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1.
Electron. j. biotechnol ; 45: 19-29, May 15, 2020. tab, ilus, graf
Article in English | LILACS | ID: biblio-1177401

ABSTRACT

BACKGROUND: Long non-coding RNAs (lncRNAs), as post-transcriptional regulators, were thought to function in the inductive property of dermal papilla cells (DPCs) in cashmere goat. Previously, lncRNA-599554 was identified in secondary hair follicle (SHF) of cashmere goat, but its functional significance is unknown. RESULTS: In the present investigation, we verified that lncRNA-599554 had significantly higher expression at the anagen dermal papilla of cashmere goat SHF than that at telogen. Based on overexpression and knockdown techniques, we found that lncRNA-599554 contributes the inductive property of DPCs of cashmere goat, which was assessed by detecting the changes in the expression of several typical indictor genes in DPCs including ET-1, SCF, Versican, ALP, Lef1 and Ptc-1. Based on RNA pull-down assay, we verified that lncRNA-599554 directly interacted with chi-miR-15a-5p. Also, we showed that lncRNA-599554 positively regulated the Wnt3a expression in DPCs but which did not appear to involve its modulating of promoter methylation. Based on the use of Dual-luciferase reporter assays, our data indicated that lncRNA-599554 regulated the Wnt3a expression through chi-miR-15a-5p-mediated post-transcriptional level. CONCLUSIONS: We showed that lncRNA-599554 contributes the inductive property of DPCs in cashmere goat which might be achieved through sponging chi-miR-15b-5p to promote the Wnt3a expression. The results from the present investigation provided a novel insight into the functional mechanism of lncRNA-599554 in the SHF regeneration of cashmere goat along with the formation and growth of cashmere fiber.


Subject(s)
Animals , Hair Follicle/cytology , Hair Follicle/metabolism , Dermis/cytology , Wnt3A Protein/metabolism , RNA, Long Noncoding/metabolism , Biological Assay/methods , Goats , RNA, Long Noncoding/genetics , Luciferases , Methylation
2.
Electron. j. biotechnol ; 43: 55-61, Jan. 2020. tab, ilus, graf
Article in English | LILACS | ID: biblio-1087522

ABSTRACT

Background: Matrix metalloproteinase 12 (MMP12), a member of MMPs, can take lots of roles including extracellular matrix component degradation, viral infection, inflammation, tissue remodeling and tumorigenesis. To explore the transcriptional regulation of MMP12 gene, a sensitive luciferase reporter HEK293 cell line for endogenous MMP12 promoter was generated by CRISPR/Cas9 technology. Results: The HEK293-MMP12-T2A-luciferase-KI cell line was successfully established by CRISPR/Cas9 technology. The sequencing results indicated that one allele of the genome was proven to have a site-directed insertion of luciferase gene and another allele of the genome was confirmed to have additional 48 bp insertion in this cell line. The cell line was further demonstrated to be a sensitive reporter of the endogenous MMP12 promoter by applying transcription factors STAT3, AP-1 and SP-1 to the cell line. The reporter cell line was then screened with bioactive small molecule library, and a small molecule Tanshinone I was found to significantly inhibit the transcriptional activity of MMP12 gene in HEK293-MMP12-T2A-luciferase-KI cell line by luciferase activity assay, which was further confirmed to inhibit the expression of MMP12 mRNA in wild-type HEK293 cells. Conclusions: This novel luciferase knock-in reporter system will be helpful for investigating the transcriptional regulation of MMP12 gene and screening the drugs targeting MMP12 gene.


Subject(s)
Humans , Matrix Metalloproteinase 12/genetics , CRISPR-Cas Systems , Luciferases/genetics , Transcription, Genetic , Cell Communication , Cell Line , Promoter Regions, Genetic/genetics , Cell Culture Techniques , Extracellular Matrix , Gene Knock-In Techniques , Clustered Regularly Interspaced Short Palindromic Repeats
4.
Yonsei Medical Journal ; : 210-217, 2020.
Article in English | WPRIM | ID: wpr-811475

ABSTRACT

PURPOSE: The goal of this study was to explore the effects of hsa-let-7g on cell proliferation and apoptosis, and elucidate its role in lung cancer development.MATERIALS AND METHODS: The expression levels of has-let-7g and HOXB1 in tissues and cells were measured by qRT-PCR. An inhibitor of hsa-let-7g or one targeting a control messenger RNA were transfected into A549 and H1944 lung cancer cells, and the effects of hsa-let-7g dysregulation on cell viability and apoptosis were analyzed using CCK-8 and apoptosis detection assays. HOXB1 was confirmed as the target gene of hsa-let-7g, based on luciferase reporter assay results. The relationship between hsa-let-7g and HOXB1 was confirmed by co-transfection of inhibitors of hsa-let-7g and HOXB1 followed by Western blot, CCK-8, and apoptosis detection assays.RESULTS: We observed high expression of hsa-let-7g in lung cancer tissues compared to the corresponding normal tissues, and generally higher expression of hsa-let-7g in patients with advanced tumor classification. The results of CCK-8 and apoptosis detection experiments showed that the inhibition of hsa-let-7g significantly inhibited proliferation of A549 and H1944 cells, but also promoted apoptosis. HOXB1 is a specific target of hsa-let-7g, and downregulation of HOXB1 in lung cancer cells reversed the suppressive effects caused by knocking down hsa-let-7g.CONCLUSION: These data collectively suggest that the expression of hsa-let-7g inhibits lung cancer cells apoptosis and promotes proliferation by down-regulating HOXB1. The results from this study demonstrate the potential of hsa-let-7g/HOXB1 axis as a therapeutic target for the treatment of lung cancer.


Subject(s)
Apoptosis , Blotting, Western , Cell Proliferation , Cell Survival , Classification , Down-Regulation , Humans , Luciferases , Lung Neoplasms , Lung , MicroRNAs , RNA, Messenger , Sincalide
5.
Article in English | WPRIM | ID: wpr-811138

ABSTRACT

BACKGROUND: Recent studies have shown that microRNAs (miRNAs) are involved in the process of cardiomyocyte apoptosis. We have previously reported that granulocyte-colony stimulating factor (G-CSF) ameliorated diastolic dysfunction and attenuated cardiomyocyte apoptosis in a rat model of diabetic cardiomyopathy. In this study, we hypothesized a regulatory role of cardiac miRNAs in the mechanism of the anti-apoptotic effect of G-CSF in a diabetic cardiomyopathy rat model.METHODS: Rats were given a high-fat diet and low-dose streptozotocin injection and then randomly allocated to receive treatment with either G-CSF or saline. H9c2 rat cardiomyocytes were cultured under a high glucose (HG) condition to induce diabetic cardiomyopathy in vitro. We examined the extent of apoptosis, miRNA expression, and miRNA target genes in the myocardium and H9c2 cells.RESULTS: G-CSF treatment significantly decreased apoptosis and reduced miR-34a expression in diabetic myocardium and H9c2 cells under the HG condition. G-CSF treatment also significantly increased B-cell lymphoma 2 (Bcl-2) protein expression as a target for miR-34a. In addition, transfection with an miR-34a mimic significantly increased apoptosis and decreased Bcl-2 luciferase activity in H9c2 cells.CONCLUSION: Our results indicate that G-CSF might have an anti-apoptotic effect through down-regulation of miR-34a in a diabetic cardiomyopathy rat model.


Subject(s)
Animals , Apoptosis , Diabetic Cardiomyopathies , Diet, High-Fat , Down-Regulation , Glucose , Granulocyte Colony-Stimulating Factor , In Vitro Techniques , Luciferases , Lymphoma, B-Cell , MicroRNAs , Models, Animal , Myocardium , Myocytes, Cardiac , Rats , Streptozocin , Transfection
6.
Biol. Res ; 53: 14, 2020. graf
Article in English | LILACS | ID: biblio-1100920

ABSTRACT

BACKGROUND: Previous studies have shown that long noncoding RNA (IncRNA) LINC00483 was aberrantly expressed in human cancers, including gastric cancer. However, the regulatory mechanism of this IncRNA in gastric cancer remains largely unknown. The present study aimed to investigate the effect of LINC00483 on gastric cancer development and explore the potential regulatory network of LINC00483/microRNA (miR)-490-3p/mitogen-activated protein kinase 1 (MAPK1). METHODS: Thirty patients with gastric cancer were recruited for tissues collection. The expression levels of LINC00483, miR-490-3p and MAPK1 were detected by quantitative real-time polymerase chain reaction or western blot. Cell viability, apoptosis, migration and invasion were determined by MTT, flow cytometry, transwell assays and western blot, respectively. The target association between miR-490-3p and LINC00483 or MAPK1 was confirmed by luciferase reporter assay. Xenograft model was established to assess the function of LINC00483 in vivo. RESULTS: LINC00483 and MAPK1 levels were increased in gastric cancer tissues and cells. Knockdown of LINC00483 or MAPK1 inhibited cells viability, migration and invasion but promoted apoptosis in gastric cancer cells. Moreover, MAPK1 overexpression attenuated the effect of LINC00483 knockdown on gastric cancer development. LINC00483 could increase MAPK1 expression by competitively sponging miR-490-3p. miR-490-3p overexpression suppressed gastric cancer development, which was abated by introduction of LINC00483. Besides, inhibition of LINC00483 decreased xenograft tumor growth by regulating miR-490-3p/MAPK1 axis. CONCLUSION: Knockdown of LINC00483 inhibited gastric cancer development in vitro and in vivo by increasing miR- 490-3p and decreasing MAPK1, elucidating a novel mechanism for understanding the development of gastric cancer.


Subject(s)
Humans , Animals , Male , Stomach Neoplasms/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , MicroRNAs/metabolism , RNA, Long Noncoding/metabolism , Stomach Neoplasms/genetics , Gene Expression Regulation, Neoplastic , Cell Movement , Cell Survival , Apoptosis , Xenograft Model Antitumor Assays , MicroRNAs/genetics , Cell Line, Tumor/metabolism , Epithelial Cells/metabolism , RNA, Long Noncoding/genetics , Carcinogenesis/metabolism , Luciferases/metabolism , Mice, Inbred BALB C
7.
Article in Chinese | WPRIM | ID: wpr-771905

ABSTRACT

OBJECTIVE@#To clone the promoter sequence of acute monocytic leukemia new antigen gene.MLAA-34 and identify its promoter core region.@*METHODS@#The full-length fragment of MLAA-34 gene promoter region was amplified by PCR, then was ligated into pGL3-Basic vector, and the recombinant plasmid was cloned. Constructed a series of MLAA-34 gene promoter 5' flanking region truncated plasmid. These recombinant plasmids were transfected into U937 and HEK293 cells, and the dual luciferase reporter gene was used to detect the promoter activity of each fragment to determine the minimum active region. Transcription factor binding sites were analyzed by bioinformatics methods.@*RESULTS@#The recombinant plasmid containing MLAA-34 promoter sequence and its truncated plasmid were successfully constructed, and the promoter activity was significantly increased as compared with the empty vector (P<0.001). The minimal active region of MLAA-34 located between 402 bp and 200 bp. It contained multiple transcription factor binding sites such as E2F1, MZF-1, SP1, USF2 and STAT3.@*CONCLUSION@#The promoter of luciferase reporter gene has been successfully constructed with different deletion fragments of MLAA-34, and its core promoter region may contain multiple transcription factor sequence.


Subject(s)
Adult , Antigens, Neoplasm , Genetics , Apoptosis Regulatory Proteins , Genetics , Cloning, Molecular , Genes, Reporter , HEK293 Cells , Humans , Leukemia, Monocytic, Acute , Genetics , Luciferases , Promoter Regions, Genetic
8.
Chinese Journal of Biotechnology ; (12): 1469-1477, 2019.
Article in Chinese | WPRIM | ID: wpr-771782

ABSTRACT

The aim of this study was to screen the active regions and transcription factor binding sites in the promoter of the CBD103 gene related to Arctic fox coat color, and to provide a basis for revealing the molecular genetic mechanism of CBD103 gene regulating the coat color formation. The 5'-flanking region fragment 2 123 bp of Arctic fox CBD103 gene was cloned, and 4 truncated promoter reporter vectors of different lengths were constructed. The promoter activity was detected by the dual-luciferase reporter assay system. Point mutations were performed on the 3 predicted specificity protein 1 (Sp1) transcription factor binding sites in the highest promoter active region, and 3 mutant vectors were constructed. The activity was then detected by the dual-luciferase reporter assay system. The results showed that the region 1 656 (-1 604/+51) had the highest activity in the 4 truncated promoters of different lengths, and the promoter activity of the three mutant vectors constructed in this region were significantly lower than that of the wild type (fragment 1 656). The region of -1 604 /+51 was the core promoter region of CBD103 gene in Arctic fox and -1 552/-1 564, -1 439/-1 454 and -329/-339 regions were positive regulatory regions. This study successfully obtained the core promoter region and positive regulation regions of the Arctic fox CBD103 gene, which laid a foundation for further study on the molecular genetic mechanism of this gene regulating Arctic fox coat color.


Subject(s)
Animals , Binding Sites , Foxes , Luciferases , Promoter Regions, Genetic , Sp1 Transcription Factor , beta-Defensins
9.
Natural Product Sciences ; : 341-347, 2019.
Article in English | WPRIM | ID: wpr-786425

ABSTRACT

Luffa cylindrica (LC) is a very fast-growing climber and its fruit have been considered as agricultural wastes. We conducted to check the comparative qualities of ethanol solvent extraction (LCE) and supercritical carbon dioxide extraction (LCS) of L. cylindrica fruit and seed. LCS had higher antioxidant and polyphenol contents than LCE. LCS were significantly increased peroxisome proliferator-activated receptor (PPAR)-a and involucrin expression as epidermal differentiation marker in 3D skin equivalent model. LCS also showed antimicrobial activity against Staphylococcus aureus, a causative bacteria in atopic dermatitis. In addition, LCS inhibited the adipocyte differentiation of 3T3-L1 cells. When treated with the extract at a concentration of 100 µg/mL, the Wnt/β-catenin pathway reporter luciferase activity of HEK 293-TOP cells was increased approximately by 2-folds compared to that of the untreated control group. These results indicate that L. cylindrica supercritical carbon dioxide extract may serve as a cosmeceutical for improving skin barrier function and the treatment of obesity.


Subject(s)
3T3-L1 Cells , Adipocytes , Bacteria , Carbon Dioxide , Carbon , Dermatitis, Atopic , Ethanol , Fruit , Luciferases , Luffa , Obesity , Peroxisomes , Skin , Staphylococcus aureus
10.
Kosin Medical Journal ; : 106-116, 2019.
Article in English | WPRIM | ID: wpr-786391

ABSTRACT

OBJECTIVES: Hypoxia—a characteristic of almost all types of solid tumors—has been associated with poor outcomes in several human malignancies. Genipin—an active constituent of Gardenia fruit— has been reported to exert an anti-tumor effect in several cancers. In this study, we investigated inhibition of angiogenesis using Genipin-mediated hypoxia-induced hypoxia inducible factor (HIF-1) and VEGF expression in human cervical cancer cells.METHODS: Under normoxic and hypoxic conditions, the expression of HIF-1α and VEGF in cervical cancer HeLa cells was detected by quantitative reverse transcription polymerase chain reaction and western blotting. Luciferase reporter assays were used to investigate the molecular mechanisms underlying the hypoxia-induced survivin activation.RESULTS: Surprisingly, we found that Genipin suppressed the HIF-1α accumulation during hypoxia in human liver cancer cell line (HepG2), human prostate cancer cell line (LNCaP), colon cancer cell line (HCT116), and breast cancer cell line (MDA231). Genipin treatment also significantly reduced hypoxia-induced secretion of VEGF.CONCLUSIONS: Suppression of HIF-1α accumulation following treatment with Genipin under hypoxia was associated with PI3K and MAPK pathways. Taken together, these results suggested that Genipin inhibits HIF-1α expression through inhibition of PI3K and MAPK signaling pathways. These results provide new insights into a potential mechanism of the anticancer properties of Genipin.


Subject(s)
Hypoxia , Blotting, Western , Breast Neoplasms , Cell Line , Colonic Neoplasms , Gardenia , HeLa Cells , Humans , Liver Neoplasms , Luciferases , Polymerase Chain Reaction , Prostatic Neoplasms , Reverse Transcription , Uterine Cervical Neoplasms , Vascular Endothelial Growth Factor A
12.
Article in Chinese | WPRIM | ID: wpr-813101

ABSTRACT

To clone human mitogen-activated protein kinase kinase 6 (MKK6) gene promoter and explore its transcription activity by ubiquitin specific peptidase 22 (USP22).
 Methods: MKK6 gene promoter was amplified by PCR and two bases mutation within USP22 binding site was subsequently introduced. The wild type and mutant MKK6 promoter were inserted into the luciferase report vector pGL3-Basic, respectively. Recombinant plasmids were co-transfected with plasmid pRL-TK into HeLa cells, and the luciferase activities were measured by dual luciferase reporter system. Furthermore, the direct interaction between USP22 and MKK6 promoter was detected by chromatin immunoprecipitation (ChIP) assay. Finally, the MKK6 transcription activity was measured after knockdown of USP22.
 Results: The recombinant luciferase report vectors containing wild or mutant type of MKK6 promoter were successfully constructed. Mutation of USP22 binding site resulted in decrease of MKK6 promoter-driven luciferase activity in HeLa cells (P<0.05). USP22 could interact directly with MKK6 promoter. Down-regulation of USP22 led to the decreased MKK6 mRNA expression (P<0.05).
 Conclusion: USP22 could regulate the transcription activity of MKK6 gene in HeLa cells.


Subject(s)
HeLa Cells , Humans , Luciferases , MAP Kinase Kinase 6 , Promoter Regions, Genetic , Thiolester Hydrolases , Metabolism , Transcription, Genetic
13.
Yonsei Medical Journal ; : 22-29, 2019.
Article in English | WPRIM | ID: wpr-719691

ABSTRACT

PURPOSE: It is well documented that natural killer (NK) cytotoxicity against hepatocellular carcinoma (HCC) cells is impaired in HCC, which might account for the failure of anti-tumor immune response. miRNAs are considered as important regulators for the development and functions of NK cells. However, the entire role of miR-506 in NK cells remains far from being addressed. MATERIALS AND METHODS: The expressions of miR-506 and signal transducer and activator of transcription 3 (STAT3) mRNA in primary NK cells from HCC patients and healthy controls were detected by quantitative real-time PCR. NK cell cytotoxicity was assessed by CFSE/7AAD cytotoxicity assay and lactate dehydrogenase assay. Luciferase reporter assay, RNA immunoprecipitation assay, and western blot were conducted to confirm the interaction between miR-506 and STAT3. RESULTS: miR-506 expression was downregulated and STAT3 mRNA was upregulated in primary NK cells from HCC patients. Primary NK cells from HCC patients showed remarkably reduced cytotoxicity against SMMC7721 or HepG2 cells. NK cell cytotoxicity was positively correlated with miR-506 expression and negatively correlated with STAT3 mRNA expression. Additionally, miR-506 overexpression enhanced NK cell cytotoxicity against HCC cells, while miR-506 inhibitor showed the reverse effect. Moreover, miR-506 could suppress STAT3 expression by directly targeting 3′-untranslated regions of STAT3. A negative correlation between miR-506 and STAT3 mRNA expression in HCC patients was observed. Mechanistically, overexpressing STAT3 greatly reversed miR-506-mediated promotion of NK cell cytotoxicity against HCC cells. CONCLUSION: miR-506 enhanced NK cell cytotoxicity against HCC cells by targeting STAT3, suggesting that modulating miR-506 expression maybe a promising approach for enhancing NK cell-based antitumor therapies.


Subject(s)
Blotting, Western , Carcinoma, Hepatocellular , Hep G2 Cells , Humans , Immunoprecipitation , Killer Cells, Natural , L-Lactate Dehydrogenase , Luciferases , MicroRNAs , Real-Time Polymerase Chain Reaction , RNA , RNA, Messenger , STAT3 Transcription Factor
14.
Yonsei Medical Journal ; : 38-47, 2019.
Article in English | WPRIM | ID: wpr-719689

ABSTRACT

PURPOSE: Helicobacter pylori (HP)-infected gastric cancer (GC) is known to be a fatal malignant tumor, but the molecular mechanisms underlying its proliferation, invasion, and migration remain far from being completely understood. Our aim in this study was to explore miR-1915 expression and its molecular mechanisms in regulating proliferation, invasion, and migration of HP-infected GC cells. MATERIALS AND METHODS: Quantitative real-time PCR and western blot analysis were performed to determine miR-1915 and receptor for advanced glycation end product (RAGE) expression in HP-infected GC tissues and gastritis tissues, as well as human gastric mucosal cell line GES-1 and human GC cell lines SGC-7901 and MKN45. CCK8 assay and transwell assay were performed to detect the proliferation, invasion, and migration capabilities. MiR-1915 mimics and miR-1915 inhibitor were transfected into GC cells to determine the target relationship between miR-1915 and RAGE. RESULTS: MiR-1915 was under-expressed, while RAGE was over-expressed in HP-infected GC tissues and GC cells. Over-expressed miR-1915 could attenuate cellular proliferation, invasion, and migration capacities. RAGE was confirmed to be the target gene of miR-1915 by bioinformatics analysis and luciferase reporter assay. Moreover, HP-infected GC cellular proliferation, invasion, and migration were inhibited after treatment with pcDNA-RAGE. CONCLUSION: MiR-1915 exerted tumor-suppressive effects on cellular proliferation, invasion, and migration of HP-infected GC cells via targeting RAGE, which provided an innovative target candidate for treatment of HP-infected GC.


Subject(s)
Blotting, Western , Cell Line , Cell Proliferation , Computational Biology , Gastritis , Helicobacter pylori , Helicobacter , Humans , Luciferases , Rage , Real-Time Polymerase Chain Reaction , Stomach Neoplasms , Up-Regulation
15.
Article in English | WPRIM | ID: wpr-719275

ABSTRACT

BACKGROUND/AIMS: Indoxyl sulfate (IS) is a uremic toxin and an important causative factor in the progression of chronic kidney disease. Recently, paricalcitol (19-nor-1,25-dihydroxyvitamin D2) was shown to exhibit protective effects in kidney injury. Here, we investigated the effects of paricalcitol treatment on IS-induced renal tubular injury. METHODS: The fluorescent dye 2ʹ,7ʹ-dichlorofluorescein diacetate was used to measure intracellular reactive oxygen species (ROS) following IS administration in human renal proximal tubular epithelial (HK-2) cells. The effects of IS on cell viability were determined using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays and levels of apoptosis-related proteins (Bcl-2-associated protein X [Bax] and B-cell lymphoma 2 [Bcl-2]), nuclear factor-κB (NF-κB) p65, and phosphorylation of mitogen-activated protein kinase (MAPK) and protein kinase B (Akt) were determined by semiquantitative immunoblotting. The promoter activity of NF-κB was measured by luciferase assays and apoptosis was determined by f low cytometry of cells stained with f luorescein isothiocyanate-conjugated Annexin V protein. RESULTS: IS treatment increased ROS production, decreased cell viability and induced apoptosis in HK-2 cells. IS treatment increased the expression of apoptosis-related protein Bax, decreased Bcl-2 expression, and activated phosphorylation of MAPK, NF-κB p65, and Akt. In contrast, paricalcitol treatment decreased Bax expression, increased Bcl-2 expression, and inhibited phosphorylation of MAPK, NF-κB p65, and Akt in HK-2 cells. NF-κB promoter activity was increased following IS, administration and was counteracted by pretreatment with paricalcitol. Additionally, flow cytometry analysis revealed that IS-induced apoptosis was attenuated by paricalcitol treatment, which resulted in decreased numbers of fluorescein isothiocyanate-conjugated Annexin V positive cells. CONCLUSIONS: Treatment with paricalcitol inhibited IS-induced apoptosis by regulating MAPK, NF-κB, and Akt signaling pathway in HK-2 cells.


Subject(s)
Annexin A5 , Apoptosis , Cell Survival , Flow Cytometry , Fluorescein , Humans , Immunoblotting , Indican , Kidney , Luciferases , Lymphoma, B-Cell , Phosphorylation , Protein Kinases , Proto-Oncogene Proteins c-akt , Reactive Oxygen Species , Renal Insufficiency, Chronic , Signal Transduction
16.
Yonsei Medical Journal ; : 352-359, 2019.
Article in English | WPRIM | ID: wpr-742548

ABSTRACT

PURPOSE: Previous studies have confirmed that microRNAs play important roles in the pathogenesis of acute aortic dissection (AAD). Here, we aimed to explore the role of miR-145 and its regulatory mechanism in the pathogenesis of AAD. MATERIALS AND METHODS: AAD tissue samples were harvested from patients with aortic dissection and normal donors. Rat aortic vascular smooth muscle cells (VSMCs) were transfected with miR-145 mimic/inhibitor or negative control mimic/inhibitor. Gene and protein expression was measured in human aortic dissection tissue specimens and VSMCs by qRT-PCR and Western blot. Luciferase reporter assay was applied to verify whether connective tissue growth factor (CTGF) was a direct target of miR-145 in VSMCs. Methyl thiazolyl tetrazolium assay was used to detect VSMC viability. RESULTS: miR-145 expression was downregulated in aortic dissection tissues and was associated with the survival of patients with AAD. Overexpression of miR-145 promoted VSMC proliferation and inhibited cell apoptosis. Moreover, CTGF, which was increased in aortic dissection tissues, was decreased by miR-145 mimic and increased by miR-145 inhibitor. Furthermore, CTGF was confirmed as a target of miR-145 and could reverse the promotion effect of miR-145 on the progression of AAD. CONCLUSION: miR-145 suppressed the progression of AAD by targeting CTGF, suggesting that a miR-145/CTGF axis may provide a potential therapeutic target for AAD.


Subject(s)
Animals , Apoptosis , Blotting, Western , Connective Tissue Growth Factor , Humans , Luciferases , MicroRNAs , Muscle, Smooth, Vascular , Rats , Tissue Donors
17.
Yonsei Medical Journal ; : 381-388, 2019.
Article in English | WPRIM | ID: wpr-742544

ABSTRACT

PURPOSE: Osteoarthritis (OA) is a commonly occurring illness without a definitive cure, at present. Long non-coding RNAs (lncRNAs) have been widely confirmed to be involved in the modulation of OA progression. This study aimed to investigate the role and mechanism of lncRNA H19 in OA. MATERIALS AND METHODS: Abundances of H19 and microRNA-130a (miR-130a) in lipopolysaccharide (LPS)-treated C28/I2 cells were measured by reverse-transcription quantitative PCR (RT-qPCR). CCK-8 and flow cytometry analyses were carried out to assess cell viability and apoptosis. Starbase online software was used to predict the putative binding sites between H19 and miR-130a. Luciferase reporter, RNA pull down, and RT-qPCR were performed to analyze the true interaction between H19 and miR-130a. RESULTS: A notably dose-dependent elevation of H19 levels was observed in LPS-treated C28/I2 cells. Knockdown of H19 ameliorated the injury of LPS-induced C28/I2 cells, reflected by induced viability, decreased apoptosis, and reduced inflammatory factor secretions. Moreover, H19 negatively regulated the expression of miR-130a via acting as a molecular sponge for miR-130a. The stimulatory effects of H19 on cell damage were abolished following the restoration of miR-130a. CONCLUSION: LncRNA H19 aggravated the injury of LPS-induced C28/I2 cells by sponging miR-130a, hinting a novel regulatory mechanism and a potential therapeutic target for OA.


Subject(s)
Apoptosis , Binding Sites , Cell Survival , Flow Cytometry , Luciferases , Osteoarthritis , Polymerase Chain Reaction , Porifera , RNA , RNA, Long Noncoding , Sincalide
18.
Yonsei Medical Journal ; : 267-276, 2019.
Article in English | WPRIM | ID: wpr-742535

ABSTRACT

PURPOSE: Hepatocellular carcinoma (HCC) is a highly aggressive malignant tumor, the prognosis of which remains poor. Recently, microRNAs have been reported to play crucial functions in multiple tumors, including HCC. However, the molecular mechanisms of miR-370 in HCC still remain largely unknown. The present study focused on the effects of miR-370 on HCC migration, invasion, and epithelial-mesenchymal transition (EMT). MATERIALS AND METHODS: We investigated the key roles and possible regulatory mechanism of miR-370 in regulating HCC metastasis with functional assays, such as transwell assay. Quantitative real-time PCR (qRT-PCR) was used to detect miR-370 and guanylylcyclase domain containing 1 (GUCD1) expression in HCC tissues and cells. Subsequently, we performed transwell assays to determine the functions of miR-370 in HCC cell invasion and migration. Western blot was used to determine protein expressions of relevant genes. Luciferase reporter assays were conducted to confirm the target gene of miR-370. RESULTS: qRT-PCR analysis demonstrated that miR-370 was dramatically downregulated in HCC. Moreover, downregulated miR-370 was found to be associated with poor survival and adverse clinicopathologic characteristics of HCC patients. Transwell assays revealed that miR-370 overexpression dramatically suppressed HCC invasion and migration. Meanwhile, miR-370 restoration prominently inhibited EMT progression in HCC cells. Luciferase reporter assays confirmed GUCD1 as a downstream target gene of miR-370. GUCD1 expression in HCC tissues was prominently increased and inversely correlated with miR-370 expression. Furthermore, GUCD1 was verified as mediating the suppressive influence of miR-370 on cell metastasis and EMT in HCC. CONCLUSION: Taken together, our study confirmed that miR-370 suppressed HCC cell metastasis and EMT via regulating GUCD1. Accordingly, the miR-370/GUCD1 axis may potentially acts as attractive therapeutic targets and novel biomarkers for HCC treatment.


Subject(s)
Biomarkers , Blotting, Western , Carcinoma, Hepatocellular , Epithelial-Mesenchymal Transition , Humans , Luciferases , MicroRNAs , Negotiating , Neoplasm Metastasis , Prognosis , Real-Time Polymerase Chain Reaction
19.
Yonsei Medical Journal ; : 298-307, 2019.
Article in English | WPRIM | ID: wpr-742531

ABSTRACT

PURPOSE: Previous study has well documented the anti-apoptotic effects of miR-590 on oxidized low-density lipoprotein (ox-LDL)-treated endothelial cells (ECs). However, the mechanism underlying the anti-apoptotic effects of miR-590 in ox-LDL-treated ECs remains to be further addressed. MATERIALS AND METHODS: ApoE(−/−) mice fed with a high-fat diet (HFD) and human aortic endothelial cells (HAECs) treated with ox-LDL were used as in vivo and in vitro models of atherosclerosis. The expressions of miR-590 and toll-like receptor 4 (TLR4) were detected by quantitative real-time PCR and Western blot, respectively. Atherosclerotic lesion analysis was performed using Evans blue and hematoxylin-eosin staining. Cell proliferation was assessed by MTT assay. Apoptosis was examined using flow cytometry analysis and Western blot analysis of Cleaved poly (ADP-ribose) polymerase (PARP) and Cleaved Caspase-3 levels. The effect of miR-590 on TLR4/nuclear factor kappa B (NF-κB) pathway was evaluated by Western blot. Binding between miR-590 and TLR4 was confirmed by luciferase reporter assay and Western blot. RESULTS: miR-590 was downregulated in the aorta tissues from HFD-fed apoE(−/−) mice and ox-LDL-treated HAECs. miR-590 overexpression inhibited atherosclerotic lesion in HFD-induced apoE(−/−) mice and promoted proliferation and inhibited apoptosis of ox-LDL-treated HAECs. Additionally, TLR4 was identified as a direct target of miR-590 in ox-LDL-treated HAECs. Moreover, anti-miR-590 reversed TLR4 knockdown-mediated promotion of cell proliferation and suppression of apoptosis in ox-LDL-treated HAECs. miR-590 overexpression suppressed the TLR4/NF-κB pathway, and inhibition of the TLR4/NF-κB pathway promoted cell proliferation and impeded apoptosis in ox-LDL-treated HAECs. CONCLUSION: miR-590 promoted proliferation and blocked ox-LDL-induced apoptosis in HAECs through inhibition of the TLR4/NF-κB pathway.


Subject(s)
Animals , Aorta , Apoptosis , Atherosclerosis , Blotting, Western , Caspase 3 , Cell Proliferation , Diet, High-Fat , Endothelial Cells , Evans Blue , Flow Cytometry , Humans , In Vitro Techniques , Lipoproteins , Luciferases , Mice , Real-Time Polymerase Chain Reaction , Toll-Like Receptor 4
20.
Yonsei Medical Journal ; : 148-157, 2019.
Article in English | WPRIM | ID: wpr-742524

ABSTRACT

PURPOSE: Breast cancer (BC) is one of the most common malignant tumors, affecting a significant number of women worldwide. MicroRNAs (miRNAs) have been reported to play important roles in tumorigenesis. The aim of this study was to determine the roles of miR-182-5p in BC progression. MATERIALS AND METHODS: The expressions of miR-182-5p and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) were measured in BC tissues and cells by quantitative real-time polymerase chain reaction or Western blot. Cell proliferation and invasion were detected by cell counting kit-8 assay and trans-well assay, respectively. The interaction between miR-182-5p and PTEN was probed by bioinformatics analysis, luciferase activity, and RNA immunoprecipitation. A murine xenograft model was established to investigate the role of miR-182-5p in BC progression in vivo. RESULTS: An abundance of miR-182-5p was noted in BC tissues and cells. High expression of miR-182-5p was associated with poor survival. Abrogation of miR-182-5p inhibited cell proliferation and invasion in BC cells. Interestingly, PTEN was indicated as a target of miR-182-5p, and its restoration reversed miR-182-5p-mediated promotion of proliferation and invasion of BC cells. Moreover, depletion of miR-182-5p suppressed tumor growth via up-regulating PTEN expression in the murine xenograft model. CONCLUSION: MiR-182-5p exhaustion blocked cell proliferation and invasion by regulating PTEN expression, providing a novel therapeutic avenue for treatment of BC.


Subject(s)
Blotting, Western , Breast Neoplasms , Breast , Carcinogenesis , Cell Count , Cell Proliferation , Chromosomes, Human, Pair 10 , Computational Biology , Female , Heterografts , Humans , Immunoprecipitation , Luciferases , MicroRNAs , Real-Time Polymerase Chain Reaction , RNA
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