scholarly journals Decreased MiR-155 Level in the Peripheral Blood of Non-Alcoholic Fatty Liver Disease Patients may Serve as a Biomarker and may Influence LXR Activity

2016 ◽  
Vol 39 (6) ◽  
pp. 2239-2248 ◽  
Author(s):  
Lei Wang ◽  
Ning Zhang ◽  
Zun Wang ◽  
Dong-mei Ai ◽  
Zhen-yu Cao ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Target Gene ◽  
Luciferase Reporter ◽  
Healthy Controls ◽  
Reporter Assay ◽  
Intracellular Lipid ◽  
Alcoholic Fatty Liver ◽  
Alcoholic Fatty Liver Disease ◽  
Dual Luciferase Reporter Assay

Background: Obesity is now a common risk factor for non-alcoholic fatty liver disease (NAFLD). Thus, it is important to explore its underlying mechanisms. Methods: Total RNA was extracted from peripheral whole blood samples from 50 NAFLD patients and 50 healthy controls. In addition, human liver specimens were obtained through liver biopsies from NAFLD patients and healthy controls. The level of miRNA was studied using real-time PCR. The expression of lipogenic genes was analyzed using western blot, and a dual luciferase reporter assay was conducted to identify the possible target gene. Adenovirus vectors were injected into the tail vein of the high fat diet (HFD)-fed mice to study the role of miR-155 on lipid accumulation in vivo. Results: The level of miR-155 was markedly reduced in the livers and peripheral blood of NAFLD patients compared with healthy controls. Upregulation of miR-155 decreased intracellular lipid content and the SREBP1 and FAS protein levels, while inhibition of miR-155 enhanced the intracellular lipid content. The dual luciferase reporter assay showed that Liver X receptor (LXR)α was the target gene of miR-155, and silencing miR-155 reduced the expression of SREBP1 and FAS. An in vivo study showed that upregulation of miR-155 decreased the hepatic lipid accumulation mainly by suppressing the LXRα-dependent lipogenic signaling pathway. Conclusions: In summary, decreased expression of miR-155 in the peripheral blood may be utilized as a potential novel biomarker for NAFLD screening mainly by targeting LXRα.

scholarly journals MiR-125b Regulates the Osteogenic Differentiation of Human Mesenchymal Stem Cells by Targeting BMPR1b

2017 ◽  
Vol 41 (2) ◽  
pp. 530-542 ◽  
Author(s):  
Huaqing Wang ◽  
Zhao Xie ◽  
Tianyong Hou ◽  
Zhiqiang Li ◽  
Ke Huang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Target Gene ◽  
Luciferase Reporter ◽  
Marker Genes ◽  
Reporter Assay ◽  
Defect Repair ◽  
Dual Luciferase Reporter Assay

Background/Aims: Osteogenic differentiation of mesenchymal stem cells (MSCs) plays a crucial role in bone regeneration and bone reparation. This complex process is regulated precisely and firmly by specific factors. Recent studies have demonstrated that miR-125b regulates osteogenic differentiation, but little is known about the molecular mechanisms of this regulation. Furthermore, how miR-125b regulates the osteogenic differentiation of MSCs still needs elucidation. Methods: In the present study, human bone marrow-derived mesenchymal stem cells (hBMSCs) were isolated and induced to osteoblasts with miR-125b inhibition or overexpression. qRT-PCR and western blot analysis were used to detect the expression of osteogenic marker genes and proteins. Alkaline phosphatase (ALP) and Alizarin Red (ARS) staining were performed to evaluate the osteoblast phenotype. TargetScan, PicTar and miRanda database were used to predict the target gene of miR-125b. Dual luciferase reporter assay and RNA interference were performed to verify the target gene. Micro-CT imaging and histochemical staining were used to investigate the bone defect repair capacity of miR-125b in vivo. Results: We observed that miR-125b was expressed at a low level during the osteogenic differentiation of hBMSCs. Then, we found that osteogenic marker genes were negatively regulated by miR-125b during the course of osteogenic differentiation, suggesting that miR-125b down regulation plays an important role in the process of osteogenic differentiation. Bioinformatics approaches using miRNA target prediction algorithms indicated that the bone morphogenetic protein type Ib receptor (BMPR1b) is a potential target of miR-125b. The results of the dual luciferase reporter assay indicated that miR-125b binds to the 3’-UTR of the BMPR1b gene. We observed that knockdown of BMPR1b by siRNA inhibited the osteogenic differentiation of hBMSCs. Furthermore, by co-transfecting cells with an miR-125b inhibitor and si-BMPR1b, we found that the osteogenic capacity of the cells transfected with miR-125b inhibitor was blocked upon knockdown of BMPR1b. In vivo, demineralized bone matrix (DBM) was composited with hBMSCs as a scaffold to repair segmental femoral defects. By inhibiting the expression of miR-125b, hBMSCs showed a better capacity to repair bone defects. Conclusions: Taken together, our study demonstrated that miR-125b regulated the osteogenic differentiation of hBMSCs by targeting BMPR1b and that inhibiting miR-125b expression could enhance the capacity of bone defect repair in vivo.

scholarly journals MiR-196b Promotes the Invasion and Migration of Lung Adenocarcinoma Cells by Targeting AQP4

2021 ◽  
Vol 20 ◽  
pp. 153303382098586
Author(s):  
Xuhui Wu ◽  
Gongzhi Wu ◽  
Huaizhong Zhang ◽  
Xuyang Peng ◽  
Bin Huang ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
Cell Invasion ◽  
Target Gene ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Reporter Assay ◽  
Invasion And Migration ◽  
And Migration ◽  
Dual Luciferase Reporter Assay

Objective: We aimed to investigate the mechanism of the regulatory axis of miR-196b/AQP4 underlying the invasion and migration of lung adenocarcinoma (LUAD) cells. Methods: LUAD miRNA and mRNA expression profiles were downloaded from TCGA database and then differential analysis was used to identify the target miRNA. Target gene for the miRNA was obtained via prediction using 3 bioinformatics databases and intersection with the differentially expressed mRNAs searched from TCGA-LUAD. Then, qRT-PCR and western blot were used to validate the expression of miR-196b and AQP4. Dual-luciferase reporter assay was performed to confirm the targeting relationship between miR-196b and AQP4. Transwell assay was used to investigate the migration and invasion of LUAD cells. Results: MiR-196b was screened out by differential and survival analyses, and the downstream target gene AQP4 was identified. In LUAD, miR-196b was highly expressed while AQP4 was poorly expressed. Besides, overexpression of miR-196b promoted cell invasion and migration, while overexpression of AQP4 had negative effects. Moreover, the results of the dual-luciferase reporter assay suggested that AQP4 was a direct target of miR-196b. In addition, we also found that overexpressing AQP4 could suppress the promotive effect of miR-196b on cancer cell invasion and migration. Conclusion: MiR-196b promotes the invasion and migration of LUAD cells by down-regulating AQP4, which helps us find new molecular targeted therapies for LUAD.

scholarly journals MiR-18a-5p Promotes NPC Cell Proliferation, Invasion, Migration, and EMT by Targeting SMAD2

2020 ◽  
Author(s):  
Pengcheng Li ◽  
Junhui Xing ◽  
Jianwu Jiang ◽  
Xinyu Tian ◽  
Xuemeng Liu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Lines ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Invasion And Migration ◽  
Metastasis Model ◽  
And Migration ◽  
Dual Luciferase Reporter Assay

Abstract Background: Nasopharyngeal carcinoma (NPC) is the most common malignant tumor in the head and neck that is characterized by high local malignant invasion and distant metastasis. miR-18a-5p reportedly plays an important role in tumorigenesis and development. However, little is known about the mechanism underlying miR-18a-5p’s role in NPC.Methods：Quantitative real-time PCR was used to detect the expression of miR-18a-5p in NPC tissues and cell lines. MTT assay and plate clone formation assay were used to detect the effect of miR-18a-5p on NPC cell proliferation. Woundhealing assays and Transwell assays were used to detect the effect of miR-18a-5p on NPC cell invasion and migration. The expressions of epithelialmesenchymal transition (EMT)-related proteins N-cadherin, Vimentin, and E-cadherin were detected by Westernblot. Bioinformatics and dual-luciferase reporter assay were used to detect the targeting interaction between miR-18a-5p and SMAD2. Xenotransplantation and metastasis model were used to detect the effect of miR-18a-5p on NPC growth and metastasis in vivo.Results:miR-18a-5p was highly expressed in NPC tissues and cell lines. Overexpression of miR-18a-5p promotedNPC cell proliferation, invasion, migration, and EMT process, whereas inhibition of miR-18a-5p expression led to the oppositeresults. Results of dual-luciferase reporter assay showed that SMAD2 was the target gene of miR-18a-5p, and SMAD2 could reverse the effect of miR-18a-5p on NPC cell line. Xenotransplantation and metastasis model experiments in nude mice showed that miR-18a-5p promotesNPC growth and metastasis in vivo.Conclusions:Targeting SMAD2 downregulated miR-18a-5p expression, thereby promoting NPC cell proliferation, invasion, migration, and EMT.

scholarly journals MicroRNA-132, Delivered by Mesenchymal Stem Cell-Derived Exosomes, Promote Angiogenesis in Myocardial Infarction

2018 ◽  
Vol 2018 ◽  
pp. 1-11 ◽  
Author(s):  
Teng Ma ◽  
Yueqiu Chen ◽  
Yihuan Chen ◽  
Qingyou Meng ◽  
Jiacheng Sun ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Endothelial Cells ◽  
Real Time ◽  
Real Time Pcr ◽  
Target Gene ◽  
Tube Formation ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Ischemic Diseases

Background. To cure ischemic diseases, angiogenesis needs to be improved by various strategies in ischemic area. Considering that microRNA-132 (miR-132) regulates endothelial cell behavior during angiogenesis and the safe and efficacious delivery of microRNAs in vivo is rarely achieved, an ideal vehicle for miR-132 delivery could bring the promise for ischemic diseases. As a natural carrier of biological molecules, exosomes are more and more developed as an ideal vehicle for miRNA transfer. Meanwhile, mesenchymal stem cells could release large amounts of exosomes. Thus, this study aimed to investigate whether MSC-derived exosomes can be used for miR-132 delivery in the treatment of myocardial ischemia. Methods. MSC-derived exosomes were electroporated with miR-132 mimics and inhibitors. After electroporation, miR-132 exosomes were labelled with DiI and added to HUVECs. Internalization of DiI-labelled exosomes was examined by fluorescent microscopy. Expression levels of miR-132 in exosomes and HUVECs were quantified by real-time PCR. The mRNA levels of miR-132 target gene RASA1 in HUVECs were quantified by real-time PCR. Luciferase reporter assay was performed to examine the targeting relationship between miR-132 and RASA1. The effects of miR-132 exosomes on the angiogenic ability of endothelial cells were evaluated by tube formation assay. Matrigel plug assay and myocardial infarction model were used to determine whether miR-132 exosomes can promote angiogenesis in vivo. Results. miR-132 mimics were effectively electroporated and highly detected in MSC-derived exosomes. The expression level of miR-132 was high in HUVECs preincubated with miR-132 mimic-electroporated exosomes and low in HUVECs preincubated with miR-132 inhibitor-electroporated exosomes. The expression level of RASA1, miR-132 target gene, was reversely correlated with miR-132 expression in HUVECs pretreated with exosomes. Luciferase reporter assay further confirmed that RASA1 was a direct target of miR-132. Exosomes loaded with miR-132, as a vehicle for miRNA transfer, significantly increased tube formation of endothelial cells. Moreover, subcutaneous injection of HUVECs pretreated with miR-132 exosomes in nude mice significantly increased their angiogenesis capacity in vivo. In addition, transplantation of miR-132 exosomes in the ischemic hearts of mice markedly enhanced the neovascularization in the peri-infarct zone and preserved heart functions. Conclusions. The findings suggest that the export of miR-132 via MSC-derived exosomes represents a novel strategy to enhance angiogenesis in ischemic diseases.

scholarly journals Long non-coding RNA BCYRN1 exerts an oncogenic role in colorectal cancer through regulating miR-204-3p/KRAS axis

2020 ◽  
Author(s):  
Liu Yang ◽  
Yinan Zhang ◽  
Jun Bao ◽  
Ji-Feng Feng
Keyword(s):  
Colorectal Cancer ◽  
Cell Lines ◽  
Reporter Gene ◽  
Target Gene ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Luciferase Reporter Gene ◽  
Tumor Tissues ◽  
Dual Luciferase Reporter Assay

Abstract Background: It has been well documented that long non-coding RNAs (lncRNAs) regulate numerous characteristics of cancer, including proliferation, migration, metastasis, apoptosis, even metabolism. LncRNA BCYRN1 (BCYRN1) is a newly identified brain cytoplasmic lncRNA with 200-nucleotide, which was discovered highly expressed in tumor tissues of cancer, including hepatocellular carcinoma, gastric cancer and lung cancer. However, the roles of BCYRN1 in colorectal cancer (CRC) remain obscure. This study was designed to reveal the acts of BCYRN1 in the occurrence and progression of CRC.Methods: RT-PCR was used to detect the expression level of BCYRN1 in tumor tissues and CRC cell lines. Knock down BCYRN1 in CRC cells, evaluate cell proliferation changes by CCK-8 test, EdU test, and Ki-67 and PCNA expression; evaluate cell migration and invasion changes by wound healing assay, Transwell assay and invasion-related protein expression . Through flow cytometry analysis to assess whether BCYRN1 regulates apoptosis of CRC cells. The dual luciferase reporter gene detects the competitive binding of BCYRN1 to miR-204-3p. In vivo experiments to evaluate the effect of BCYRN1 on tumor development. TargetScan analysis and dual luciferase reporter gene detect the target gene of miR-204-3p. Rescue experiments verified the effect of BCYRN1 on CRC by regulating the effect of miR-204-3p on KRAS.Results: We found that compared with normal tissues and human intestinal epithelial cells (HIECs), BCYRN1 levels were significantly increased in tumor tissues and cell lines of CRC. We further determined that knockdown of BCYRN1 inhibited proliferation, migration, invasion, and promoted apoptosis of CRC cells. In addition, bioinformatics analysis and dual luciferase reporter assay showed that BCYRN1 served as a competitive endogenous RNA (ceRNA) to regulate the development of CRC through competitively binding to miR-204-3p. Further studies proved that overexpression of miR-204-3p reversed the effects of BCYRN1 on CRC. Next, TargetScan analysis and dual luciferase reporter assay indicated that KRAS was a target gene of miR-204-3p and negatively regulated by miR-204-3p. A series of rescue experiments showed that BCYRN1 affected the occurrence and development of CRC by regulating the effects of miR-204-3p on KRAS. In addition, tumorigenic experiments in CRC model mice confirmed that down-regulated BCYRN1 effectively inhibited tumor growth. Conclusions: Our findings suggested that BCYRN1 plays a carcinogenic role in CRC by regulating the miR-204-3p/KRAS axis.

scholarly journals CircRIMKLB Promotes Myoblasts Proliferation and Inhibits Differentiation via Sponging miR-29c to Release KCNJ12

2020 ◽  
Author(s):  
Jian Wang ◽  
Yifan Wen ◽  
Jiawei Xu ◽  
Binglin Yue ◽  
Jialin Zhong ◽  
...  
Keyword(s):  
Muscle Regeneration ◽  
Muscle Development ◽  
Rna Binding ◽  
Circular Rna ◽  
Normal Function ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Dual Luciferase Reporter Assay

Abstract BackgroundMuscle development is a complex progress that regulated by many factors, and non-coding RNA (ncRNA) is found to act as a vital part of performing normal function of muscle cells. Circular RNA(circRNA), a kind ncRNA with closed RNA, is reported to affect life process. However, there are limited studies about how circRNAs affect muscle development and this study is aimed to find how circ RNA Ribosomal modification protein rimK like family member B (circRIMKLB) affects muscle development.ResultsWe found circular circRIMKLB expressed differentially at different stages of muscle development. The results revealed that circRIMKLB could promote myoblasts proliferation and inhibits differentiation. MicroRNA-29c (miR-29c) was identified as a downstream of circRIMKLB by dual-luciferase reporter assay and RNA binding proteinImmunoprecipitation (RIP) assay. Besides, Channel subfamily J member 12 (KCNJ12) was proved to be a novel target of miR-29c via dual-luciferase reporter assay, quantitative real time polymerase chain reaction(qRT-PCR) and western blot. We also explored the effect of circRIMKLB and KCNJ12 on muscle regeneration after injury in vivo and found that circRIMKLB and KCNJ12 could participate in regulating cell cycle.ConclusionsIn conclusion, we proved circRIMKLB could sponge miR-29c to affect the expression level of KCNJ12 and eventually affect myoblast proliferation and differentiation and participate in cell cycle regulation in muscle regeneration after injury in vivo.

scholarly journals MC-LR induced overproduction of progesterone via inhibiting miR-3473g: in vitro and in vivo evidence

Reproduction ◽  
2020 ◽  
Vol 159 (1) ◽  
pp. 81-89
Author(s):  
Xiaoyan Li ◽  
Jinling Zhu ◽  
Jie Tian ◽  
Dongmei Li ◽  
Xiaodong Han ◽  
...  
Keyword(s):  
Health Risk ◽  
Granulosa Cells ◽  
Human Exposure ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Inner Membrane ◽  
Premature Luteinization ◽  
Dual Luciferase Reporter Assay

Health risk of human exposure to microcystin-leucine arginine (MC-LR) has drawn more and more attention in recent years. In the present study, MC-LR inhibited miR-3473g expression of mouse granulosa cells both in vitro and in vivo. By dual-luciferase reporter assay, we confirmed miR-3473g is able to bind the 3′-UTR of StAR protein (StAR) mRNA and suppress StAR expression. Thus, downregulation of miR-3473g after MC-LR exposure led to StAR overexpression. Excessive StAR probably transported much more cholesterol into the inner membrane of the mitochondria and finally resulted in overproduction of progesterone. Our results revealed that MC-LR exposure was associated with premature luteinization of granulosa cells and may adversely affect women’s fertility.

MiR-877-5p down-regulation of PDK-1 involving in aspirin-induced gastric epithelial cells damage

2020 ◽  
Author(s):  
zongdan jiang ◽  
Ran Dan ◽  
Zou Jianjun ◽  
Wang Zhibing ◽  
Wang Zhi ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Target Gene ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Gastric Epithelial Cells ◽  
Reporter Assay ◽  
Qrt Pcr ◽  
Proliferation And Apoptosis ◽  
Mucosal Erosion ◽  
Dual Luciferase Reporter Assay

Abstract It has been found that the expression of miR-877-5p is increased in serum of patients taking NSAIDs drugs. However, whether miR-877-5p play a role in aspirin-induced gastrointestinal mucosal erosion remains largely unknown. In this study, we investigated the effects of miR-877-5p on gastric epithelial cells (GES-1) proliferation and apoptosis in vitro. MiR-877-5p mimic/inhibitor and their oligonucleotides were transfected into GES-1 cells, then GES-1 cells were treated with different concentrations of aspirin (1.25, 2.5, 5 and 10 mmol/L). The bioinformatics software and dual-luciferase reporter assay were used to predict and verify the target gene of miR-877-5p. qRT-PCR and Western Blotting were employed to assess gene and protein expression, and CCK-8 assay and flow cytometry analysis were used to detect cell proliferation and apoptosis, respectively. qRT-PCR data showed that miR-877-5p level was significantly increased in aspirin incubated GES-1 cells. The proliferation of GES-1 cells were markedly inhibited and apoptosis was significantly induced in the miR-877-5p mimic groups compared to control groups. Using PITA, Targetscan and miRWalk database, the three databases indicated that PDK1 was a target gene of miR-miR-877-5p. Dual luciferase reporter assay confirmed that the existence of a direct interaction between miR-877-5p and PDK1 mRNA. Importantly, miR-877-5p knockdown resulted in a significant upregulation of PDK1 mRNA and its encoded protein in GES-1 cells. miR-877-5p plays a role in aspirin-induced gastrointestinal mucosal erosion, which may via down-regulation of targeting PDK1 gene.

scholarly journals Long non-coding RNA BCYRN1 exerts an oncogenic role in colorectal cancer through regulating miR-204-3p/KRAS axis

2020 ◽  
Author(s):  
Liu Yang ◽  
Yinan Zhang ◽  
Jun Bao ◽  
Ji-Feng Feng
Keyword(s):  
Colorectal Cancer ◽  
Cell Lines ◽  
Reporter Gene ◽  
Target Gene ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Reporter Assay ◽  
Luciferase Reporter Gene ◽  
Dual Luciferase Reporter Assay

Abstract Background: It has been well documented that long non-coding RNAs (lncRNAs) regulate numerous characteristics of cancer, including proliferation, migration, metastasis, apoptosis, and even metabolism. LncRNA BCYRN1 (BCYRN1) is a newly identified brain cytoplasmic lncRNA with 200 nucleotides that was discovered to be highly expressed in tumour tissues, including those of hepatocellular carcinoma, gastric cancer and lung cancer. However, the roles of BCYRN1 in colorectal cancer (CRC) remain obscure. This study was designed to reveal the role of BCYRN1 in the occurrence and progression of CRC.Methods: RT-PCR was used to detect the expression level of BCYRN1 in tumour tissues and CRC cell lines. BCYRN1 was knocked down in CRC cells, and cell proliferation changes were evaluated by cell counting kit-8 (CCK-8), 5-ethynyl-2’-deoxyuridine (EdU), and Ki-67 and proliferating cell nuclear antigen (PCNA) expression assays. Cell migration and invasion changes were evaluated by wound healing, Transwell and invasion-related protein expression assays. Flow cytometry analysis was used to assess whether BCYRN1 regulates the apoptosis of CRC cells. The dual luciferase reporter gene detects the competitive binding of BCYRN1 to miR-204-3p. In vivo experiments were performed to evaluate the effect of BCYRN1 on tumour development. TargetScan analysis and dual luciferase reporter gene assays were applied to detect the target gene of miR-204-3p. Rescue experiments verified that BCYRN1 affects CRC by regulating the effect of miR-204-3p on KRAS.Results: We found that compared with normal tissues and human intestinal epithelial cells (HIECs), CRC tumour tissues and cell lines had significantly increased BCYRN1 levels. We further determined that knockdown of BCYRN1 inhibited the proliferation, migration, and invasion and promoted the apoptosis of CRC cells. In addition, bioinformatics analysis and dual luciferase reporter assay showed that BCYRN1 served as a competitive endogenous RNA (ceRNA) to regulate the development of CRC through competitively binding to miR-204-3p. Further studies proved that overexpression of miR-204-3p reversed the effects of BCYRN1 on CRC. Next, TargetScan analysis and dual luciferase reporter assay indicated that KRAS is a target gene of miR-204-3p and is negatively regulated by miR-204-3p. A series of rescue experiments showed that BCYRN1 affected the occurrence and development of CRC by regulating the effects of miR-204-3p on KRAS. In addition, tumorigenesis experiments in a CRC mouse model confirmed that BCYRN1 downregulation effectively inhibited tumour growth.Conclusions: Our findings suggest that BCYRN1 plays a carcinogenic role in CRC by regulating the miR-204-3p/KRAS axis.

scholarly journals LncRNA KLF3-AS1 in human mesenchymal stem cell-derived exosomes ameliorates pyroptosis of cardiomyocytes and myocardial infarction through miR-138-5p/Sirt1 axis

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Qing Mao ◽  
Xiu-Lin Liang ◽  
Chuan-Long Zhang ◽  
Yi-Heng Pang ◽  
Yong-Xiang Lu
Keyword(s):  
Myocardial Infarction ◽  
Severe Disease ◽  
Human Mesenchymal Stem Cells ◽  
Human Mesenchymal Stem Cell ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
The Relationship ◽  
Dual Luciferase Reporter Assay

Abstract Aim Myocardial infarction (MI) is a severe disease with increased mortality and disability rates, posing heavy economic burden for society. Exosomes were uncovered to mediate intercellular communication after MI. This study aims to explore the effect and mechanism of lncRNA KLF3-AS1 in exosomes secreted by human mesenchymal stem cells (hMSCs) on pyroptosis of cardiomyocytes and MI. Methods Exosomes from hMSCs were isolated and identified. Exosomes from hMSCs with transfection of KLF3-AS1 for overexpression were injected into MI rat model or incubated with hypoxia cardiomyocytes. Effect of KLF3-AS1 on MI area, cell viability, apoptosis, and pyroptosis was determined. The relationship among miR-138-5p, KLF3-AS1, and Sirt1 was verified by dual-luciferase reporter assay. Normal cardiomyocytes were transfected with miR-138-5p inhibitor or sh-Sirt1 to clarify whether alteration of miR-138-5p or sh-Sirt1 can regulate the effect of KLF3-AS1 on cardiomyocytes. Results Exosomes from hMSCs were successfully extracted. Transfection of KLF3-AS1 exosome in rats and incubation with KLF3-AS1 exosome in hypoxia cardiomyocytes both verified that overexpression of KLF3-AS1 in exosomes leads to reduced MI area, decreased cell apoptosis and pyroptosis, and attenuated MI progression. KLF3-AS1 can sponge miR-138-5p to regulate Sirt1 expression. miR-138-5p inhibitor transfection and KLF3-AS1 exosome incubation contribute to attenuated pyroptosis and MI both in vivo and in vitro, while transfection of sh-Sirt1 could reverse the protective effect of exosomal KLF3-AS1 on hypoxia cardiomyocytes. Conclusion LncRNA KLF3-AS1 in exosomes secreted from hMSCs by acting as a ceRNA to sponge miR-138-5p can regulate Sirt1 so as to inhibit cell pyroptosis and attenuate MI progression.

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