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OBJECTIVE@#To explore the expression level of exosome derived miR-181b-5p in different disease stages of children with acute lymphoblastic leukemia and its relationship with clinical characteristics.@*METHODS@#Bone marrow plasma samples of 86 children with ALL were collected. Exosomes were extracted by exosome extraction kit, and RNA in exosomes was extracted by TRIzol method. The levels of miR-181b-5p in the blood plasma exosomes of the patients in the newly diagnosed group, relapse group, remission group and control group were detected by qRT- PCR. The difference of miR-181b-5p expression level in each group was compared and analyzed, and the relationship between miR-181b-5p expression level and clinical characteristics was analyzed.@*RESULTS@#The expression level of exosomal miR-181b-5p in the newly diagnosed group and the relapsed group was significantly lower than that in the remission group and the control group (P< 0.05). The expression level of exosomal miR-181b-5p in T-ALL children was higher than that in B-ALL children (P<0.05). The expression level of plasma exosomal miR-181b-5p in male children was higher than that in female children (P<0.01).@*CONCLUSION@#Exosome derived miR-181b-5p changes dynamically in the course of ALL children, and can be used as a marker miRNA to monitor disease status. Exosomes can transmit information in the tumor microenvironment and serve as a potential carrier for biomolecular targeted therapy.
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Humans , Male , Female , Child , Exosomes/metabolism , Clinical Relevance , MicroRNAs/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Tumor MicroenvironmentABSTRACT
Objective:To explore whether microRNA (miRNA) -181b-5p inhibits the proliferation and invasion of cutaneous melanoma cells by targeting pleckstrin (PLEK) .Methods:Bioinformatics methods were used to analyze cutaneous melanoma-associated core genes; dual-luciferase reporter assay was performed to verify the targeted interaction between miRNA-181b-5p and PLEK. Oligo RNA and small interfering RNA (siRNA) were used to regulate the expression of miRNA-181b-5p and PLEK in A375 cells respectively in this experiment, and A375 cells were divided into the following groups in detail: mimic negative control group, miRNA-181b-5p mimic group, inhibitor negative control group, miRNA-181b-5p inhibitor group, PLEK siRNA group, siRNA negative control group, miRNA-181b-5p inhibitor + control siRNA co-transfection group and miRNA-181b-5p inhibitor + PLEK siRNA3 co-transfection group. After 48-hour treatment, qPCR was performed to determine the mRNA expression of miRNA-181b-5p and PLEK in A375 cells, Western blot analysis to determine the PLEK protein expression, and Transwell assay to assess the invasive ability of A375 cells; after additional 24-96 hours of culture, cell counting kit-8 (CCK8) assay was conducted to assess the proliferative ability of A375 cells.Results:PLEK was the core gene for cutaneous melanoma. PLEK expression in the cutaneous melanoma in situ tissues was significantly higher than that in the paracancerous tissues ( P = 0.031) , but lower than that in the metastatic tissues ( P = 0.001) . Compared with human epidermal melanocytes HEMa-LP, the mRNA and protein expression of PLEK significantly increased in A375 cells (mRNA: 3.884 ± 0.156 vs. 0.997 ± 0.010, t = 18.48, P < 0.001; protein: 2.840 ± 0.301 vs. 1.029 ± 0.094, t = 5.47, P = 0.005) , but the miRNA-181b-5p expression significantly decreased in A375 cells (0.333 ± 0.042 vs. 0.967 ± 0.069, t = 7.83, P = 0.001) . Dual-luciferase reporter assay showed targeted binding of miRNA-181b-5p to PLEK. Compared with the mimic negative control group, the miRNA-181b-5p mimic group showed significantly decreased survival rate of A375 cells (48 hours: t = 7.96, P = 0.015; 72 hours: t = 7.50, P = 0.002; 96 hours: t = 7.96, P = 0.001) , and significantly decreased invasive ability of A375 cells ( t = 5.07, P = 0.007) ; on the contrary, the survival rate and invasive ability of A375 cells were significantly higher in the miRNA-181b-5p inhibitor group than in the inhibitor negative control group (survival rate: 24 hours, t =5.38, P = 0.013; 48 hours, t = 5.36, P = 0.013; 72 hours, t =7.63, P = 0.005; 96 hours, t = 5.99, P = 0.004; invasive ability: t = 7.24, P = 0.002) ; compared with the siRNA negative control group, the proliferative and invasive ability of A375 cells significantly decreased in the PLEK siRNA group (proliferative ability: 48, 72, 96 hours, P = 0.015, 0.011, 0.001, respectively; invasive ability: t = 4.93, P = 0.008) ; compared with the miRNA-181b-5p inhibitor + control siRNA co-transfection group, the miRNA-181b-5p inhibitor + PLEK siRNA co-transfection group showed significantly decreased proliferation rate and invasive ability of A375 cells (proliferation rate: 24, 48, 72, 96 hours, P = 0.042, 0.042, 0.037, 0.017, respectively; invasive ability: t = 8.52, P = 0.001) . Conclusion:miRNA-181b-5p can inhibit the proliferation and invasion of cutaneous melanoma A375 cells, likely by down-regulating the PLEK expression.
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Objective To investigate the expression of miR-181b-5p in peripheral blood of patients with esophageal cancer and to develop a guidance for the diagnosis and prognosis of esophageal cancer.Methods Blood samples were collected from 34 cases of esophageal cancer and 26 cases of healthy volunteers.The single candidate miRNA from WBC was selected by RNA miRNAs chip screening analysis.Then the expression of the candidate miRNA was detected by RT-qPCR.Results Compares with the normal group,the expression of miR-181b-5p was down-regula-ted in esophageal cancer group.Conclusions The expression of miR-181b-5p in esophageal cancer is down regula-ted,which may be related to the occurrence and development of esophageal cancer.
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Objective To investigate the effect of miR-181b on the proliferation and apoptosis of thyroid papillary carcinoma cells by down-regulating CYLD protein and its mechanism. Methods qPCR was used to detect the expression and difference of miR-181b in papilla-ry thyroid carcinoma and normal thyroid tissue. Western blotting was used to detect the regulation between miR-181b and CYLD protein. Clone formation assay was used to detect the proliferation of thyroid cancer cells after inhibition of miR-181b. Flow cytometry was used to detect the apoptosis of thyroid carcinoma cells after inhibition of miR-181b. Results Compared with normal thyroid tissue,the expression of miR-181b in papillary thyroid carcinoma was up-regulated,the difference was statistically significant(P<0. 05). The expression level of miR-181b in FTC-133 cell line was relatively high. Western blotting confirmed that miR-181b could directly regulate the expression of CYLD protein. Inhi-bition of miR-181b expression can inhibit the proliferation of thyroid cancer cells,and to some extent promote its apoptosis behavior. Conclu-sion miR-181b can regulate the expression of CLYD and affect the proliferation and apoptosis of thyroid cancer cells.
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Background:Fusobacterium nucleatum (Fn)is a common oral pathogen. Studies have shown that Fn is closely related to the occurrence and development of colorectal cancer,especially the inflammation-related colorectal cancer. Aims:To investigate the mechanism of Fn in forming an inflammatory microenvironment in colon cancer cells. Methods:An inflammation model of Caco-2 cells infected by Fn was constructed,and miRNA sequencing was performed. miR-181b mimics or inhibitor was transfected into Fn infected Caco-2 cells. mRNA and protein expressions of TNF-α were determined by qRT-PCR and Western blotting,respectively,and concentration of TNF-α in supernatant was measured by ELISA, number of lymphocyte penetrating the membrane was measured by Transwell chamber. Results:Compared with control group,mRNA and protein expressions of TNF-α were significantly increased (P < 0. 05),concentration of TNF-α in supernatant was significantly increased (P < 0. 05),and number of lymphocyte penetrating the membrane was significantly increased in Fn group (P < 0. 05). miRNA sequencing and qRT-PCR results showed that expression of miR-181b was significantly decreased in Fn group than in control group (P < 0. 05). Compared with control group,mRNA and protein expressions of TNF-α were significantly decreased in miR-181b mimics + Fn group (P < 0. 05),however,mRNA and protein expressions of TNF-α were significantly increased in miR-181b inhibitor group (P < 0. 05). Bioinformatics tools and Luciferase assay confirmed that TNF-α might be the target gene of miR-181b in Caco-2 cells. Conclusions:Fn can up-regulate the expression of TNF-α by inhibiting miR-181b in Caco-2 cells and recruiting lymphocytes to form an inflammatory microenvironment.
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Background:Fusobacterium nucleatum (Fn)is a common oral pathogen. Studies have shown that Fn is closely related to the occurrence and development of colorectal cancer,especially the inflammation-related colorectal cancer. Aims:To investigate the mechanism of Fn in forming an inflammatory microenvironment in colon cancer cells. Methods:An inflammation model of Caco-2 cells infected by Fn was constructed,and miRNA sequencing was performed. miR-181b mimics or inhibitor was transfected into Fn infected Caco-2 cells. mRNA and protein expressions of TNF-α were determined by qRT-PCR and Western blotting,respectively,and concentration of TNF-α in supernatant was measured by ELISA, number of lymphocyte penetrating the membrane was measured by Transwell chamber. Results:Compared with control group,mRNA and protein expressions of TNF-α were significantly increased (P < 0. 05),concentration of TNF-α in supernatant was significantly increased (P < 0. 05),and number of lymphocyte penetrating the membrane was significantly increased in Fn group (P < 0. 05). miRNA sequencing and qRT-PCR results showed that expression of miR-181b was significantly decreased in Fn group than in control group (P < 0. 05). Compared with control group,mRNA and protein expressions of TNF-α were significantly decreased in miR-181b mimics + Fn group (P < 0. 05),however,mRNA and protein expressions of TNF-α were significantly increased in miR-181b inhibitor group (P < 0. 05). Bioinformatics tools and Luciferase assay confirmed that TNF-α might be the target gene of miR-181b in Caco-2 cells. Conclusions:Fn can up-regulate the expression of TNF-α by inhibiting miR-181b in Caco-2 cells and recruiting lymphocytes to form an inflammatory microenvironment.
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Burkitt lymphoma (BL) is a highly malignant non-Hodgkin's lymphoma that is closely related to the abnormal expression of genes. Familial acute myelogenous leukemia related factor (FAMLF; GenBank accession No. EF413001.1) is a novel gene that was cloned by our research group, and miR-181b is located in the intron of the FAMLF gene. To verify the role of miR-181b and FAMLF in BL, RNAhybrid software was used to predict target site of miR-181b on FAMLF and real-time quantitative PCR (RQ-PCR) was used to detect expression of miR-181b and FAMLF in BL patients, Raji cells and unaffected individuals. miR-181b was then transfected into Raji and CA46 cell lines and FAMLF expression was examined by RQ-PCR and western blotting. Further, Raji cells viability and proliferation were detected by MTT and clone formation, and Raji cell cycle and apoptosis were detected by flow cytometry. The results showed that miR-181b can bind to bases 21–42 of the FAMLF 5′ untranslated region (UTR), FAMLF was highly expressed and miR-181b was lowly expressed in BL patients compared with unaffected individuals. FAMLF expression was significantly and inversely correlated to miR-181b expression, and miR-181b negatively regulated FAMLF at posttranscriptional and translational levels. A dual-luciferase reporter gene assay identified that the 5′ UTR of FAMLF mRNA contained putative binding sites for miR-181b. Down-regulation of FAMLF by miR-181b arrested cell cycle, inhibited cell viability and proliferation in a BL cell line model. Our findings explain a new mechanism of BL pathogenesis and may also have implications in the therapy of FAMLF-overexpressing BL.
Subject(s)
Humans , Male , Female , Infant , Child, Preschool , Child , Adolescent , Adult , Young Adult , Burkitt Lymphoma/genetics , Burkitt Lymphoma/metabolism , Gene Expression Regulation, Neoplastic , MicroRNAs/metabolism , Proteins/metabolism , Apoptosis/genetics , Cell Line, Tumor , Cell Proliferation/genetics , Cell Survival/genetics , Down-Regulation/genetics , MicroRNAs/genetics , Proteins/geneticsABSTRACT
Background:miR-181b is related to the progression of many tumors,however,its expression in colitis-associated colon cancer is not clear. Aims:To investigate miR-181b expression and its potential target genes in the process of colitis-associated colon carcinogenesis. Methods: Colitis-associated colon cancer model was established by combined administration of azoxymethane(AOM)and dextran sulfate sodium(DSS)in mice. The expression of miR-181b in colon tissue at different time points was measured by real time fluorescent quantitative PCR(qPCR). Target genes of miR-181b were screened preliminarily by genome microarray combined with miRNA target gene prediction softwares TargetScan, PicTar and miRDB. Expressions of target genes in colitis-associated colon cancer mice were detected by qPCR. Results:The expression of miR-181b was gradually elevated during the development of colitis-associated colon cancer. However, AOM or DSS alone did not change expression of miR-181b. Genome microarray combined with miRNA target gene prediction softwares showed that Ipmk,E2f5,Klf6,Prkcd,Bai3,Hic2 might be the target genes of miR-181b. qPCR showed that expressions of Ipmk,Prkcd,Bai3,Hic2 were significantly decreased in colitis-associated colon cancer than in control group. Conclusions:Expression of miR-181b in colon is upregulated with the development of colitis-associated colon cancer,but is irrelevant with simple chronic inflammation.
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Programmed cell death 4 (PDCD4) is a RNA-binding protein that acts as a tumor suppressor in many cancer types, including colorectal cancer (CRC). During CRC carcinogenesis, PDCD4 protein levels remarkably decrease, but the underlying molecular mechanism for decreased PDCD4 expression is not fully understood. In this study, we performed bioinformatics analysis to identify miRNAs that potentially target PDCD4. We demonstrated miR-181b as a direct regulator of PDCD4. We further showed that activation of IL6/STAT3 signaling pathway increased miR-181b expression and consequently resulted in downregulation of PDCD4 in CRC cells. In addition, we investigated the biological effects of PDCD4 inhibition by miR-181b both in vitro and in vivo and found that miR-181b could promote cell proliferation and migration and suppress apoptosis in CRC cells and accelerate tumor growth in xenograft mice, potentially through targeting PDCD4. Taken together, this study highlights an oncomiR role for miR-181b in regulating PDCD4 in CRC and suggests that miR-181b may be a novel molecular therapeutic target for CRC.
Subject(s)
Animals , Humans , Male , Mice , Apoptosis Regulatory Proteins , Genetics , Metabolism , Caco-2 Cells , Cell Proliferation , Colorectal Neoplasms , Genetics , Metabolism , Pathology , Heterografts , Mice, Nude , Mice, SCID , MicroRNAs , Genetics , Metabolism , Neoplasm Proteins , Genetics , Metabolism , Neoplasm Transplantation , RNA, Neoplasm , Genetics , Metabolism , RNA-Binding Proteins , Genetics , MetabolismABSTRACT
Objective: To investigate the effects of increasing the expression of miR-181b on the function of human breast cancer cell lines MDA-MB-231 and MCF-7, and to explore its possible target gene. Methods: miR-181b mimics were transfected into MDA-MB-231 and MCF-7 cells by LipofectAMINE 2000 and then the capabilities of proliferation and migration as well as the cell cycle distribution of MDA-MB-231 and MCF-7 cells were evaluated by MTT, Transwell and flow cytometry methods, respectively. Recombinant vector psiCHECK-2/cyclin- dependent kinase 8 (CDK8)-3'-untranslated region (3'-UTR) and miR-181b mimics were co-transfected into human embryonic kidney HEK293T cells and then the targeting of miR-181b combined with 3'-UTR of CDK8 gene was detected by dual luciferase report system. The expression of CDK8 in MDA-MB-231 and MCF-7 cells after transfection with miR-181b mimics was determined by Western blotting. Results: As compared with the miR-181b mimics-negative control (NC) tranfection group, the capabilities of proliferation and migration in MDA-MB-231 and MCF-7 cells after transfection with miR-181b mimics were significantly inhibited (P < 0.05), and the cell cycle was arrested at G0/G1 phase (P < 0.01). The luciferase activity of HEK293T cells after co-transfection with psiCHECK-2/CDK8-3'UTR and miR-181b mimics was reduced (P < 0.01), and it confirmed that miR-181b specifically targeted with CDK8 3'-UTR. As compared with the miR-181b mimics-NC transfection group, the expression level of CDK8 in MDA-MB-231 and MCF-7 cells after transfection with miR-181b mimics was down-regulated (P < 0.05). Conclusion: The up-regulation of miR-181b expression can inhibit the proliferation and migration of human breast cancer cells. This effect may be related to miR-181b targeting of the expression of CDK8. Copyright © 2014 by TUMOR.