scholarly journals LncRNA PVT1 Promotes Hypoxia-Induced Cardiomyocyte Injury by Inhibiting miR-214-3p

2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Chuanliang Liu ◽  
Jieqiong Zhang ◽  
Xuejie Lun ◽  
Lei Li
Keyword(s):  
Cell Viability ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
H9c2 Cell ◽  
H9c2 Cells ◽  
Flow Cytometry Analysis ◽  
Chain Reaction ◽  
Cell Vitality ◽  
Gene Assay ◽  
Polymerase Chain

Objective. To explore the effect and related mechanism of LncRNA PVT1 on hypoxia-induced cardiomyocyte injury. Methods. PVT1RNA and miR-214-3p levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell vitality and apoptosis were, respectively, evaluated by Cell Counting Kit-8 (CCK-8) and flow cytometry analysis. Starbase and Dual luciferase reporter (DLR) gene assay was employed to validate the interaction between miR-214-3p and PVT1. Results. PVT1 was statistically upregulated, and miR-214-3p was statistically downregulated in hypoxia-induced H9c2 cells. The survival rate of H9c2 cells induced by hypoxia decreased statistically, while the apoptosis rate increased statistically ( P < 0.05 ). PVT1 knockdown upregulated the hypoxia-induced H9c2 cell viability and inhibited apoptosis. DLR assay verified the targeting relationship between PVT1 and miR-214-3p. In addition, miR-214-3p inhibitors reversed the viability of H9c2 cells with PVT1 knockout and promoted apoptosis. Conclusion. Silencing PVT1 can enhance the hypoxia-induced H9c2 cell viability and inhibit apoptosis, providing a potential target for the treatment of cardiovascular diseases.

scholarly journals MiR-887 Promotes the Progression of Hepatocellular Carcinoma via Targeting VHL

2020 ◽  
Vol 19 ◽  
pp. 153303382094042
Author(s):  
Wei Zou ◽  
Jun Cheng
Keyword(s):  
Hepatocellular Carcinoma ◽  
Polymerase Chain Reaction ◽  
Real Time ◽  
Luciferase Activity ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Chain Reaction ◽  
Gene Assay ◽  
Proliferation And Metastasis ◽  
Polymerase Chain

Background: MiR-887 has been proved to promote the tumorigenesis in diverse cancers, but its function and downstream mechanism in hepatocellular carcinoma remain obscure. Methods: Quantitative real-time polymerase chain reaction was performed to detect the expression levels of miR-887 in hepatocellular carcinoma tissues and cell lines. MiR-887 mimics and miR-887 inhibitor were transfected into Huh7 and MHCC97H to establish miR-887 overexpression or inhibition models. Cell Counting Kit-8 and colony formation experiment were conducted to monitor cell proliferation. Subcutaneous xenotransplanted tumor model and tail vein injection model in mice were also established to further verify the effect of miR-887 on hepatocellular carcinoma in vivo. The targeting relationship between miR-887 and von Hippel-Lindau tumor suppressor (VHL) was determined by quantitative real-time polymerase chain reaction, Western blot, and luciferase reporter gene assay. Results: miR-887 expression in hepatocellular carcinoma tissues was significantly upregulated. Compared with the control cells, the proliferation and metastasis of cancer cells were enhanced by miR-887 mimics and suppressed by miR-887 inhibitor. Compared with control mice, the volume and weight of subcutaneous tumors of mice in the miR-887 mimics group were significantly elevated, and the significant increase was found in the occurrence of lung metastasis. Moreover, bioinformatics tools showed that miR-887 and VHL had 2 binding sites. Luciferase activity assay demonstrated that miR-887 can inhibit the luciferase activity of VHL, and miR-887 mimics could reduce the expressions of VHL at both messenger RNA and protein levels to increase hypoxia-inducible factor α expression. Conclusion: The upregulation of miR-887 could facilitate the proliferation and metastasis of hepatocellular carcinoma cells via targeting VHL.

scholarly journals Knockdown of Circ_SLC39A8 protects against the progression of osteoarthritis by regulating miR-591/IRAK3 axis

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Jizhe Yu ◽  
Yushuang Qin ◽  
Naxin Zhou
Keyword(s):  
Cell Viability ◽  
Molecular Mechanisms ◽  
Interleukin 1 ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Circular Rnas ◽  
Oa Chondrocytes ◽  
Polymerase Chain ◽  
Apoptosis And Inflammation ◽  
The Relationship

Abstract Background The dysregulation of circular RNAs (circRNAs) has been identified in various human diseases, including osteoarthritis (OA). The purpose of this study was to identify the role and mechanism of circ_SLC39A8 in regulating the progression of OA. Methods The expression levels of circ_SLC39A8, miR-591, and its potential target gene, interleukin-1-receptor-associated kinase 3 (IRAK3), were identified by quantitative real-time polymerase chain reaction (qRT-PCR). Cell viability and apoptosis were determined by Cell Counting Kit-8 (CCK-8) assay and flow cytometry, respectively. The relationship between miR-591 and circ_SLC39A8 or IRAK3 was predicted by bioinformatics tools and verified by dual-luciferase reporter. Results Circ_SLC39A8 and IRAK3 were upregulated and miR-591 was downregulated in OA cartilage tissues. Knockdown of circ_SLC39A8 inhibited apoptosis and inflammation in OA chondrocytes, while these effects were reversed by downregulating miR-591. Promotion cell viability effects of miR-591 were partially reversed by IRAK3 overexpression. Conclusion Our findings indicated that knockdown of circ_SLC39A8 delayed the progression of OA via modulating the miR-591-IRAK3 axis, providing new insight into the molecular mechanisms of OA pathogenesis.

LncRNA XIST Relieves Hypoxia-Induced Damage in H9C2 Cells by Downregulating miR-429

2021 ◽  
Vol 7 (5) ◽  
pp. 1245-1253
Author(s):  
Na Yu ◽  
Xue Han ◽  
Xueqin Wang ◽  
Wanling Yu ◽  
Liqiu Yan
Keyword(s):  
Caspase 3 ◽  
Luciferase Reporter ◽  
Control Group ◽  
H9c2 Cell ◽  
H9c2 Cells ◽  
Atp Content ◽  
Xist Expression ◽  
Gene Assay ◽  
Lncrna Xist ◽  
Group A

This paper aimed to investigate LncRNA XIST relieving hypoxia-induced damage in H9C2 cells by downregulating miR-429. Rat H9C2 cell lines were selected and divided into a normal control group, a hypoxia group, a XIST expression group, a XIST blank expression group, a miR-429 interference group and a blank interference group. qPCR was adopted for detecting LncRNA XIST and miR-429 expression. Western blot (WB) was adopted for detecting the expression of AMPK, PDH, FAT, MCPT-1, Caspase-3, Bax and Bcl-2, ATP content, and levels of SOD, MDA and LDH. Dual luciferase reporter gene assay (DLRGA) and RNA pull-down were adopted for verifying the correlation of LncRNA XIST with miR-429. Hypoxia-induced H9C2 cells had low LncRNA XIST expression and high miR-429 expression. LncRNA XIST upregulation or miR-429 downregulation could inhibit AMPK, PDH, Caspase-3 and Bax, upregulate FAT, MCPT-1 and Bcl-2, and increase ATP content and SOD activity, as well as reduce MDA content and LDH activity. miR-429 was the target gene of LncRNA XIST. LncRNA XIST can relieve hypoxia-induced damage in H9C2 cells via binding to and downregulating miR-429

scholarly journals MiR-218 Promotes Adriamycin-Induced H9C2 Apoptosis by Inhibiting Stress-Associated Endoplasmic Reticulum Protein 1

2022 ◽  
Vol 2022 ◽  
pp. 1-10
Author(s):  
Qinghua Chen ◽  
Gang Chen ◽  
Shuofang Zhao
Keyword(s):  
Signaling Pathway ◽  
P38 Mapk ◽  
Cell Apoptosis ◽  
Cell Activity ◽  
Mapk Signaling ◽  
Luciferase Reporter ◽  
Tunel Staining ◽  
H9c2 Cell ◽  
H9c2 Cells ◽  
Gene Assay

Objective. Adriamycin is a clinically important chemotherapeutic drug, but its use is restricted due to its myocardial toxicity. Therefore, it is especially important to explore the toxicity mechanism of Adriamycin (ADR) to cardiomyocytes. Methods. The myocardial toxicity model of ADR was constructed in vitro, and the effect of miR-218 inhibitor and sh-Serp1 on the activity of H9C2 cells induced by ADR was detected by MTT method. Also, flow cytometry, real-time polymerase chain reaction (RT-PCR), and TUNEL staining were used to detect the cell apoptosis. The activity of LDH was detected by colorimetry, and the interaction of miR-218 with Serp1 was detected by double-luciferase reporter gene assay. Western blotting technique was used to detect the expression level of caspase3 and p38 MAPK signal pathway. Results. miR-218 inhibitor can obviously inhibit ADR-induced decrease in cell activity of H9C2 cells, inhibit cell apoptosis, and inhibit p38 MAPK signaling pathway activation. Conversely, sh-Serp1 aggravated the decrease in H9C2 cell activity and promoted cell apoptosis. Conclusion. Upregulation of miR-218 expression will promote ADR-induced apoptosis of H9C2 cells. At the same time, we confirmed that the mechanism by which miR-218 promotes myocardial apoptosis was through the Serp1/p38 MAPK/caspase-3 signaling pathway.

miR-27b-3p Down-Regulation Prevents Hypoxia-Induced Cardiomyocyte Apoptosis Through Regulating Yes-Associated Protein 1 (YAP1) Expression

2021 ◽  
Vol 11 (5) ◽  
pp. 948-956
Author(s):  
Lilin Wang ◽  
Bo Feng ◽  
Shu Zhu
Keyword(s):  
Cell Viability ◽  
Target Genes ◽  
Luciferase Reporter ◽  
Pcr Analysis ◽  
H9c2 Cells ◽  
Luciferase Reporter Gene ◽  
Western Blot Assay ◽  
Heart Protection ◽  
Gene Assay ◽  
Related Proteins

Background: Congenital heart disease (CHD) is one of the most common birth defects. MicroR-NAs (miRNAs) are a group of endogenous, non-coding small RNAs and mediate the target genes expression. An increasing evidence showed that in recent years, miRNAs have given rise to more and more attention in heart protection and development. In our research, the main purpose was to determine the effect of miR-27b-3p in CHD and analyze related mechanisms. Methods: We performed qRT-PCR analysis to examine miR-27b-3p expression in myocardial tissue from 30 patients with CHD and hypoxia-induced H9C2 cells. Then, we performed biological software TargetScan to predict the relationship of miR-27b-3p and YAP1, and dual luciferase reporter gene assay was used to verify the results. H9C2 cells were transfected with inhibitor control, miR-27b-3p inhibitor, miR-27b-3p inhibitor + control-siRNA or miR-27b-3p inhibitor + YAP1-siRNA for 6 hours and then induced by hypoxia for 72 hours. Subsequently, we performed MTT and FCM analysis to detect cell viability and apoptosis. Finally, we used western blot assay to measure the expression of apoptosis-related proteins. Results: Our study indicated that miR-27b-3p expression in myocardial samples of cyanotic CHD patients was significantly higher than that of the acyanotic CHD patients. miR-27b-3p expression was gradually up-regulated with the increase of hypoxia induction time in H9C2 cells. Besides, we confirmed that YAP1 was a target gene of miR-27b-3p. Moreover, our results showed that miR-27b-3p inhibitor improved cell viability, decreased apoptosis, and affected apoptosis-related proteins expression in hypoxia induced H9C2 cells. These changes were reversed by YAP1-siRNA. All data demonstrated that miR-27b-3p/YAP1 might be new potential bio-marker and therapeutic target for CHD treatment.

scholarly journals Long noncoding RNA FTX ameliorates hydrogen peroxide-induced cardiomyocyte injury by regulating the miR-150/KLF13 axis

2020 ◽  
Vol 15 (1) ◽  
pp. 1000-1012
Author(s):  
Yamin Zhang ◽  
Xiaoying Fan ◽  
Hua Yang
Keyword(s):  
Hydrogen Peroxide ◽  
Cell Viability ◽  
Cell Apoptosis ◽  
Noncoding Rna ◽  
Ischemia Reperfusion ◽  
Myocardial Reperfusion ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
H9c2 Cells

AbstractBackgroundMyocardial reperfusion is an effective therapy for acute myocardial infarction (AMI). However, ischemia/reperfusion (I/R) injury following myocardial reperfusion is a significant limitation for AMI treatment. Five prime to Xist (FTX) was recognized as a biomarker of multiple diseases, including heart disease. However, the molecular mechanism of FTX in I/R injury is unclear.MethodsCell viability was evaluated by using cell counting kit-8 (CCK-8) assay. Apoptosis was analyzed by using a caspase-3 activity detection kit and flow cytometry. The expression of FTX, microRNA (miR)-150, and Kruppel-like factor 13 (KLF13) was measured by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The interaction of miR-150 and FTX or KLF13 was confirmed by a dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. Protein expression of KLF13 was examined by Western blot. The role of FTX was detected in I/R-injured heart tissues in vivo.ResultsHydrogen peroxide (H2O2) induced cardiomyocyte injury by decreasing cell viability and expediting cell apoptosis. However, FTX alleviated cardiomyocyte injury by promoting cell proliferation and restricting cell apoptosis of H9C2 cells that were treated with H2O2. In addition, we discovered that FTX directly interacted with miR-150, while KLF13 was a target of miR-150. Rescue experiments showed that miR-150 neutralized the FTX-mediated promotion of cell progression and restriction of cell apoptosis in H9C2 cells treated with H2O2. KLF13 knockdown restored the effect of miR-150 on increased proliferation and decrease in apoptosis in H2O2-treated cardiomyocytes. Furthermore, FTX enhanced the expression of KLF13 protein through interaction with miR-150. Upregulation of FTX repressed apoptosis in I/R-injured heart tissues in vivo.ConclusionFTX relieves H2O2-induced cardiomyocyte injury by increasing KLF13 expression via depletion of miR-150, thus providing a novel therapeutic target for the alleviation of I/R injury.

scholarly journals Breviscapine Participates in the Progression of Prostate Cancer by Inhibiting ZFP91 Expression through Upregulation of MicroRNA-129-5p

2021 ◽  
Vol 2021 ◽  
pp. 1-8
Author(s):  
Jie Yang ◽  
Wanjun Jin ◽  
Xiaokang Zhang ◽  
Pengcheng Chang ◽  
Duo Zheng
Keyword(s):  
Prostate Cancer ◽  
Inhibitory Effect ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Migration Ability ◽  
Luciferase Reporter Gene Assay ◽  
Gene Assay ◽  
Polymerase Chain ◽  
Diphenyl Tetrazolium Bromide

Objective. To investigate the effect of breviscapine (BVP) on the development of prostate cancer and its molecular mechanism. Materials and Methods. After treatment with breviscapine and microRNA-129-5p, MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) and cell counting kit-8 (CCK-8) tests were performed to examine the proliferation rate of cells, while Transwell was used to analyze cell migration ability; at the same time, quantitative real-time polymerase chain reaction (qRT-PCR) was applied to detect the expression of microRNA-129-5p and ZFP91 in prostate cancer cells. In addition, the binding of microRNA-129-5p and ZFP91 was confirmed by dual-luciferase reporting assay; meanwhile, cell reverse experiment verified that breviscapine can regulate ZFP91 via upregulating microRNA-129-5p. Results. The results of MTT, CCK-8, and Transwell experiments demonstrated that breviscapine inhibited the proliferation as well as the migration capacities of PC cells; meanwhile, it upregulated the level of microRNA-129-5p in PC cells while downregulated that of ZFP91. Furthermore, dual-luciferase reporter gene assay verified that ZFP91 was a potential target of microRNA-129-5p. Finally, cell reverse experiment confirmed that breviscapine downregulated ZFP91 expression by upregulating microRNA-129-5p, while downregulation of microRNA-129-5p partially reversed the inhibitory effect of breviscapine on cell proliferation ability. Conclusions. Breviscapine may inhibit the expression of ZFP91 through upregulating microRNA-129-5p and thus participating in the progression of PC.

scholarly journals MicroRNA-550a-3-5p controls the brain metastasis of lung cancer by directly targeting YAP1

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Liang Wei ◽  
Guangxue Wang ◽  
Cheng Yang ◽  
Yanfei Zhang ◽  
Yiming Chen ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Brain Metastasis ◽  
Cell Viability ◽  
Western Blotting ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Lung Cancer Patients ◽  
Gene Assay ◽  
And Migration

Abstract Background This study aimed to explore the potential regulatory mechanisms of brain metastasis and to identify novel underlying targets of lung cancer with brain metastasis. Methods Exosomes were isolated from the plasma of lung cancer patients with or without brain metastasis and low or high metastatic lung cancer cells, and small RNA from plasma-derived exosomes were sequenced. Differentially expressed miRNAs (DE-miRNAs) were identified. Human brain microvascular endothelial cells (HBMECs) were transfected with miR-550a-3-5p mimics or inhibitors and exosomes. Cell viability, migration, and apoptosis/cycle were determined using Cell Counting Kit-8 (CCK-8), Transwell, and flow cytometry, respectively. Western blotting was used to measure the expression of the associated proteins. Finally, a dual-luciferase reporter gene assay was performed to confirm the miR-550a-3-5p target. Results Transmission electron microscopy, NanoSight, and western blotting showed that exosomes were successfully isolated and cell-derived exosomes could be taken up by HBMECs. Sequencing identified 22 DE-miRNAs which were enriched in the MAPK, chemokine, PPAR, and Wnt signaling pathways. MiR-550a-3-5p was significantly enriched in brain metastatic exosomes. Cellular experiments showed that miR-550a-3-5p and exosome enrichment significantly inhibited cell viability and migration, promoted apoptosis, and regulated the cell cycle of HBMECs compared with the controls (P  <  0.05). Compared with the controls, high levels of both miR-550a-3-5p and exosomes markedly upregulated cleaved-PARP expression, but downregulated the expression of pRB, CDK6, YAP1, CTGF, and CYR61 (P  <  0.05). Finally, YAP1 was confirmed to bind directly to miR-550a-3-5p. Conclusion Our results indicate that miR-550a-3-5p and YAP1 may be novel potential targets for controlling brain metastasis.

Inhibition of microRNA-181a-5p Protects H9c2 Cells from Hypoxia-Induced Injury Through Up-Regulating Sirtuin 1 Expression

2020 ◽  
Vol 10 (5) ◽  
pp. 682-689
Author(s):  
Qin He ◽  
Rui Dai ◽  
Xiaoju Xiong ◽  
Jinhua Liu ◽  
Zhonghan He ◽  
...  
Keyword(s):  
Cell Injury ◽  
Sirtuin 1 ◽  
Luciferase Reporter ◽  
H9c2 Cell ◽  
Inflammatory Factor ◽  
H9c2 Cells ◽  
Creatine Kinase Mb ◽  
Gene Assay ◽  
Hypoxia Treatment

Myocardial infarction (MI), a life-threatening cardiac event, results in extreme damage to the heart muscle. In this study, we were committed to exploring the role and related mechanisms of microRNA-181a-5p (miR-181a-5p) in MIin vitro. Firstly, we established the MIin vitro cell model by subjecting H9c2 cells to hypoxia. We found that miR-181a-5p was significantly increased in hypoxia-induced H9c2 cells. Then, TargetScan and dual luciferase reporter gene assay confirmed the binding sites between Sirtuin 1 (SIRT1) and miR-181a-5p. SIRT1 was significantly reduced in hypoxia-induced H9c2 cells. Next, we explored the effect of miR-181a-5p inhibitor on hypoxiainduced H9c2 cell injury. The findings indicated that miR-181a-5p inhibitor significantly reduced creatine kinase-MB (CK-MB) and cardiac troponin I (cTnI) production enhanced by hypoxia treatment. Moreover, miR-181a-5p inhibitor increased mitochondrial viability in hypoxia-induced H9c2 cells. MTT assay showed that miR-181a-5p inhibitor enhanced hypoxia-induced H9c2 cell viability, and flow cytometry assay indicated that miR-181a-5p inhibitor reduced H9c2 cell apoptosis. ELISA assay indicated that compared with hypoxia treatment group, miR-181a-5p inhibitor decreased the secretion of inflammatory factor such as IL-6, TNF-α and IL-1β . Finally, Western blot assay showed that miR-181a-5p inhibitor decreased the expression of p-p65, indicating the inhibition on NF- κB signaling pathway activation. However, all these effects of miR-181a-5p inhibitor on hypoxia-induced H9c2 cells were reversed by SIRT1-siRNA. Taken together, miR-181a-5p inhibitor protected against hypoxia-induced H9c2 cell injury by targeting SIRT1.

scholarly journals miR-146a-5p Mediates Intermittent Hypoxia-Induced Injury in H9c2 Cells by Targeting XIAP

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Guofu Lin ◽  
Jiefeng Huang ◽  
Qingshi Chen ◽  
Lida Chen ◽  
Dehuai Feng ◽  
...  
Keyword(s):  
Cell Apoptosis ◽  
Intermittent Hypoxia ◽  
Target Genes ◽  
Cardiac Injury ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
H9c2 Cell ◽  
H9c2 Cells ◽  
Obstructive Sleep ◽  
Apoptosis Protein

MicroRNAs (miRNAs) have emerged as key modulators in the pathophysiologic processes of cardiovascular diseases. However, its function in cardiac injury induced by obstructive sleep apnea (OSA) remains unknown. The aim of the current study was to identify the effect and potential molecular mechanism of miR-146a-5p in intermittent hypoxia(IH)- induced myocardial damage. We exposed H9c2 cells to IH condition; the expression levels of miR-146a-5p were detected by RT-qPCR. Cell viability, cell apoptosis, and the expressions of apoptosis-associated proteins were assessed via Cell Counting Kit-8 (CCK-8), flow cytometry, and western blotting, respectively. Target genes of miR-146a-5p were confirmed by dual-luciferase reporter assay. IH remarkably lowered viability but enhanced cell apoptosis. Concomitantly, the miR-146a-5p expression level was increased in H9c2 cells after IH. Subsequent experiments showed that IH-induced injury was alleviated through miR-146a-5p silence. X-linked inhibitor of apoptosis protein (XIAP) was predicted by bioinformatics analysis and further confirmed as a direct target gene of miR-146a-5p. Surprisingly, the effect of miR-146a-5p inhibition under IH may be reversed by downregulating XIAP expression. In conclusion, our results demonstrated that miR-146a-5p could attenuate viability and promote the apoptosis of H9c2 by targeting XIAP, thus aggravating the H9c2 cell injury induced by IH, which could enhance our understanding of the mechanisms for OSA-associated cardiac injury.

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