过表达GATA-4骨髓间充质干细胞外泌体中miR-330-3p的时-量变化
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摘要
背景:前期实验已经证明过表达GATA-4骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)分泌的外泌体(exosomes)可以通过抑制心肌细胞凋亡,有效修复心肌梗死引起的心肌损伤,进一步发现在其外泌体中miRNA-330-3p明显高表达且功能涉及抗凋亡,提示miRNA-330-3p可能是外泌体修复心肌损伤的关键分子。目的:探讨过表达GATA-4小鼠BMSCs分泌的外泌体(BMSCs~(GATA-4)-exosomes)中miR-330-3p的时-量变化。方法:在过表达GATA-4小鼠BMSCs培养体系内加入miR-330-3p模拟剂(miR-330-3p-mimic)及抑制剂(miR-330-3p-inhibitor)作为BMSCs~(GATA-4-miR-330-3p-mimic)组及BMSCs~(GATA-4-miR-330-3p-inhibitor)组,将BMSCs~(GATA-4)组、BMSCs~(GATA-4-空载体)组、BMSCs组、BMSCs~(GATA-4-miR-330-3p-mimic-空载体)组及BMSCs~(GATA-4-miR-330-3p-inhibitor-空载体)组作为混杂因素对照组。上述各组与基因开启剂强力霉素共培养24,48 h,提取各组细胞分泌的外泌体。采用RT-PCR检测各组细胞及其外泌体内miR-330-3p的表达量。采用光镜评估BMSCs~(GATA-4-miR-330-3p-mimic)组、BMSCs~(GATA-4-miR-330-3p-inhibitor)组与BMSCs组细胞在24,48 h的形态变化。结果与结论:①BMSCs~(GATA-4-miR-330-3p-mimic)组细胞与外泌体内miR-330-3p的表达率最高,且随着时间推移其miR-330-3p的表达逐渐增多;②BMSCs~(GATA-4-miR-330-3p-inhibitor)组细胞与外泌体内miR-330-3p的表达率最低,且随着时间推移其miR-330-3p的表达逐渐减少;③BMSCs~(GATA-4-miR-330-3p-mimic)组、BMSCs~(GATA-4-miR-330-3p-inhibitor)组与BMSCs组在24,48 h的形态无变化;④结果表明,BMSCs内miR-330-3p表达与外泌体内miR-330-3p表达正相关,BMSCs过表达miR-330-3p可以有效提高外泌体中miR-330-3p的表达,而静默miR-330-3p可以有效减少外泌体中miR-330-3p的表达。
BACKGROUND: Previous experiments have demonstrated that exosomes secreted by bone marrow mesenchymal stem cells overexpressing GATA-4(BMSCsGATA-4) can effectively repair myocardial injury induced by myocardial infarction through inhibiting cardiomyocyte apoptosis. We further discovered that miRNA-330-3 p is highly expressed in BMSCs~(GATA-4) exosomes and involved in anti-apoptosis, suggesting that miRNA-330-3 p may be a key molecule for exosomes to repair myocardial injury.OBJECTIVE: To investigate the time-volume variation in miR-330-3 p expression in BMSCs~(GATA-4) exosomes.METHODS: Experimental group 1(BMSCs~(GATA-4–miR-330-3 p-mimic)) and experimental group 2(BMSCs~(GATA-4–miR-330-3 p-inhibitor)) were established by adding miR-330-3 p-mimic or miR-330-3 p-inhibitor to the culture systems of BMSCs~GATA-4 exosomes, respectively. The BMSCsGATA-4,BMSCsGATA-4–empty vector, BMSCs, BMSCs~(GATA-4–miR-330-3 p-mimic–empty) vector, and BMSCs~(GATA-4–miR-330-3 p-inhibitor–empty) vector groups were taken as confounding factors and incubated for 24 or 48 hours with doxycycline, a gene opener. Exosomes secreted by each group were extracted.RT-PCR was used to detect the expression level of miR-330-3 p in each group of cells and secreted exosomes. Morphological changes in the BMSCs~(GATA-4-microRNA-330-3 p-mimic), BMSCs~(GATA-4-microRNA-330-3 p-inhibitor), and BMSCs groups at 24 and 48 hours of incubation with doxycycline were observed under light microscopy.RESULTS AND CONCLUSION: The expression of miR-330-3 p was highest in the cells and exosomes of the BMSCs~(GATA-4–miR-330-3p-mimic) group(P < 0.05), and increased gradually with time. Conversely, the expression of miR-330-3 p was lowest in the cells exosomes of the BMSCs~(GATA-4-miR-330-3 p-inhibitor) group, and moreover, it decreased gradually with time. There were no morphological changes in the BMSCs~(GATA-4–miR-330-3 p-mimic), BMSCs~(GATA-4–miR-330-3 p-inhibitor), and BMSC group at 24 and 48 hours of incubation with doxycycline. Therefore,miR-330-3 p expression in BMSCs is positively correlated with that in exosomes. Expression of miR-330-3 p in exosomes can be effectively increased through miR-330-3 p overexpression while reduced by silencing.
BACKGROUND: Previous experiments have demonstrated that exosomes secreted by bone marrow mesenchymal stem cells overexpressing GATA-4(BMSCsGATA-4) can effectively repair myocardial injury induced by myocardial infarction through inhibiting cardiomyocyte apoptosis. We further discovered that miRNA-330-3 p is highly expressed in BMSCs~(GATA-4) exosomes and involved in anti-apoptosis, suggesting that miRNA-330-3 p may be a key molecule for exosomes to repair myocardial injury.OBJECTIVE: To investigate the time-volume variation in miR-330-3 p expression in BMSCs~(GATA-4) exosomes.METHODS: Experimental group 1(BMSCs~(GATA-4–miR-330-3 p-mimic)) and experimental group 2(BMSCs~(GATA-4–miR-330-3 p-inhibitor)) were established by adding miR-330-3 p-mimic or miR-330-3 p-inhibitor to the culture systems of BMSCs~GATA-4 exosomes, respectively. The BMSCsGATA-4,BMSCsGATA-4–empty vector, BMSCs, BMSCs~(GATA-4–miR-330-3 p-mimic–empty) vector, and BMSCs~(GATA-4–miR-330-3 p-inhibitor–empty) vector groups were taken as confounding factors and incubated for 24 or 48 hours with doxycycline, a gene opener. Exosomes secreted by each group were extracted.RT-PCR was used to detect the expression level of miR-330-3 p in each group of cells and secreted exosomes. Morphological changes in the BMSCs~(GATA-4-microRNA-330-3 p-mimic), BMSCs~(GATA-4-microRNA-330-3 p-inhibitor), and BMSCs groups at 24 and 48 hours of incubation with doxycycline were observed under light microscopy.RESULTS AND CONCLUSION: The expression of miR-330-3 p was highest in the cells and exosomes of the BMSCs~(GATA-4–miR-330-3p-mimic) group(P < 0.05), and increased gradually with time. Conversely, the expression of miR-330-3 p was lowest in the cells exosomes of the BMSCs~(GATA-4-miR-330-3 p-inhibitor) group, and moreover, it decreased gradually with time. There were no morphological changes in the BMSCs~(GATA-4–miR-330-3 p-mimic), BMSCs~(GATA-4–miR-330-3 p-inhibitor), and BMSC group at 24 and 48 hours of incubation with doxycycline. Therefore,miR-330-3 p expression in BMSCs is positively correlated with that in exosomes. Expression of miR-330-3 p in exosomes can be effectively increased through miR-330-3 p overexpression while reduced by silencing.
引文
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[16]Guan A,Wang H,Li X,et al.MiR-330-3p inhibits gastric cancer progression through targeting MSI1.Am J Transl Res.2016;8(11):4802-4811.
[2]He JG,Li HR,Han JX,et al.GATA-4-expressing mouse bone marrow mesenchymal stem cells improve cardiac function after myocardial infarction via secreted exosomes.Sci Rep.2018;8(1):9047.
[3]Golpanian S,Wolf A,Hatzistergos KE,et al.Rebuilding the Damaged Heart:Mesenchymal Stem Cells,Cell-Based Therapy,and Engineered Heart Tissue.Physiol Rev.2016;96(3):1127-1168.
[4]Cai M,Shen R,Song L,et al.Bone Marrow Mesenchymal Stem Cells(BM-MSCs)Improve Heart Function in Swine Myocardial Infarction Model through Paracrine Effects.Sci Rep.2016;6:28250.
[5]Purushothaman A,Bandari SK,Liu J,et al.Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions.J Biol Chem.2016;291(4):1652-1663.
[6]Lee KH,Chen YL,Yeh SD,et al.MicroRNA-330 acts as tumor suppressor and induces apoptosis of prostate cancer cells through E2F1-mediated suppression of Akt phosphorylation.Oncogene.2009;28(38):3360-3370.
[7]Qu S,Yao Y,Shang C,et al.MicroRNA-330 is an oncogenic factor in glioblastoma cells by regulating SH3GL2 gene.PLoSOne.2012;7(9):e46010.
[8]Li Y,Zhu X,Xu W,et al.miR-330 regulates the proliferation of colorectal cancer cells by targeting Cdc42.Biochem Biophys Res Commun.2013;431(3):560-565.
[9]Mao Y,Chen H,Lin Y,et al.microRNA-330 inhibits cell motility by downregulating Sp1 in prostate cancer cells.Oncol Rep.2013;30(1):327-333.
[10]Liang J,Liu X,Xue H,et al.MicroRNA-103a inhibits gastric cancer cell proliferation,migration and invasion by targeting c-Myb.Cell Prolif.2015;48(1):78-85.
[11]Mesci A,Huang X,Taeb S,et al.Targeting of CCBE1 by miR-330-3p in human breast cancer promotes metastasis.Br J Cancer.2017;116(10):1350-1357.
[12]Arora S,Ranade AR,Tran NL,et al.MicroRNA-328 is associated with(non-small)cell lung cancer(NSCLC)brain metastasis and mediates NSCLC migration.Int J Cancer.2011;129(11):2621-31.
[13]Meng H,Wang K,Chen X,et al.MicroRNA-330-3p functions as an oncogene in human esophageal cancer by targeting programmed cell death 4.Am J Cancer Res.2015;5(3):1062-1075.
[14]Liu X,Shi H,Liu B,et al.miR-330-3p controls cell proliferation by targeting early growth response 2 in non-small-cell lung cancer.Acta Biochim Biophys Sin(Shanghai).2015;47(6):431-440.
[15]Yao Y,Xue Y,Ma J,et al.MiR-330-mediated regulation of SH3GL2 expression enhances malignant behaviors of glioblastoma stem cells by activating ERK and PI3K/AKTsignaling pathways.PLoS One.2014;9(4):e95060.
[16]Guan A,Wang H,Li X,et al.MiR-330-3p inhibits gastric cancer progression through targeting MSI1.Am J Transl Res.2016;8(11):4802-4811.