Exploration of inhibitory mechanisms of curcumin in lung cancer metastasis using a miRNA- transcription factor-target gene network.
ABSTRACT: The present study was aimed to unravel the inhibitory mechanisms of curcumin for lung cancer metastasis via constructing a miRNA-transcription factor (TF)-target gene network. Differentially expressed miRNAs between human high-metastatic non-small cell lung cancer 95D cells treated with and without curcumin were identified using a TaqMan human miRNA array followed by real-time PCR, out of which, the top 6 miRNAs (miR-302b-3p, miR-335-5p, miR-338-3p, miR-34c-5p, miR-29c-3p and miR-34a-35p) with more verified target genes and TFs than other miRNAs as confirmed by a literature review were selected for further analysis. The miRecords database was utilized to predict the target genes of these 6 miRNAs, TFs of which were identified based on the TRANSFAC database. The findings of the above procedure were used to construct a miRNA-TF-target gene network, among which miR-34a-5p, miR-34c-5p and miR-302b-3p seemed to regulate CCND1, WNT1 and MYC to be involved in Wnt signaling pathway through the LEF1 transcription factor. Therefore, we suggest miR-34a-5p/miR-34c-5p/miR-302b-3p -LEF1-CCND1/WNT1/MYC axis may be a crucial mechanism in inhibition of lung cancer metastasis by curcumin.
Project description:The micro RNA (miR)-34 family is composed of 5p and 3p strands of miR-34a, miR-34b, and miR-34c. The 5p strand's expression and function is studied in cervical cancer. The 3p strand's function and regulation remain to be elucidated. To study the function of the passenger strands of miR-34 family members, we overexpressed 5p and 3p strands using a synthetic miRNA in cervical cell lines. Cell proliferation was evaluated using crystal violet. Migration and invasion were tested using transwell assays, Western blot, and zymography. Possible specific targets and cell signaling were investigated for each strand. We found that miR-34a-5p inhibited proliferation, migration, and cell invasion accompanied by matrix metalloproteinase 9 (MMP9) activity and microtubule-associated protein 2 (MAP2) protein reduction. We also found that miR-34b-5p and miR-34c-5p inhibit proliferation and migration, but not invasion. In contrast, miR-34c-5p inhibits MMP9 activity and MAP2 protein, while miR-34b-5p has no effect on these genes. Furthermore, miR-34a-3p and miR-34b-3p inhibit proliferation and migration, but not invasion, despite the later reducing MMP2 activity, while miR-34c-3p inhibit proliferation, migration, and cell invasion accompanied by MMP9 activity and MAP2 protein inhibition. The difference in cellular processes, MMP2 and MMP9 activity, and MAP2 protein inhibition by miR-34 family members suggests the participation of other regulated genes. This study provides insights into the roles of passenger strands (strand*) of the miR-34 family in cervical cancer.
Project description:An in depth investigation at the genomic level is needed to identify early human-relevant cardiotoxicity biomarkers that are induced by drugs and environmental toxicants. The main objective of this study was to investigate the role of microRNAs (miRNAs) as cardiotoxicity biomarkers using human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) that were exposed to doxorubicin (DOX) as a "gold standard" cardiotoxicant. hiPSC-CMs were exposed to 156 nM DOX for 2 days or for 6 days of repeated exposure, followed by drug washout and incubation in drug-free culture medium up to day 14 after the onset of exposure. The induced miRNAs were profiled using miRNA microarrays, and the analysis of the data was performed using the miRWalk 2.0 and DAVID bioinformatics tools. DOX induced early deregulation of 14 miRNAs (10 up-regulated and 4 down-regulated) and persistent up-regulation of 5 miRNAs during drug washout. Computational miRNA gene target predictions suggested that several DOX-responsive miRNAs might regulate the mRNA expression of genes involved in cardiac contractile function. The hiPSC-CMs exposed to DOX in a range from 39 to 156 nM did not show a significant release of the cytotoxicity marker lactate dehydrogenase (LDH) compared to controls. Quantitative real-time PCR analyses confirmed the early deregulation of miR-187-3p, miR-182-5p, miR-486-3p, miR-486-5p, miR-34a-3p, miR-4423-3p, miR-34c-3p, miR-34c-5p and miR-1303, and also the prolonged up-regulation of miR-182-5p, miR-4423-3p and miR-34c-5p. Thus, we identified and validated miRNAs showing differential DOX-responsive expression before the occurrence of cytotoxicity markers such as LDH, and these miRNAs also demonstrated the significant involvement in heart failure in patients and animal models. These results suggest that the DOX-induced deregulated miRNAs in human CMs may be used as early sensitive cardiotoxicity biomarkers for screening potential drugs and environmental cardiotoxicants with a similar mechanism of action.
Project description:Myotonic Dystrophy Type-2 (DM2) is an autosomal dominant disease caused by the expansion of a CCTG tetraplet repeat. It is a multisystemic disorder, affecting skeletal muscles, the heart, the eye, the central nervous system and the endocrine system. Since microRNA (miRNA) expression is disrupted in Myotonic Dystrophy Type-1 and many other myopathies, miRNAs deregulation was studied in skeletal muscle biopsies of 13 DM2 patients and 13 controls. Eleven miRNAs were deregulated: 9 displayed higher levels compared to controls (miR-34a-5p, miR-34b-3p, miR-34c-5p, miR-146b-5p, miR-208a, miR-221-3p and miR-381), while 4 were decreased (miR-125b-5p, miR-193a-3p, miR-193b-3p and miR-378a-3p). To explore the relevance of DM2 miRNA deregulation, the predicted interactions between miRNA and mRNA were investigated. Global gene expression was analyzed in DM2 and controls and bioinformatic analysis identified more than 1,000 miRNA/mRNA interactions. Pathway and function analysis highlighted the involvement of the miRNA-deregulated mRNAs in multiple aspects of DM2 pathophysiology. In conclusion, the observed miRNA dysregulations may contribute to DM2 pathogenetic mechanisms.
Project description:Hepatocyte nuclear factor (HNF)-4? is a key member of the transcription factor network regulating hepatocyte differentiation and function. Genetic and molecular evidence suggests that expression of HNF-4? is mainly regulated at the transcriptional level. Activation of HNF-4A gene involves the interaction of distinct sets of transcription factors and co-transcription factors within enhancer and promoter regions. Here we study the inhibitory effect of microRNAs (miRNAs) on the 3'-untranslated region (3'-UTR) of HNF-4A mRNA. The potential recognition elements of a set of miRNAs were identified utilizing bioinformatics analysis. The family members of miR-34 and miR-449, including miR-34a, miR-34c-5p and miR-449a, share the same target elements located at two distinct locations within the 3'-UTR of HNF-4A. The over-expression of miR-34a, miR-34c-5p or miR-449a in HepG2 cells led to a significant decrease in the activity of luciferase reporter carrying 3'-UTR of HNF-4A. The repressive effect on reporter activity was partially or fully eliminated when one or two of the binding site(s) for miR-34a/miR-34c-5p/miR-449a were deleted within the 3'-UTR. The protein level of HNF-4? was dramatically reduced by over-expression of miR-34a, miR-34c-5p and miR-449a, which correlates with a decrease in the binding activity of HNF-4? and transactivation of HNF-4? target genes. These results suggest that the recognition sites of miR-34a, miR-34c-5p and miR-449a within 3'-UTR of HNF-4A are functional. The mechanism of down-regulation of the binding activity and transactivation of HNF-4? by the miRNAs involves the decrease in HNF-4? protein level via miRNAs selectively targeting HNF-4A 3'-UTR, leading to the translational repression of HNF-4? expression.
Project description:This study aimed to identify specific microRNAs (miRNAs) related to postmenopausal osteoporosis (OP) in human. A total of 67 conserved miRNAs, including 50 miRNAs significantly up-regulated and 17 miRNAs significantly downregulated, showed differential expression between OP group and control group. 180 hairpin structures were predicted and 199 potential novel miRNA candidates with 18 to 25 nt in length, which will greatly enrich the human miRBase. 4 miRNAs (miR-518b, miR-582-3p, miR-148a-3p and miRNA-223-3p) had upregulated expression and 4 (miR-7d-5p, miR-210-3p, miR-324-5p and miR-654-3p) showed down-regulated expression. Target genes of these miRNAs were involved in bone development, cell proliferation in bone marrow, osteoblast development, negative regulation of osteoblast differentiation, and negative regulation of osteoclast development, as well as several osteogenesis related pathways. Canonical Wnt signaling pathway was selected for verification and function analysis. The expression of Wnt1, FZD10, LRP5, DVL2 and LEF1 was down-regulated significantly, while that of SFRP1, DKK1, and CHD8 was up-regulated markedly. In conclusion, these genes play important roles in OP, which improves our understanding of pathogenesis of OP.
Project description:WNT signaling is fundamental to bone health and its aberrant activation leads to skeletal pathologies. A heterozygous missense mutation p.C218G in WNT1, a key WNT pathway ligand, leads to severe early-onset osteoporosis with multiple peripheral and spinal fractures. Despite the severe skeletal manifestations, conventional bone markers are normal in mutation-positive patients. The objective of this study was to find novel biomarkers that differentiate between WNT1 mutation-positive and -negative subjects. We evaluated serum levels of 192 miRNAs in 12 mutation-positive (median age 39 years, range 11-76 years) and 12 mutation-negative (35 years, range 9-59 years) subjects from two Finnish families. The results indicate significant differences in circulating miRNA profiles with 2 upregulated (miR-18a-3p, miR-223-3p) and 6 downregulated miRNAs (miR-22-3p, miR-31-5p, miR-34a-5p, miR-143-5p miR-423-5p, miR 423-3p) in the mutation-positive subjects. Three of these (miR-22-3p, miR-34a-5p, and miR-31-5p) are known inhibitors of WNT signaling: miR-22-3p and miR-34a-5p target WNT1 mRNA and miR-31-5p is predicted to bind to the WNT1 3’UTR. Our results suggest that the WNT1 mutation disrupts a feed-back regulation between these miRNAs and WNT1, providing new insights into the pathogenesis of WNT-related bone disorders. Future studies are warranted to explore the potential diagnostic and therapeutic applications of these findings in osteoporosis. Overall design: Cross-sectional design comparing serum microRNA profiles of 12 subjects with heterozygous missense mutation p.C218G in WNT1 to a control group with wildtype WNT1.
Project description:Influenza is a serious worldwide disease that captures global attention in the past few years after outbreaks. The recent discoveries of microRNA (miRNA) and its unique expression profile in influenza patients have offered a new method for early influenza diagnosis. The aim of this study was to examine the utility of miRNAs for the diagnosis of influenza.Thirteen selected miRNAs were investigated with the hosts' throat swabs (25 H1N1, 20 H3N2, 20 influenza B and 21 healthy controls) by real-time quantitative polymerase chain reaction (RT-qPCR) using U6 snRNA as endogenous control for normalization, and receiver operating characteristic (ROC) curve/Area under curve (AUC) for analysis.miR-29a-3p, miR-30c-5p, miR-34c-3p and miR-181a-5p are useful biomarkers for influenza A detection; and miR-30c-5p, miR-34b-5p, miR-205-5p and miR-449b-5p for influenza B detection. Also, use of both miR-30c-5p and miR-34c-3p (AUC=0.879); and miR-30c-5p and miR-449b-5p (AUC=0.901) are better than using one miRNA to confirm influenza A and influenza B infection, respectively.Given its simplicity, non-invasiveness and specificity, we found that the throat swab-derived miRNAs miR-29a-3p, miR-30c-5p, miR-34b-5p, miR-34c-3p, miR-181a-5p, miR-205-5p and miR-449b-5p are a useful tool for influenza diagnosis on influenza A and B.
Project description:MicroRNAs are known to play essential role in the gene expression regulation in cancer. In our research, next-generation sequencing technology was applied to explore the abnormal miRNA expression of oral squamous cell carcinoma (OSCC) in Chinese hamster. A total of 3 novel miRNAs (Novel-117, Novel-118, and Novel-135) and 11 known miRNAs (crg-miR-130b-3p, crg-miR-142-5p, crg-miR-21-3p, crg-miR-21-5p, crg-miR-542-3p, crg-miR-486-3p, crg-miR-499-5p, crg-miR-504, crg-miR-34c-5p, crg-miR-34b-5p and crg-miR-34c-3p) were identified. We conducted functional analysis, finding that 340 biological processes, 47 cell components, 46 molecular functions were associated with OSCC. Meanwhile the gene expression of Caspase-9, Caspase-3, Bax, and Bcl-2 were determined by qRT-PCR and the protein expression of PTEN and p-AKT by immunohistochemistry. Our research proposed further insights to the profiles of these miRNAs and provided a basis for investigating the regulatory mechanisms involved in oral cancer research.
Project description:<b>Background</b>: Our previous studies reported that lymphoid enhancer-binding factor 1 (LEF1) was upregulated in esophageal squamous cell carcinoma (ESCC) and the positive expression of LEF1 was correlated with aberrant clinicopathological characteristics in ESCC patients. However, the upstream mechanism of regulating LEF1 is not clear fully. In this study, we explored the role of miR-34a-5p in ESCC and the possible regulatory mechanism. <b>Methods</b>: In this study, we applied western blotting, quantitative real-time polymerase chain reaction (qRT-PCR), bioinformatics analysis, a luciferase reporter assay, and a series of functional assays to show the potential role of miR-34a-5p in regulating LEF1 in ESCC. <b>Results</b>: By various functional assays, we demonstrated that LEF1 promoted proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) in ESCC cells. By bioinformatics analysis and luciferase reporter assay, miR-34a-5p was identified for directly targeting LEF1. Then we investigated the expression of miR-34a-5p and LEF1 in ESCC. As a result, miR-34a-5p was downregulated while LEF1 was upregulated in ESCC tissue and cell lines. Overexpression of miR-34a-5p could inhibit proliferation, migration, invasion and EMT of ESCC cells. The rescue experiment showed that re-expression of LEF1 reversed the suppressive effect caused by miR-34a-5p. At last, we found that miR-34a-5p could suppress Hippo-YAP1/TAZ signaling pathway in ESCC. <b>Conclusion</b>: Our results indicate miR-34a-5p inhibits proliferation, migration, invasion and EMT in ESCC by targeting LEF1 and suppressing the Hippo-YAP1/TAZ signaling pathway, which may provide a new antitumor strategy to delay ESCC progress.
Project description:<b>Background: </b>Untreated nephropathy can progress to renal failure. The traditional Mongolian remedy Narenmandula regulates the kidney "yang." This study aimed to identify key microRNAs (miRNAs) targeted by Narenmandula in a rat model of nephropathy.<br><br><b>Methods: </b>Fifteen rats exhibiting normal renal function were randomized to three study arms. Nephropathy was induced in <i>n</i>?=?10 rats using doxorubicin hydrochloride, followed by either Narenmandula treatment (treatment group) or no treatment (control group). In <i>n</i>?=?5 rats, no doxorubicin was given and renal function remained unchanged (healthy group). Microarray analysis identified miRNAs which were differentially expressed (DE-miRNAs) between groups. Target genes of DE-miRNAs were predicted using miRWalk version 2.0, followed by enrichment analysis using DAVID, and construction of the miRNA coregulatory network using Cytoscape.<br><br><b>Results: </b>Nephropathy was successfully induced, with doxorubicin resulting in differential expression of 3645 miRNAs (1324 upregulated and 2321 downregulated). Narenmandula treatment induced differential expression of a total of 159 miRNAs (102 upregulated and 57 downregulated). Upregulated DE-miRNAs (e.g., miR-497-5p, miR-195-5p, miR-181a-5p, miR-181c-5p, and miR-30e-5p) and downregulated DE-miRNAs (e.g., miR-330-3p and miR-214-3p) regulated a high number of target genes. Moreover, the miRNA pairs (e.g., miR-195-5p-miR-497-5p, miR-181a-5p-miR-181c-5p, and miR-30e-5p-miR-30a-5p) coregulated a high number of genes. Enrichment analysis indicated functional synergy between miR-30e-5p-miR-30a-3p, miR-34a-5p-miR-30e-5p, miR-30e-5p-miR-195-3p, and miR-30a-3p-miR-195-3p pairs.<br><br><b>Conclusion: </b>Narenmandula may modulate doxorubicin-induced nephropathy via targeting miR-497-5p, miR-195-5p, miR-181a-5p, miR-181c-5p, miR-30e-5p, miR-330-3p, miR-214-3p, miR-34a-5p, miR-30a-3p, and miR-30a-5p.