Target Prediction Programs Research Articles

Dysregulated genes targeted by microRNAs and metabolic pathways in bladder cancer revealed by bioinformatics methods.

The present study aimed to identify bladder cancer-associated microRNAs (miRNAs) and target genes, and further analyze the potential molecular mechanisms involved in bladder cancer. The mRNA and miRNA expression profiling dataset GSE40355 was downloaded from the Gene Expression Omnibus database. The Limma package in R was used to identify differential expression levels. The Human microRNA Disease Database was used to identify bladder cancer-associated miRNAs and Target prediction programs were used to screen for miRNA target genes. Enrichment analysis was performed to identify biological functions. The Database for Annotation, Visualization and Integration Discovery was used to perform OMIM_DISEASE analysis, and then protein-protein interaction (PPI) analysis was performed to identify hubs with biological essentiality. ClusterONE plugins in cytoscape were used to screen modules and the InterPro database was used to perform protein domain enrichment analysis. A group of 573 disease dysregulated genes were identified in the present study. Enrichment analysis indicated that the muscle organ development and vascular smooth muscle contraction pathways were significantly enriched in terms of disease dysregulated genes. miRNAs targets (frizzled class receptor 8, EYA transcriptional coactivator and phosphatase 4, sacsin molecular chaperone, calcium voltage-gated channel auxiliary subunit β2, peptidase inhibitor 15 and catenin α2) were mostly associated with bladder cancer. PPI analysis revealed that calmodulin 1 (CALM1), Jun proto-oncogene, AP-1 transcription factor subunit (JUN) and insulin like growth factor 1 (IGF1) were the important hub nodes. Additionally, protein domain enrichment analysis indicated that the serine/threonine protein kinase active site was enriched in module 1 extracted from the PPI network. Overall, the results suggested that the IGF signaling pathway and RAS/MEK/extracellular signal-regulated kinase transduction signaling may exert vital molecular mechanisms in bladder cancer, and that CALM1, JUN and IGF1 may be used as novel potential therapeutic targets.

Identifying Regulatory Targets of the Small RNA MtlS in Vibrio cholerae

In the facultative pathogen Vibrio cholerae, MtlS is a small RNA (sRNA) transcribed antisense to the 5′ untranslated region of mtlA, which codes for the transporter of the sugar alcohol mannitol. MtlS works in cis as a translational repressor of mtlA and it is postulated that the sRNA allows the bacteria to respond rapidly to changes in environmental carbon sources. Because bacterial sRNAs can target multiple genes, we hypothesized that MtlS may regulate other genes in addition to mtlA. Previous work in the lab used RNA target‐prediction programs and mass spectrometry‐based proteomics to identify additional MtlS targets. The results identified 6 potential targets: VCA1045 (mtlA), VCA0820, VCA1046, VC1560, VC1899, and VC2683. The objective of this study was to determine if the putative targets were post‐transcriptionally regulated by MtlS. We constructed gfp translational reporter fusions of the candidate genes in a V. cholerae ΔmtlS strain carrying either a control vector or a plasmid ectopically expressing mtlS. An mtlA‐GFP fusion served as a positive control and exhibited the expected down‐regulation of gfp expression when mtlS was expressed. We have tested reporter fusions to all the candidate genes for regulation by MtlS by monitoring total cell fluorescence and western blot analysis. In an experiment done in duplicate in the presence of MtlS, during stationary phase growth we observed 20% down‐regulation of VC1899, encoding a protein with homology to nucleic acid‐binding proteins. In an experiment done in duplicate, we have also observed 30% logarithmic and stationary phase downregulation of VCA1046, encoding the mannitol dehydrogenase mtlD, in the presence of MtlS. Results based on another experiment conducted in duplicate indicate 20% logarithmic and stationary phase down‐regulation of VC1560, encoding a protein with homology to a catalase‐peroxidase, in the presence of MtlS. Tests with VC2683, encoding a putative cystathionine gamma‐synthase, have been inconclusive, as both down‐regulation and up‐regulation by MtlS has been observed; further experiments will need to be conducted with the VC2683‐gfp fusion. The VCA0820‐gfp fusion does not exhibit any fluorescence, in the presence or absence of MtlS. This work positively contributes to the notion that a single sRNA can have multiple regulatory targets. Ultimately, this work will expand our knowledge on MtlS's role in helping V. cholerae adapt quickly to changes in environmental carbon sources and on sRNAs in general.Support or Funding InformationNIH R15 AI090606This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Validation of a microRNA target site polymorphism in H3F3B that is potentially associated with a broad schizophrenia phenotype.

Despite much progress, few genetic findings for schizophrenia have been assessed by functional validation experiments at the molecular level. We previously reported evidence for genetic linkage of broadly defined schizophrenia to chromosome 17q25 in a sample of 24 multiplex families. 2,002 SNPs under this linkage peak were analyzed for evidence of linkage disequilibrium using the posterior probability of linkage (PPL) framework. SNP rs1060120 produced the strongest evidence for association, with a PPLD|L score of 0.21. This SNP is located within the 3'UTR of the histone gene H3F3B and colocalizes with potential gene target miR-616. A custom miRNA target prediction program predicted that the binding of miR-616 to H3F3B transcripts would be altered by the allelic variants of rs1060120. We used dual luciferase assays to experimentally validate this interaction. The rs1060120 A allele significantly reduced luciferase expression, indicating a stronger interaction with miR-616 than the G allele (p = 0.000412). These results provide functional validation that this SNP could alter schizophrenia epigenetic mechanisms thereby contributing to schizophrenia-related disease risk.

Identification of microRNAs that target PD-L1 in mismatch repair-deficient colorectal cancer.

85 Background: Programmed cell death 1 (PD-1) / PD-ligand 1 (PD-L1) immune checkpoint blockade has emerged as a promising therapeutic strategy in various types of cancer. Here we report that certain microRNAs (miRNAs) are involved in immunosuppressive microenvironment by directly targeting PD-L1 in mismatch repair deficient (dMMR) colorectal cancer (CRC). Methods: We identified candidate miRNAs by using RNA-sequence analyses for mRNA and miRNA expression obtained from The Cancer Genome Atlas (TCGA) Colon Adenocarcinoma combined with miRNA target prediction programs. HCT116 and SW837 CRC cell lines were transfected with miRNA mimics and inhibitors, and PD-L1 expression was examined by qRT-PCR, western blotting and flow cytometry. The CRC cell lines were co-cultured with activated T cells and then cytotoxicity for T cells were evaluated. Results: Using miRNA expression profiles of 260 tumors obtained through TCGA Colon Adenocarcinoma, 47 miRNA probes were found to be inversely correlated with PD-L1 expression. Among them, 19 mature miRNAs were down-regulated in dMMR tumors. Furthermore, eight in silico miRNA-target prediction programs were utilized to identify candidate miRNAs that target the 3’UTR of PD-L1 mRNA. We found that forced miRNA expression decreased PD-L1 expression in protein (total and surface) and mRNA levels, and they also dramatically decreased IFN-induced cell surface PD-L1 expression by 32% in CRC cell lines. Furthermore, we found that forced miRNA expression decreased PD-L1 associated apoptotic cell death in co-cultured activated T cells. Conclusions: Our findings suggest that PD-L1 expression is at least in part regulated by miRNAs and may also suggest potential miRNAs to serve as biomarkers and therapeutic targets for cancer immunotherapy.

LINC01121 Inhibits Cell Apoptosis While Facilitating Proliferation, Migration, and Invasion Though Negative Regulation of the Camp/PKA Signaling Pathway via GLP1R

Background/Aims: Pancreatic cancer is an aggressive malignancy as a result of highly metastatic potential. The current study was carried out to alter the expression of LINC01121 in pancreatic cancer, with the aim of elucidating its effects on the biological processes of cell proliferation, migration, invasion, and apoptosis. We hypothesized that both the GLP1R gene and cAMP/PKA signaling pathway participate in the aforementioned process. Methods: Microarray data (GSE14245, GSE27890 and GSE16515) and annotating probe files linked to pancreatic cancer were downloaded through the GEO database. The Multi Experiment Matrix (MEM) site was used to predict the target gene of lncRNA. Both pancreatic cancer tissues (n = 56) and paracancerous tissues (n = 45) were collected from patients diagnosed with pancreatic cancer. Immunohistochemistry was applied to identify the positive expression rate of GLP1R protein. Isolated pancreatic cancer cells and PANC-1 cells were independently classified into the blank, negative control (NC), LINC01121 vector, siRNA-LINC01121, siRNA-GLP1R and siRNA-LINC01121 + siRNA-GLP1R groups. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis were applied to detect the expressions of LINC01121, GLP1R, cAMP, PKA, CREB, Bcl-2, Bad and PCNA. Cell proliferation, migration, invasion, cycle progression, and apoptosis were examined by MTT assay, scratch test, Transwell assay and flow cytometry analyses of Annexin V-FITC/PI staining. Results: Observations were made indicating that LINC01121 was highly expressed, while low expressions of GLP1R in pancreatic cancer were detected based on microarray data, which was largely in consistent with the data collected of LINC01121 and GLP1R within the tissues. The target prediction program and luciferase activity analysis was testament to the notion suggesting that GLP1R was indeed a target of LINC01121. In contrast to the blank and NC groups, the LINC01121 vector group exhibited increased expressions of LINC01121; decreased mRNA and protein levels of GLP1R, Bad, cAMP, and PKA; increased protein levels of CREB, Bcl-2, PCNA, p-PKA and p-CREB; increased cell proliferation, migration and invasion; and decreased cell apoptosis. There was no significant difference detected among the blank, NC, and siRNA-LINC01121 + siRNA-GLP1R groups, except that decreased LINC01121 expression was determined in the siRNA-LINC01121 + siRNA-GLP1R group. Parallel data were observed in the pancreatic cancer cells and PANC-1 cells. Conclusion: The current study presents evidence indicating that LINC01121 might inhibit apoptosis while acting to promote proliferation, migration, and invasion of pancreatic cancer cells, supplementing the stance held that LINC01121 functions as a tumor promoter by means of its involvement in the process of translational repression of the GLP1R and inhibition of the cAMP/PKA signaling pathway.

Inhibition of microRNA-376b Protects Against Renal Interstitial Fibrosis via Inducing Macrophage Autophagy by Upregulating Atg5 in Mice with Chronic Kidney Disease

Background/Aims: Renal interstitial fibrosis (RIF) is a common feature that facilitates the progression of chronic kidney disease (CKD), and emerging lines of evidence suggest that microRNA-376b (miR-376b) is capable of promoting RIF. In this study, we examined collagen deposition in kidney tissues, the autophagy and mitochondrial reactive oxygen species (ROS) of macrophages, and the apoptosis of kidney fibroblasts (KFBs) after the promotion or suppression of endogenous miR-376b in cultured macrophages and renal fibroblasts obtained from mice with CKD. Methods: FVB/N mice were prepared to establish a CKD model. A target prediction program and luciferase activity determination were used to confirm that autophagy-related gene 5 (Atg5) was a direct target of miR-376b. Macrophages and KFBs were isolated after the treatment to study the mechanisms and functions of miR-376b in relation to Atg5 in CKD. The autophagy level was determined, and KFB proliferation and apoptosis were assessed through MTT and EdU assays and flow cytometry, respectively. Results: Atg5 was confirmed as a direct target of miR-376b. miR-376b and Atg5 exhibited high and low expression in kidney tissues from mice with CKD. The mice treated with a miR-376b inhibitor exhibited reduced collagen deposition, suppressed interstitial fibrosis, a higher level of autophagy, higher ROS production, enhanced apoptosis, and inhibited proliferation of KFBs, which suggested that the downregulation of miR-376b could exert beneficial effects on CKD through Atg5. Conclusion: miR-376b downregulation promotes macrophage autophagy to relieve RIF by negatively regulating Atg5 in mice with CKD. Thus, miR-376b might represent a potential focus of future investigations on treatments for CKD.

Recent Molecular Genetic Explorations of Caenorhabditis elegans MicroRNAs.

MicroRNAs are small, noncoding RNAs that regulate gene expression at the post-transcriptional level in essentially all aspects of Caenorhabditis elegans biology. More than 140 genes that encode microRNAs in C. elegans regulate development, behavior, metabolism, and responses to physiological and environmental changes. Genetic analysis of C. elegans microRNA genes continues to enhance our fundamental understanding of how microRNAs are integrated into broader gene regulatory networks to control diverse biological processes, including growth, cell division, cell fate determination, behavior, longevity, and stress responses. As many of these microRNA sequences and the related processing machinery are conserved over nearly a billion years of animal phylogeny, the assignment of their functions via worm genetics may inform the functions of their orthologs in other animals, including humans. In vivo investigations are especially important for microRNAs because in silico extrapolation of their functions using mRNA target prediction programs can easily assign microRNAs to incorrect genetic pathways. At this mezzanine level of microRNA bioinformatic sophistication, genetic analysis continues to be the gold standard for pathway assignments.

Role of microRNA-33a in regulating the expression of PD-1 in lung adenocarcinoma

BackgroundMiRNAs are vital in functioning as either oncogenes or tumor suppressors in the cell cycle. Target transcripts for immune checkpoint molecules such as PD-1/PD-L1 and (programmed cell death-1/its ligand and cytotoxic T-lymphocyte antigen 4) have proven to be beneficial against several solid tumors, including lung adenocarcinoma.MethodsSimultaneous quantification of the expression level of miR-33a and PD-1, PD-L1 and CTLA4 mRNAs with NanoString technology was performed in 88 lung adenocarcinoma specimens. A cohort of 323 lung adenocarcinoma patients from the cancer genome atlas (TCGA) database was further analyzed, in order to test our hypothesis. Potential interference of PD-1, PD-L1 and CTLA4 gene expression by miR-33a was predicted using the microRNA target prediction program RNA22.ResultsHigh miR-33a expression was significantly associated with younger (p = 0.005), female (p = 0.04), patients with low grade (p < 0.0001), early stage (p = 0.03) tumors, and better survival. The hypothesis of the involvement of miR-33a in PD-1/PD-L1/CTLA4 mechanisms was corroborated by the finding of putative miR-33a binding sites in all three genes using the RNA22 method. We found an inverse correlation between miR-33a and PD-1 levels (p = 0.01), as well as for PD-L1 (p = 0.01) and CTLA4 (p = 0.03) expression, and a significant better prognosis for patients with high miR-33a/low PD-1. TCGA database analysis confirmed that miR-33a high levels were associated with low PD-1 expression and with longer survival on a larger population.ConclusionsOur study emphasizes the notion of a potential value of miR-33a as a favorable prognostic marker through PD-1 regulation.

Arsenic Induces Members of the mmu-miR-466-669 Cluster Which Reduces NeuroD1 Expression.

Chronic arsenic exposure can result in adverse development effects including decreased intellectual function, reduced birth weight, and altered locomotor activity. Previous in vitro studies have shown that arsenic inhibits stem cell differentiation. MicroRNAs (miRNAs) are small noncoding RNAs that regulate multiple cellular processes including embryonic development and cell differentiation. The purpose of this study was to examine whether altered miRNA expression was a mechanism by which arsenic inhibited cellular differentiation. The pluripotent P19 mouse embryonal carcinoma cells were exposed to 0 or 0.5 μM sodium arsenite for 9 days during cell differentiation, and changes in miRNA expression was analyzed using microarrays. We found that the expression of several miRNAs important in cellular differentiation, such as miR-9 and miR-199 were decreased by 1.9- and 1.6-fold, respectively, following arsenic exposure, while miR-92a, miR-291a, and miR-709 were increased by 3-, 3.7-, and 1.6-fold, respectively. The members of the miR-466-669 cluster and its host gene, Scm-like with 4 Mbt domains 2 (Sfmbt2), were significantly induced by arsenic from 1.5- to 4-fold in a time-dependent manner. Multiple miRNA target prediction programs revealed that several neurogenic transcription factors appear to be targets of the cluster. When consensus anti-miRNAs targeting the miR-466-669 cluster were transfected into P19 cells, arsenic-exposed cells were able to more effectively differentiate. The consensus anti-miRNAs appeared to rescue the inhibitory effects of arsenic on cell differentiation due to an increased expression of NeuroD1. Taken together, we conclude that arsenic induces the miR-466-669 cluster, and that this induction acts to inhibit cellular differentiation in part due to a repression of NeuroD1.

MiR-422a inhibits cell proliferation in colorectal cancer by targeting AKT1 and MAPK1

BackgroundmiRNAs are regarded as molecular biomarkers and therapeutic targets for colorectal cancer (CRC), a series of miRNAs have been proven to involve into CRC carcinogenesis, invasion and metastasis. Aberrant miR-422a expression and its roles have been reported in some cancers. However, the function and underlying mechanism of miR-422a in the progression of CRC remain largely unknown.MethodsReal-time PCR were used to quantify miR-422a expression in CRC tissues. Both vivo and vitro functional assays showed miR-422a inhibits CRC cell proliferation. Target prediction program (miRBase) and luciferase reporter assays were conducted to confirm the target genes AKT1 and MAPK1 of miR-422a. Specimens from 50 patients with CRC were analyzed for the correlation between the expression of miR-422a and the expression of the target genes AKT1 and MAPK1 by real-time PCR.ResultsMiR-422a was down‑regulated in CRC tissues and cell lines. Ectopic expression of miR-422a inhibited cell proliferation and tumor growth ability; inhibition of endogenous miR-422a, by contrast, promoted cell proliferation and tumor growth ability of CRC cells. MiR-422a directly targets 3′-UTR of the AKT1 and MAPK1, down-regulation of miR-422a led to the activation of Raf/MEK/ERK and PI3K/AKT signaling pathways to promote cell proliferation in CRC. In addition, miR-422a expression was negatively correlated with the expressions of AKT1 and MAPK1 in CRC tissues.ConclusionmiR-422a inhibits cell proliferation in colorectal cancer by targeting AKT1 and MAPK1.

An integrated bioinformatical analysis of miR-19a target genes in multiple myeloma.

MicroRNA (miR)-19a, as an oncomiR, has been studied in several types of cancer; however, its role in the development and progression of multiple myeloma (MM) remains unclear. The present study used a bioinformatics approach to investigate the involvement of miR-19a in MM. miR-19a targets were predicted using target prediction programs, followed by screening for differentially expressed genes in MM. The function of these genes was then annotated using gene ontology term enrichment, signaling pathway enrichment and protein-protein interaction (PPI) analysis. In addition, natural language processing (NLP) was performed to identify genes associated with MM. A total of 715 putative targets of miR-19a were identified in the present study, of which 40 were experimentally validated. A total of 121 genes were identified to be differentially expressed in MM, including 80 upregulated genes and 41 downregulated genes. Among the differentially expressed genes, ras homolog family member B, clathrin heavy chain, prosaposin and protein phosphatase 6 regulatory subunit 2 were predicted target genes of miR-19a. The results of NLP revealed that 2 of the differentially expressed genes, Y-box binding protein 1 and TP53 regulated inhibitor of apoptosis 1, were reported to be associated with MM. In addition, 41 target genes of miR-19a were identified to be associated with the development and progression of MM. These results may aid in understanding the molecular mechanisms of miR-19a in the development and progression of MM. In addition, the results of the present study indicate that targets genes of miR-19a are potential candidate biomarkers for MM.

Abstract 2522: MiRNA-652 induces neuroendocrine-like differentiation in LNCaP prostate cancer cells through decreased PP2A function

Abstract Background: MicroRNA (miRNA) dysregulation has been shown to contribute to prostate cancer progression. Recently, we identified a panel of five miRNAs associated with prostate cancer recurrence and metastasis using next generation miRNA sequencing. Here, we examine the clinical implications of dysregulation of miR-652 and its biological mechanism. Methods: Using a cohort of 585 patients treated with radical prostatectomy, we examined the prognostic significance of miR-652 using the Kaplan Meier method and Cox proportional hazard models. We examined miR-652 expression in prostate cancer cells and created cell lines that overexpressed miR-652 for functional assays. Finally, we examined pathways through which miR-652 may function using prediction algorithms, and confirmed by Western blotting, IHC and luciferase reporter assays. Results: Patients overexpressing miR-652 had significantly increased rates of biochemical recurrence (p&amp;lt;0.0001). In multivariable models adjusting for known clinical prognostic factors, patients with high miR-652 expression had an increased risk of biochemical recurrence (HR 1.47, 95% CI 1.09-1.98). Overexpression of miR-652 in PC3 and LNCaP cells resulted in increased growth, migration and invasion. Prostate cancer cell xenografts overexpressing miR-652 had increased tumorigenicity and metastases. Using the miRNA target prediction program miRanda, we identified the B” regulatory subunit, PPP2R3A, of the tumor suppressor PP2A as a potential target of miR-652. PC3 and LNCaP cells transfected with miR-652 mimic dowregulated the PR72 isoform of PPP2R3A. Mutation of the miR-652 binding site in the PPP2R3A gene negated this effect. Western blotting demonstrated that mir-652 induced PPR2R3A inhibition induced epithelial-mesenchymal transition (EMT) in PC3 cells. Prolonged miR-652 expression in LNCaP cells induced a neurite-like morphology, suggesting neurodocrine-like differentiation (NED). Western blotting of LNCaP cells after prolonged miR-652 exposure, resulted in increased phospho-AKT and phospho-β-catenin (phosphorylated at Serine 552), reduced AR expression, and upregulation of the NED marker, neuron specific enolase (NSE), consistent with NED. Expression of NED markers, chromogranin A, NSE, and synaptophysin were also observed in a xenografted tumor derived from LNCaP-652 overexpressing cells. Conclusion: These observations suggest that increased levels of miR-652 found in prostate cancer may contribute to tumor progression by promoting NED, tumor cell survival and cell migration/invasion through decreased PP2A function which may provide an opportunity for novel therapy in prostate cancer. Citation Format: Tania Benatar, Yutaka Amemiya, Christopher J. Wallis, Linda Sugar, Christopher Sherman, Robert Nam, Arun K. Seth. MiRNA-652 induces neuroendocrine-like differentiation in LNCaP prostate cancer cells through decreased PP2A function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2522. doi:10.1158/1538-7445.AM2017-2522

Puerarin promotes the viability and differentiation of MC3T3‑E1 cells by miR‑204‑regulated Runx2 upregulation.

Puerarin has attracted increasing attention because of its beneficial effects on anti‑osteoporosis, but the molecular mechanisms underlying its actions on osteoblasts are not fully understood. The current study aimed to investigate the effect of puerarin on the cell viability and differentiation of mouse MC3T3‑E1 osteoblast‑like cells invitro and its underlying mechanisms. The results indicated that 0.01, 0.1 and 1mg/ml puerarin significantly promoted the viability of osteoblasts, enhanced alkaline phosphatase (ALP) activity and increased the expression of transforming growth factor‑β1, Smad2, Smad3 and Runt‑related transcription factor (Runx)2. Micro (mi)RNA target prediction programs predicted that miR‑204 may directly target Runx2. Following treatment with 0.1mg/ml puerarin for 48h, the expression level of miR‑204 was downregulated. Besides, miR‑204 dramatically repressed the luciferase activity of wildtype Runx2 3'‑UTR transfected cells, but not that of the mutant ones. Overexpression of miR‑204 in osteoblasts significantly decreased the protein expression of Runx2, while inhibition of miR‑204 enhanced Runx2's expression. In addition, overexpression of miR‑204 inhibited the cell viability and ALP activity of osteoblasts, while inhibition of miR‑204 had the opposite effect. The results suggested that puerarin may promote MC3T3‑E1 osteoblast‑like cell viability and differentiation, which may be related to the downregulation of miR‑204 and the following activation of Runx2.

Evaluation of circulating miRNAs during late pregnancy in the mare.

MicroRNAs (miRNAs) are small, non-coding RNAs which are produced throughout the body. Individual tissues tend to have a specific expression profile and excrete many of these miRNAs into circulation. These circulating miRNAs may be diagnostically valuable biomarkers for assessing the presence of disease while minimizing invasive testing. In women, numerous circulating miRNAs have been identified which change significantly during pregnancy-related complications (e.g. chorioamnionitis, eclampsia, recurrent pregnancy loss); however, no prior work has been done in this area in the horse. To identify pregnancy-specific miRNAs, we collected serial whole blood samples in pregnant mares at 8, 9, 10 m of gestation and post-partum, as well as from non-pregnant (diestrous) mares. In total, we evaluated a panel of 178 miRNAs using qPCR, eventually identifying five miRNAs of interest. One miRNA (miR-374b) was differentially regulated through late gestation and four miRNAs (miR-454, miR-133b, miR-486-5p and miR-204b) were differentially regulated between the pregnant and non-pregnant samples. We were able to identify putative targets for the differentially regulated miRNAs using two separate target prediction programs, miRDB and Ingenuity Pathway Analysis. The targets for the miRNAs differentially regulated during pregnancy were predicted to be involved in signaling pathways such as the STAT3 pathway and PI3/AKT signaling pathway, as well as more endocrine-based pathways, including the GnRH, prolactin and insulin signaling pathways. In summary, this study provides novel information about the changes occurring in circulating miRNAs during normal pregnancy, as well as attempting to predict the biological effects induced by these miRNAs.

Down-Regulation of MicroRNA-210 Confers Sensitivity towards 1'S-1'-Acetoxychavicol Acetate (ACA) in Cervical Cancer Cells by Targeting SMAD4.

MicroRNAs (miRNAs) are short non-coding RNAs that regulate genes posttranscriptionally. Past studies have reported that miR-210 is up-regulated in many cancers including cervical cancer, and plays a pleiotropic role in carcinogenesis. However, its role in regulating response towards anti-cancer agents has not been fully elucidated. We have previously reported that the natural compound 1’S-1’-acetoxychavicol acetate (ACA) is able to induce cytotoxicity in various cancer cells including cervical cancer cells. Hence, this study aims to investigate the mechanistic role of miR-210 in regulating response towards ACA in cervical cancer cells. In the present study, we found that ACA down-regulated miR-210 expression in cervical cancer cells, and suppression of miR-210 expression enhanced sensitivity towards ACA by inhibiting cell proliferation and promoting apoptosis. Western blot analysis showed increased expression of mothers against decapentaplegic homolog 4 (SMAD4), which was predicted as a target of miR-210 by target prediction programs, following treatment with ACA. Luciferase reporter assay confirmed that miR-210 binds to sequences in 3′UTR of SMAD4. Furthermore, decreased in SMAD4 protein expression was observed when miR-210 was overexpressed. Conversely, SMAD4 protein expression increased when miR-210 expression was suppressed. Lastly, we demonstrated that overexpression of SMAD4 augmented the anti-proliferative and apoptosis-inducing effects of ACA. Taken together, our results demonstrated that down-regulation of miR-210 conferred sensitivity towards ACA in cervical cancer cells by targeting SMAD4. These findings suggest that combination of miRNAs and natural compounds could provide new strategies in treating cervical cancer.

Suppression of microRNA-629 enhances sensitivity of cervical cancer cells to 1'S-1'-acetoxychavicol acetate via regulating RSU1.

BackgroundCervical cancer is the fourth most frequent malignancy affecting women worldwide, but drug resistance and toxicities remain a major challenge in chemotherapy. The use of natural compounds is promising because they are less toxic and able to target multiple signaling pathways. The 1′S-1′-acetoxychavicol acetate (ACA), a natural compound isolated from wild ginger Alpinia conchigera, induced cytotoxicity on various cancer cells including cervical cancer. MicroRNAs (miRNAs) are short noncoding RNAs that regulate numerous biological processes, such as apoptosis and chemosensitivity. Past studies reported that miR-629 is upregulated in many cancers, and its expression was altered in ACA-treated cervical cancer cells. However, the role of miR-629 in regulating sensitivity toward ACA or other anticancer agents has not been reported. Hence, this study aims to investigate the role of miR-629 in regulating response toward ACA on cervical cancer cells.MethodsThe miR-629 expression following transfection with miR-629 hairpin inhibitor and hairpin inhibitor negative control was measured using quantitative real-time polymerase chain reaction (RT-qPCR). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to investigate sensitivity toward ACA. Apoptosis was detected using Annexin V/propidium iodide and Caspase 3/7 assays. The gene target for miR-629 was identified using miRNA target prediction programs, luciferase reporter assay and Western blots. Gene overexpression studies were performed to evaluate its role in regulating response toward ACA.ResultsTransfection with miR-629 hairpin inhibitor downregulated its expression in both cervical cancer cell lines. Suppression of miR-629 increased sensitivity toward ACA by reducing cell proliferation and inducing apoptosis. Luciferase reporter assay confirmed RSU1 as a direct target of miR-629. Overexpression of miR-629 decreased RSU1 protein expression, while inhibition of miR-629 increased RSU1 protein expression. Overexpression of RSU1 augmented antiproliferative and apoptosis-inducing effects of ACA.ConclusionOur findings showed that combination of ACA with miR-629 and RSU1 may provide a potential strategy in treating cervical cancer.

MiR-455-5p functions as a potential oncogene by targeting galectin-9 in colon cancer.

Although there is evidence that galectin-9 is a critical factor in health and disease, the upstream regulatory microRNA (miRNA or miR) of the protein remains poorly defined. miR-455-5p is characterized as a tumor-associated miRNA in cancer research. However, the actual role of miR-455-5p with respect to inhibiting or promoting tumorigenesis in colon cancer is unclear. The present study aimed to investigate the expression, role and target regulation association of galectin-9 and miR-455-5p in colon cancer. Western blot analysis and reverse transcription-quantitative polymerase chain reaction were used for the detection of the expression levels of galectin-9 and miRNAs. Cell Counting kit-8 test was used for the evaluation of cell proliferation, while flow cytometry was used for cell apoptosis analysis. A potential interaction between galectin-9 and miR-455-5p was predicted by target prediction programs and confirmed by luciferase assay and transfection with miRNA mimics. The present study revealed that elevated expression of galectin-9 and miR-455-5p in colon cancer was associated with HT29 cell proliferation and apoptosis. Furthermore, the present study demonstrated that miR-455-5p reduced galectin-9 expression by directly targeting its 3'-untranslated region. These data suggest that miR-455-5p functions as a potential oncogene in colon cancer by targeting galectin-9.

Epigallocatechin-3-gallate Modulates MicroRNA Expression Profiles in Human Nasopharyngeal Carcinoma CNE2 Cells.

Background:Epigallocatechin-3-gallate (EGCG) has exhibited antitumor properties in several types of cancers, including nasopharyngeal carcinoma (NPC), but the molecular mechanisms underlying this function remain incompletely understood. The aim of the present study was to characterize the global impact of EGCG on the expression of microRNAs (miRNAs) in NPC cells.Methods:Using microarray analysis, the alterations of miRNA expression profiles were investigated in EGCG-treated CNE2 cells. Furthermore, the target genes and signaling pathways regulated by EGCG-specific miRNAs were identified using target prediction program and gene ontology analysis.Results:A total of 14 miRNAs exhibited >2-fold expression changes in a dose-dependent manner after treatment with 20 μmol/L and 40 μmol/L EGCG. Totally 43, 49, and 52 target genes from these differentially expressed miRNAs were associated with the apoptosis, cell cycle regulation, and cell proliferation, respectively. A total of 66 signaling pathways, primarily involved in cancer development and lipid and glucose metabolism, were shown to be regulated by EGCG-specific miRNAs.Conclusion:EGCG induces considerable alterations of miRNA expression profiles in CNE2 cells, which provides mechanistic insights into cellular responses and antitumor activity mediated by EGCG.

Expression profile of long noncoding RNAs in human cerebral aneurysms: a microarray analysis.

OBJECTIVE The pathogenesis of cerebral aneurysms (CAs) remains largely unknown. Long noncoding RNAs (lncRNAs) were reported recently to play crucial roles in many physiological and biological processes. Here, the authors compared the gene-expression profiles of CAs and their control arteries to investigate the potential functions of lncRNAs in the formation of CAs. METHODS A prospective case-control study was designed to identify the changes in expression of lncRNAs and mRNAs between 12 saccular CA samples (case group) and 12 paired superficial temporal artery samples (control group). Microarray analysis was performed to investigate the expression of lncRNAs and messenger RNAs (mRNAs), and reverse-transcription quantitative polymerase chain reaction was used to validate the microarray analysis findings. Then, an lncRNA target-prediction program and gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were applied to explore potential lncRNA functions. RESULTS A comparison between the case and control groups revealed that 1518 lncRNAs and 2545 mRNAs were expressed differentially. By using target-prediction program analysis, the authors constructed a complex network consisting of 2786 matched lncRNA-mRNA pairs, in which ine1 mRNA was potentially targeted by one to tens of lncRNAs, and vice versa. The results of further gene ontology and KEGG pathway analyses indicated that lncRNAs were involved mainly in regulating immune/inflammatory processes/pathways and vascular smooth muscle contraction, both of which are known to have crucial pathobiological relevance in terms of CA formation. CONCLUSIONS By comparing CAs with their control arteries, the authors created an expression profile of lncRNAs in CAs and propose here their possible roles in the pathogenesis of CAs. The results of this study provide novel insight into the mechanisms of CA pathogenesis and shed light on developing new therapeutic intervention for CAs in the future.

MicroRNA-194 suppresses prostate cancer migration and invasion by downregulating human nuclear distribution protein.

Human NudC nuclear distribution protein(hNUDC) is differentially expressed between normal and cancer cells. Based on its marked altered expression and its roles in modulating cell division, cytokineses and migration, a detailed understanding of the mechanisms regulating hNUDC expression in cancer cells is critical. In this study, we identified miR-194 as a downstream target of hNUDC and linked its expression to reduced metastatic capacity and tumorigenicity of prostate cancer(PCa) cells. Using miRNA target prediction programs, hNUDC mRNA was found to contain a potential binding site for miR-194 within its 3'UTR. A Reporter assay confirmed that post-transcriptional regulation of hNUDC was dependent on the miR-194 binding site. Forced expression of miR-194 in PCa cell lines, PC-3 and DU-145, led to a decrease in the mRNA and protein levels of hNUDC. Overexpression of miR-194 in these cells inhibited cell migration and invasion, and induced multinucleated cells. Our data showed that hNUDC knockdown by siRNA significantly reduced the migration and invasion in the PC-3 and DU-145 cells, phenocopying the results of miR-194 overexpression. Furthermore, lentivirus-mediated stable expression of miR-194 in PCa cells reduced the ability of colony formation as detected by a soft agar assay and exhibited significantly less tumorigenic ability invivo. Our results suggest a novel role for miR-194 in effectively controlling cell metastatic processes in PCa cells via the regulation of hNUDC expression.