MicroRNA-5p and -3p co-expression and cross-targeting in colon cancer cells.
ABSTRACT: Two mature miRNA species may be generated from the 5' and 3' arms of a pre-miRNA precursor. In most cases, only one species remains while the complementary species is degraded. However, co-existence of miRNA-5p and -3p species is increasingly being reported. In this work, we aimed to systematically investigate co-expression of miRNA-5p/3p in colon cancer cells in a genome-wide analysis, and to examine cross-targeting of the dysregulated miRNAs and 5p/3p species.Four colon cancer cell lines were examined relative to two normal colon tissues. Of the 1,190 miRNAs analyzed, 92 and 36 were found to be up- or down-regulated, respectively, in cancer cells. Nineteen co-expressed miRNA-5p/3p pairs were further identified suggesting frequent 5p/3p co-accumulation in colon cancer cells. Of these, 14 pairs were co-up-regulated and 3 pairs were co-down-regulated indicating concerted 5p/3p dysregulation. Nine dysregulated miRNA pairs fell into three miRNA gene families, namely let-7, mir-8/200 and mir-17, which showed frequent cross-targeting in the metastasis process. Focusing on the let-7d-5p/3p pair, the respectively targeted IGF1R and KRAS were shown to be in a reverse relationship with expression of the respective miRNA, which was confirmed in transient transfection assays using let-7d mimic or inhibitor. Targeting of KRAS by let-7d was previous reported; targeting of IGF1R by let-7d-5p was confirmed in luciferase assays in this study. The findings of let-7d-5p/3p and multiple other miRNAs targeting IGF1R, KRAS and other metastasis-related factors suggest that 5p/3p miRNAs contribute to cross-targeting of multiple cancer-associated factors and processes possibly to evade functional abolishment when any one of the crucial factors are inactivated.miRNA-5p/3p species are frequently co-expressed and are coordinately regulated in colon cancer cells. In cancer cells, multiple cross-targeting by the miRNAs, including the co-existing 5p/3p species, frequently occurs in an apparent safe-proof scheme of miRNA regulation of important tumorigenesis processes. Further systematic analysis of co-existing miRNA-5p/3p pairs in clinical tissues is important in elucidating 5p/3p contributions to cancer pathogenesis.
Project description:<h4>Background</h4>A miRNA precursor generally gives rise to one major miRNA species derived from the 5' arm, and are called miRNA-5p. However, more recent studies have shown co-expression of miRNA-5p and -3p, albeit in different concentrations, in cancer cells targeting different sets of transcripts. Co-expression and regulation of the -5p and -3p miRNA species in stem cells, particularly in the reprogramming process, have not been studied.<h4>Methods</h4>In this work, we investigated co-expression and regulation of miRNA-5p and -3p species in human induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs) and embryonic stem cells (ESC) using a nanoliter-scale real-time PCR microarray platform that included 1,036 miRNAs.<h4>Results</h4>In comparing iPSC and ESC, only 32 miRNAs were found to be differentially expressed, in agreement of the ESC-like nature of iPSC. In the analysis of reprogramming process in iPSCs, 261 miRNAs were found to be differentially expressed compared with the parental MSC and pre-adipose tissue, indicating significant miRNA alternations in the reprogramming process. In iPSC reprogrammed from MSC, there were 88 miRNAs (33.7%), or 44 co-expressed 5p/3p pairs, clearly indicating frequent co-expression of both miRNA species on reprogramming. Of these, 40 pairs were either co-up- or co-downregulated indicating concerted 5p/3p regulation. The 5p/3p species of only 4 pairs were regulated in reverse directions. Furthermore, some 5p/3p species of the same miRNAs were found to target the same transcript and the same miRNA may cross-target different transcripts of proteins of the G1/S transition of the cell cycle; 5p/3p co-targeting was confirmed in stem-loop RT-PCR.<h4>Conclusion</h4>The observed cross- and co-regulation by paired miRNA species suggests a fail-proof scheme of miRNA regulation in iPSC, which may be important to iPSC pluripotency.
Project description:Renal cell carcinoma (RCC) is the third most frequent urinary malignancy and one of the most lethal. Current diagnostic and follow-up techniques are harmful and unspecific in low-grade tumors. Novel minimally invasive markers such as urine microRNAs (miRNAs) are under study. However, discrepancies arise among studies in part due to lack of consent regarding normalization. We aimed to identify the best miRNA normalizer for RCC studies performed in urine samples together with a miRNA profile with diagnostic value and another for follow-up. We evaluated the performance of 120 candidate miRNAs in the urine of 16 RCC patients and 16 healthy controls by RT-qPCR followed by a stability analysis with RefFinder. In this screening stage, miR-20a-5p arose as the most stably expressed miRNA in RCC and controls, with a good expression level. Its stability was validated in an independent cohort of 51 RCC patients and 32 controls. Using miR-20a-5p as normalizer, we adjusted and validated a diagnostic model for RCC with three miRNAs (miR-200a-3p, miR-34a-5p and miR-365a-3p) (AUC = 0.65; Confidence Interval 95% [0.51, 0.79], <i>p</i> = 0.043). let-7d-5p and miR-205-5p were also upregulated in patients compared to controls. Comparing RCC samples before surgery and fourteen weeks after, we identified let-7d-5p, miR-152-3p, miR-30c-5p, miR-362-3p and miR-30e-3p as potential follow-up profile for RCC. We identified validated targets of most miRNAs in the <i>renal cell carcinoma</i> pathway. This is the first study that identifies a robust normalizer for urine RCC miRNA studies, miR-20a-5p, which may allow the comparison of future studies among laboratories. Once confirmed in a larger independent cohort, the miRNAs profiles identified may improve the non-invasive diagnosis and follow-up of RCC.
Project description:INTRODUCTION:Amyotrophic lateral sclerosis (ALS) is a debilitating neurologic disorder with poor survival rates and no clear biomarkers for disease diagnosis and prognosis. METHODS:We compared serum microRNA (miRNA) expression from patients with ALS with healthy controls and patients with multiple sclerosis and Alzheimer disease. We also correlated miRNA expression in cross-sectional and longitudinal cohorts of ALS patients with clinical parameters. RESULTS:We identified 7 miRNAs (miR-192-5p, miR-192-3p, miR-1, miR-133a-3p, miR-133b, miR-144-5p, miR-19a-3p) that were upregulated and 6 miRNAs (miR-320c, miR-320a, let-7d-3p, miR-425-5p, miR-320b, miR-139-5p) that were downregulated in patients with ALS compared with healthy controls, patients with Alzheimer disease, and patients with multiple sclerosis. Changes in 4 miRNAs (miR-136-3p, miR-30b-5p, miR-331-3p, miR-496) correlated positively and change in 1 miRNA (miR-2110) correlated negatively with changes in clinical parameters in longitudinal analysis. DISCUSSION:Our findings identified serum miRNAs that can serve as biomarkers for ALS diagnosis and progression. Muscle Nerve 58: 261-269, 2018.
Project description:Increasing evidence has demonstrated that microRNAs (miRNAs) are involved in colon cancer initiation and progression, and may serve as diagnostic and prognostic biomarkers for colon cancer. Here, we investigated the levels of miR-9-1, miR-203-3p, miR-221-3p, miR-342-3p, miR-491-5p and miR-503-5p in 90 pairs of colon cancer and adjacent normal tissues, and explored the relationship between their expression and clinical outcome of colon cancer. Five miRNAs (miR-203-3p, miR-221-3p, miR-342-3p, miR-491-5p and miR-503-5p) were dysregulated in colon cancer tissue (P < 0.05). The levels of miR-503-5p in larger tumors (? 6 cm) were higher than those in smaller ones (< 6 cm) (P = 0.031), while the levels of miR-203-3p and miR-491-5p in patients aged 70 years and older were higher than those in patients aged younger than 70 years (P = 0.019 and 0.049, respectively). The high levels of miR-221-3p (HR = 2.416, 95% CI 1.314-4.445, P = 0.005), miR-342-3p (HR = 1.807, 95% CI 1.003-3.253, P = 0.049) and miR-491-5p (HR = 1.868, 95% CI 1.032-3.384, P = 0.039) were significantly associated with worse survival time. Moreover, combination analysis of miR-221-3p, miR-342-3p and miR-491-5p expression revealed that patients with 3 highly expressed miRNAs had lower survival rates compared with those with zero-to-two highly expressed miRNAs (HR = 2.100, 95% CI 1.157-3.813, P = 0.015), especially those with TNM stages I and II (HR = 4.204,95% CI 1.762-10.030, P = 0.001). Our results suggest that the three-miRNA signature may help doctors better predict prognosis and guide treatment decisions for colon cancer.
Project description:This study explored the expression of several miRNAs reported to be deregulated in age-related macular degeneration (AMD). Total RNA was isolated from sera from patients with dry AMD (<i>n</i> = 12), wet AMD (<i>n</i> = 14), and controls (<i>n</i> = 10). Forty-two previously investigated miRNAs were selected based on published data and their role in AMD pathogenesis, such as angiogenic and inflammatory effects, and were co-analysed using a miRCURY LNA miRNA SYBR<sup>®</sup> Green PCR kit via quantitative real-time polymerase chain reaction (qRT-PCR) to validate their presence. Unsupervised hierarchical clustering indicated that AMD serum specimens have a different miRNA profile to healthy controls. We successfully validated the differentially regulated miRNAs in serum from AMD patients versus controls. Eight miRNAs (hsa-let-7a-5p, hsa-let-7d-5p, hsa-miR-23a-3p, hsa-miR-301a-3p, hsa-miR-361-5p, hsa-miR-27b-3p, hsa-miR-874-3p, hsa-miR-19b-1-5p) showed higher expression in the serum of dry AMD patients than wet AMD patients and compared with healthy controls. Increased quantities of certain miRNAs in the serum of AMD patients indicate that these miRNAs could potentially serve as diagnostic AMD biomarkers and might be used as future AMD treatment targets. The discovery of significant serum miRNA biomarkers in AMD patients would provide an easy screening tool for at-risk populations.
Project description:BACKGROUND Hypertension is one of the most widespread health conditions in the world, and the molecular mechanism of it is still unclear. In this study, we identified the hub genes (hub miRNA genes) associated with hypertension and explored the relationship between hypertension miRNA-gene by constructing a mRNA co-expression network and a miRNA co-expression network, which can help to reveal the mechanism and predict the prognosis of hypertension progression. MATERIAL AND METHODS Based on gene expression profile data of hypertensive samples from the Gene Expression Omnibus database, WGCNA was used to detect hypertension-related biomarkers and key mRNA and miRNA modules. Then, DAVID was used to perform gene-annotation enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) and miRPath were used for pathway analysis of mRNA and miRNAs genes. RESULTS We identified 3 key modules relating to hypertension, 2 mRNA modules named Msaddlebrown and Mgreenyellow and 1 miRNA module named Msalmon. In addition, 12 hub genes (RPL21, RPS28, LOC442727/PTGAP10, LOC100129599/RPS29P14, TBXAS1, FCER1G, CFP, FURIN, PECAM1, IGSF6, NCF1C, and LOC285296/UNC93B3) and 7 hub miRNAs (hsa-miR-1268a/b, hsa-miR-513c-3p, hsa-miR-4799-5p, hsa-miR-296-3p, hsa-miR-5195-5p, hsa-miR-219-2-3p, and hsa-miR-548d-5p) relating to hypertension were identified. HIF-1 signaling pathway and insulin signaling pathway were closely related to the 3 key modules. We also discovered 4 miRNAs (hsa-miR-548am-3p, hsa-miR-513c-3p, hsa-miR-182-5p, and hsa-miR-548d-5p) and 6 genes (IGF1R, GSK3B, FOXO1, PRKAR2B, HIF1A, and PIK3R1) were the core nodes in the hypertension-related miRNA-gene network, and hsa-miR-548am-3p was at the center of the network. CONCLUSIONS These findings will help improve the understanding of the pathogenesis of hypertension, and the discovered genes can serve as signatures for early diagnosis of hypertension.
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.
Project description:(1) Background: Lymph node (LN) status is an indubitable prognostic factor for survival among colon cancer patients. MicroRNAs (miRNAs) have been implicated in the development and progression of many cancers and are potential biomarkers for cancer diagnosis and prognosis. Therefore, we validated candidate biomarkers using circulating miRNAs by analyzing the plasma miRNA concentrations from patients with colon cancer to predict LN metastasis. (2) Methods: This study included 79 blood samples from patients diagnosed with colon cancer. The NanoString assay was used for screening, and TaqMan miRNA assays for quantitative real-time polymerase chain reaction (RT-PCR) test was used for validation. In a discovery set, we compared the expression of 800 circulating miRNAs in 24 samples (stage 0/I/IIA versus IIIB/IIIC). For validation, a total 79 samples were tested using quantitative RT-PCR. (3) Results: In the discovery set, 10 candidate circulating miRNAs were detected (4 up-regulated miRNAs: miR-323a-3p, miR-382-5p, miR-29a-3p, and miR-376a-3p; 6 down-regulated miRNAs: miR-26a-5p, let-7g-5p, miR-15b-5p, miR-142-3p, miR-374a-5p, and let-7b-5p). In the validation set, higher expression of three circulating miRNAs (miR-323a-3p, miR-382-5p, and miR-376a-3p) was significantly associated with LN metastasis (<i>p</i> = 0.0063, 0.0107, and 0.0022). (4) Conclusions: High expression of circulating miR-323a-3p, miR-382-5p, and miR-376a-3p was significantly associated with LN metastasis in colon cancer patients. These miRNAs could be circulating biomarker candidates that predict the presence of LN metastasis.
Project description:Specific types of human papillomaviruses (HPVs) cause cervical cancer. Cervical cancers exhibit aberrant cellular microRNA (miRNA) expression patterns. By genome-wide analyses, we investigate whether the intracellular and exosomal miRNA compositions of HPV-positive cancer cells are dependent on endogenous E6/E7 oncogene expression. Deep sequencing studies combined with qRT-PCR analyses show that E6/E7 silencing significantly affects ten of the 52 most abundant intracellular miRNAs in HPV18-positive HeLa cells, downregulating miR-17-5p, miR-186-5p, miR-378a-3p, miR-378f, miR-629-5p and miR-7-5p, and upregulating miR-143-3p, miR-23a-3p, miR-23b-3p and miR-27b-3p. The effects of E6/E7 silencing on miRNA levels are mainly not dependent on p53 and similarly observed in HPV16-positive SiHa cells. The E6/E7-regulated miRNAs are enriched for species involved in the control of cell proliferation, senescence and apoptosis, suggesting that they contribute to the growth of HPV-positive cancer cells. Consistently, we show that sustained E6/E7 expression is required to maintain the intracellular levels of members of the miR-17~92 cluster, which reduce expression of the anti-proliferative p21 gene in HPV-positive cancer cells. In exosomes secreted by HeLa cells, a distinct seven-miRNA-signature was identified among the most abundant miRNAs, with significant downregulation of let-7d-5p, miR-20a-5p, miR-378a-3p, miR-423-3p, miR-7-5p, miR-92a-3p and upregulation of miR-21-5p, upon E6/E7 silencing. Several of the E6/E7-dependent exosomal miRNAs have also been linked to the control of cell proliferation and apoptosis. This study represents the first global analysis of intracellular and exosomal miRNAs and shows that viral oncogene expression affects the abundance of multiple miRNAs likely contributing to the E6/E7-dependent growth of HPV-positive cancer cells.
Project description:In C. elegans, miRNAs are genetic biomarkers of aging. Similarly, multiple miRNAs are differentially expressed between younger and older persons, suggesting that miRNA-regulated biological mechanisms affecting aging are evolutionarily conserved. Previous human studies have not considered participants' lifespans, a key factor in identifying biomarkers of aging. Using PCR arrays, we measured miRNA levels from serum samples obtained longitudinally at ages 50, 55, and 60 from 16 non-Hispanic males who had documented lifespans from 58 to 92. Numerous miRNAs showed significant changes in expression levels. At age 50, 24 miRNAs were significantly upregulated, and 73 were significantly downregulated in the long-lived subgroup (76-92 years) as compared with the short-lived subgroup (58-75 years). In long-lived participants, the most upregulated was miR-373-5p, while the most downregulated was miR-15b-5p. Longitudinally, significant Pearson correlations were observed between lifespan and expression of nine miRNAs (p value<0.05). Six of these nine miRNAs (miR-211-5p, 374a-5p, 340-3p, 376c-3p, 5095, 1225-3p) were also significantly up- or downregulated when comparing long-lived and short-lived participants. Twenty-four validated targets of these miRNAs encoded aging-associated proteins, including PARP1, IGF1R, and IGF2R. We propose that the expression profiles of the six miRNAs (miR-211-5p, 374a-5p, 340-3p, 376c-3p, 5095, and 1225-3p) may be useful biomarkers of aging.