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ObjectiveTo explore the mechanism and pathway of Gandou Fumu decoction （GDFMD） in the development of liver fibrosis in Wilson's disease （WD）. MethodFirst， 30 TX-j mice were randomly divided into the model group， high-dose， medium-dose， and low-dose GDFMD groups， and penicillamine group， with six mice in each group， and another six wild-type mice were used as the normal group. The high-dose， medium-dose， and low-dose GDFMD groups were intragastrically administered drugs of 13.92， 6.96， 3.48 g·kg-1. In the penicillamine group， 0.1 g·kg-1 of penicillamine was given by intragastric administration. The model group and the normal group were given equal volume of normal saline， once a day， for four consecutive weeks. Samples were collected four weeks after gavage， and enzyme-linked immunosorbent assay （ELISA） was used to detect type Ⅲ procollagen peptide （PCⅢ）， collagen type Ⅳ （Col Ⅳ）， hyaluronic acid （HA）， and laminin （LN）. Hematoxylin-eosin （HE）， Masson， and picric acid-Sirus red collagen （Sirus Red） staining were used to observe the histopathological changes of liver fibrosis. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）， immunohistochemistry， and Western blot were used to observe the expressions of α-smooth muscle actin （α-SMA） and collagen type Ⅰ （Col Ⅰ）， which were related to the activation of hepatic stellate cells （HSCs）. The expression of miR-29b-3p was observed by Real-time PCR. The expression of Unc-51-like kinase 1 （ULK1） and its downstream-related factors were observed by Western blot. The downstream genes of miR-29b-3p were verified by the dual luciferase reporter gene detection method. ResultCompared with the normal group， the four items of liver fibrosis （PCⅢ， Col Ⅳ， HA， and LN） in the model group were significantly abnormal （P<0.01）， and the pathology was significantly abnormal. The expression of HSC activation-related indicators including α-SMA and Col Ⅰ， as well as α-SMA mRNA and Col Ⅰ mRNA was up-regulated （P<0.05， P<0.01）， and miR-29b-3p expression was down-regulated （P<0.01）. ULK1， p-ULK1， autophagy-related gene 13 （Atg13）， p-Atg13， Beclin-1， FAK family kinase-interacting protein of 200 kDa （FIP200）， activating molecule in BECN1-regulated autophagy protein 1 （AMBKA1）， and microtubule-associated protein 1 light chain 3Ⅱ/Ⅰ（LC3Ⅱ/Ⅰ） were up-regulated （P<0.05， P<0.01）. p62 protein expression was down-regulated （P<0.01）. Compared with the model group， the four items of liver fibrosis in the high-dose， medium-dose， and low-dose GDFMD groups and the penicillamine group were significantly improve （P<0.01）， and the pathological conditions were improved. The expression of HSC activation-related indicators including α-SMA and Col Ⅰ， as well as α-SMA mRNA and Col Ⅰ mRNA was down-regulated （P<0.05， P<0.01）， and the expression of miR-29b-3p was up-regulated （P<0.01）. ULK1， p-ULK1， Atg13， p-Atg13， Beclin-1， FIP200， AMBKA1， and LC3Ⅱ/Ⅰ were down-regulated （P<0.05， P<0.01）， and p62 protein expression was up-regulated （P<0.01）. The prediction software predicted that there was a binding site between miR-29b-3p and ULK1. The dual-luciferase reporter gene detection method indicated that the luciferase activity of the ULK1-WT plasmid-transfected cell group was reduced when miR-29b-3p mimics were co-cultured （P<0.01）. ConclusionGDFMD can regulate ULK1-mediated autophagy by up-regulating miR-29b-3p and further exert its anti-hepatic fibrosis effect in Wilson's disease.
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@# miR-29b是最近生物医学界所关注的研究热点之一，尤其在人类癌症中。越来越多的研究发现，miR-29b在多种癌症 中异常表达，与肿瘤细胞增殖、分化、凋亡、侵袭、转移及药物耐受相关，有望成为癌症的新型诊断标志物及治疗靶点。本文重点 就miR-29b在人类癌症中的表达、作用及其调控机制和临床应用的研究进展进行综述，以促进miR-29b在临床诊断和治疗中的转 化应用。
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BACKGROUND: Breast cancer is the second common malignant cancer among females worldwide. Accumulating studies have indicated that deregulation of miRNA expression in breast cancer will contribute to tumorigenesis and form different cancer subtypes. However, the reported studies on miR-29b-3p-regulated breast cancer are limited so far. Herein, we investigated the role and mechanism of miR-29b-3p in the triple negative breast cancer cell line MDA-MB-231. METHODS: The relative miR-29b-3p expression in different breast cancer cell lines were determined by qRT-PCR. CCK8 and colony formation assay were used to determine the influence of miR-29b-3p on cell proliferation. Migration assay and invasion assay were performed for cell migration and invasion respectively. To study the cell integrity immunofluorescence was performed. TUNEL assay, flow cytometry assay, hoechst staining and western blot were conducted to determine the influence of miR-29b-3p inhibitor on cell apoptosis. TRAF3 was found to be the target gene of miR-29b-3p using bioinformatics predictions. Dual-luciferase assay was performed to determine the relative luciferase activity in NC, miR-29b-3p mimic, miR-29b-3p inhibitor with TRAF3 3'-UTR wt or TRAF3 3'-UTR mt reporter plasmids. The proteins expression of NF-κB signaling pathway in MDA-MB-231 after transfection with NC, miR-29b-3p mimic, miR-29b-3p inhibitor were determined by western blot. RESULTS: The miR-29b-3p expression was significantly increased in MDA-MB-231 compare with MCF-10A. miR-29b-3p inhibitor reduced the cell viability of MDA-MB-231 and inhibited cell migration and invasion. Cell cytoskeleton integrity destroyed after miR-29b-3p inhibitor treatment. Furthermore, we identified the mechanism and found miR-29b-3p targets the TRAF3 and activates NF-κB signaling pathway. CONCLUSIONS: From the above studies, our results indicated that miR-29b-3p acts as a promoter for the development of MDA-MB-231.
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Humans , Female , Down-Regulation/genetics , Apoptosis/drug effects , MicroRNAs/metabolism , TNF Receptor-Associated Factor 3/metabolism , Triple Negative Breast Neoplasms/genetics , Blotting, Western , Cell Line, Tumor , TNF Receptor-Associated Factor 3/genetics , Cell Proliferation , Triple Negative Breast Neoplasms/pathology , Luciferases/metabolismABSTRACT
Background: Long noncoding RNAs (lncRNAs) have been reported to be associated with dermis process during burn wound healing. This study aimed to investigate the role of lncRNA X-inactive specific transcript (XIST) in human skin fibroblasts (HSF) and extracellular matrix (ECM) as well as the regulatory network of XIST/microRNA-29b-3p (miR-29b-3p)/collagen 1 alpha 1 (COL1A1). Methods: The wound samples were collected from 25 patients with deep partial thickness burn at day 5 after burn. The thermal injured model was established using HSF cells. The expressions of XIST, miR-29b-3p and COL1A1 were measured by quantitative real-time polymerase chain reaction and western blot. ECM synthesis, cell proliferation and migration were detected by western blot, cell counting kit-8 and trans-well assays, respectively. The interaction between miR-29b-3p and XIST or COL1A1 was explored by bioinformatics analysis and luciferase reporter assay. Results: The expressions of XIST and COL1A1 were enhanced but miR-29b-3p expression was decreased after thermal injury. XIST overexpression promoted ECM synthesis, cell proliferation and migration in thermal injured HSF cells. However, XIST knockdown played an opposite effect. miR-29b-3p overexpression inhibited ECM synthesis, cell proliferation and migration, which was reversed by XIST. COL1A1 silence suppressed ECM synthesis, cell proliferation and migration by miR-29b-3p targeting. Moreover, COL1A1 up-regulation weakened the effect of XIST silence on ECM synthesis and HSF cell function. Conclusion: XIST promoted ECM synthesis, cell proliferation and migration by sponging miR-29b-3p and targeting COL1A1 in HSF cells after thermal injury, indicating the promoting role of XIST in wound healing.
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<p><b>OBJECTIVE</b>To investigate the expression of miR-29b in cholangiocarcinoma and explore its effects on cell proliferation and apoptosis of cholangiocarcinoma cells.</p><p><b>METHODS</b>Real-time PCR was used to detect the expression of miR-29b in cholangiocarcinoma cells line QBC939 and cholangiocarcinoma tissues. The lentiviral vector LV-hsa-miR-29b and blank vector were constructed to infect QBC939 cells. MTT assay and cell clone formation assay were performed to assess the changes in the cell proliferation and clone formation, respectively; flow cytometry was employed to evaluate the effect of miR-29b overexpression on cell cycle and apoptosis.</p><p><b>RESULTS</b>The expression of miR-29b was significantly down-regulated in QBC939 cells and cholangiocarcinoma tissues as compared with H-69 cells and normal tissues ( < 0.01). Compared with the blank vector, the lentiviral vector LV-hsa-miR-29b caused significantly increased expression of miR-29b in QBC939 cells ( < 0.01), which exhibited suppressed cell proliferation and clone formation ( < 0.01 or 0.05), cell cycle arrest at the S phase ( < 0.05), and significantly increased cell apoptosis ( < 0.01).</p><p><b>CONCLUSIONS</b>As a tumor-suppressing miRNA, miR-29b is down-regulated in cholangiocarcinoma, and its overexpression can suppress the proliferation and induce apoptosis of cholangiocarcinoma cells.</p>
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MicroRNA (miRNA) is a kind of highly conserved microRNA molecules that regulate gene expression after transcriptional levels in eukaryotes. Numerous evidences indicate that miRNA can regulate malignant phenotypes of neoplasms by repressing the expressions of critical oncogenes or tumor suppressor genes, and abnormal expression of miRNA becomes one of the most important features of cancer. The expression of miR-29b was low in multiple types of tumors and its up-regulated expression will depress the expression of some target genes, which plays a crucial role in the occurrence and development of various neoplasms and is expected to serve as a biomarker for early diagnosis and prognosis assessment becoming a novel target of drug development and new treatment.
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MicroRNA (miRNA) is a kind of highly conserved microRNA molecules that regulate gene expression after transcriptional levels in eukaryotes. Numerous evidences indicate that miRNA can regulate malignant phenotypes of neoplasms by repressing the expressions of critical oncogenes or tumor suppressor genes, and abnormal expression of miRNA becomes one of the most important features of cancer. The expression of miR-29b was low in multiple types of tumors and its up-regulated expression will depress the expression of some target genes, which plays a crucial role in the occurrence and development of various neoplasms and is expected to serve as a biomarker for early diagnosis and prognosis assessment becoming a novel target of drug development and new treatment.
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Objective To investigate the expression of miR-29s in the glioma stem cells,and explore how the members of miR-29s affect the bio-logical behaviors of glioma stem cells. Methods Eight patient specimens were used to culture glioma stem cells. Real-time PCR was adopted to test the expression of miR-29s. CCK-8 analysis was performed to test the proliferation ,Transwell was used to test cell migration and invasion ,and flow-cytometry analysis was carried out to test apoptosis. Results miR-29a,miR-29b and miR-29c were decreased in glioma stem cells. Over-ex-pression of miR-29s could inhibit the proliferation,cell migration and invasion,but promote apoptosis of glioma stem cells. Conclusion miR-29s acts as a cancer suppressor gene in the glioma stem cells ,and miR-29a plays the dominant functional role in the family.
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OBJECTIVE: To investigate the effect of α-lipoic acid on the oxidative stress of wound tissues and diabetic wound healing in mice with diabetic feet.
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Osteosarcoma is the most common primary sarcoma of bone, and it is a leading cause of cancer death among adolescents and young adults. However, the molecular mechanism underlying osteosarcoma carcinogenesis remains poorly understood. Recently, cyclin-dependent kinase 6 (CDK6) was identified as an important oncogene. We found that CDK6 protein level, rather than CDK6 mRNA level, is much higher in osteosarcoma tissues than in normal adjacent tissues, which indicates a post-transcriptional mechanism involved in CDK6 regulation in osteosarcoma. MiRNAs are small non-coding RNAs that repress gene expression at the post-transcriptional level and have widely been shown to play important roles in many human cancers. In this study, we investigated the role of miR-29b as a novel regulator of CDK6 using bioinformatics methods. We demonstrated that CDK6 can be downregulated by miR-29b via binding to the 3'-UTR region in osteosarcoma cells. Furthermore, we identified an inverse correlation between miR-29b and CDK6 protein levels in osteosarcoma tissues. Finally, we examined the function of miR-29b-driven repression of CDK6 expression in osteosarcoma cells. The results revealed that miR-29b acts as a tumor suppressor of osteosarcoma by targeting CDK6 in the proliferation and migration processes. Taken together, our results highlight an important role for miR-29b in the regulation of CDK6 in osteosarcoma and may open new avenues for future osteosarcoma therapies.
Subject(s)
Animals , Humans , Mice , Rats , 3' Untranslated Regions , Base Sequence , Bone Neoplasms , Metabolism , Pathology , Cell Line, Tumor , Cell Movement , Cell Proliferation , Cyclin-Dependent Kinase 6 , Genetics , Metabolism , MicroRNAs , Metabolism , Osteosarcoma , Metabolism , Pathology , RNA Interference , RNA, Messenger , Metabolism , RNA, Small Interfering , Metabolism , Sequence Alignment , Up-RegulationABSTRACT
Objective:This paper aims to bioinformatically analyze the target genes of miR-29b and to provide clues for cancer research targeting miR-29b. Methods:The differential expression levels of miRNAs in CD133+and CD133-A549 cells were detected using the miRNA PCR chip. Real-time polymerase chain reaction was performed to verify the partially differential expression of miR-NAs. Target genes of miR-29b were predicted by miRecords and analyzed by gene ontology and signal transduction pathway enrich-ment analysis. Results: The miR-29b expression was significantly decreased in the CD133+A549 cells compared with that in the CD133-cells. The number of miR-29b target genes was 106. The functions of these target genes were enriched in binding and extracel-lular matrix structural constituent (P<0.01). The JAK-STAT and TGF-βsignal transduction pathways were significantly enriched (P<0.05). Conclusion:The abnormal expression of miR-29b may be related to metastasis. Some of the predicted target genes of miR-29b were significantly enriched in the signaling pathways in relation to the tumors.
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Objective: To analyze the expression level of microRNA-29b (miR-29b) in human cervical cancer tissues, and to investigate the function of miR-29b in regulating the genesis of human cervical cancer and its mechanism. Methods: The expressions of miR-29b in 50 tissue specimens of cervical cancer and the corresponding adjacent paracancerous tissues were detected by real-time fluorescent quantitative PCR. The relationship of miR-29b expression and clinicopathological features of patients with cervical cancer was analyzed. After the transfection of miR-29b mimics into cervival cancer SiHa cells, the effects of miR-29b overexpression on proliferation, cell cycle distribution and migration of SiHa cells were detected by cell count kit-8 (CCK-8), flow cytometry, wound-healing and Transwell assays, respectively. The target gene of miR-29b was predicted by Targetscan software. Then the interaction between miR-29b and its target gene in human cervical cancer cells was confirmed by dual-luciferase reporter gene system and Western blotting. The expression of target gene of miR-29b in cervical cancer tissues was detected by real-time fluorescent quantitative PCR. Results: Compared with the corresponding adjacent paracancerous tissues, the expression of miR-29b was down-regulated in cervical cancer tissues (P 2 = 0.225, P < 0.05) in cervical cancer SiHa cells. The expression level of CCND2 protein in cervical cancer tissues was higher than that in the corresponding adjacent paracancerous tissues (P < 0.05). Conclusion: MiR-29b is down-regulated in human cervical cancer tissues. MiR-29b may be served as a tumor-suppressor through down-regulating CCND2 expression, so it may be used as a new target for therapy and diagnosis of human cervical cancer.
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Os tumores de mama são caracterizados pela sua alta heterogeneidade. O câncer de mama é uma doença complexa, que possui o seu desenvolvimento fortemente influenciado por fatores ambientais, combinada a uma progressiva acumulação de mutações genéticas e desregulação epigenética de vias críticas. Alterações nos padrões de expressão gênica podem ser resultado de uma desregulação no controle de eventos epigenéticos, assim como, na regulação pós-transcricional pelo mecanismo de RNA de interferência endógeno via microRNA (miRNA). Estes eventos são capazes de levar à iniciação, à promoção e à manutenção da carcinogênese, como também ter implicações no desenvolvimento da resistência à terapia Os miRNAs formam uma classe de RNAs não codificantes, que durante os últimos anos surgiram como um dos principais reguladores da expressão gênica, através da sua capacidade de regular negativamente a atividade de RNAs mensageiros (RNAms) portadores de uma seqüencia parcialmente complementar. A importância da regulação mediada por miRNAs foi observada pela capacidade destas moléculas em regular uma vasta gama de processos biológicos incluindo a proliferação celular, diferenciação e a apoptose. Para avaliar a expressão de miRNAs durante a progressão tumoral, utilizamos como modelo experimental a série 21T que compreende 5 linhagens celulares originárias da mesma paciente diagnosticada com um tumor primário de mama do tipo ErbB2 e uma posterior metástase pulmonar. Essa série é composta pela linhagem obtida a partir do tecido normal 16N, pelas linhagens correspondentes ao carcinoma primário 21PT e 21NT e pelas linhagens obtidas um ano após o diagnóstico inicial, a partir da efusão pleural no sítio metastatico 21MT1 e 21MT2. O miRNAoma da série 21T revelou uma redução significativa nos níveis de miR-205 e nos níveis da proteina e-caderina e um enriquecimento do fator pró-metastático ZEB-1 nas células 21MT...
Breast tumors are characterized by their high heterogeneity. It is a complex disease, which has its development strongly influenced by environmental factors, combined with a progressive accumulation of genetic mutations and epigenetic dysregulation of critical pathways. Changes in gene expression patterns may be a result of a deregulation in epigenetic events as well as in post-transcriptional regulation driven by RNA interference endogenously represented by microRNA (miRNA) these mechanisms are capable to promote the initiation, maintenance and progression of carcinogenesis; they are also implicated on the development of therapy resistance. miRNAs form a class of non-coding RNAs which have emerged in recent years as one of the major regulators of gene expression through its capacity to silence messenger RNAs (mRNAs) containing a partially complementary sequence. The importance of regulation mediated by miRNAs was observed on their ability to regulate a wide range of biological processes including cell proliferation, differentiation and apoptosis.To gain insights into the mechanisms involved in breast cancer initiation and progression conducted a miRNA global expression on 21T series that are an in vitro model of breast cancer progression comprising cell lines derived from the same patient which include a normal epithelia (16N), primary in situ ductal carcinoma (21PT and 21NT) and cells derived from pleural effusion of lung metastasis (21MT-1 and 21MT-2)...