Overexpression Of miR-21 Research Articles

MicroRNA Expression in Chronic Venous Leg Ulcers and Implications for Wound Healing: A Scoping Review.

Purpose: Chronic venous leg ulcers (CVLUs) comprise the majority of lower-extremity wounds, yet their pathophysiology is not fully understood. While research has shown that microRNAs are an important component of wound inflammation, few have explored the role of microRNAs (miRNAs) in the healing of CVLUs. This scoping review examines miRNAs in CVLUs and the association with wound healing. Methods: In December 2023, we searched MEDLINE/PubMed, Embase, Scopus, and CINAHL for studies published in 2013-2023 examining miRNAs in CVLU healing. Results: Six studies met inclusion criteria. MicroRNAs were extracted from various specimens including serum, skin biopsy samples, and adipose tissue-derived mesenchymal cells from individuals with CVLUs. Overexpression of miR-221, miR-222, miR-92a, and miR-301a-3p hindered angiogenesis, while overexpression of miR-296, miR-126, miR-378, and miR-210 facilitated angiogenesis. Overexpression of miR-34a/c, miR-301a-3p, miR-450-5p, miR-424-5p, miR-516-5p, and miR-7704 increased local inflammatory responses and inhibited keratinocytes proliferation, impairing healing, while overexpression of miR-19a/b and miR-20 downregulated keratinocytes' inflammatory response, promoting healing. Downregulation of miR-205, miR-96-5p, and miR-218-5p enhanced cellular proliferation and promoted wound healing. Downregulation of miR-17-92 was linked with impaired healing. Discussion: MicroRNAs play a role in regulating angiogenesis, inflammatory responses, and cell migration in chronic-wound healing. However, studies of miRNAs in CVLUs are limited and lack a standardized approach to measurement and quantification. Further research is warranted to elucidate the mechanisms underlying microRNA involvement in CVLU healing to better understand the pathophysiology and for the future development of targeted therapies.

MiR-326 overexpression inhibits colorectal cancer cell growth and proteasome activity by targeting PNO1: unveiling a novel therapeutic intervention strategy

Proteasome inhibition emerges as a promising strategy for cancer prevention. PNO1, pivotal for colorectal cancer (CRC) progression, is involved in proteasome assembly in Saccharomyces cerevisiae. Hence, we aimed to explore the role of PNO1 in proteasome assembly and its up- and down-streams in CRC. Here, we demonstrated that PNO1 knockdown suppressed CRC cells growth, proteasome activities and assembly, as well as CDKN1B/p27Kip1 (p27) degradation. Moreover, p27 knockdown partially attenuated the inhibition of HCT116 cells growth by PNO1 knockdown. The up-stream studies of PNO1 identified miR-326 as a candidate miRNA directly targeting to CDS-region of PNO1 and its overexpression significantly down-regulated PNO1 protein expression, resulting in suppression of cell growth, decrease of proteasome activities and assembly, as well as increasing the stability of p27 in CRC cells. These findings indicated that miR-326 overexpression can suppress CRC cell growth, acting as an endogenous proteasome inhibitor by targeting PNO1.

MiR-210 as a therapeutic target in diabetes-associated endothelial dysfunction.

MicroRNA (miR)-210 function in endothelial cells and its role in diabetes-associated endothelial dysfunction are not fully understood. We aimed to characterize the miR-210 function in endothelial cells and study its therapeutic potential in diabetes. Two different diabetic mouse models (db/db and Western diet-induced), miR-210 knockout and transgenic mice, isolated vessels and human endothelial cells were used. miR-210 levels were lower in aortas isolated from db/db than in control mice. Endothelium-dependent relaxation (EDR) was impaired in aortas from miR-210 knockout mice, and this was restored by inhibiting miR-210 downstream protein tyrosine phosphatase 1B (PTP1B), mitochondrial glycerol-3-phosphate dehydrogenase 2 (GPD2), and mitochondrial oxidative stress. Inhibition of these pathways also improved EDR in both diabetic mouse models. High glucose reduced miR-210 levels in endothelial cells and impaired EDR in mouse aortas, effects that were reversed by overexpressing miR-210. However, plasma miR-210 levels were not affected in individuals with type 2 diabetes (T2D) following improved glycaemic status. Of note, genetic overexpression using miR-210 transgenic mice and pharmacological overexpression using miR-210 mimic in vivo ameliorated endothelial dysfunction in both diabetic mouse models by decreasing PTP1B, GPD2 and oxidative stress. Genetic overexpression of miR-210 altered the aortic transcriptome, decreasing genes in pathways involved in oxidative stress. miR-210 mimic restored decreased nitric oxide production by high glucose in endothelial cells. This study unravels the mechanisms by which down-regulated miR-210 by high glucose induces endothelial dysfunction in T2D and demonstrates that miR-210 serves as a novel therapeutic target.

FOXO1-mediated autophagy regulation by miR-223 in sepsis-induced immunosuppression.

Immunosuppression is the main cause of the high mortality rate in patients with sepsis. The decrease in the number and dysfunction of CD4+ T lymphocytes is crucial to the immunosuppressed state of sepsis, in turn affecting the development and prognosis of sepsis. Autophagy has been shown to play an important role in the immune imbalance exhibited during sepsis. In this study, we modulate the expression of miR-223 in CD4+ T lymphocytes, via the transfection of a mimic or an inhibitor of miR-223 to establish cell models of miR-223 overexpression and knockdown, respectively. Levels of autophagy were monitored using a double-labeled lentivirus (mRFP-GFP-LC3) and electron microscopy, and western blot analysis was used to estimate the levels of autophagy-related proteins and FOXO1 in the two cell models after co-treatment with lipopolysaccharide (LPS) and siRNA against FOXO1. We found that when the expression of miR-223 increased, FOXO1 expression decreased and autophagy decreased; whereas, when FOXO1 expression was inhibited, autophagy decreased significantly in different cell models after LPS induction. Thus, this study proved that miR-223 participate in the regulation of LPS-induced autophagy via the regulation of FOXO1 expression in CD4+ T lymphocytes which shed a new light for the diagnosis and treatment of sepsis.

MiRNA-153 attenuates progression of non-small cell lung cancer through targeting positive regulatory/SET domain 2

Background: To explore whether micro ribonucleic acid (miR)-153 regulates positive regulatory/SET domain 2 (PRDM2) in a targeted manner and affects the proliferation and apoptosis of non-small cell lung cancer (NSCLC) A549 cells. Methodology: The expressions of miR-153 and PRDM2 in NSCLC tissues and A549 cells were detected by quantitative real-time polymerase chain reaction (qRT-PCR). TargetScan was utilized to predict miR-153 target genes. Methyl thiazolyl tetrazolium (MTT) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were carried out to study the cell proliferation and apoptosis. Western blotting was performed to examine the changes in the proteins in the Janus kinase-signal transducer and activator of transcription (JAK/STAT) signaling pathway following the miR-153 overexpression. Results: The expression of miR-153 was decreased and that of PRDM2 was increased in NSCLC tissues and cells. Target genes regulated by miR-153 participated in self-vascular development, miRNA metabolic process and the Wnt signaling pathway. The overexpression of miR-153 led to an obvious reduction in the proliferation ability of A549 cells, a notable increase in apoptotic cells, and significant decreases in phosphorylated (p)-JAK2 and p-STAT3 proteins. Dual-luciferase reporter gene assay revealed that miR-153 could directly modulate the expression level of PRDM2. Conclusion: MiR-153 directly targets PRDM2 and affects A549 cell proliferation and apoptosis through the JAK/STAT pathway. Keywords: Non-small cell lung cancer; miR-153; positive regulatory/SET domain 2; Janus kinase-signal transducer and activator of transcription (JAK/STAT) signaling pathway.

Sphingosine Kinase 2 Controls the Aggressive Phenotype of Oral Squamous Cell Carcinoma by Regulating miR-205 and miR-296 through p53

Alterations in microRNAs, p53, and sphingolipid metabolism have been associated with head and neck squamous cell carcinoma (HNSCC). However, sphingosine kinase 2, a critical enzyme in sphingolipid metabolism, is poorly understood in HNSCC. Our aim was to investigate how SK2 and p53 interact to regulate miR-205 and miR-296. Analysis of small-RNA-seq data from non-tumor oral keratinocytes with SK2 overexpression (NOK-SK2) compared to NOK-control (NOK- Ø) revealed differential expression of more than 100 miRNAs being half regulated by p53. The expression of miR-205 was downregulated, and miR-296 was upregulated in NOK-SK2 cells; however, cells with SK2 knockdown and p53 overexpression showed an opposite profile. Proteins involved in miRNA biogenesis were increased in NOK-SK2 cells while their levels were decreased in NOK-SK2 cells with p53 overexpression. miR-205 mimic and miR-296 inhibitor decreased the aggressiveness and cancer stem-like cells in oral keratinocytes and oral carcinoma cells with SK2 deregulation. Overexpression of miR-205 in HN12-SK2 cells decreased tumor formation capacity and NOK-SK2 cells abrogated the tumor growth in mice. Our results indicate crosstalk between SK2 and p53 in regulating miR-205 and miR-296, which could be potential targets for HNSCC therapy.

Tectorigenin Inhibits Glycolysis-induced Cell Growth and Proliferation by Modulating LncRNA CCAT2/miR-145 Pathway in Colorectal Cancer.

Colorectal cancer (CRC) places a heavy burden on global health. Tectorigenin (Tec) is a type of flavonoid-based compound obtained from the Chinese medical herb Leopard Lily Rhizome. It was found to exhibit remarkable anti-tumor properties in previous studies. However, the effect and molecular mechanisms of Tec in colorectal cancer have not been reported. The objective of this study was to explore the action of Tec in proliferation and glycolysis in CRC and the potential mechanism with regard to the long non-coding RNA (lncRNA) CCAT2/micro RNA-145(miR-145) pathway in vitro and in vivo . The anti-tumor effect of Tec in CRC was examined in cell and animal studies, applying Cell Counting Kit-8 (CCK-8) assay as well as xenograft model experiments. Assay kits were utilized to detect glucose consumption and lactate production in the supernatant of cells and animal serum. The expression of the glycolysis-related proteins was assessed by Western Blotting, and levels of lncRNA CCAT2 and miR-145 in CRC tissue specimens and cells were assessed by realtime quantitative PCR (RT-qPCR). Tec significantly suppressed cell glycolysis and proliferative rate in CRC cells. It could decrease lncRNA CCAT2 in CRC cells but increase the expression of miR-145. LncRNA CCAT2 overexpression or inhibition of miR-145 could abolish the inhibitive effects of Tec on the proliferation and glycolysis of CRC cells. The miR-145 mimic rescued the increased cell viability and glycolysis levels caused by lncRNA CCAT2 overexpression. Tec significantly inhibited the growth and glycolysis of CRC xenograft tumor. The expression of lncRNA CCAT2 decreased while the expression of miR-145 increased after Tec treatment in vivo. Tec can inhibit the proliferation and glycolysis of CRC cells through the lncRNA CCAT2/miR-145 axis. Altogether, the potential targets discovered in this research are of great significance for CRC treatment and new drug development.

Autophagy-associated biomarkers ULK2, UVRAG, and miRNAs miR-21, miR-126, and miR-374: Prognostic significance in glioma patients.

As the pioneering study from Pakistan, our research distinctly focuses on validating the roles of autophagy-associated genes and MicroRNAs (miRs) in the unique context of our population for glioma prognosis. The study delves into the nuanced interplay of autophagy within a miR-modulated environment, prompting an exploration of its potential impact on glioma development and survival. Employing real-time PCR (qPCR), we meticulously assessed the expression profiles of autophagy genes and miRs in glioma tissues, complemented by immunohistochemistry on Formalin-fixed paraffin-embedded tissues from the same patients. Our comprehensive statistical analyses, including the data normality hypothesis Shapiro-Wilk test, the Mann-Whitney U-test, Spearman correlation test, and Kaplan-Meier survival analysis, were tailored to unravel the intricate associations specific to low- and high-grade glioma within our population. Clinicopathological analysis revealed a predominance of male patients (66%) with a median age of 35 years. Glioblastoma (32%) and Astrocytoma (36%) were the most prevalent histopathological subtypes. Molecular analysis showed significant correlations between prognostic markers (Ki-67, IDH-1, p53) and clinicopathological factors, including age, histological type, radiotherapy, and chemotherapy. In high-grade glioma, increased expression of AKT and miR-21, coupled with reduced ULK2 and LC3 expression was distinctly observed. While correlation analysis identified a strong positive correlation between ULK2 and UVRAG, PTEN, miR-7, and miR-100 in low-grade glioma, unveiling distinctive molecular signatures unique to our study. Furthermore, a moderate positive correlation emerged between ULK2 and mTOR, miR-7, miR-30, miR-100, miR-204, and miR-374, also between miR-21 and miR-126. Similarly, a positive correlation appeared between ULK2 and AKT, LC3, PI3K, PTEN, ULK1, VPS34, mTOR, Beclin1, UVRAG, miR-7 and miR-374. AKT positively correlated with LC3, PI3K, PTEN, ULK1, VPS34, mTOR, Beclin1, UVRAG, miR-7, miR-30, miR-204, miR-374, miR-126 and miR-21 weakly correlated with AKT and miR-30 in high-grade glioma, providing further insights into the autophagy pathway within our population. The enrichment analysis for miR-21, miR-126, and miR-374 showed MAPK pathway as a common pathway along with Ras, PI3K, and mTOR pathway. The low ULK2, UVRAG, and miR-374 expression group exhibited significantly poor overall survival in glioma, while miR-21 over-expression indicated a poor prognosis in glioma patients, validating it in our population. This study provides comprehensive insights into the molecular landscape of gliomas, highlighting the dysregulation of autophagy genes ULK2, and UVRAG and the associated miR-21, miR-126 and miR-374 as potential prognostic biomarkers and emphasizing their unique significance in shaping survival outcomes in gliomas within the specific context of the Pakistani population.

MicroRNA-375 modulates neutrophil chemotaxis via targeting Cathepsin B in zebrafish

Neutrophils are crucial for defense against numerous infections, and their migration and activations are tightly regulated to prevent collateral tissue damage. We previously performed a neutrophil-specific miRNA overexpression screening and identified several microRNAs, including miR-375, as potent modulators for neutrophil activity. Overexpression of miR-375 decreases neutrophil motility and migration in zebrafish and human neutrophil-like cells. We screened the genes downregulated by miR-375 in zebrafish neutrophils and identified that Cathepsin B (Ctsba) is required for neutrophil motility and chemotaxis upon tail wounding and bacterial infection. Pharmacological inhibition or neutrophil-specific knockout of ctsba significantly decreased the neutrophil chemotaxis in zebrafish and survival upon systemic bacterial infection. Notably, Ctsba knockdown in human neutrophil-like cells also resulted in reduced chemotaxis. Inhibiting integrin receptor function using RGDS rescued the neutrophil migration defects and susceptibility to systemic infection in zebrafish with either miR-375 overexpression or ctsba knockout. Our results demonstrate that miR-375 and its target Ctsba modulate neutrophil activity during tissue injury and bacterial infection in vivo, providing novel insights into neutrophil biology and the overall inflammation process.

Extracellular vesicle transfer of miR-1 to adipose tissue modifies lipolytic pathways following resistance exercise.

Extracellular vesicles (EVs) have emerged as important mediators of inter-tissue signaling and exercise adaptations. In this human study (n = 32), we provide evidence that muscle-specific microRNA-1 (miR-1) was transferred to adipose tissue via EVs following an acute bout of resistance exercise. Using a multi-model machine learning automation tool, we discovered muscle primary miR-1 transcript and CD63+ EV count in circulation as top explanatory features for changes in adipose miR-1 levels in response to resistance exercise. RNA-sequencing (RNA-seq) and in-silico prediction of miR-1 target genes identified caveolin 2 (CAV2) and tripartite motif containing 6 (TRIM6) as miR-1 target genes downregulated in the adipose tissue of a subset of participants with the highest increases in miR-1 levels following resistance exercise (n = 6). Overexpression of miR-1 in differentiated human adipocyte-derived stem cells downregulated these miR-1 targets and enhanced catecholamine-induced lipolysis. These data identify a potential EV-mediated mechanism by which skeletal muscle communicates to adipose tissue and modulates lipolysis via miR-1.

MiR-206 Suppresses Triacylglycerol Accumulation via Fatty Acid Elongase 6 in Dairy Cow Mammary Epithelial Cells.

Cow milk possesses high nutritional value due to its rich array of beneficial fatty acids. It is important to understand the mechanisms involved in lipid metabolism in dairy cows. These mechanisms are driven by a complex molecular regulatory network. In addition, there are many regulatory factors involved in the process of fatty acid metabolism, including transcription factors and non-coding RNAs, amongst others. MicroRNAs (miRNAs) can regulate the expression of target genes and modulate various biological processes, including lipid metabolism. Specifically, miR-206 has been reported to impair lipid accumulation in nonruminant hepatocytes. However, the effects and regulatory mechanisms of miR-206 on lipid metabolism in bovine mammary cells remain unclear. In the present study, we investigated the effects of miR-206 on lipid-related genes and TAG accumulation. The direct downstream gene of miR-206 was subsequently determined via a dual-luciferase assay. Finally, the fatty acid content of bovine mammary epithelial cells (BMECs) upon ELOVL6 inhibition was examined. The results revealed that miR-206 overexpression significantly decreased triacylglycerol (TAG) concentration and abundances of the following: acetyl-coenzyme A carboxylase alpha (ACACA); fatty acid synthase (FASN); sterol regulatory element binding transcription factor 1 (SREBF1); diacylglycerol acyltransferase 1 (DGAT1); 1-acylglycerol-3-phosphate O-acyltransferase 6 (AGPAT6); lipin 1 (LPIN1); and fatty acid elongase 6 (ELOVL6). Overexpression of miR-206 was also associated with an increase in patatin-like phospholipase domain-containing 2 (PNPLA2), while inhibition of miR-206 promoted milk fat metabolism in vitro. In addition, we found that ELOVL6 is a direct target gene of miR-206 through mutation of the binding site. Furthermore, ELOVL6 intervention significantly decreased the TAG levels and elongation indexes of C16:0 and C16:1n-7 in BMECs. Finally, ELOVL6 siRNA partially alleviated the increased TAG accumulation caused by miR-206 inhibition. In summary, we found that miR-206 inhibits milk fatty acid synthesis and lipid accumulation by targeting ELOVL6 in BMECs. The results presented in this paper may contribute to the development of strategies for enhancing the quality of cow milk and its beneficial fatty acids, from the perspective of miRNA-mRNA networks.

The magnesium-doped CSH/BCP promotes alveolar bone regeneration by mediating M2 macrophage polarization via miR-21–5p/Smad2 axis

The repair of alveolar bone defects is a complex biological process that involves the interplay between implant materials, bone marrow mesenchymal stem cells (BMSCs), and the immune cells present in the surrounding area. Moreover, the polarization of macrophages induced by implant materials has been shown to play a crucial role in regulating bone regeneration. Recent studies have discovered that M2 macrophages promote the osteogenic differentiation of BMSCs by activating the TGF-β/BMP signaling pathway. However, the molecular mechanisms underlying the regulation of bone regeneration in BMSCs by macrophages remain unclear. Our in vivo study demonstrated that a 10 % magnesium (Mg2+)-doped calcium sulfate hemihydrate -biphasic calcium phosphate (CSH/BCP) composite ceramic material was effective in repairing alveolar bone defects in rabbits. In our in vitro study, we observed that this ceramic material could induce M2 macrophage polarization and reduce inflammation. Moreover, the conditioned media obtained from M2 macrophages promoted the osteogenic differentiation of BMSCs. Additionally, our research showed that overexpression of miR-21–5p inhibited M2 macrophage polarization and reduced the osteogenic differentiation capacity of BMSCs. Co-overexpression of miR-21–5p and Smad2 in macrophages partially reversed the effects of miR-21–5p on the osteogenic differentiation of BMSCs and macrophage polarization. These findings suggested that Mg2+-doped CSH/BCP composite ceramic materials could enhance M2 macrophage polarization through the miR-21–5p/Smad2 axis, and the microenvironment of M2 macrophages could further promote the osteogenic differentiation of BMSCs. The findings presented in this work offer valuable insights into the molecular mechanisms that regulate bone regeneration in BMSCs and the role of macrophages in this process.

Regulation of KIF23 by miR-107 controls replicative tumor cell fitness in mouse and human hepatocellular carcinoma

Regulation of KIF23 by miR-107 controls replicative tumor cell fitness in mouse and human hepatocellular carcinoma

Involvement of oncomiRs miR-23, miR-24, and miR-27 in the regulation of alternative polyadenylation in glioblastoma via CFIm25 cleavage factor.

Glioblastoma multiforme (GBM) is a highly aggressive brain tumor with a poor prognosis. The cleavage factor Im 25 (CFIm25), a crucial component of the CFIm complex, plays a key role in regulating the length of the mRNA 3'-UTR and has been implicated in various cancers, including GBM. This study sought to investigate the regulatory influence of specific microRNAs (miRNAs) on CFIm25 expression in GBM, a highly aggressive brain tumor. Bioinformatics analysis identified miRNA candidates targeting CFIm25 mRNA, and gene expression profiles from the NCBI database (GSE90603) were used for further analysis. Expression levels of CFIm25 and selected miRNAs were assessed using qRT-PCR in GBM clinical samples (n = 20) and non-malignant brain tissues (n = 5). Additionally, the MTT assay was performed to examine the effect of miRNA overexpression on U251 cell viability. Lentivectors expressing the identified miRNAs were employed to experimentally validate their regulatory role on CFIm25 in U251 cell lines, and Western blot analysis was conducted to determine CFIm25 protein levels. We observed significantly increased levels of miR-23, miR-24, and miR-27 expression, associated with a marked reduction in CFIm25 expression in GBM samples compared to non-malignant brain tissues. In particular, overexpression of miR-23, miR-24, and miR-27 in U251 cells resulted in CFIm25 downregulation at both the mRNA and protein levels, while their inhibition increased CFIm25 and reduced cell proliferation. These observations strongly implicate miR-23, miR-24, and miR-27 in regulating CFIm25 expression in GBM, emphasizing their potential as promising therapeutic targets for enhancing treatment responses in glioblastoma.

CircRNA18_46222157_46248185 inhibits melanogenesis by targeting miR-211/EP300 pathway in goat melanocytes.

This study investigated the effects of circRNA18_46222157_46248185 (named circRNA18) on goat melanogenesis, which differs significantly in goat skins isolated from white and brown coat-colored skins. Expression patterns of circRNA18 in goat skin and melanocytes were determined by qRT-PCR and In situ hybridization. The circRNA18 interference vector was designed and synthesized to transfect melanocytes and detect the effect of circRNA18 interference on melanin production. Bioinformatics software was used to predict the targeted adsorption miRNAs of circRNA18, verified by luciferase assay. miRNA expression vector was constructed and transfected into melanocytes to detect the effect of miRNA on melanin production, and the targeted regulatory genes were detected by luciferase assay. Target gene interference vector was constructed to detect the influence of target gene interference on melanin production. qRT-PCR results unveiled distinct expression patterns of circRNA18 in diverse tissues of male and female goats, while in situ hybridization assays showed that circRNA18 is expressed in the cytoplasm of melanocytes. Functional analysis demonstrated that the downregulation of circRNA18 in melanocytes leads to a significant increase (P<0.01) in melanin production. Bioinformatics analysis identified a potential miR-211 binding site on circRNA18, and luciferase assay confirmed their interaction. Overexpression of miR-211 in melanocytes significantly augmented (P<0.01) melanin production. There were two potential miR-211 binding sites on adenoviral E1A-binding protein (EP300), and the overexpression of miR-211 in melanocytes significantly decreased (P<0.001) EP300 expression, with luciferase assay confirming their interaction. Downregulation of EP300 expression in melanocytes through siRNA-EP300 transfection results in a substantial increase (P<0.05) in melanin production. qRT-PCR results indicated that overexpression of mimics-circRNA18 in melanocytes markedly suppressed (P<0.0001) miR-211 expression, significantly elevated (P<0.01) EP300 expression, and significantly inhibited (P<0.001) melanin production. circRNA18_46222157_46248185 acted as a negative regulator of melanogenesis in goat melanocytes by targeting the miR-211/EP300 pathway, and guiding animal hair color breeding strategies.

CHO cell engineering via targeted integration of circular miR-21 decoy using CRISPR/RMCE hybrid system.

Chinese hamster ovary (CHO) cells, widely acknowledged as the preferred host system for industrial recombinant protein manufacturing, play a crucial role in developing pharmaceuticals, including anticancer therapeutics. Nevertheless, mammalian cell-based biopharmaceutical production methods are still beset by cellular constraints such as limited growth and poor productivity. MicroRNA-21 (miR-21) has a major impact on a variety of malignancies, including glioblastoma multiforme (GBM). However, reduced productivity and growth rate have been linked to miR-21 overexpression in CHO cells. The current study aimed to engineer a recombinant CHO (rCHO) cell using the CRISPR-mediated precise integration into target chromosome (CRIS-PITCh) system coupled with the Bxb1 recombinase-mediated cassette exchange (RMCE) to express a circular miR-21 decoy (CM21D) with five bulged binding sites for miR-21 sponging. Implementing the ribonucleoprotein (RNP) delivery method, a landing pad was inserted into the genome utilizing the CRIS-PITCh technique. Subsequently, the CM21D cassette flanked by Bxb1 attB was then retargeted into the integrated landing pad using the RMCE/Bxb1 system. This strategy raised the targeting efficiency by 1.7-fold, and off-target effects were decreased. The miR-21 target genes (Pdcd4 and Atp11b) noticed a significant increase in expression upon the miR-21 sponging through CM21D. Following the expression of CM21D, rCHO cells showed a substantial decrease in doubling time and a 1.3-fold increase in growth rate. Further analysis showed an increased yield of hrsACE2, a secretory recombinant protein, by 2.06-fold. Hence, we can conclude that sponging-induced inhibitionof miR-21 may lead to a growth rate increase that could be linked to increased CHO cell productivity. For industrial cell lines, including CHO cells, an increase in productivity is crucial. The results of our research indicate that CM21D is an auspicious CHO engineering approach. KEY POINTS: • CHO is an ideal host cell line for producing industrial therapeutics manufacturing, and miR-21 is downregulated in CHO cells, which produce recombinant proteins. • The miR-21 target genes noticed a significant increase in expression upon the miR-21 sponging through CM21D. Additionally, sponging of miR-21 by CM21D enhanced the growth rate of CHO cells. • Productivity and growth rate were increased in CHO cells expressing recombinant hrs-ACE2 protein after CM21D knocking in.

Nutrology and molecular state of art in muscle recovery and sports performance: a systematic review

Introduction: Nutrition makes it possible to recover from the negative impact of an injury induced by physical exercise. Recent progress has been made regarding gut microbiota, regenerative nutrition, and skeletal muscle metabolism. MicroRNAs (miRs) have emerged as critical regulators of numerous biological processes, modulating gene expression at the post-transcriptional level. Objective: It was to carry out a systematic review to elucidate the main considerations of nutrology and molecular state of art through exosomes, microRNAs, and nutrients in muscle recovery and sports performance. Methods: The systematic review rules of the PRISMA Platform were followed. The research was carried out from April to June 2023 in Scopus, PubMed, Science Direct, Scielo, and Google Scholar databases. The quality of the studies was based on the GRADE instrument and the risk of bias was analyzed according to the Cochrane instrument. Results and Conclusion: A total of 237 articles were found. A total of 107 articles were evaluated in full and 52 were included and developed in this systematic review study. Considering the Cochrane tool for risk of bias, the overall assessment resulted in 47 studies with a high risk of bias and 70 studies that did not meet GRADE. Based on the findings, it was concluded that dietary manipulations and metabolites may affect tissue stem cell fate decisions. Self-renewal and differentiation of mesenchymal stem cells can be regulated by manipulating vitamin C, A, or D levels and valine restriction. miRs play an important role as regulatory molecules during the muscle healing process. Myoblasts are known to secrete exosomes enriched with miRs into the inflammatory environment, whereby miR-224 is transferred to macrophages to inhibit M2 polarization. Additional data demonstrate that WNT-9a may be a direct target of miR224 for macrophage polarization. The results showed that miR-122 and myogenic markers were down-regulated in C2C12 cells after TGF-β stimulation, and overexpression of miR-122 can restore myogenesis inhibited by TGF-β. Evidence suggests that the exosome derived from mesenchymal stem cells exhibits functions similar to mesenchymal stem cells with low immunogenicity and without tumorization. High rates of intestinal self-renewal are enabled by intestinal stem cells (LGR5+) at the base of intestinal crypts. LGR5+ activity, including proliferation and differentiation rates, is affected by large shifts in nutrient availability, as occurs on a high-fat diet or fasting. The practice of physical activity, endogenous metabolites, and dietary nutrients can directly influence epigenetic enzymes.

AB060. Autophagy-associated biomarkers ULK2, UVRAG, and miRNAs miR-21, miR-126, and miR-374: prognostic significance in glioma patients.

Autophagy is a self-renewing process of the cell having a dual role in gliomagenesis depending on the tumor stage. Several microRNAs play a key role in the regulation of autophagy and the outcome of cancer. We investigated the potential relevance of autophagy in gliomagenesis and survival by exploring the association of the basal gene expression of autophagy-associated markers LC3, ULK1/2, UVRAG, Beclin1, mTOR, UVRAG, PI3K, AKT, PTEN and their target microRNAs miR-126, miR-374, miR-21, miR-7, miR-204 and miR-100 in low- and high-grades of gliomas. A total of 50 fresh glioma tissues were used for the extraction of RNA using TRIzol-Chloroform method and reverse transcribed cDNA. The cDNA was used to determine the expression of genes and microRNAs using quantitative real-time polymerase chain reaction (qPCR). Mann-Whitney U-test was used to determine the statistical significance. In high-grade glioma, increased expression of AKT and miR-21, coupled with reduced ULK2 and LC3 expression was distinctly observed. While correlation analysis identified a strong positive correlation between ULK2 and UVRAG, PTEN, miR-7, and miR-100 and a moderate positive correlation emerged between ULK2 and mTOR, miR-7, miR-30, miR-100, miR-204, and miR-374, also between miR-21 and miR-126 in low-grade glioma. Similarly, a positive correlation appeared between ULK2 and AKT, LC3, PI3K, PTEN, ULK1, VPS34, mTOR, Beclin1, UVRAG, miR-7 and miR-374. AKT positively correlated with LC3, PI3K, PTEN, ULK1, VPS34, mTOR, Beclin1, UVRAG, miR-7, miR-30, miR-204, miR-374, miR-126 and miR-21 weakly correlated with AKT and miR-30 in high-grade glioma. The low ULK2, UVRAG, and miR-374 expression group exhibited significantly poor overall survival in glioma, while miR-21 over-expression indicated a poor prognosis in glioma patients. This study provides comprehensive insights into the molecular landscape of gliomas, highlighting the dysregulation of autophagy genes ULK2, and UVRAG and the associated miR-21, miR-126 and miR-374 as potential prognostic biomarkers and emphasizing their unique significance in shaping survival outcomes in gliomas patients.

Screening and identification of miRNAs negatively regulating FAM83A/Wnt/β-catenin signaling pathway in non-small cell lung cancer

The prevalence of non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancers, with the Wnt/β-catenin signaling pathway exhibiting robust activation in this particular subtype. The expression of FAM83A (family with sequence similarity 83, member A) has been found to be significantly upregulated in lung cancer, leading to the stabilization of β-catenin and activation of the Wnt signaling pathway. In this study, we conducted a screening of down-regulated miRNAs in lung cancer with FAM83A as the target. Ultimately, we identified miR-1 as a negative regulator of FAM83A and confirmed that FAM83A is a direct target gene of miR-1 through dual luciferase reporter assays. The overexpression of miR-1 significantly attenuated the expression level of FAM83A and suppressed the Wnt signaling pathway, leading to a reduction in the expression levels of downstream target genes AXIN2, CyclinD1, and C-MYC. Additionally, it decreased the nuclear translocation of β-catenin. In addition, overexpression of miR-1 accelerated the degradation of β-catenin by inhibiting FAM83A, promoted the assembly of β-catenin degradation complex, and inhibited the proliferation, migration and invasion of NSCLC cells. In summary, miR-1 may be a potential candidate miRNA for the treatment of NSCLC.

MiR-203 secreted in extracellular vesicles mediates the communication between neural crest and placode cells required for trigeminal ganglia formation.

While interactions between neural crest and placode cells are critical for the proper formation of the trigeminal ganglion, the mechanisms underlying this process remain largely uncharacterized. Here, by using chick embryos, we show that the microRNA (miR)-203, whose epigenetic repression is required for neural crest migration, is reactivated in coalescing and condensing trigeminal ganglion cells. Overexpression of miR-203 induces ectopic coalescence of neural crest cells and increases ganglion size. By employing cell-specific electroporations for either miR-203 sponging or genomic editing using CRISPR/Cas9, we elucidated that neural crest cells serve as the source, while placode cells serve as the site of action for miR-203 in trigeminal ganglion condensation. Demonstrating intercellular communication, overexpression of miR-203 in the neural crest in vitro or in vivo represses an miR-responsive sensor in placode cells. Moreover, neural crest-secreted extracellular vesicles (EVs), visualized using pHluorin-CD63 vector, become incorporated into the cytoplasm of placode cells. Finally, RT-PCR analysis shows that small EVs isolated from condensing trigeminal ganglia are selectively loaded with miR-203. Together, our findings reveal a critical role in vivo for neural crest-placode communication mediated by sEVs and their selective microRNA cargo for proper trigeminal ganglion formation.