scholarly journals miR-16-5p Regulates PTPN4 and Affects Cardiomyocyte Apoptosis and Autophagy Induced by Hypoxia/Reoxygenation

2021 ◽  
Vol 2021 ◽  
pp. 1-12
Author(s):  
Zheng Cao ◽  
Jinglan Liu ◽  
Zhanqing Zhao ◽  
Qiao Wang
Keyword(s):  
Reporter Gene ◽  
Cell Apoptosis ◽  
Cell Damage ◽  
Negative Control Group ◽  
Negative Control ◽  
Expression Level ◽  
Luciferase Reporter ◽  
Control Group ◽  
Luciferase Reporter Gene ◽  
Hypoxia Reoxygenation

Objectives. To explore the effects of miR-16-5p and PTPN4 on the apoptosis and autophagy of AC16 cardiomyocytes after hypoxia/reoxygenation treatment. Methods. AC16 cells were divided into the control group (NC), hypoxia/reoxygenation group (H/R), knockdown miR-16-5p negative control group (NC inhibitor), knockdown miR-16-5p group (miR-16-5p inhibitor), overexpression miR-16-5p negative control group (NC mimics), overexpression miR-16-5p group (miR-16-5p mimics), silent PTPN4 negative control group (sh-NC), silent PTPN4 group (sh-PTPN4), and silent PTPN4 + knockdown miR-16-5p group (sh-PTPN4 + miR-16-5p inhibitor). Real-time fluorescent quantitative PCR (RT-qPCR) and western blotting (WB) were used to measure the expression level of miR-16-3p, miR-16-5p, protein tyrosine phosphatase nonreceptor type 4 (PTPN4), and autophagy-related proteins (beclin-1, LC3 II/I, and P26) in AC16 cells. The apoptosis level of AC16 cells in each group was measured by flow cytometry and TUNEL. The dual-luciferase reporter gene experiment was also used to verify the targeting relationship between miR-16-5p and PTPN4. Results. After H/R treatment, the levels of myocardial injury markers including LDH and CK-MB in AC16 cells were increased significantly ( P < 0.05 ), and the levels of cell apoptosis and autophagy also increased significantly ( P < 0.05 ). The level of miR-16-3p in AC16 cells did not change significantly after H/R treatment, whereas the level of miR-16-5p was increased significantly ( P < 0.05 ). After miR-16-5p was knocked down, the levels of LDH and CK-MB in AC16 cells treated with H/R were significantly reduced ( P < 0.05 ), and the rates of cell apoptosis and autophagy were also significantly reduced ( P < 0.05 ). miR-16-5p negatively regulated the expression level of PTPN4 protein in AC16 cells ( P < 0.05 ), and the dual-luciferase reporter gene experiment confirmed that PTPN4 was the downstream target of miR-16-5p. Silencing of PTPN4 significantly increased the damage of AC16 cells induced by H/R treatment ( P < 0.05 ), but simultaneously inhibiting the expression of PTPN4 and miR-16-5p reversed the protective effect of miR-16-5p knockdown on AC16 cells ( P < 0.05 ). Conclusions. The expression of miR-16-5p is upregulated in AC16 cells after H/R treatment and the knockdown which can protect AC16 cells from H/R-induced cell damage that may be due to its regulation on the expression of PTPN4.

scholarly journals Transcriptional Regulation of CYP3A4/2B6/2C9 Mediated via Nuclear Receptor PXR by Helicid and Its Metabolites

2015 ◽  
Vol 2015 ◽  
pp. 1-9
Author(s):  
Qun Chen ◽  
Hai-tang Xie ◽  
Yan Li ◽  
Guo Wang ◽  
Zhe Xu ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Nuclear Receptor ◽  
Reporter Gene ◽  
Negative Control Group ◽  
Negative Control ◽  
Luciferase Reporter ◽  
Control Group ◽  
Luciferase Reporter Gene ◽  
Expression Plasmid

Objective. This study aims at establishing and validating an in vitro system to screen drug inducers of CYPs mediated via hPXR, as well as studying transcriptional regulation of CYPs mediated via hPXR by helicid and its two metabolites.Methods. Cloning the nuclear receptor hPXR and the promoters of CYP3A4, CYP2B6, CYP2C9, and inserting the trans-element to the upstream of firefly luciferase reporter gene of the pGL4.17 vectors, then cotransfecting the report vectors and hPXR expression plasmid to HepG2 cell line. After 24 hours, the transfected cells were treated with helicid (0.004, 0.04, and 0.4 μmol/L) and its metabolite I and metabolite II (0.0004, 0.004, and 0.04 μmol/L) for 48 h, while rifampin (10 μmol/L) was included as the positive control and 0.1% DMSO as the negative control group. Cells were lysized and luciferase activity was determined using a dual luciferase reporter assay kit.Results. Helicid and its metabolites did not significantly increase promoter activities of CYP3A4, CYP2B6, and CYP2C9 in HepG2 cells transfected with PXR expression plasmid (P>0.05).Conclusion. PXR-expressed CYP3A4, CYP2B6, and CYP2C9 dual luciferase reporter gene platforms were successfully established, and helicid and its metabolites I, II do not significantly induce the transcription of CYP3A4, CYP2B6, and CYP2C9.

scholarly journals MiR-206 regulates the progression of osteoporosis via targeting HDAC4

2021 ◽  
Vol 26 (1) ◽  
Author(s):  
Zhiyuan Lu ◽  
Dawei Wang ◽  
Xuming Wang ◽  
Jilong Zou ◽  
Jiabing Sun ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Apoptosis ◽  
Diagnostic Value ◽  
Expression Level ◽  
Luciferase Reporter ◽  
Control Group ◽  
Luciferase Reporter Gene ◽  
Mineral Density ◽  
Gene Assay

Abstract Background More and more studies have confirmed that miRNAs play an important role in maintaining bone remodeling and bone metabolism. This study investigated the expression level of miR-206 in the serum of osteoporosis (OP) patients and explored the effect and mechanism of miR-206 on the occurrence and development of osteoporosis. Methods 120 postmenopausal women were recruited, including 63 cases with OP and 57 women without OP. The levels of miR-206 were determined by qRT-PCR technology. Spearman correlation coefficient was used to evaluate the correlation of miR-206 with bone mineral density (BMD). An ROC curve was used to evaluate the diagnostic value of miR-206 in osteoporosis. The effects of miR-206 on cell proliferation and cell apoptosis of hFOBs were measured by CCK-8 assay and flow cytometry, respectively. Luciferase reporter gene assay was used to confirm the interaction of miR-206 and the 3′UTR of HDAC4. Results Serum miR-206 had low expression level in osteoporosis patient group compared with control group. The expression level of serum miR-206 had diagnostic value for osteoporosis, and the serum miR-206 levels were positively correlated with BMD. The down-regulated miR-206 could inhibit cell proliferation and promote cell apoptosis. Luciferase analysis indicated that HDAC4 was the target gene of miR-206. Conclusions MiR-206 could be used as a new potential diagnostic biomarker for osteoporosis, and in in vitro cell experiments, miR-206 may regulate osteoblast cell proliferation and apoptosis by targeting HDAC4.

scholarly journals Mir-204 Regulates LPS-Induced A549 Cell Damage by Targeting FOXK2

2021 ◽  
Vol 2021 ◽  
pp. 1-10
Author(s):  
Shufen Li ◽  
Lifen Zhao ◽  
Xujiong Li ◽  
Gaiping Shang ◽  
Lijing Gao ◽  
...  
Keyword(s):  
A549 Cell ◽  
Cell Apoptosis ◽  
Cell Injury ◽  
Cell Damage ◽  
A549 Cells ◽  
Inflammatory Factors ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Downstream Target ◽  
Apoptosis Related Protein

Objective. To assess whether miR-204 and HA affect A549 cell injury induced by lipopolysaccharide. Material and Methods. A549 cells were treated with hirsutanol A, and cell damage was induced by LPS followed by analysis of cell proliferation by CCK-8, cell apoptosis by flow cytometry, apoptosis-related protein expression by western blot, downstream target of miR-20 by dual-luciferase reporter gene, and inflammatory factors by ELISA and PCR. Results. LPS can significantly inhibit the viability of A549 cells, induce cell apoptosis, and promote the release of IL-6, IL-1β, and TNF-α, while HA pretreatment can target FOXK2 by upregulating miR-204 levels, thereby alleviating apoptosis and promoting cell viability and at the same time inhibiting the release of inflammatory factors by inhibiting the activation of NF-κB. Conclusions. miR-204 participates in the protection of HA acute lung injury by targeting FOXK2.

scholarly journals Overexpression of miRNA-21 Promotes the Proliferation and Invasion in Hepatocellular Carcinoma Cells via Suppressing SMAD7

2019 ◽  
Vol 18 ◽  
pp. 153303381987868 ◽  
Author(s):  
Yan Wang ◽  
Ping Zhang ◽  
Mei Yuan ◽  
Xiaojie Li
Keyword(s):  
Hepatocellular Carcinoma ◽  
Family Member ◽  
Reporter Gene ◽  
Aspartate Aminotransferase ◽  
Alanine Aminotransferase ◽  
Hepatoma Cell ◽  
Luciferase Reporter ◽  
Control Group ◽  
Luciferase Reporter Gene ◽  
Proliferation And Invasion

Purpose: This study aimed to explore the molecular mechanism of microRNA-21 and smad family member 7 in hepatocellular carcinoma. Method: A total of 57 participants were divided into control group (healthy participants, n = 10) and hepatocellular carcinoma group (hepatocellular carcinoma patients, n = 37). The expression of microRNA-21 levels were first detected in these two groups. Cell transfection was performed on hepatoma cell lines, followed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and Transwell assay to reveal proliferation and invasion ability. Furthermore, the relation between microRNA-21 and smad family member 7 was revealed by luciferase reporter gene and RNA immunoprecipitation assay. Finally, a transplantation tumor model of breast cancer in mice was constructed. Results: The serum indicators including α-alanine aminotransferase, aspartate aminotransferase, and albumin were differentially expressed between hepatocellular carcinoma group and control group. Compared to the control group, there was a high expression of microRNA-21 in hepatocellular carcinoma group. Low expression of microRNA-21 inhibited the proliferation and invasion of HepG2.2.15 and Huh7-1.3 cells. Luciferase reporter gene and RNA innumoprecipitation assay showed that smad family member 7 was the target gene of microRNA-21. Moreover, mice model analysis showed that microRNA-21 might regulate the growth of the transplanted tumors in mice by targeting smad family member 7. Conclusion: The upregulated microRNA-21 might participate in the proliferation and migration in cells of hepatocellular carcinoma via suppression of smad family member 7. Furthermore, serum indicators such as alanine aminotransferase, aspartate aminotransferase, and albumin might be used as serum diagnostic markers for hepatocellular carcinoma.

scholarly journals The effect of miR-23b-3p on growth hormone in pituitary cells of Yanbian yellow cattle

2020 ◽  
Author(s):  
Jiuxiu Ji ◽  
Angang Lou ◽  
Rui Zhang ◽  
Taihua Jin ◽  
Siyu Xiang ◽  
...  
Keyword(s):  
Protein Expression ◽  
Expression Level ◽  
Luciferase Reporter ◽  
Control Group ◽  
Pituitary Cells ◽  
Mrna Transcription ◽  
Luciferase Reporter Gene ◽  
Protein Expression Level ◽  
Yellow Cattle ◽  
Reporter Gene System

Abstract Background There is a relationship between miR-23b-3p and GH in pituitary of Yanbian yellow cattle. However, the specific mechanism of the effect of miR-23b-3p on GH in pituitary of Yanbian yellow cattle is still unclear.This study aimed to evaluate the effect of miR-23b-3p on growth hormone (GH) in pituitary cells of Yanbian yellow cattle. Methods The primary culture of Yanbian yellow cattle pituitary cells was carried out, and mimics (miR-23b-3p-mi group), mimics reference substance (NC group), inhibitor (miR-23b-3p-in group) and inhibitor reference substance (iNC group) of miR-23b-3p were transfected into the established pituitary primary cells. After 48 h, the cells were collected and the total RNA and protein were extracted.The mRNA transcription and protein expression level of GH and miR-23b-3p target genes were detected by real time fluorescence quantitative PCR (qPCR) and Western blot, respectively. The target relationship of miR-23b-3p was validated by double luciferase reporter gene system. Results Compared with the NC control group, GH mRNA transcription and protein expression level in pituitary cells of Yanbian yellow cattle was significantly decreased by adding miR-23b-3p minics ( P <0.01), while compared with the iNC control group, GH mRNA transcription and protein expression level were significantly increased by adding miR-23b-3p inhibitor( P <0.05). The result of bioinformatics analysis and double luciferase reporter gene system validation proved that miR-23b-3p targeted 3'UTR of pituitary specific transcription factor 1 (POU1F1). Compared with the NC control group, POU1F1 mRNA transcription and protein expression level were significantly inhibited by the addition of miR-23b-3p minics ( P <0.01), while compared with the iNC control group, POU1F1 mRNA transcription and protein expression level were significantly increased by the addition of miR-23b-3p inhibitor ( P <0.01). Conclusions miR-23b-3p could regulate GH in pituitary cells by regulating POU1F1 gene.

scholarly journals THE STUDY OF ORGANOPHOSPHATE (DIAZINON) TOXICITY TOWARD LIVER HISTOPATHOLOGY AND MALONDIALDEHYDE (MDA) SERUM LEVELS ON RATS (Rattus norvegicus)

2019 ◽  
Vol 1 (2) ◽  
pp. 15-23
Author(s):  
Dyah Ayu Oktavianie Pratama ◽  
◽  
Zulfa Aulia ◽  
Aulanni'am Aulanni'am ◽  
Fajar Shodiq Permata ◽  
...  
Keyword(s):  
Free Radicals ◽  
Rattus Norvegicus ◽  
Cell Damage ◽  
Inflammatory Cells ◽  
Serum Levels ◽  
Negative Control Group ◽  
Negative Control ◽  
Control Group ◽  
Exact Test ◽  
Liver Histopathology

Diazinon is an insecticide that has a higher toxicity than other insecticides. Normally, insecticides are detoxified by liver, but this process produced free radicals which causes cell damage. Free radicals also increase lipid peroxidation which directly increase malondialdehyde levels. This research was aimed to determine the influence of diazinon toxicity to the liver histopathology and levels of malondialdehyde (MDA) in the serum of rats (Rattus norvegicus). The rats were divided into 4 groups which consist of negative control group and three experimental groups which were given diazinon 20 mg/kgBW (P1), 40 mg/ kgBW (P2), and 60 mg/ kgBW (P3). The rats were given diazinon for 8 weeks orally. The parameters used in this research was liver histopathology with hematoxilin eosin stain and Malondialdehyde levels with Thiobarbituric test. Liver histopathology was analyzed in descriptive qualitative and the level of MDA was analyzed quantitatively using ANOVA and Tukey's exact test with α = 0.05. The result of this research showed the influence of organophosphate (diazinon) on liver histolopathology was shown by inflammatory cells infiltration in hepatic parenchyma, sinusoidal congestion, and cytoplasmic vacuolation of the hepatocytes. Statistical analysis proved that the diazinon was able to increase the level of MDA serum significantly (p<0,05) up to 71% in experimental group P1 (dose 20 mg/kgBW), which the increase level of MDA serum depend on dose of diazinon. This research concluded that diazinon is one of the organophosphate pesticide that toxic based on liver histopathology and MDA serum levels.

scholarly journals Effect of miRNA-200b on the proliferation and apoptosis of cervical cancer cells by targeting RhoA

Open Medicine ◽  
2020 ◽  
Vol 15 (1) ◽  
pp. 1019-1027
Author(s):  
Lijie He ◽  
Jing Wang ◽  
Dandan Chang ◽  
Dandan Lv ◽  
Haina Li ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Cancer Cells ◽  
Negative Control Group ◽  
Negative Control ◽  
Luciferase Reporter ◽  
Control Group ◽  
Rt Pcr ◽  
Blank Control Group ◽  
Cervical Cancer Cells ◽  
Mrna And Protein Expression

AbstractObjectiveThis article aims to investigate the effect of miRNA-200b on the proliferation and apoptosis of cervical cancer cells by targeting RhoA.MethodsHeLa cells of cervical cancer were divided into five groups: blank control group, negative control group (miRNA-200b mimic NC), miRNA-200b mimic group, RhoA-negative control group, and RhoA overexpression group. Cells were collected 48 h after transfection. The expression levels of miRNA-200b were detected by RT-PCR. Target relationship between miRNA-200b and RhoA was verified by the dual-luciferase reporter assay. RhoA mRNA and protein expression were detected by western blot and RT-PCR methods. Flow cytometry was used to detect the apoptosis of cells in each group, and the CCK8 method was used to detect the proliferation of cells in each group. The mRNA and protein expression of Bax and cyclin D1 were detected by RT-PCR and western blot.ResultsThe results of the dual luciferase reporter assay showed that RhoA was the target gene of microRNA 200b. Compared with the blank control group and the miRNA-200b mimic-NC group, the proportion of apoptotic cells increased significantly in the miRNA-200b mimic group, and the proliferation of cells was inhibited (P < 0.05). After overexpression of RhoA, the percentage of apoptotic cells decreased and the ability of cell proliferation increased significantly (P < 0.05).ConclusionmiRNA-200b can inhibit the proliferation and promote the apoptosis of cervical cancer cells by targeting the RhoA gene.

scholarly journals A Novel Mechanism of bta-miR-210 in Bovine Early Intramuscular Adipogenesis

Genes ◽  
2020 ◽  
Vol 11 (6) ◽  
pp. 601
Author(s):  
Ling Ren ◽  
Qian Li ◽  
Xin Hu ◽  
Qiyuan Yang ◽  
Min Du ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Adipogenic Differentiation ◽  
Growth Factor Receptor ◽  
Negative Control Group ◽  
Negative Control ◽  
Luciferase Reporter ◽  
Control Group ◽  
Intracellular Lipid Accumulation ◽  
Major Factors

Intramuscular fat (IMF) is one of the major factors determining beef quality. IMF formation is influenced by multiple conditions including genetic background, age and nutrition. In our previous investigation, bta-miR-210 was found to be increased during adipogenesis using miRNA-seq. In this study, we validated the upregulation of bta-miR-210 in platelet-derived growth factor receptor α positive (PDGFRα+) progenitor cells during adipogenic differentiation in vitro. To investigate its role in adipogenesis, bta-miR-210 mimics were introduced into progenitor cells, which resulted in enhanced intracellular lipid accumulation. Accordingly, the expression of adipocyte-specific genes significantly increased in the bta-miR-210 mimic group compared to that in the negative control group (p < 0.01). Dual-luciferase reporter assays revealed that WISP2 is a target of bta-miR-210. WISP2 knockdown enhanced adipogenesis. In conclusion, bta-miR-210 positively regulates the adipogenesis of PDGFRα+ cells derived from bovine fetal muscle by targeting WISP2.

MicroRNA-150 regulates steroidogenesis of mouse testicular Leydig cells by targeting STAR

Reproduction ◽  
2017 ◽  
Vol 154 (3) ◽  
pp. 229-236 ◽  
Author(s):  
Xu-Jing Geng ◽  
Dong-Mei Zhao ◽  
Gen-Hong Mao ◽  
Li Tan
Keyword(s):  
Leydig Cells ◽  
Reproductive Development ◽  
Negative Control Group ◽  
Negative Control ◽  
Sex Steroid ◽  
Luciferase Reporter ◽  
Control Group ◽  
Reporter Assay ◽  
Group A

Leydig cells are essential for male reproductive development throughout life. Production of androgens as well as intermediate steroids is tightly regulated. Although microRNAs (miRNAs) are suggested to play important roles in spermatogenesis, little is currently known regarding the regulation of steroidogenesis by miRNAs in Leydig cells. Here, we found that miR-150 was predominantly expressed in Leydig cells within mouse testis. Therefore, we determined steroidogenesis of the Leydig cells in which miR-150 was knocked down or overexpressed using miR-150 antagomir and agomir, respectively. Compared with negative control group, a significant increase of STAR expression was observed in miR-150 antagomir-treated Leydig cells. Conversely, STAR expression was significantly reduced in miR-150 agomir-transfected Leydig cells. Production of sex-steroid precursors and testosterone of Leydig cells was also negatively controlled by miR-150. We further identifiedStaras a target of miR-150 using luciferase reporter assay. Finally, we confirmed that miR-150 was necessary for steroidogenesis and spermatogenesisin vivovia intratesticular injection of miR-150 antagomir or agomir. Taken together, our studies suggest that miR-150 negatively regulates the expression of STAR and steroidogenesis of Leydig cells in mice.

scholarly journals MicroRNA miR-24-3p Reduces Apoptosis and Regulates Keap1-Nrf2 Pathway in Mouse Cardiomyocytes Responding to Ischemia/Reperfusion Injury

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Xu Xiao ◽  
Zhigang Lu ◽  
Victor Lin ◽  
Adam May ◽  
Daniel H. Shaw ◽  
...  
Keyword(s):  
Reperfusion Injury ◽  
Reporter Gene ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Myocardial Cell ◽  
Cardiac Cells ◽  
Expression Level ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene

In recent years, microRNAs (miRNAs) have received increasing attention for their role in ischemia/reperfusion injury (I/RI), and many miRNAs have been demonstrated to play a very important role in cardiac I/RI. The miRNA miR-24-3p is a tumor suppressor that regulates multiple tumors; however, it remains unclear whether the expression level of miR-24-3p is altered in cardiac cells under I/RI. In this study, we used mouse primary cardiomyocytes and the H9C2 cardiomyocyte cell line to perform in vitro stimulated ischemia/reperfusion (SI/R) and then detected miR-24-3p expression level using quantitative real-time PCR (qRT-PCR). We discovered that the expression of miR-24-3p was significantly increased in cardiomyocytes following SI/R, and that the miR-24-3p level was inversely correlated to the ischemia marker HIF-1a. Furthermore, we transfected cardiomyocytes with miR-24-3p mimic or inhibitor to explore the role of miR-24-3p in cardiomyocyte ischemia/reperfusion injury in vitro. We performed flow cytometry to detect the apoptotic rate of H9C2 cardiomyocytes and found that the transfection of miR-24-3p mimic resulted in the decrease of the apoptosis rate of cardiomyocytes after SI/R, whereas the transfection of miR-24-3p inhibitor increased the number of apoptotic cardiomyocytes. These data suggest that the overexpression of miR-24-3p could reduce in vitro myocardial cell apoptosis induced by I/R injury. Finally, we applied the dual luciferase reporter gene system to verify whether miR-24-3p targets the Keap1 gene, and found that the luciferase signal intensity from a vector carrying the Keap1 wild-type reporter gene was significantly reduced after transfection with miR-24-3p mimic. The Keap1 protein level was also reduced following the transfection of miR-24-3p. The results from this study suggest a novel function of miR-24-3p in protecting cardiomyocytes from ischemia/reperfusion injury by the activation of the Nrf2-Keap1 pathway.

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