Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 115
Filter
1.
Chinese Medical Journal ; (24): 829-839, 2021.
Article in English | WPRIM | ID: wpr-878056

ABSTRACT

BACKGROUND@#MicroRNAs are closely associated with the progression and outcomes of multiple human diseases, including sepsis. In this study, we examined the role of miR-23a in septic injury.@*METHODS@#Lipopolysaccharide (LPS) was used to induce sepsis in a rat model and H9C2 and HK-2 cells. miR-23a expression was evaluated in rat myocardial and kidney tissues, as well as H9C2 and HK-2 cells. A miR-23a mimic was introduced into cells to identify the role of miR-23a in cell viability, apoptosis, and the secretion of inflammatory cytokines. Furthermore, the effect of Rho-associated kinase 1 (ROCK1), a miR-23a target, on cell damage was evaluated, and molecules involved in the underlying mechanism were identified.@*RESULTS@#In the rat model, miR-23a was poorly expressed in myocardial (sham vs. sepsis 1.00 ± 0.06 vs. 0.27 ± 0.03, P < 0.01) and kidney tissues (sham vs. sepsis 0.27 ± 0.03 vs. 1.00 ± 0.06, P < 0.01). Artificial overexpression of miR-23a resulted in increased proliferative activity (DNA replication rate: Control vs. LPS vs. LPS + Mock vs. LPS + miR-23a: H9C2 cells: 34.13 ± 3.12 vs. 12.94 ± 1.21 vs. 13.31 ± 1.43 vs. 22.94 ± 2.26, P < 0.05; HK-2 cells: 15.17 ± 1.43 vs. 34.52 ± 3.46 vs. 35.19 ± 3.12 vs. 19.87 ± 1.52, P < 0.05), decreased cell apoptosis (Control vs. LPS vs. LPS + Mock vs. LPS + miR-23a: H9C2 cells: 11.39 ± 1.04 vs. 32.57 ± 2.29 vs. 33.08 ± 3.12 vs. 21.63 ± 2.35, P < 0.05; HK-2 cells: 15.17 ± 1.43 vs. 34.52 ± 3.46 vs. 35.19 ± 3.12 vs. 19.87 ± 1.52, P < 0.05), and decreased production of inflammatory cytokines, including interleukin-6 (Control vs. LPS vs. LPS + Mock vs. LPS + miR-23a: H9C2 cells: 59.61 ± 5.14 vs. 113.54 ± 12.30 vs. 116.51 ± 10.69 vs. 87.69 ± 2.97 ng/mL; P < 0.05, F = 12.67, HK-2 cells: 68.12 ± 6.44 vs. 139.65 ± 16.62 vs. 143.51 ± 13.64 vs. 100.82 ± 9.74 ng/mL, P < 0.05, F = 9.83) and tumor necrosis factor-α (Control vs. LPS vs. LPS + Mock vs. LPS + miR-23a: H9C2 cells: 103.20 ± 10.31 vs. 169.67 ± 18.84 vs. 173.61 ± 15.91 vs. 133.36 ± 12.32 ng/mL, P < 0.05, F = 12.67, HK-2 cells: 132.51 ± 13.37 vs. 187.47 ± 16.74 vs. 143.51 ± 13.64 vs. 155.79 ± 15.31 ng/mL, P < 0.05, F = 9.83) in cells. However, ROCK1 was identified as a miR-23a target, and further up-regulation of ROCK1 mitigated the protective function of miR-23a in LPS-treated H9C2 and HK-2 cells. Moreover, ROCK1 suppressed sirtuin-1 (SIRT1) expression to promote the phosphorylation of nuclear factor-kappa B (NF-κB) p65, indicating the possible involvement of this signaling pathway in miR-23a-mediated events.@*CONCLUSION@#Our results indicate that miR-23a could suppress LPS-induced cell damage and inflammatory cytokine secretion by binding to ROCK1, mediated through the potential participation of the SIRT1/NF-κB signaling pathway.


Subject(s)
Animals , Apoptosis/genetics , Cell Line , Cytokines , Inflammation/genetics , Lipopolysaccharides , MicroRNAs/genetics , NF-kappa B , Rats , Sirtuin 1 , rho-Associated Kinases/genetics
2.
Acta Physiologica Sinica ; (6): 491-500, 2021.
Article in Chinese | WPRIM | ID: wpr-887684

ABSTRACT

Many studies have shown that circular RNAs (circRNAs) play a key regulatory role in the whole biological process of tumors. The purpose of this study was to explore the biological function and molecular mechanism of circ_0001666 in non-small cell lung cancer (NSCLC), so as to provide new targets for the diagnosis and treatment of NSCLC. Gene expression profiles were downloaded from Gene Expression Omnibus (GEO, GSE101586) and the differential genes were obtained by using GEO2R analysis. The quantitative real time polymerase chain reaction (qRT-PCR) was used to detect the expression level of circ_0001666 in NSCLC cells. Cell counting kit-8 (CCK-8) and Annexin V-FITC apoptosis detection kit were respectively used to assess the cell proliferation and apoptosis, where circ_0001666 was knockdown in NSCLC cells. The targeted relationship among mircoRNA 330-5p (miR-330-5p), circ_0001666, and high mobility group A2 protein (HMGA2) was verified by bioinformatics prediction, dual-luciferase reporter gene, RNA immunoprecipitation (RIP) and RNA pull down assay. The results showed that the expression of circ_0001666 in NSCLC cells was significantly up-regulated than that in normal lung epithelial cells. Circ_0001666 knockdown reduced the cell viability and promoted the apoptosis of NSCLC cells, which could be reversed by miR-330-5p inhibitors. MiR-330-5p is the downstream target of circ_0001666 and can be adsorbed by circ_0001666. HMGA2 is a target gene of miR-330-5p, which can be indirectly regulated by circ_0001666. The results suggest that circ_0001666 promotes the proliferation and inhibits apoptosis of NSCLC cells via miR-330-5p/HMGA2 axis.


Subject(s)
Apoptosis/genetics , Carcinoma, Non-Small-Cell Lung/genetics , Cell Proliferation/genetics , HMGA2 Protein , Humans , Lung Neoplasms/genetics , MicroRNAs/genetics , RNA, Circular
3.
Article in English | WPRIM | ID: wpr-922584

ABSTRACT

OBJECTIVES@#To explore the molecular mechanism for thyroid cancer metastasis via analyzing the role of microRNA (miR)-21-5p and its target gene recombinant sclerostin domain containing protein 1 (SOSTDC1) in thyroid cancer.@*METHODS@#The target miR-21-5p was screened through bioinformatics analysis and cell verification, and the thyroid cancer cell lines was transfected with miR-21-5p inhibitor. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) test, flow cytometry, and cell scratch test were used to detect the proliferation, apoptosis and migration of thyroid cancer cells in the miR-21-5p inhibitor group and the inhibitor control group, respectively. The luciferase report experiment was used to verify the relationship between miR-21-5p and SOSTDC1, Western blotting was used to detect the expression levels and phosphorylation levels of SOSTDC1,phosphatidylinositol 3 kinase (PI3K), protein kinase B (Akt) and mitogen-activated protein kinases (MAPK), extracellular regulated protein kinases (ERK) in thyroid cancer cells.@*RESULTS@#MiR-21-5p was significantly increased in thyroid cancer cells,which was negatively correlated with SOSTDC1 (@*CONCLUSIONS@#MiR-21-5p in thyroid cancer cells can target the expression of SOSTDC1 and affect the activities of PI3K/Akt and MAPK/ERK, thereby inhibiting the apoptosis of thyroid cancer cells and promoting cell proliferation and migration.


Subject(s)
Adaptor Proteins, Signal Transducing , Apoptosis/genetics , Cell Line, Tumor , Cell Movement , Cell Proliferation , Humans , MicroRNAs/genetics , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Thyroid Neoplasms/genetics
4.
Article in Chinese | WPRIM | ID: wpr-922023

ABSTRACT

OBJECTIVE@#To study the effect of down-regulating miR-488 targeting Jag1 on the injury of hypoxia-reoxygenation myocardial H9c2 cells.@*METHODS@#A hypoxic-reoxygenated myocardial H9c2 cell injury model was constructed. miR-488 inhibitor was used to transfect the cells. CCK-8 method and flow cytometry were used to detect cell proliferation and apoptosis in each group. Lactate dehydrogenase (LDH), superoxide dismutase (SOD), malonaldehyde (MDA), catalase (CAT) levels were detected. Western blotting was used to detect the expression of Bcl-2 associated X Protein (Bax) and B cell lymphoma/lewkmia-2 (Bcl-2). Target genes of miR-488 were predicted, and a luciferase reporter system was used to verify the targeting relationship between the two. Myocardial H9c2 cells were co-transfected with miR-488 inhibitor and Jag1 siRNA, and treated with hypoxia and reoxygenation, cell proliferation, apoptosis, LDH, SOD, MDA, CAT levels, and Bax, Bcl-2 protein expression were detected.@*RESULTS@#The expression of miR-488 in the hypoxia-reoxygenated myocardial H9c2 cells was increased, along with reduced cell proliferation, increased apoptosis, increased Bax protein expression, decreased Bcl-2 protein expression, increased MDA, decreased CAT and SOD, and increased LDH level in the supernatant of cell culture. When myocardial H9c2 cells were transfected with miR-488 inhibitor and treated with hypoxia and reoxygenation, the expression of miR-488 was decreased, along with increased cell proliferation, decreased apoptosis, decreased Bax protein expression, increased Bcl-2 protein expression, decreased MDA, increased CAT and SOD, and decreased LDH level in the supernatant of cell culture. Down-regulation of miR-488 could target and down-regulate Jag1 expression. And Jag1 siRNA could reverse the effect of miR-488 inhibitor on the proliferation, apoptosis, LDH, SOD, MDA, CAT levels and the expression of Bax and Bcl-2 of hypoxic-reoxygenated myocardial H9c2 cells.@*CONCLUSION@#Down-regulating miR-488 targeted Jag1 can attenuate hypoxia-reoxygenation induced myocardial H9c2 cell injury.


Subject(s)
Apoptosis/genetics , Down-Regulation , Humans , Hypoxia/genetics , Jagged-1 Protein/genetics , MicroRNAs/genetics , Myocardial Reperfusion Injury , Myocytes, Cardiac
5.
Article in Chinese | WPRIM | ID: wpr-921928

ABSTRACT

OBJECTIVE@#To explore the effects of siRNA hsa-circ-0000885 modified bone marrow mesenchymal stem cells (BMSCs) on osteogenic differentiation, cell proliferation and apoptosis in order to provide new ideas and methods for the clinical treatment of osteoporosis (OP).@*METHODS@#From September 2018 to February 2020, 13 patients with osteoporosis admitted to our hospital were selected as the research objects, including 11 females and 2 males, with an age of (65.45±10.77) years old. After obtaining the informed consent of patients, peripheral blood tissues were extracted. Then the expression level of cir-cRNA in peripheral blood mononuclear cells(PBMC) was detected by circ RNA chip. The expression of circ RNA was silenced by siRNA technology. The BMSCs were transfected with lentivirus. According to the siRNA interference plasmid hsa-circ-0000885, the cells were divided into the blank group, the empty vector group and the siRNA interference group. After 72 hours of treatment, the cell cycle was detected by flow cytometry, the apoptosis level was detected by AV-PI kit, and the osteogenic differentiation ability of BMSCs was detected by ALP staining.@*RESULTS@#The expression of hsa-circ-0000885 in PBMC of patients with osteoporosis was significantly higher than that of healthy controls (@*CONCLUSION@#The lentivirus mediated siRNA hsa-circ-0000885 plasmid transfected into BMSCs and osteoclast co culture system can promote cell proliferation, inhibit apoptosis and promote osteogenic differentiation of BMSCs, which can be used as a potential therapeutic target for OP patients.


Subject(s)
Aged , Apoptosis/genetics , Cell Differentiation , Cell Proliferation/genetics , Cells, Cultured , Coculture Techniques , Female , Humans , Lentivirus , Leukocytes, Mononuclear , Mesenchymal Stem Cells , Middle Aged , Osteoclasts , Osteogenesis/genetics , RNA, Small Interfering/genetics , Transfection
6.
Article in Chinese | WPRIM | ID: wpr-921525

ABSTRACT

Objective To explore the effect of miR-145-5p on the proliferation and apoptosis of human ovarian cancer cells and the possible molecular mechanisms involved.Methods Real-time quantitative PCR was performed to detect the expression of miR-145-5p in ovarian epithelial cells and ovarian cancer cells.CCK-8 and flow cytometry were used to detect the effects of miR-145-5p overexpression on the proliferation and apoptosis of ovarian cancer cells.TargetScan was employed to predict the target genes of miR-145-5p.Western blotting,dual luciferase reporter assay and rescue experiment were employed to predict and verify the underlying molecular mechanism of miR-145-5p function.Results The expression of miR-145-5p in ovarian cancer cells was significantly lower than that in normal ovarian epithelial cells(


Subject(s)
Apoptosis/genetics , Carcinoma, Ovarian Epithelial/genetics , Cell Line, Tumor , Cell Proliferation/genetics , Female , Gene Expression Regulation, Neoplastic , Humans , MicroRNAs/genetics , Ovarian Neoplasms/genetics
7.
Article in English | WPRIM | ID: wpr-921331

ABSTRACT

Objective@#The expression patterns of ribosomal large subunit protein 23a (RPL23a) in mouse testes and GC-1 cells were analyzed to investigate the potential relationship between RPL23a expression and spermatogonia apoptosis upon exposure to X-ray.@*Methods@#Male mice and GC-1 cells were irradiated with X-ray, terminal dUTP nick end-labelling (TUNEL) was performed to detect apoptotic spermatogonia @*Results@#Ionizing radiation (IR) increased spermatogonia apoptosis, the expression of RPL11, MDM2 and p53, and decreased RPL23a expression in mice spermatogonia @*Conclusion@#These results suggested that IR reduced RPL23a expression, leading to weakened the RPL23a-RPL11 interactions, which may have activated p53, resulting in spermatogonia apoptosis. These results provide insights into environmental and clinical risks of radiotherapy following exposure to IR in male fertility. The graphical abstract was available in the web of www.besjournal.com.


Subject(s)
Animals , Apoptosis/genetics , Gene Expression Regulation , Male , Mice , Ribosomal Proteins/metabolism , Signal Transduction , Spermatogonia/radiation effects
8.
Chinese Medical Journal ; (24): 2611-2618, 2021.
Article in English | WPRIM | ID: wpr-921137

ABSTRACT

BACKGROUND@#Nucleolar protein 6 (NOL6) is a nucleolar RNA-associated protein that is highly conserved between species. It has been proved to be associated with the prognosis of liver cancer. However, the underlying mechanism has not been fully established. This study aimed to assess the relationship between NOL6 and liver cancer prognosis.@*METHODS@#We constructed an NOL6-short hairpin RNA (shRNA)-expressing lentivirus. Through viral transfection, cell growth assay and fluorescence-activated cell sorting, we evaluated the effect of shRNA-mediated NOL6 knockdown on the proliferation, colony formation, and apoptosis of hepatocellular carcinoma (HCC) cells. The relationship between NOL6 expression and HCC patient survival has been established through bioinformatics analysis. We also explored the downstream molecular regulatory network of NOL6 in HCC by performing an Ingenuity Pathway Analysis in the database.@*RESULTS@#Increased NOL6 expression was detected in HCC cells compared to normal controls; HCC patients with high NOL6 expression had poorer prognoses than those with low expression. NOL6 knockdown inhibited HCC cell proliferation, apoptosis, and colony formation. Also, MAPK8, CEBPA, and FOSL1 were selected as potential downstream genes of NOL6.@*CONCLUSIONS@#NOL6 up-regulates HCC cell proliferation and affects downstream expression of related genes. Moreover, NOL6 is considered to be associated with poor prognosis in HCC patients.


Subject(s)
Apoptosis/genetics , Carcinoma, Hepatocellular/genetics , Cell Line, Tumor , Cell Proliferation/genetics , Gene Expression Regulation, Neoplastic , Humans , Liver Neoplasms/genetics , Nuclear Proteins , Prognosis
9.
Int. j. morphol ; 38(3): 523-529, June 2020. graf
Article in English | LILACS | ID: biblio-1098282

ABSTRACT

This study aimed to investigate the morphometric and the pattern of protein and gene expression related to the extrinsic apoptotic pathway in experimental focal cerebral ischemia and the hole of neuroprotection with hypothermia and ketoprofen. For this analysis, 120 rats were randomly divided into 3 groups (20 animals each): control - no surgery (20 animals); sham - simulation of surgery (20 animals); ischemic - focal ischemia for 1 hour, without reperfusion (80 animals) and divided into four subgroups with 20 animals each: ischemic + intraischemic hypothermia; ischemic + previous intravenous ketoprofen, and ischemic + hypothermia and ketoprofen. The infarct volume was measured using morphometric analysis of infarct areas defined by triphenyl tetrazolium chloride and the patterns of expression of the apoptosis genes (Fas, c-Flip, caspase-8 and caspase-3) and the apoptosis protein caspase-3 were evaluated by quantitative real-time PCR and immunohistochemistry, respectively. Hypo expression of genes of extrinsic pathway of apoptosis was observed: Fas receptor, c-Flip and caspase-8 in the ischemics areas. Increases in the gene and protein caspase-3 in the ischemic areas were also observed, and these increases were reduced by hypothermia and ketoprofen, also noted in the morphometric study. The caspases-3 increase suggests that this gene plays an important role in apoptosis, probably culminating in cell death and that the neuroprotective effect of hypothermia and ketoprofen is involved.


Este estudio tuvo como objetivo investigar la morfometría y el patrón de expresión de proteínas y genes relacionados con la vía apoptótica extrínseca en la isquemia cerebral focal experimental y el agujero de neuroprotección con hipotermia y ketoprofeno. Se dividieron aleatoriamente 120 ratas en 3 grupos (20 animales cada uno): control - sin cirugía (20 animales); simulación - simulación de cirugía (20 animales); isquemia isquemia focal durante 1 hora, sin reperfusión (80 animales) y dividida en cuatro subgrupos con 20 animales cada uno: isquemia + hipotermia intraisquémica; isquemia + ketoprofeno intravenoso previo, e isquemia + hipotermia y ketoprofeno. El volumen del infarto se midió utilizando un análisis morfométrico de áreas de infarto definidas por cloruro de trifenil tetrazolio y los patrones de expresión de los genes de apoptosis (Fas, c-Flip, caspase-8 y caspase-3) y la proteína de apoptosis caspase-3 fueron evaluados por PCR cuantitativa en tiempo real e inmunohistoquímica, respectivamente. Se observó hipoexpresión de genes de la vía extrínseca de la apoptosis: receptor Fas, c-Flip y caspasa-8 en las áreas isquémicas. También se observaron aumentos en el gen y la proteína caspasa-3 en las áreas isquémicas y estos aumentos se redujeron por hipotermia y ketoprofeno, también observado por estudio morfométrico. El aumento de caspasas-3 sugiere que este gen tiene un papel importante en la apoptosis, y probable causa de muerte celular, involucrando el efecto neuroprotector de la hipotermia y el ketoprofeno.


Subject(s)
Animals , Rats , Brain Ischemia/genetics , Brain Ischemia/metabolism , Immunohistochemistry , Brain Ischemia/pathology , Brain Ischemia/therapy , Ketoprofen/pharmacology , Apoptosis/genetics , Neuroprotective Agents/pharmacology , Disease Models, Animal , Caspase 3/genetics , Caspase 8/genetics , Real-Time Polymerase Chain Reaction , Hypothermia, Induced
10.
Braz. j. med. biol. res ; 53(1): e8883, Jan. 2020. tab, graf
Article in English | LILACS | ID: biblio-1055486

ABSTRACT

Opa-interacting protein 5 antisense transcript 1 (OIP5-AS1) is one kind of cytoplasmic long non-coding RNA (lncRNA), which has been demonstrated to play a critical function in multiple cancers. However, the detailed mechanism of OIP5-AS1 in the regulation of cervical cancer progression is still obscure. Here, we demonstrated that lncRNA OIP5-AS1 was upregulated in cervical cancer and was correlated with poor prognosis by bioinformatics studies. OIP5-AS1 depletion inhibited cell proliferation and promoted cell apoptosis in cervical cancer cells. Furthermore, we clarified that ROCK1 was the downstream effector of OIP5-AS1 and OIP5-AS1 acted as a molecular sponge of miR-143-3p. Finally, we verified that OIP5-AS1 exerted its function in the regulation of cervical cancer progression via interacting with miR-143-3p to regulate ROCK1 expression. Our study revealed novel mechanisms about how lncRNA OIP5-AS1 executed its function in cervical cancer and thus provided potential therapeutic targets for the disease.


Subject(s)
Humans , Female , Uterine Cervical Neoplasms/pathology , Apoptosis/physiology , MicroRNAs/metabolism , Cell Proliferation/physiology , rho-Associated Kinases/metabolism , RNA, Long Noncoding/metabolism , Gene Expression Regulation, Neoplastic , Up-Regulation , Uterine Cervical Neoplasms/metabolism , Blotting, Western , Apoptosis/genetics , Reverse Transcriptase Polymerase Chain Reaction , MicroRNAs/genetics , Cell Line, Tumor , Cell Proliferation/genetics , rho-Associated Kinases/genetics , RNA, Long Noncoding/genetics
11.
Biol. Res ; 53: 09, 2020. graf
Article in English | LILACS | ID: biblio-1100915

ABSTRACT

BACKGROUND: Osteoarthritis (OA) is one of the most common rheumatic diseases of which clinical symptoms includes swelling, synovitis and inflammatory pain, affect patients' daily life. It was reported that non-coding RNAs play vital roles in OA. However, the regulation mechanism of ncRNA in OA pathogenesis has not been fully elucidated. METHODS: The expression of SNHG7, miR-34a-5p and SYVN1 was detected using qRT-PCR in tissues, serum and cells. The protein expression of SYVN1, PCNA, cleavage-caspase 3, beclinl and LC3 were measured using western blot. The RNA immunoprecipitation (RIP), RNA pulldown, and luciferase reporter assays were used to verify the relationship between SNHG7, miR-34a-5p and SYVN1. The MTT and flow cytometry assay was performed to detected cell proliferation and cell apoptosis respectively. RESULTS: In this study, SNHG7 and SYVN1 expression were down-regulated, but miR-34a-5p was up-regulated in OA tissues and IL-1P treated cells compared with normal tissues and chondrocyte. Functional investigation revealed that up-regulated SNHG7 or down-regulated miR-34a-5p could promote cell proliferation and inhibit cell apoptosis and autophagy in OA cells. More than that, RIP, pulldown and luciferase reporter assay was applied to determine that miR-34a-5p was a target miRNA of SNHG7 and SYVN1 was a target mRNA of miR-34-5p. Rescue experiments showed that overexpression of miR-34a reversed high expression of SNHG7-mediated suppression of apoptosis and autophagy as well as promotion of proliferation, while its knockdown inhibited cell apoptosis and autophagy and promoted cell proliferation which could be impaired by silencing SYVN1. In addition, SNHG7 regulated SYVN1 through sponging miR-34a-5p. CONCLUSION: SNHG7 sponged miR-34a-5p to affect cell proliferation, apoptosis and autophagy through targeting SYVN1 which provides a novel sight into the pathogenesis of OA.


Subject(s)
Humans , Osteoarthritis/metabolism , Autophagy/physiology , Apoptosis/physiology , MicroRNAs/metabolism , Ubiquitin-Protein Ligases/metabolism , RNA, Long Noncoding/metabolism , Osteoarthritis/genetics , Autophagy/genetics , Enzyme-Linked Immunosorbent Assay , Down-Regulation , Up-Regulation , Blotting, Western , Apoptosis/genetics , MicroRNAs/genetics , Ubiquitin-Protein Ligases/genetics , Cell Proliferation , Real-Time Polymerase Chain Reaction , RNA, Long Noncoding/genetics
12.
Biol. Res ; 53: 44, 2020. graf
Article in English | LILACS | ID: biblio-1131888

ABSTRACT

BACKGROUND: Atherosclerosis (AS) is the main pathological basis of coronary heart disease, cerebral infarction and peripheral vascular disease, which seriously endanger people's life and health. In recent years, long non-coding RNA (lncRNA) has been found to be involved in gene expression regulation, but the research on AS is still in the initial stage. In this study, we mainly studied the role of HCG11 in patients with AS. Quantitative Real-time Polymerase Chain Reaction (QRT-PCR) was used to detect the expression of HCG11 and miR-144 in the serum of AS patients and healthy volunteers. Oxidation Low Lipoprotein (Ox-LDL), interleukin-6 (IL-6) and tumor necrosis factor α (TNF α) radiation were used to establish human vascular smooth muscle cells (VSMCs) in vitro model. Cell proliferation was determined by Cell Counting Kit-8 (CCK-8) assay. The apoptosis rate was determined by flow cytometry (FACS) and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL) staining. The expression levels of Forkhead box protein F1 (FOXF1), B cell lymphoma-2 (Bcl-2) and BCL2-Associated X (Bax) were detected by qRT-PCR. Luciferase gene reporter and RNA pull down experiments confirmed the relationship between HCG11 and miR-144, miR-144 and FOXF1. RESULTS: This study showed that HCG11 was significantly upregulated in patients with AS, while miR-144 was down-regulated in patients with AS. Ox-LDL and IL-6 in VSMCs induced up-regulation of HCG11 and down-regulation of miR-144. Overexpression of HCG11 promoted the proliferation and inhibited apoptosis of VSMCs. Luciferase gene reporter gene assay showed that HCG11 could bind to miR-144, and miR-144 could bind to FOXF1. Overexpression of miR-144 reversed the effect of HCG11 on VSMCs. CONCLUSIONS: LncRNA HCG11 regulates proliferation and apoptosis of vascular smooth muscle cell through targeting miR-144-3p/FOXF1 axis.


Subject(s)
Humans , Myocytes, Smooth Muscle/cytology , MicroRNAs/genetics , Atherosclerosis/genetics , Forkhead Transcription Factors/genetics , RNA, Long Noncoding/genetics , Apoptosis/genetics , Cell Proliferation/genetics , Muscle, Smooth, Vascular/cytology
13.
Chinese Medical Journal ; (24): 2829-2839, 2020.
Article in English | WPRIM | ID: wpr-877939

ABSTRACT

BACKGROUND@#Acute myeloid leukemia (AML) is a malignant hematological disease, originating from hematopoiesis stem cell differentiation obstruction and clonal proliferation. New reagents or biologicals for the treatment of AML are urgently needed, and exosomes have been identified as candidate biomarkers for disease diagnosis and prognosis. This study aimed to investigate the effects of exosomes from bone marrow mesenchymal stem cells (BMSCs) on AML cells as well as the underlying microRNA (miRNA)-mediated mechanisms.@*METHODS@#Exosomes were isolated using a precipitation method, followed by validation using marker protein expression and nanoparticle tracking analysis. Differentially expressed miRNAs were identified by deep RNA sequencing and confirmed by quantitative real-time polymerase chain reaction (qPCR). Cell proliferation was assessed by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt method, and cell cycle progression and apoptosis were detected by flow cytometry. Functional gene expression was analyzed by qPCR and Western blotting (WB). Significant differences were determined using Student's t test or analysis of variance.@*RESULTS@#BMSCs-derived exosomes effectively suppressed cell proliferation (both P < 0.0001 at 10 and 20 μg/mL) and cell cycle progression (P < 0.01 at G0-G1 stage), and also significantly enhanced cell apoptosis (P < 0.001) in KG-1a cells. There were 1167 differentially expressed miRNAs obtained from BMSCs-derived exosomes compared with KG-1a cell-derived exosomes (P < 0.05). Knockdown of hsa-miR-124-5p in BMSCs abrogated the effects of BMSCs-derived exosomes in regulating KG-1a such as the change in cell proliferation (both P < 0.0001 vs. normal KG-1a cell [NC] at 48 and 72 h). KG-1a cells treated with BMSCs-derived exosomes suppressed expression of structural maintenance of chromosomes 4 (P < 0.001 vs. NC by qPCR and P < 0.0001 vs. NC by WB), which is associated with the progression of various cancers. This BMSCs-derived exosomes effect was significantly reversed with knockdown of hsa-miR-124-5p (P < 0.0001 vs. NC by WB).@*CONCLUSIONS@#BMSCs-derived exosomes suppress cell proliferation and cycle progression and promote cell apoptosis in KG-1a cells, likely acting through hsa-miR-124-5p. Our study establishes a basis for a BMSCs-derived exosomes-based AML treatment.


Subject(s)
Apoptosis/genetics , Cell Proliferation/genetics , Exosomes/genetics , Humans , Leukemia, Myeloid, Acute/genetics , Mesenchymal Stem Cells , MicroRNAs/genetics
14.
Chinese Journal of Biotechnology ; (12): 2151-2161, 2020.
Article in Chinese | WPRIM | ID: wpr-878474

ABSTRACT

Kidney is one of the most important organs of the body and the mammalian kidney development is essential for kidney unit formation. The key process of kidney development is metanephric development, where mesenchymal-epithelial transition (MET) plays a crucial role. Here we investigated the biological function of PPP3CA in metanephric mesenchyme (MM) cells. qRT-PCR and Western blotting were used to detect PPP3CA and MET makers expression in mK3, mK4 cells respectively at mRNA and protein level. Subsequently, PPP3CA was stably knocked down via lentivirus infection in mK4 cells. Flow cytometry, EdU/CCK-8 assay, wound healing assay were conducted to clarify the regulation of PPP3CA on cell apoptosis, proliferation and migration respectively. PPP3CA was expressed higher in epithelial-like mK4 cells than mesenchyme-like mK3 cells. Thus, PPP3CA was silenced in mK4 cells and PPP3CA deficiency promoted E-cadherin expression, cell apoptosis. Moreover, PPP3CA knock down attenuated cell proliferation and cell migration in mK4 cell. The underlying mechanism was associated with the dephosphorylation of PPP3CA on ERK1/2. Taken together, our results indicated that PPP3CA mediated MET process and cell behaviors of MM cells, providing new foundation for analyzing potential regulator in kidney development process.


Subject(s)
Animals , Apoptosis/genetics , Cell Line , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Epithelial-Mesenchymal Transition/genetics , Gene Silencing , Mesenchymal Stem Cells/cytology , Mesoderm , Mice
15.
Biol. Res ; 52: 33, 2019. tab, graf
Article in English | LILACS | ID: biblio-1019498

ABSTRACT

BACKGROUND: Studies have shown that cancer susceptibility candidate 11 (CASC11), a newly discovered long non-coding RNA (lncRNA), was aberrantly overexpressed in hepatic carcinoma, gastric cancer and colorectal cancer. However, its effects on cervical cancer has been kept unknown up to now. The present study was aimed to investigate the relationship between lncRNA CASC11 and cervical cancer and further explore the mechanism of CASC11 effect on cervical cancer progression. MATERIALS: Quantitative real-time polymerase chain reaction (RT-qPCR) was used to detect the expressions of CASC11 in cancerous and adjacent normal tissues of patients with cervical cancer as well as in cell lines. The proliferation, migration, invasion and apoptosis were assayed after transfecting the cell with si-CASC11 or pcDNA3.1-CASC11. TOP/FOP-Flash luciferase reporter assay and western blot were used to analysis the activation of Wnt/ß-catenin signaling pathway. Si-CASC11-transfected HeLa cells were subcutaneously inoculated into male athymic (nude) mice to investigate the effect of CASC11 on the tumor formation. RESULTS: We discovered that CASC11, the expression of which was positively associated with the tumor size and the FIGO staging and negatively related to the patients' survival rate, was up-regulated in the cervical cancer tissues and cell lines. Silencing CASC11 inhibited the proliferation, migration as well as invasion and promoted the cell apoptosis. Conversely, overexpression of CASC11 facilitated the cancer cell's proliferation, migration and invasion ability and suppressed the apoptosis. Further study showed that CASC11 promoted the migration and invasion of cervical cancer cells by activating Wnt/ß-catenin signaling pathway and silencing CASC11 inhibited the tumor growth in vivo. CONCLUSION: Our study demonstrated that CASC11 promoted the cervical cancer progression by activating Wnt/ß-catenin signaling pathway for the first time, which provides a new target or a potential diagnostic biomarker of the treatment for cervical cancer.


Subject(s)
Humans , Animals , Female , Mice , Uterine Cervical Neoplasms/genetics , Uterine Cervical Neoplasms/pathology , Genetic Predisposition to Disease , MicroRNAs/genetics , beta Catenin/genetics , Wnt Signaling Pathway/genetics , Uterine Cervical Neoplasms/virology , Apoptosis/genetics , Disease Progression , Papillomavirus Infections/complications , Cell Proliferation/genetics , Genome-Wide Association Study , Flow Cytometry
16.
Acta cir. bras ; 34(5): e201900501, 2019. tab, graf
Article in English | LILACS | ID: biblio-1010875

ABSTRACT

Abstract Purpose: To analyze the effects of ischemic preconditioning (IPC) in the expression of apoptosis-related genes in rat small intestine subjected to ischemia and reperfusion. Methods: Thirty anesthetized rats underwent laparotomy and were drive into five groups: control (CG); ischemia (IG); ischemia and reperfusion (IRG); IPC and ischemia (IG+IPC); IPC and ischemia and reperfusion (I/RG+IPC). Intestinal ischemia was performed by clamping the superior mesenteric artery for 60 minutes, whereas reperfusion lasted for 120 minutes. IPC was carried out by one cycle of 5 minutes of ischemia followed by 10 minutes of reperfusion prior to the prolonged 60-minutes-ischemia and 120-minutes-reperfusion. Thereafter, the rats were euthanized and samples of small intestine were processed for histology and gene expression. Results: Histology of myenteric plexus showed a higher presence of neurons presenting pyknotic nuclei and condensed chromatin in the IG and IRG. IG+IPC and I/RG+IPC groups exhibited neurons with preserved volume and nuclei, along with significant up-regulation of the anti-apoptotic protein Bcl2l1 and down-regulation of pro-apoptotic genes. Moreover, Bax/Bcl2 ratio was lower in the groups subjected to IPC, indicating a protective effect of IPC against apoptosis. Conclusion: Ischemic preconditioning protect rat small intestine against ischemia/reperfusion injury, reducing morphologic lesions and apoptosis.


Subject(s)
Animals , Male , Reperfusion Injury/prevention & control , Apoptosis/genetics , Ischemic Preconditioning/methods , Apoptosis Regulatory Proteins/analysis , Jejunum/blood supply , Jejunum/pathology , Reference Values , Random Allocation , Down-Regulation , Gene Expression , Reproducibility of Results , Rats, Wistar , Mesenteric Artery, Superior , Constriction , Endothelial Cells/pathology , Apoptosis Regulatory Proteins/genetics , Real-Time Polymerase Chain Reaction , Mesenteric Ischemia/genetics , Mesenteric Ischemia/pathology
17.
Biol. Res ; 52: 12, 2019. graf
Article in English | LILACS | ID: biblio-1011414

ABSTRACT

BACKGROUND/AIMS: Hypoxia microenvironment plays a crucial role during tumor progression and it tends to exhibit poor prognosis and make resistant to various conventional therapies. HIF-1α acts as an important transcriptional regulator directly or indirectly associated with genes involved in cell proliferation, angiogenesis, apoptosis and energy metabolism during tumor progression in hypoxic microenvironment. This study was aimed to investigate the expression pattern of the hypoxia associated genes and their association during breast cancer progression under hypoxic microenvironment in breast cancer cells. METHODS: Cell proliferation in MCF-7 and MDA-MB-231 cell lines treated with different concentration of CoCl2 was analyzed by MTT assay. Flow cytometry was performed to check cell cycle distribution, whereas cell morphology was examined by phase contrast microscopy in both the cells during hypoxia induction. Expression of hypoxia associated genes HIF-1α, VEGF, p53 and BAX were determined by semiquantitative RT-PCR and real-time PCR. Western blotting was performed to detect the expression at protein level. RESULTS: Our study revealed that cell proliferation in CoCl2 treated breast cancer cells were concentration dependent and varies with different cell types, further increase in CoCl2 concentration leads to apoptotic cell death. Further, accumulation of p53 protein in response to hypoxia as compare to normoxia showed that induction of p53 in breast cancer cells is HIF-1α dependent. HIF-1α dependent BAX expression during hypoxia revealed that after certain extent of hypoxia induction, over expression of BAX conquers the effect of anti-apoptotic proteins and ultimately leads to apoptosis in breast cancer cells. CONCLUSION: In conclusion our results clearly indicate that CoCl2 simulated hypoxia induce the accumulation of HIF-1α protein and alter the expression of hypoxia associated genes involved in angiogenesis and apoptosis.


Subject(s)
Humans , Cell Hypoxia/drug effects , Cobalt/pharmacology , Apoptosis/drug effects , Transfection , Cell Hypoxia/genetics , Gene Expression Regulation, Neoplastic , Blotting, Western , Apoptosis/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , MCF-7 Cells , Flow Cytometry
18.
Biol. Res ; 52: 58, 2019. graf
Article in English | LILACS | ID: biblio-1100910

ABSTRACT

BACKGROUND: Our previous study showed that knockdown of long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) attenuated myocardial apoptosis in mouse acute myocardial infarction (AMI). This study aims to explore whether MALAT1 enhanced cardiomyocyte apoptosis via autophagy regulation and the underlying mechanisms of MALAT1 regulating autophagy. METHODS: Cardiomyocytes were isolated from neonatal mice and then stimulated with hypoxia/reoxygenation (H/R) injury to mimic AMI. The autophagy level was assessed using GFP-LC3 immunofluorescence and western blot analysis of autophagy-related proteins. RNA pull-down and RNA immunoprecipitation (RIP) was performed to analyze the binding of MALAT1 and EZH2. Chromatin immunoprecipitation (ChIP) assay was performed to analyze the binding of TSC2 promoter and EZH2. The cell apoptosis was evaluated using TUNEL staining and western blot analysis of apoptosis-related proteins. RESULTS: H/R injury increased MALAT1 expression in cardiomyocytes. Furthermore, MALAT1 overexpression inhibited, whereas MALAT1 knockdown enhanced the autophagy of cardiomyocytes. Moreover, MALAT1 overexpression recruited EZH2 to TSC2 promoter regions to elevate H3K27me3 and epigenetically inhibited TSC2 transcription. Importantly, TSC2 overexpression suppressed mTOR signaling and then activated the autophagy. Further results showed that MALAT1 inhibited proliferation and enhanced apoptosis of cardiomyocytes through inhibiting TSC2 and autophagy. CONCLUSION: These findings demonstrate that the increased MALAT1 expression induced by H/R injury enhances cardiomyocyte apoptosis through autophagy inhibition by regulating TSC2-mTOR signaling.


Subject(s)
Animals , Mice , Autophagy/physiology , Apoptosis/physiology , Myocytes, Cardiac/metabolism , TOR Serine-Threonine Kinases/genetics , RNA, Long Noncoding/genetics , Tuberous Sclerosis Complex 2 Protein/genetics , Autophagy/genetics , Signal Transduction , Blotting, Western , Fluorescent Antibody Technique , Apoptosis/genetics , Reverse Transcriptase Polymerase Chain Reaction , Chromatin Immunoprecipitation , TOR Serine-Threonine Kinases/metabolism , RNA, Long Noncoding/metabolism , Tuberous Sclerosis Complex 2 Protein/metabolism
19.
Braz. j. med. biol. res ; 52(11): e8657, 2019. tab, graf
Article in English | LILACS | ID: biblio-1039263

ABSTRACT

Although Taxol has improved the survival of cancer patients as a first-line chemotherapeutic agent, an increasing number of patients develop resistance to Taxol after prolonged treatment. The potential mechanisms of cancer cell resistance to Taxol are not completely clear. It has been reported that microRNAs (miRNAs) are involved in regulating the sensitivity of cancer cells to various chemotherapeutic agents. In this study, we aimed to explore the role of miR-129-5p in regulating the sensitivity of breast cancer cells to Taxol. Cell apoptosis and autophagy, and the sensitivity of MCF-7 cells to Taxol were assessed with a series of in vitro assays. Our results showed that the inhibition of autophagy increased the Taxol-induced apoptosis and the sensitivity of MCF-7 cells to Taxol. Up-regulation of miR-129-5p also inhibited autophagy and induced apoptosis. Furthermore, miR-129-5p overexpression increased the sensitivity of MCF-7 cells to Taxol. High mobility group box 1 (HMGB1), a target gene of miR-129-5p and a regulator of autophagy, was negatively regulated by miR-129-5p. We found that interference of HMGB1 enhanced the chemosensitivity of Taxol by inhibiting autophagy and inducing apoptosis in MCF-7 cells. Taken together, our findings suggested that miR-129-5p increased the chemosensitivity of MCF-7 cells to Taxol through suppressing autophagy and enhancing apoptosis by inhibiting HMGB1. Using miR-129-5p/HMGB1/autophagy-based therapeutic strategies may be a potential treatment for overcoming Taxol resistance in breast cancer.


Subject(s)
Humans , Female , Breast Neoplasms/metabolism , Paclitaxel/metabolism , HMGB1 Protein/metabolism , MicroRNAs/metabolism , MCF-7 Cells/metabolism , Antineoplastic Agents, Phytogenic/metabolism , Autophagy/genetics , Breast Neoplasms/genetics , Breast Neoplasms/drug therapy , Gene Expression Regulation, Neoplastic/genetics , Up-Regulation/genetics , Paclitaxel/therapeutic use , Apoptosis/genetics , Drug Resistance, Neoplasm/genetics , HMGB1 Protein/genetics , MicroRNAs/genetics , Antineoplastic Agents, Phytogenic/therapeutic use
20.
Braz. j. med. biol. res ; 52(10): e8631, 2019. tab, graf
Article in English | LILACS | ID: biblio-1039247

ABSTRACT

The long non-coding RNA (lncRNA) maternally expressed gene 3 (MEG3), a tumor suppressor, is critical for the carcinogenesis and progression of different cancers, including hepatocellular carcinoma (HCC). To date, the roles of lncRNA MEG3 in HCC are not well illustrated. Therefore, this study used western blot and qRT-PCR to evaluate the expression of MEG3, miR-9-5p, and Sex determining Region Y-related HMG-box 11 (SOX11) in HCC tissues and cell lines. RNA pull-down and luciferase reporter assay were used to evaluate these molecular interactions. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and flow cytometry detected the viability and apoptosis of HCC cells, respectively. The results showed that MEG3 and SOX11 were poorly expressed but miR-9-5p was highly expressed in HCC. The expression levels of these molecules suggested a negative correlation between MEG3 and miR-9-5p and a positive correlation with SOX11, confirmed by Pearson's correlation analysis and biology experiments. Furthermore, MEG3 could combine with miR-9-5p, and SOX11 was a direct target of miR-9-5p. Moreover, MEG3 over-expression promoted cell apoptosis and growth inhibition in HCC cells through sponging miR-9-5p to up-regulate SOX11. Therefore, the interactions among MEG3, miR-9-5p, and SOX11 might offer a novel insight for understanding HCC pathogeny and provide potential diagnostic markers and therapeutic targets for HCC.


Subject(s)
Humans , Male , Female , Middle Aged , Carcinoma, Hepatocellular/genetics , MicroRNAs/genetics , SOXC Transcription Factors/genetics , RNA, Long Noncoding/genetics , Liver Neoplasms/genetics , Transfection , Gene Expression Regulation, Neoplastic , Transcriptional Activation , Up-Regulation , Apoptosis/genetics , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , MicroRNAs/metabolism , Cell Line, Tumor , Cell Proliferation/genetics , SOXC Transcription Factors/metabolism , Real-Time Polymerase Chain Reaction , RNA, Long Noncoding/metabolism , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Neoplasm Staging
SELECTION OF CITATIONS
SEARCH DETAIL