously reported in CC, its significant association with the FoxO signaling pathway, which is one of the major pathways regulating skeletal muscle atrophy, underscores the relevance of miR-141-5p in the context of CC. Citation Format: Mao Okada, Shunsuke Kondo, Yukiko Igawa, Tatsuya Yoshida, Milos Havelka, Mika Mizunuma, Yuki Ichikawa, Noboru Yamamoto. Urinary microRNA expression in pancreatic cancer with cachexia [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 5690.

Abstract 2277: Understanding the impact of clonal hematopoiesis on multiple myeloma: Insights from exosomal RNA analysis and survival analysis

Abstract Clonal Hematopoiesis of Indeterminate Potential (CHIP) is a trait characterized by the accumulation of somatic mutations in hematopoietic stem cells in certain adults. It has been observed that the presence of CHIP can have significant implications for the development and prognosis of various blood cancers. Multiple myeloma (MM), a cancer involving the abnormal proliferation of malignant plasma cells in the bone marrow, is one such condition. Exosomes, a type of extracellular vesicles, play a role in cell communication by carrying information between cells. Analysis of UK Biobank data confirmed the poor prognosis of multiple myeloma patients with CHIP and identified a potential of paracrine effect on plasma cancer cells. To gain deeper insights into this paracrine effect on plasma cells, we attempted to analyze cell-to-cell interaction, such as the expression of miRNAs in exosomes, through exosome RNA sequencing. In our research, exosomes were extracted from bone marrow samples of 30 multiple myeloma patients, and exosome RNA sequencing was performed. Differential expression of miRNAs was identified. A total of 14 down-regulated miRNAs were identified based on adjusted p value &amp;lt; 0.05, log2 fold change &amp;lt; -1. We found that hsa-let-7f-5p, hsa-let-7a-5p, and hsa-miR-148a-3p, which are mainly miRNAs targeting MAP kinase, were significantly down-regulated in the CHIP cases. A computational tool was used to predict and integrate the target genes and pathways regulated by these DE miRNAs. The results suggested that certain inflammatory and oncogenic pathways including the MAPK signaling pathways, were not effectively suppressed in cases with CHIP (KEGG, p &amp;lt; 0.005). In addition, the chemokine signaling pathway and cytokine-cytokine receptor interaction (KEGG, p &amp;lt; 0.005) related to the paracrine effect, integrins in angiogenesis (PID, p &amp;lt; 0.005) related to poor prognosis and metastasis of cancer were also not adequately inhibited in the CHIP cases. Using UK Biobank data, we called somatic CHIP variants excluding those with VAF &amp;lt; 0.02 and analyzed them against ICD-10 codes. Hazard ratios (HR) were computed through Cox proportional regression with covariates. CHIP presence significantly correlated with MM diagnosis (HR: 1.64, CI: 1.35-2, p &amp;lt; 0.001), notably in TET2 mutation carriers (HR: 2.35, CI: 1.63-3.4, p &amp;lt; 0.001). TET2 mutations intensified MGUS to MM progression (HR: 5.0, CI: 2.27-11.0, p &amp;lt; 0.001). CHIP in MM impacted EFS (HR: 1.61, CI: 1.25-2.1, p &amp;lt; 0.001), with TET2 mutation carriers facing higher hazards (HR: 1.66, CI: 1.05-2.6, p = 0.031). CHIP, especially TET2 mutations, significantly contribute to adverse MM outcomes and MGUS to MM evolution. Our findings provide insight into how CHIP influences the tumorigenesis and cancer progression of MM. Citation Format: Jeongmin Park, GangPyo Ryu, Daniel Nachun, Maggie Maurer, Siddhartha Jaiswal, Dong-Yeop Shin, Ja Min Byun, Junshik Hong, Youngil Koh, Sung-Soo Yoon. Understanding the impact of clonal hematopoiesis on multiple myeloma: Insights from exosomal RNA analysis and survival analysis [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 2277.

Abstract 7020: Prognostic implications of microRNA 181a 2 3p expression in association with tumor budding in patients with stage III lung squamous cell carcinoma

Abstract Introduction: Tumor budding (Bd) has been known as an independent prognostic factor in various malignancies. MicroRNA (miRNA) are known to play an important role in regulating Bd. However, understanding the relationship between Bd and miRNA in Lung Squamous cell carcinoma (LUSC) remains limited. Methods: 115 LUSC patients who underwent curative resection were included in the discovery cohort. Bd was verified through immunohistochemical (IHC) stain. Bd was classified according to a three tier system: Bd1 with 0-4 buds, Bd2 with 5-9 buds, and Bd3 with 10 or more buds. MiRNA expression profiles were analyzed through RNA sequencing. Differences in survival outcomes for miRNA, as identified in the discovery cohort, were confirmed using LUSC data of The Cancer Genome Atlas (TCGA) as a validation cohort. Results: The relationship between Bd2 and the prolongation of recurrence-free survival (RFS) was statistically significant (p=0.0113), whereas Bd1 was correlated with Overall Survival (OS) (p=0.0468). Multivariate analysis indicated that both a high T stage (HR: 5.211, 95% CI: 1.168-23.250, p = 0.031; and HR: 5.107, 95% CI: 1.447-18.030, p &amp;lt; 0.011) and Bd3 (HR: 3.683, 95% CI: 1.370-9.900, p = 0.010; and HR: 1.600, 95% CI: 0.800-3.201, p = 0.018) are independently significant poor prognostic factors for both RFS and OS. MiRNA-181a-2-3p was identified through Differentially Expressed miRNA (DER) analysis. A higher expression of miRNA-181a-2-3p in stage III LUSC of the TCGA dataset was associated with OS (p=0.0463). Conclusions: Our study demonstrates that the miRNA 181a 2 3p expression is associated with improved OS. This association may be attributed to its inhibitory effect on Bd in advanced stages of LUSC. Citation Format: Eunhyo Lee, Seoree Kim, Jeana Kim, Minho Lee, Yoonho Ko, Sang Hoon Chun. Prognostic implications of microRNA 181a 2 3p expression in association with tumor budding in patients with stage III lung squamous cell carcinoma [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 7020.

Abstract 2436: In-depth analysis of microRNA signature in breast cancer derived extracellular vesicles: A potential biomarker repository for breast cancer diagnosis

Abstract Breast cancer remains one of the leading causes of cancer-related mortality among women worldwide. With the rising incidence of breast cancer, early and accurate diagnosis is pivotal in improving survival rates and patient outcomes. Our study addresses to identify vesicles associated with breast cancer in scope of miRNA signature. We focused on isolating breast cancer-derived extracellular vesicles (BEVs) from the bloodstream with immune affinity capture techniques. Then, we analyzed the miRNA profiles of BEVs in plasma samples from 120 breast cancer patients, 46 individuals with benign tumors, and 45 healthy controls. In this study, we analyzed to discover miRNA candidates for diagnosis of breast cancer with The Gene Expression Omnibus (GEO) database and found 379 down-regulated and 584 up-regulated differentially expressed miRNAs (DEMs) in tissue samples across 5 GEO datasets, and 28 down-regulated and 80 up-regulated DEMS in blood samples. Subsequently, we measured the expression levels of these 10 candidate miRNAs in BEVs from breast cancer cells and compared them with EVs from human mammary cells (MCF10a), using our BEV isolation method. The expression profiles of these miRNAs in BEVs were significantly elevated, supporting their potential as surrogate markers for breast cancer diagnosis. When comparing the AUC values of 10 candidate miRNAs through ROC analysis, 5 EV miRNAs (miR-21, miR-106b, miR-181a, miR-484, and miR-1260b) showed a high AUC values above 0.8. Subsequently, we verified a distinct signature of 5 EV miRNAs that effectively differentiate breast cancer patients from normal controls with 57.14% of sensitivity and 95% of specificity. Overall, our study not only complements existing diagnostic methods with high accuracy but also provides a deeper understanding of the molecular aspects of breast cancer, heralding a significant advancement in precision medicine and personalized cancer care. Citation Format: Jee Ye Kim, Min Woo Kim, Young Kim, Sol Moon, Suji Lee, Hyojung Lee, Joon Ye Kim, Seung Il Kim. In-depth analysis of microRNA signature in breast cancer derived extracellular vesicles: A potential biomarker repository for breast cancer diagnosis [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 2436.

Abstract 460: Fecal microRNA and microbiome profiles in colorectal cancer patients: Are they predictor of survival and how do behave in longitudinal samples

Abstract Background: Colorectal cancer (CRC) is characterized by a high complexity that significantly influences treatment response and survival. There is still a lack of prognostic factors after surgery that could help predict postoperative survival. Currently, lymph node metastases are the single most important prognostic factor but it gives no clue on tumor aggressiveness and recurrence. Our group recently contributed to the identification of a fecal microRNA (miRNA) signature that accurately discriminates sporadic CRC and aimed at improving non-invasive diagnosis (Pardini et al, Gastroenterology 2023) as well as altered gut microbiome species associated with CRC (Thomas et al, Nat. Medicine 2019), but there are scanty evidence on the potentiality of such markers for prognosis. Aims: In the same cohort, we investigated if the fecal miRNome and gut microbiome at diagnosis are associated with CRC patients’ prognosis. Moreover, we assessed fecal miRNA in a new set of CRC patients collected longitudinally before and after the surgery to investigate whether such associations reflect miRNA profiles over time. Methods. We performed small RNA and shotgun metagenomic sequencing in stool samples in 3 cohorts from: Italy (IT, 128 CRC), Czech Republic (CZ, 69 CRC), and Turkey (TU, 34 CRC, 40 controls). Stool samples from TU CRC subjects were collected before surgery and after 6 months. Preliminary Results: In the IT and CZ cohorts, an average of 479 fecal miRNAs were detected with 209 differentially expressed (DE) miRNAs significantly associated with survival, including miR-1246 which was present in the CRC-associated signature identified by us. Two miRNAs (miR-197-5p and miR-6081-5p) were significantly down-regulated in patients with the worst prognosis (adj.p&amp;lt;0.01, HR&amp;lt;0.50) in both cohorts. In TU cohort, 494 miRNAs were DE in CRC (pre-surgery) vs controls. Among them, the 25 DE miRNAs detected in our previous work were also significantly altered, including the 5-miRNA CRC signature (miR-149-3p, miR-607-5p, miR-1246, miR-4488, and miR-6777-5p) identified by machine learning. Notably, after 6 months from surgery, levels of such miRNAs reverted to those measured in controls (for miR-1246 p&amp;lt;0.001 in both controls vs pre- and vs post-surgery comparisons). More analyses on the longitudinal fecal miRNA data are ongoing as well as for the metagenome data assessing the abundancies of CRC-associated microbial species before and after tumor resection and their possible association with survival. Conclusions: Fecal miRNAs may serve as biomarkers for CRC prognosis. Following tumor resection, CRC levels of altered miRNAs return in a range comparable with those of healthy controls in a time-dependent manner. These data further support the use of fecal miRNAs as accurate diagnostic CRC biomarkers and provide preliminary information on their possible translation as a prognostic tool. Citation Format: Barbara Pardini, Nazim Arda Keles, Giulio Ferrero, Sonia Tarallo, Giulia Francescato, Amedeo Gagliardi, Virginia Alberini, Carla Di Battista, Veronika Vymetalkova, Fatma Turkoglu, Ozgul Duzgun, Omer Faruk Ozkan, Bilge Guvenc Tuna, Pavel Vodicka, Soner Dogan, Alessio Naccarati. Fecal microRNA and microbiome profiles in colorectal cancer patients: Are they predictor of survival and how do behave in longitudinal samples [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 460.

Abstract 5691: Identification of miRNA related to triple-negative breast cancer in African and European American women with obesity

Abstract Triple-negative breast cancer (TNBC) represents 10-20% of all invasive breast cancers and is characterized by a lack of expression of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. TNBC typically manifests as high-grade tumors often diagnosed at advanced stages, and affects younger patients, especially African American (AA) women. Dysregulation of miRNA expression levels contributes to TNBC pathogenesis and prognosis. Dysregulated miRNAs in advanced-stage cancers alter tumor progression, migration, and metastasis. Obesity is associated with miRNA dysregulation in adipose tissue, which is a driver of BC development and growth. This study aimed at identifying differentially expressed (DE) miRNA in TNBC patients of European American (EA) and AA women with obesity, compared to adjacent non-tumor tissues. miRNAseq data from 48 tissue samples (TNBC tumor and adjacent non-tumor) were analyzed for differential expression. Samples were from 12 EA and 12 AA patients, with 6 patients in each ethnic group having obesity and 6 normal weight, based on BMI estimates. Stringent comparisons, with and without obesity, were conducted to identify TNBC and obesity-associated miRNAs within each ethnic group. Prediction of miRNA-target genes was performed in miRWalk and Key Pathway Advisor (KPA) was employed for pathway enrichment analysis. Differential expression analysis between obese and normal-weight patients in each ethnic group revealed 79 DE miRNAs in EA and 40 in AA. We identified 55 miRNAs associated exclusively with tumors in obese patients within the EA group and 33 miRNAs within the AA group. Target gene analysis showed that in EA, 9 out of the 55 miRNAs interacted with 68 mRNAs, with miR-204-5p and miR-181b-5p accounting for 75% of interactions. In AA, 20 out of the 33 miRNAs interacted with 284 mRNAs with miR-20a-5p, -195-5p, -34a-5p, and -130a-3p with 65% of the interactions. KPA identified 84 and 330 pathways for EA and AA, respectively, with 43 common pathways,15 of them related to cancer processes sharing the gene targets GSKIP, TLBR1, TCF4, and TCF. Our study outlined unique miRNA profiles in TNBC among AA and EA women with obesity. Although common mRNAs were identified between ethnic groups, leading to the identification of common pathways on KPA, more than 50% of the identified pathways are related to unique biological processes associated with TNBC and obesity for each ethnic group. These findings underscore the importance of considering ethnic-specific miRNA signatures for understanding TNBC in the context of obesity, offering insights for targeted therapeutic strategies. Citation Format: Fokhrul Hossain, Martha Isabel González-Ramírez, Jone Garai, Xiao-Cheng Wu, Chindo Hicks, Luis Del Valle, Augusto Ochoa, Denise Danos, Lucio Miele, Jovanny Zabaleta. Identification of miRNA related to triple-negative breast cancer in African and European American women with obesity [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 5691.

Construction of a novel mRNA–miRNA–lncRNA/circRNA triple subnetwork associated with immunity and aging in intervertebral disc degeneration

Objective Intervertebral disk degeneration (IVDD) is one of the most common causes of low back pain. However, in the etiology of IVDD, the specific method by which nucleus pulposus (NP) cell senescence and the immune response induce disease is uncertain. Methods Gene Expression Omnibus database was used to find differentially expressed genes (DEGs), differentially expressed miRNAs (DE miRNAs), differentially expressed lncRNAs (DE lncRNAs), and differentially expressed circRNAs (DE circRNAs). Functional enrichment analysis was performed through Enrichr database. Potential regulatory miRNAs, lncRNAs and circRNAs of mRNAs were predicted by ENCORI and circBank, respectively. Results We identified 198 upregulated and 131 downregulated genes, 39 upregulated and 22 downregulated miRNAs, 2152 upregulated and 564 downregulated lncRNAs, and 352 upregulated and 279 downregulated circRNAs as DEGs, DE miRNAs, DE lncRNAs, DE circRNAs, respectively. Functional enrichment analysis revealed that they were significantly enriched in Toll-like receptor signaling route and the NF-kappa B signaling pathway. An mRNA–miRNA–lncRNA/circRNA network linked to the pathogenesis of NP cells in IVDD was constructed based on node degree and differential expression level. Eight immune-related DEGs (6 upregulated and 2 downregulated genes) and five aging-related DEGs (3 upregulated and 2 downregulated genes) were identified in the critical network. Conclusion We established a novel immune-related and aging-related triple regulatory network of mRNA–miRNA–lncRNA/circRNA ceRNA, among which all RNAs may be utilized as the pathogenesis biomarker of NP cells in IVDD.

Exploring the miRNAs Profile in Dark-Cutting Beef.

In an animal production system, different stressors may cause the depletion of muscle glycogen stores, resulting in an elevated pH at 24 h post mortem (pH24), which leads to cell metabolism alterations that affect the conversion of muscle into meat, causing meat quality defects, such as dark-cutting beef, also known as dark, firm, and dry (DFD) beef. This process may involve the alteration of small non-coding RNAs (miRNAs), which play critical regulatory roles in cellular processes. Here, we determined whether differential miRNA expression in the Longissimus thoracis et lumborum muscle from the Asturiana de los Valles breed at 24 h post mortem could serve as an early indicator of beef quality defects. Following total RNA extraction, complete miRNAome sequencing revealed 12 miRNAs that were significantly upregulated (p < 0.001) in DFD beef compared to the levels in CONTROL beef. These miRNAs are mainly involved in the cellular responses to redox imbalances and apoptosis. Among these, four miRNAs known to be related to oxidative stress (bta-miR-1246, bta-miR-2332, bta-miR-23b-5p, and bta-miR-2411-3p) were validated via quantitative RT-PCR. Some of their target proteins were also analyzed using Western blotting. High 70 kDa heat shock protein and low Caspase-9 expressions (p < 0.01) were found in DFD beef, suggesting the downregulation of apoptosis. These results suggest the importance of miRNAs in regulating stress in muscle cells during early post mortem, as differences in the abundance of some of these miRNAs are still observed at 24 h post mortem. These changes lead to an inadequate conversion of muscle into meat, resulting in meats with quality defects.

Hsa‑miR‑455‑3P as a predictive biomarker of anemia in patients with non‑small cell lung cancer treated with carboplatin plus paclitaxel.

Lung cancer is the leading cause of cancer-related morbidity and mortality worldwide. The initial treatment of lung cancer depends on the definition of the tumor type and its staging. The most common treatment is chemotherapy, and the first-line treatment is a combination of carboplatin and paclitaxel. Although this treatment has good efficacy, there is a high prevalence of adverse events, particularly hematological reactions. Studies on new biomarkers related to these adverse events, such as circulating microRNAs (miRNAs/miRs), are important for optimizing the quality of life of patients. miRNAs have high stability in several biological fluids and they have specific expressions in different tissues or pathologies. Thus, the present study aimed to assess the relationship between circulating miRNAs and adverse hematologic reactions caused by treatment with carboplatin + paclitaxel in patients with lung cancer. Blood was collected from patients before and 15 days after chemotherapy for hematological adverse reaction analysis, microarray and quantitative (q)PCR validation. Adverse reactions were classified according to the Common Terminology Criteria for Adverse Events v4.0. Microarray analysis was performed using plasma from six patients without anemia and six patients with anemia, and nine miRNAs were differentially expressed. miR-1273g-3p, miR-3613-5p and miR-455-3p, identified using microarray, were assessed using qPCR in 20 patients without anemia and 26 patients with anemia. Bioinformatic analyses of miR-455-3p were performed using miRWalk, the Database for Annotation, Visualization and Integrated Discovery and GeneMania software. Microarray analysis of patients with and without anemia revealed nine significant differentially-expressed plasma miRNAs among these patients. Of these, miR-1273g-3p, miR-3613-5p and miR-455-3p were chosen for further assessment. Only miR-455-3p demonstrated a significant reduction in expression (P=0.04) between the groups before chemotherapy with carboplatin + paclitaxel. Bioinformatics analysis of miR-455-3p revealed a relationship between this miRNA and the hematopoietic pathway, particularly with respect to the RUNX family transcription factor 1 (RUNX1) and TAL bHLH transcription factor 1, erythroid differentiation factor (TAL1) genes. The most prevalent adverse reactions in patients with lung cancer treated with carboplatin + paclitaxel were hematological, particularly anemia. This adverse reaction, caused by dysfunction of the hematopoietic system, may be explained by a possible association between the important genes in this system, RUNX1 and TAL1, and hsa-miR-455-3p.

A comprehensive analysis of the prognostic characteristics of microRNAs in breast cancer.

Both overall survival (OS) and disease-specific survival (DSS) are significant when determining a patient's prognosis for breast cancer (BC). The effect of DSS-related microRNAs on BC susrvival, however, is not well understood. Here, we spotted differentially expressed miRNAs (DEMs) in the TCGA database of BC DSS, identified eight DSS-related miRNAs, and constructed a risk model. AUC values at 1, 3, and 5years were 0.852, 0.861, and 0.868, respectively, indicating a risk model's excellent prognostic prediction ability. Then, we validated miRNA roles in BC OS and finally defined miR-551b as an independently prognostic miRNA in BC. According to function analysis, miR-551b is strongly linked with the emergence and spread of cancer, including protein ubiquitination, intracellular protein transport, metabolic pathways, and cancer pathways. Moreover, we confirmed the low expression of miR-551b in BC tissue and cells. After miR-551b inhibition or overexpression, cell function was either dramatically increased or diminished, respectively, indicating that miR-551b could regulate BC proliferation, invasion, and migration. In conclusion, we thoroughly clarified BC-related miRNAs on DSS and OS and verified miR-551b as a crucial regulator in the development and prognosis of cancer. These results can offer fresh ideas for BC therapy.

Four-week inhibition of the renin-angiotensin system in spontaneously hypertensive rats results in persistently lower blood pressure with reduced kidney renin and changes in expression of relevant gene networks.

Prevention of human hypertension is an important challenge and has been achieved in experimental models. Brief treatment with renin-angiotensin system (RAS) inhibitors permanently reduces the genetic hypertension of the spontaneously hypertensive rat (SHR). The kidney is involved in this fascinating phenomenon, but relevant changes in gene expression are unknown. In SHR, we studied the effect of treatment between 10 and 14 weeks of age with the angiotensin receptor blocker, losartan, or the angiotensin-converting enzyme inhibitor, perindopril [with controls for non-specific effects of lowering blood pressure (BP)], on differential RNA expression, DNA methylation, and renin immunolabelling in the kidney at 20 weeks of age. RNA sequencing revealed a six-fold increase in renin gene (Ren) expression during losartan treatment (P < 0.0001). Six weeks after losartan, arterial pressure remained lower (P = 0.006), yet kidney Ren showed reduced expression by 23% after losartan (P = 0.03) and by 43% after perindopril (P = 1.4 × 10-6) associated with increased DNA methylation (P = 0.04). Immunolabelling confirmed reduced cortical renin after earlier RAS blockade (P = 0.002). RNA sequencing identified differential expression of mRNAs, miRNAs, and lncRNAs with evidence of networking and co-regulation. These included 13 candidate genes (Grhl1, Ammecr1l, Hs6st1, Nfil3, Fam221a, Lmo4, Adamts1, Cish, Hif3a, Bcl6, Rad54l2, Adap1, Dok4), the miRNA miR-145-3p, and the lncRNA AC115371. Gene ontogeny analyses revealed that these networks were enriched with genes relevant to BP, RAS, and the kidneys. Early RAS inhibition in SHR resets genetic pathways and networks resulting in a legacy of reduced Ren expression and BP persisting for a minimum of 6 weeks.

Ferroptosis Altered microRNAs Expression in HT-1080 Fibrosarcoma Cells Based on Small RNA Sequencing and Bioinformatics Analysis.

Iron is an essential trace element in the human body. However, excess iron is harmful and may cause ferroptosis. The expression and role of microRNAs (miRNAs) in ferroptosis remain largely unknown. A model of ferroptosis induced by ferric ammonium citrate in HT-1080 cells was established in this study. The miRNAs expression profiles of the control and iron groups were obtained using small RNA sequencing and verified using qRT-PCR. A total of 1346 known miRNAs and 80 novel miRNAs were identified, including 12 up-regulated differentially expressed miRNAs (DE-miRNAs) and 16 down-regulated DE-miRNAs. SP1 was the most important upstream transcription factor regulating DE-miRNAs. The downstream target genes of DE-miRNAs were predicted based on miRDB, TargetScan, and miRBase databases, and 403 common target genes were screened. GO annotation and KEGG analysis revealed that the target genes were mainly involved in various biological processes and regulatory pathways, especially the MAPK signaling pathway and PI3K-Akt signaling pathway. Afterwards, a target genes network was constructed using STRING and Cytoscape, and the hub genes were compared with the ferroptosis database (FerrDb V2) to discover the hub genes related to ferroptosis. EGFR, GSK3B, PARP1, VCP, and SNCA were screened out. Furthermore, a DE-miRNAs-target genes network was constructed to explore key DE-miRNAs. hsa-miR-200c-3p, hsa-miR-26b-5p, and hsa-miR-7-5p were filtered out. Comprehensive bioinformatics analysis of miRNAs and its upstream and downstream regulation in ferroptosis in HT-1080 cells using small RNA sequencing is helpful for understanding the role of miRNAs in iron overload-related diseases and ferroptosis-targeted therapy for cancer.

Extraction and identification of exosomes from three different sources of human ovarian granulosa cells and analysis of their differential miRNA expression profiles.

As important functional cells in the ovary, ovarian granulosa cells are involved in the regulation of oocyte growth and development and play an important role in the study of female fertility preservation. Based on the importance of granulosa cell functionalism, in this study, we analyzed the exosome secretion capacity of human ovarian granulosa cells (SVOG/KGN-cell line, PGC-primary cells) and the differences in their miRNA expression. Cells were identified by hematoxylin-eosin staining (HE) and FSHR immunofluorescence staining; CCK8 and colony-forming assay were performed to compare cell proliferation capacity; exosomes were extracted and identified by ultra-high speed centrifugation, transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blot analysis (WB), and the expression profile of each cellular exosomal miRNA was analyzed by miRNA high-throughput sequencing. The proliferative abilities of the three granulosa cells differed, but all had the ability to secrete exosomes. In the exosomes of SVOG, KGN, and PGC cells, 218, 327, and 471 miRNAs were detected, respectively. When compared to the exosomal miRNAs of PGC cells, 111 miRNAs were significantly different in SVOG, and 70 miRNAs were washed two significantly different in KGN cells. These differential miRNA functions were mainly enriched in the cell cycle, cell division/differentiation, multicellular biogenesis, and protein binding. Human ovarian granulosa cells of different origins are capable of secreting exosomes, but there are still some differences in their exosomes and exosomal miRNAs, and experimental subjects should be selected rationally according to the actual situation.

MicroRNA regulation of islet and enteroendocrine peptides: Physiology and therapeutic implications for type 2 diabetes

The pathogenesis of type 2 diabetes (T2D) is associated with dysregulation of glucoregulatory hormones, including both islet and enteroendocrine peptides. Microribonucleic acids (miRNAs) are short noncoding RNA sequences which post transcriptionally inhibit protein synthesis by binding to complementary messenger RNA (mRNA). Essential for normal cell activities, including proliferation and apoptosis, dysregulation of these noncoding RNA molecules have been linked to several diseases, including diabetes, where alterations in miRNA expression within pancreatic islets have been observed. This may occur as a compensatory mechanism to maintain beta-cell mass/function (e.g., downregulation of miR-7), or conversely, lead to further beta-cell demise and disease progression (e.g., upregulation of miR-187). Thus, targeting miRNAs has potential for novel diagnostic and therapeutic applications in T2D. This is reinforced by the success seen to date with miRNA-based therapeutics for other conditions currently in clinical trials. In this review, differential expression of miRNAs in human islets associated with T2D will be discussed along with further consideration of their effects on the production and secretion of islet and incretin hormones. This analysis further unravels the therapeutic potential of miRNAs and offers insights into novel strategies for T2D management.