scholarly journals Porcine Milk-Derived Small Extracellular Vesicles Promote Intestinal Immunoglobulin Production through pIgR

Animals ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1522
Author(s):  
Bin Zeng ◽  
Hailong Wang ◽  
Junyi Luo ◽  
Meiying Xie ◽  
Zhengjiang Zhao ◽  
...  
Keyword(s):  
Small Intestine ◽  
Extracellular Vesicles ◽  
Immunoglobulin A ◽  
Luciferase Reporter ◽  
Epithelial Cell Line ◽  
Intestinal Immunity ◽  
In Vivo Experiments ◽  
Porcine Milk

Secretory immunoglobulin A (SIgA) plays an important role in gut acquired immunity and mucosal homeostasis. Breast milk is the irreplaceable nutritional source for mammals after birth. Current studies have shown the potential functional role of milk-derived small extracellular vesicles (sEVs) and their RNAs cargo in intestinal health and immune regulation. However, there is a lack of studies to demonstrate how milk-derived sEVs affect intestinal immunity in recipient. In this study, through in vivo experiments, we found that porcine milk small extracellular vesicles (PM-sEVs) promoted intestinal SIgA levels, and increased the expression levels of polymeric immunoglobulin receptor (pIgR) both in mice and piglet. We examined the mechanism of how PM-sEVs increased the expression level of pIgR in vitro by using a porcine small intestine epithelial cell line (IPEC-J2). Through bioinformatics analysis, dual-luciferase reporter assays, and overexpression or knockdown of the corresponding non-coding RNAs, we identified circ-XPO4 in PM-sEVs as a crucial circRNA, which leads to the expression of pIgR via the suppression of miR-221-5p in intestinal cells. Importantly, we also observed that oral administration of PM-sEVs increased the level of circ-XPO4 and decreased the level of miR-221-5p in small intestine of piglets, indicating that circRNAs in milk-derived sEVs act as sponge for miRNAs in recipients. This study, for the first time, reveals that PM-sEVs have a capacity to stimulate intestinal SIgA production by delivering circRNAs to receptors and sponging the recipient’s original miRNAs, and also provides valuable data for insight into the role and mechanism of animal milk sEVs in intestinal immunity.

scholarly journals Plant-Derived Extracellular Vesicles: Current Findings,Challenges, and Future Applications

Membranes ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 411
Author(s):  
Nader Kameli ◽  
Anya Dragojlovic-Kerkache ◽  
Paul Savelkoul ◽  
Frank R. Stassen
Keyword(s):  
Human Health ◽  
Extracellular Vesicles ◽  
Preliminary Results ◽  
In Vivo Experiments ◽  
Health And Disease ◽  
Conducting Research ◽  
Protocol Standardization ◽  
Insight Into

In recent years, plant-derived extracellular vesicles (PDEVs) have gained the interest of many experts in fields such as microbiology and immunology, and research in this field has exponentially increased. These nano-sized particles have provided researchers with a number of interesting findings, making their application in human health and disease very promising. Both in vitro and in vivo experiments have shown that PDEVs can exhibit a multitude of effects, suggesting that these vesicles may have many potential future applications, including therapeutics and nano-delivery of compounds. While the preliminary results are promising, there are still some challenges to face, such as a lack of protocol standardization, as well as knowledge gaps that need to be filled. This review aims to discuss various aspects of PDEV knowledge, including their preliminary findings, challenges, and future uses, giving insight into the complexity of conducting research in this field.

scholarly journals Oncogene APOL1 promotes proliferation and inhibits apoptosis via activating NOTCH1 signaling pathway in pancreatic cancer

2021 ◽  
Vol 12 (8) ◽  
Author(s):  
Jiewei Lin ◽  
Zhiwei Xu ◽  
Junjie Xie ◽  
Xiaxing Deng ◽  
Lingxi Jiang ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Signaling Pathway ◽  
Density Lipoprotein ◽  
Human Pancreatic Cancer ◽  
Luciferase Reporter ◽  
Notch1 Signaling ◽  
In Vivo Experiments ◽  
Notch1 Signaling Pathway

AbstractAPOL1 encodes a secreted high-density lipoprotein, which has been considered as an aberrantly expressed gene in multiple cancers. Nevertheless, the role of APOL1 in the regulatory mechanisms of pancreatic cancer remains unknown and should be explored. We identified APOL1 was abnormally elevated in human pancreatic cancer tissues compared with that in adjacent tissues and was associated with poor prognosis. The effects of APOL1 in PC cell proliferation, cell cycle, and apoptosis was verified via functional in vitro and in vivo experiments. The results showed that knockdown of APOL1 significantly inhibited the proliferation and promoted apoptosis of pancreatic cancer. In addition, we identified APOL1 could be a regulator of NOTCH1 signaling pathway using bioinformatics tools, qRT-PCR, dual-luciferase reporter assay, and western blotting. In summary, APOL1 could function as an oncogene to promote proliferation and inhibit apoptosis through activating NOTCH1 signaling pathway expression in pancreatic cancer; therefore, it may act as a novel therapeutic target for pancreatic cancer.

Radiation activates the NORAD expression to promote ESCC radioimmunotherapy resistance via EEPD1/ATR/Chk1 signaling by inhibiting the pri-miR-199 processing and exosomal transfer of miR-199a-5p

2021 ◽  
Author(s):  
Yuchen Sun ◽  
Jizhao Wang ◽  
Xuanzi Sun ◽  
Jing Li ◽  
Xu Zhao ◽  
...  
Keyword(s):  
Its Region ◽  
Luciferase Reporter ◽  
In Vivo Experiments ◽  
Cycle Arrest ◽  
Non Coding Rna ◽  
Irradiation Treatment ◽  
Recombination Repair ◽  
Reporter Assays

Abstract Background Radioresistance, a poorly understood phenomenon, results in the failure of radiotherapy and consequent local recurrence, threatening a large proportion of ESCC patients. To date, lncRNAs have been found to be involved in diverse biological processes, including radioresistance.Methods ELISA was used to evaluated the H3 modifications in radio-resistant ESCC cells. FISH and qRT-PCR were adopted to examine the expression and localization of lncRNA-NORAD, pri-miR-199a and miR-199a. Electron microscopy and Nanoparticle tracking analysis (NTA) was conducted to observe and identify exosomes. High-throughput RNA sequencing and TMT mass spectrometry were performed to identify the functional lncRNAs and proteins involved in ESCC radioresistance. A series of in vitro and in vivo experiments were performed to investigate the biological effect of NORAD. CHIP, qPCR-RIP, co-IP and dual-luciferase reporter assays were used to explore the interaction of related RNAs and proteins. Results We show here that a DNA damage activated non-coding RNA-NORAD, which is critical for ESCC radio-resistance. NORAD was highly expressed in radio-resistant ESCC cells and tissues. Irradiation treatment promotes NORAD expression via enhancing H3K4me2 enrichment on its region. NORAD knockdown cells exhibit significantly hypersensitivity to irradiation in vivo and in vitro. NORAD is required for initiating repair and restart of stalled forks, G2 cycle arrest and homologous recombination repair upon irradiation treatment. Mechanistically, NORAD inhibits miR-199a expression by competitively binding PUM1 from pri-miR-199a, inhibiting the process of pri-miR-199a. Mature miR-199a in NORAD-knockdown cells can be packaged into exosomes; miR-199a restores the radiosensitivity of radioresistant cells by targeting EEPD1, then inhibiting ATR/Chk1 signaling pathway. Simultaneously, NORAD knockdown blocks the ubiquitination of PD-L1, leads to the better response for radiation and anti-PD-1 treatment in mouse model.Conclusion This study raises the possibility that LncRNA-NORAD could be a potential treatment target for improving the efficiency of immunotherapy in combination with radiation in ESCC.

scholarly journals Postprandial transfer of colostral extracellular vesicles and their protein and miRNA cargo in neonatal calves

2019 ◽  
Author(s):  
Benedikt Kirchner ◽  
Dominik Buschmann ◽  
Vijay Paul ◽  
Michael W. Pfaffl
Keyword(s):  
Extracellular Vesicles ◽  
Expression Profiles ◽  
Bovine Milk ◽  
Cell Types ◽  
Specific Protein ◽  
In Vivo Experiments ◽  
Neonatal Calves ◽  
Almost All

Abstract Background Extracellular vesicles (EVs) such as exosomes are key regulators of intercellular communication that can be found in almost all bio fluids. Although studies in the last decade have made great headway in discerning the role of EVs in many physiological and pathophysiological processes, the bioavailability and impact of dietary EVs and their cargo still remain to be elucidated. Due to its widespread consumption and high content of EV-associated microRNAs and proteins, a major focus in this field has been set on EVs in bovine milk and colostrum. Despite promising in vitro studies in recent years that show high resiliency of milk EVs to degradation and uptake of milk EV cargo in a variety of intestinal and blood cell types, in vivo experiments continue to be inconclusive and sometimes outright contradictive. Results To resolve this discrepancy, we assessed the potential postprandial transfer of colostral EVs to the circulation of newborn calves by analysing colostrum-specific protein and miRNAs, including specific isoforms (isomiRs) in cells, EV isolations and unfractionated samples from blood and colostrum. Our findings reveal distinct populations of EVs in colostrum and blood from cows that can be clearly separated by density, particle concentration and protein content (BTN1A1, MFGE8). Postprandial blood samples of calves show a time-dependent increase in EVs that share morphological and protein characteristics of colostral EVs. Analysis of miRNA expression profiles by Next-Generation Sequencing gave a different picture however. Although significant postprandial expression changes could only be detected for calf EV samples, expression profiles show very limited overlap with highly expressed miRNAs in colostral EVs or colostrum in general. Conclusions Taken together our results indicate a selective uptake of membrane-associated protein cargo but not luminal miRNAs from colostral EVs into the circulation of neonatal calves.

CircRNA_0092516 regulates chondrocyte proliferation and apoptosis in osteoarthritis through the miR-337-3p/PTEN axis

2020 ◽  
Author(s):  
Zhihui Huang ◽  
Wenming Ma ◽  
Jinhuai Xiao ◽  
Xiaoyu Dai ◽  
Weiqi Ling
Keyword(s):  
Cell Apoptosis ◽  
Luciferase Reporter ◽  
Circular Rnas ◽  
Mechanism Analysis ◽  
Potential Mechanism ◽  
Luciferase Reporter Gene ◽  
Chondrocyte Proliferation ◽  
In Vivo Experiments

Abstract The dysregulation of circular RNAs (circRNAs) has been identified in various human diseases. Here, we probed into the potential mechanism of circRNA_0092516 in osteoarthritis (OA). The expression of circRNA_0092516 was tested by quantitative real-time PCR. MTT, flow cytometry and western blot were applied to confirm the functions of circRNA_0092516 in vitro. Besides, RNA pull-down and dual-luciferase reporter gene experiments were applied to probe into the mechanism. circRNA_0092516 was raised in the tissues of OA patients and chondrocytes stimulated by IL-1β. The potential mechanism analysis expounded that circRNA_0092516 bound to miR-337-3p, and the interference with circRNA_0092516 boosted chondrocyte proliferation and restrained cell apoptosis through the miR-337-3p/phosphatase and tensin homolog (PTEN) axis, thereby improving OA. In-vivo experiments expounded that circRNA_0092516 regulated cartilage production through miR-337-3p. Overall, our data expounded that the interference with circRNA_0092516 boosted chondrocyte proliferation and restrained cell apoptosis through the miR-337-3p/PTEN axis, eventually slowed down the progress of OA.

scholarly journals miR-30e targets GLIPR-2 to modulate diabetic nephropathy: in vitro and in vivo experiments

2017 ◽  
Vol 59 (2) ◽  
pp. 181-190 ◽  
Author(s):  
Dong Zhao ◽  
Jinhua Jia ◽  
Hong Shao
Keyword(s):  
High Glucose ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Luciferase Reporter Gene ◽  
Related Factors ◽  
Qrt Pcr ◽  
In Vivo Experiments ◽  
Renal Tubular

The objectives of this study are to investigate the effect of miR-30e targeting GLIPR-2 on the pathological mechanism of DN. The renal tissues of db/db and db/m mice at different age of weeks were stained with PAS. qRT-PCR was applied to detect the expression of miR-30e and GLIPR-2, not only in the renal tissues of mice but also in the renal tubular epithelial cells (RTECs). By luciferase reporter gene assays, we found the 3′-UTR of the GLIPR-2 mRNA as a direct target of miR-30e. The RTECs cultured in high glucose were divided into blank control, NC, miR-30e mimics, miR-30e inhibitors, miR-30e inhibitor + si-GLIPR-2 and si-GLIPR-2 groups. MTT and flow cytometry were utilized to measure the proliferation and apoptosis of RTECs, while qRT-PCR and Western blot to detect the expression of GLIPR-2- and EMT-related factors. The following results were obtained: In the renal tissues of over 8-week-old db/db mice and the RTECs cultured for 6 h in high glucose, miR-30e was downexpressed while GLIPR-2 was upregulated in a time-dependent manner. Besides, overexpression of miR-30e and si-GLIPR-2 can not only greatly improve the proliferation of RTECs cultured in high glucose, but also downregulate the apoptosis rate of RTECs and the expressions of GLIPR-2, vimentin, α-SMA, Col-I and FN and upregulate E-cadherin. Moreover, si-GLIPR-2 can reverse the proliferation reduction, GLIPR-2 and EMT occurrence caused by the downexpression of miR-30e in RTECs. In conclusion, miR-30e is downregulated in DN, and the overexpression of miR-30e can inhibit GLIPR-2, promote the proliferation of RTECs and inhibit EMT, ultimately avoid leading to renal fibrosis in DN.

Liver X receptor inhibits the growth of hepatocellular carcinoma cells via regulating HULC/miR-134-5p/FOXM1 axis

2020 ◽  
Author(s):  
Yan Zhang ◽  
Jintao He ◽  
Teng Yang ◽  
Wenhui He ◽  
Shan Jiang ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cyclin D1 ◽  
Gene Promoter ◽  
Liver X Receptor ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
In Vivo Experiments ◽  
Hcc Cells

Abstract Background Highly upregulated in liver cancer (HULC), the specifically overexpressed long non-coding RNA (lncRNA) in human hepatocellular carcinoma (HCC), can promote the growth and metastasis of HCC cells. Therefore, it will be benefit to HCC treatment by effectively downregulating HULC. Liver X receptor (LXR), a member of nuclear receptor superfamily, exerts anti-tumor effects on various human malignancies including HCC. However, it is unclear whether the anti-HCC function of LXR is involved in the regulation of HULC. Methods Quantitative real-time PCR and Western blot were used to separately examine RNA and protein levels in HCC cells. Cell counting kit-8 assay was used to detect the growth of HCC cells in vitro . Dual-luciferase reporter assays were performed to analyze the regulation of forkhead box M1 (FOXM1) by miR-134-5p and the regulation of miR-134-5p by HULC. Xenograft models were engaged to evaluate the growth of HCC cells in vivo . Results In this study, we found that activation of LXR could inhibit the growth of HCC cells by downregulating HULC. Mechanistically, LXR decreased HULC via suppressing its gene promoter activity. Moreover, HULC and FOXM1 were highly expressed while miR-134-5p was lowly expressed in HCC tissues, and the level of HULC was positively correlated with that of FOXM1 while negatively correlated with that of miR-134-5p. Additionally, miR-134-5p downregulated FOXM1 by targeting 3′-untranslated region (UTR) of its mRNA, and HULC upregulated FOXM1 and its downstream target molecule cyclin D1 through sequestrating miR-134-5p. Furthermore, activation of LXR increased miR-134-5p while decreased FOXM1 by reducing HULC in HCC cells. The in vivo experiments showed that activation of LXR repressed the growth of HCC xenografts, and decreased HULC, FOXM1 and cyclin D1 while increased miR-134-5p in the xenografts. Conclusions Our results for the first time reveal that LXR can inhibit the growth of HCC cells by regulating HULC/miR-134-5p/FOXM1 axis. The novel pathway LXR/HULC/miR-134-5p/FOXM1 may serve as a promising target in HCC treatment.

scholarly journals Renal Regenerative Potential of Extracellular Vesicles Derived from miRNA-Engineered Mesenchymal Stromal Cells

2019 ◽  
Vol 20 (10) ◽  
pp. 2381 ◽  
Author(s):  
Marta Tapparo ◽  
Stefania Bruno ◽  
Federica Collino ◽  
Gabriele Togliatto ◽  
Maria Chiara Deregibus ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Therapeutic Interventions ◽  
In Vivo Models ◽  
Regenerative Effect ◽  
In Vivo Experiments ◽  
Renal Regeneration

Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) possess pro-regenerative potential in different animal models with renal injury. EVs contain different molecules, including proteins, lipids and nucleic acids. Among the shuttled molecules, miRNAs have a relevant role in the pro-regenerative effects of EVs and are a promising target for therapeutic interventions. The aim of this study was to increase the content of specific miRNAs in EVs that are known to be involved in the pro-regenerative effect of EVs, and to assess the capacity of modified EVs to contribute to renal regeneration in in vivo models with acute kidney injuries. To this purpose, MSCs were transiently transfected with specific miRNA mimics by electroporation. Molecular analyses showed that, after transfection, MSCs and derived EVs were efficiently enriched in the selected miRNAs. In vitro and in vivo experiments indicated that EVs engineered with miRNAs maintained their pro-regenerative effects. Of relevance, engineered EVs were more effective than EVs derived from naïve MSCs when used at suboptimal doses. This suggests the potential use of a low amount of EVs (82.5 × 106) to obtain the renal regenerative effect.

scholarly journals Radiation induces NORAD expression to promote ESCC radiotherapy resistance via EEPD1/ATR/Chk1 signalling and by inhibiting pri-miR-199a1 processing and the exosomal transfer of miR-199a-5p

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Yuchen Sun ◽  
Jizhao Wang ◽  
Yuan Ma ◽  
Jing Li ◽  
Xuanzi Sun ◽  
...  
Keyword(s):  
Noncoding Rna ◽  
Radiation Treatment ◽  
Luciferase Reporter ◽  
Treatment Target ◽  
In Vivo Experiments ◽  
Cycle Arrest ◽  
Recombination Repair ◽  
Reporter Assays

Abstract Background Radioresistance, a poorly understood phenomenon, results in the failure of radiotherapy and subsequent local recurrence, threatening a large proportion of patients with ESCC. To date, lncRNAs have been reported to be involved in diverse biological processes, including radioresistance. Methods FISH and qRT–PCR were adopted to examine the expression and localization of lncRNA-NORAD, pri-miR-199a1 and miR-199a-5p. Electron microscopy and nanoparticle tracking analysis (NTA) were conducted to observe and identify exosomes. High-throughput microRNAs sequencing and TMT mass spectrometry were performed to identify the functional miRNA and proteins. A series of in vitro and in vivo experiments were performed to investigate the biological effect of NORAD. ChIP, RIP-qPCR, co-IP and dual-luciferase reporter assays were conducted to explore the interaction of related RNAs and proteins. Results We show here that DNA damage activates the noncoding RNA NORAD, which is critical for ESCC radioresistance. NORAD was expressed at high levels in radioresistant ESCC cells. Radiation treatment promotes NORAD expression by enhancing H3K4me2 enrichment in its sequence. NORAD knockdown cells exhibit significant hypersensitivity to radiation in vivo and in vitro. NORAD is required to initiate the repair and restart of stalled forks, G2 cycle arrest and homologous recombination repair upon radiation treatment. Mechanistically, NORAD inhibits miR-199a-5p expression by competitively binding PUM1 from pri-miR-199a1, inhibiting the processing of pri-miR-199a1. Mature miR-199a-5p in NORAD knockdown cells is packaged into exosomes; miR-199a-5p restores the radiosensitivity of radioresistant cells by targeting EEPD1 and then inhibiting the ATR/Chk1 signalling pathway. Simultaneously, NORAD knockdown inhibits the ubiquitination of PD-L1, leading to a better response to radiation and anti-PD-1 treatment in a mouse model. Conclusions Based on the findings of this study, lncRNA-NORAD represents a potential treatment target for improving the efficiency of immunotherapy in combination with radiation in ESCC.

scholarly journals Tumor-Derived miR-378a-3p-Containing Extracellular Vesicles Promote Osteolysis by Activating a Dyrk1a/Nfatc1/Angptl2 Axis for Bone Metastasis

2021 ◽  
Author(s):  
Jialin Wang ◽  
Xinxing Du ◽  
Xiao Wang ◽  
Huixiang Xiao ◽  
Nan Jing ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Bone Metastasis ◽  
Extracellular Vesicles ◽  
Nuclear Translocation ◽  
Intracellular Concentration ◽  
Luciferase Reporter ◽  
Metastatic Niche ◽  
Mesenchymal Transition

Abstract Background The majority of the deaths of prostate cancer (PCa) are caused by progression to bone metastatic PCa. The importance of extracellular vesicles (EVs) in the formation of the pre-metastatic niche has been demonstrated in recent years. However, whether and how tumor-derived EVs interact with bone marrow macrophages (BMMs) to release EV-delivered microRNAs to promote osteolysis and to activate pre-metastatic niche formation for PCa bone metastasis remain unclear. Methods Bioinformatics and qRT-PCR analyses were used to screen microRNAs and to identify the elevated expression of miR-378a-3p in both serum-derived EVs from PCa patients and in culture medium-derived EVs from PCa cell lines. Functional assays in vitro and in vivo were performed to investigate the functions of miR-378a-3p during PCa progression. IF staining and Dual-luciferase reporter, co-IP, western blot, RIP and ChIP assays were conducted to reveal the underlying mechanism. Results We found that EV-mediated release of miR-378a-3p from tumor cells was upregulated in bone-metastatic PCa which keeps a low intracellular concentration of miR-378a-3p, to promote proliferation and the MAOA-mediated epithelial-to-mesenchymal transition (EMT) in PCa cells. In addition, we demonstrated that the enrichment of miR-378a-3p in tumor derived EVs was induced by overexpression of hnRNPA2B1 as a transfer chaperone. After miR-378a-3p-enriched EVs were taken in by BMMs, elevated intracellular concentration of miR-378a-3p promoted osteolytic progression by targeting the Dyrk1a/Nfatc1 pathway. Mechanistically, inhibition of Dyrk1a by miR-378a-3p improved the nuclear translocation of Nfatc1 to promote expression of the downstream target gene Angptl2. As a feedback, increased secretion of Angptl2 into the tumor environment promoted PCa progression. Conclusions Our findings indicate that tumor-derived miR-378a-3p-containing EVs play a significant role in promoting prostate cancer bone metastasis by activating a Dyrk1a/Nfatc1/Angptl2 axis in BMMs to induce osteolytic progression, which implicates that miR-378a-3p may be a potential predictor of metastatic PCa. Moreover, reducing the release of miR-378a-3p-containing EVs or inhibiting the recruitment of miR-378a-3p into tumor-derived EVs might be a potential therapeutic strategy for PCa metastasis.

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