Abstract:MicroRNAs (miRNAs) have gradually been recognized as regulators of embryonic development; however, relatively few miRNAs have been identified that regulate cardiac development. A series of recent papers have established an essential role for the miRNA-17-92 (miR-17-92) cluster of miRNAs in the development of the heart. Previous research has shown that the Friend of Gata-2 (FOG-2) is critical for cardiac development. To investigate the possibility that the miR-17-92 cluster regulates FOG-2 expression and inhibi… Show more
“…Forty‐eight hours after being transfected with plasmid or miRNA mimic, mK3 cells were briefly rinsed with PBS and lysed with 200 µl of radioimmunoprecipitation assay cell lysis buffer per well of a six‐well plate. Western blotting was performed as previously described . The lysate was resolved in 12% sodium dodecyl sulphate PAGE gel, and protein bands were transferred to the polyvinylidene difluoride membrane.…”
Section: Methodsmentioning
confidence: 99%
“…Western blotting was performed as previously described. 15 The lysate was resolved in 12% sodium dodecyl sulphate PAGE gel, and protein bands were transferred to the polyvinylidene difluoride membrane. The membrane was hatched with primary antibody (rabbit anti-Six2, 1:600 dilution, Proteintech, USA, and mouse anti-β-tubulin, 1:3000 dilution, TransGen, China) at 4°overnight.…”
Increasingly recognized importance has been assumed for microRNA (miRNA) in the regulation of the delicate balance of gene expression. In our study, we aimed to explore the regulation role of miR181c towards Six2 in metanephric mesenchyme (MM) cells. Bioinformatics analysis, luciferase assay and semi-quantitative real-time (RT) PCR, subsequently RT PCR, Western blotting, 5-ethynyl-2'-deoxyuridine cell proliferation assay, Cell Counting Kit-8 assay, immunofluorescence and flow cytometry, were employed to verify the modulation function of miR181c on Six2 in the mK3 MM cell line that is one kind of MM cells. miR181c was predicted to bind the 3' untranslated region of Six2 by bioinformatics analysis, which was subsequently validated by the in vitro luciferase reporter assay. Moreover, transfection of miR181c mimic can decrease the expression of Six2 both in mRNA and protein levels in mK3 cells. Still, ectopic expression of miR181c inhibits the proliferation, promotes the apoptosis and even makes the nephron progenitor phenotype lose mK3 cells. These results revealed the ability of a single miRNA-miR181c to downregulate the expression of Six2, restrain the proliferation and promote the apoptosis that even makes the nephron progenitor phenotype lose MM cells, suggesting a potential role of miR181c during the kidney development.
“…Forty‐eight hours after being transfected with plasmid or miRNA mimic, mK3 cells were briefly rinsed with PBS and lysed with 200 µl of radioimmunoprecipitation assay cell lysis buffer per well of a six‐well plate. Western blotting was performed as previously described . The lysate was resolved in 12% sodium dodecyl sulphate PAGE gel, and protein bands were transferred to the polyvinylidene difluoride membrane.…”
Section: Methodsmentioning
confidence: 99%
“…Western blotting was performed as previously described. 15 The lysate was resolved in 12% sodium dodecyl sulphate PAGE gel, and protein bands were transferred to the polyvinylidene difluoride membrane. The membrane was hatched with primary antibody (rabbit anti-Six2, 1:600 dilution, Proteintech, USA, and mouse anti-β-tubulin, 1:3000 dilution, TransGen, China) at 4°overnight.…”
Increasingly recognized importance has been assumed for microRNA (miRNA) in the regulation of the delicate balance of gene expression. In our study, we aimed to explore the regulation role of miR181c towards Six2 in metanephric mesenchyme (MM) cells. Bioinformatics analysis, luciferase assay and semi-quantitative real-time (RT) PCR, subsequently RT PCR, Western blotting, 5-ethynyl-2'-deoxyuridine cell proliferation assay, Cell Counting Kit-8 assay, immunofluorescence and flow cytometry, were employed to verify the modulation function of miR181c on Six2 in the mK3 MM cell line that is one kind of MM cells. miR181c was predicted to bind the 3' untranslated region of Six2 by bioinformatics analysis, which was subsequently validated by the in vitro luciferase reporter assay. Moreover, transfection of miR181c mimic can decrease the expression of Six2 both in mRNA and protein levels in mK3 cells. Still, ectopic expression of miR181c inhibits the proliferation, promotes the apoptosis and even makes the nephron progenitor phenotype lose mK3 cells. These results revealed the ability of a single miRNA-miR181c to downregulate the expression of Six2, restrain the proliferation and promote the apoptosis that even makes the nephron progenitor phenotype lose MM cells, suggesting a potential role of miR181c during the kidney development.
“…Another study based on embryonic cardiomyocytes reported that overexpression of miR-17-92 would reduce the cell proliferation through posttranscriptional repression of Friend of Gata-2 (FOG-2, a nuclear corepressor protein), which is a critical factor for cardiac development. Moreover, FOG-2 could partially rescue the abnormal proliferation induced by miR-17-92 [12]. In addition, miR-17-92 cluster member miR-19 promoted cardiomyocyte proliferation through direct downregulation of PTEN in vitro [21].…”
Section: The Role Of Mir-17-92 Cluster In Cardiovascular Diseasesmentioning
confidence: 99%
“…Among all miRNA clusters, miR-17-92, which was called oncomir-1 at first, is a highly conserved cluster that is essential in adipocyte differentiation [8], lung development [9], angiogenesis [10], tumorigenesis [11], and especially heart development [12, 13]. miR-17-92 cluster has been confirmed to be involved in normal cardiac development and its change took part in the genesis of many cardiovascular diseases, including heart failure [14], cardiomyopathy, arrhythmias, and cardiac hypertrophy [15], while its mechanisms of action in these contexts are not fully understood (Figure 1).…”
MicroRNAs (miRNAs and miRs) are a large class of noncoding, single-stranded, small RNA molecules. The precise control of their expression is essential for keeping tissue homeostasis and normal development of organisms. Thus, unbalanced expression of miRNAs is a hallmark of many diseases. Two to dozens of miRNAs can form into a miRNA cluster, and the miR-17-92 cluster is one of them. Although firstly described as an oncogenic miRNA cluster, the miR-17-92 cluster has also been found to play critical role in normal cardiac development and cardiovascular disease. This review focuses on the characteristics and functions of miR-17-92 cluster in heart.
“…miRNA clusters perform important functions by regulating various cellular processes. For example, the most frequently studied miR-17-92 cluster is involved in the development of the heart, lungs, and immune system and in the formation of tumors [13]–[15]. Clustered miRNAs are usually derived from a common primary transcript and coexpressed [16].…”
BackgroundMicroRNAs (miRNAs) are a class of small non-coding RNAs generated from endogenous transcripts that form hairpin structures. The hairpin precursor is processed into two mature miRNAs that form major/minor duplexes. Mature miRNAs regulate gene expression by cleaving mRNA or repressing protein translation. Numerous miRNAs have been discovered via deep sequencing. Many miRNAs are produced from multiple genome sites. These miRNAs are grouped into paralogous families of miRNAs that generate the same major mature form within organisms. Currently, no method of distinguishing the expression of these miRNAs is available.ResultsIn the present study, strategies were developed to discriminate and quantify the expression of paralogous miRNA precursors. First, paralogous miRNA precursors that were differentially expressed in tissues were identified through analysis of the coexpression scores of their major and minor forms based on deep sequencing data. Then the precursors were identified by monitoring the expression of their host gene or minor form using real-time PCR. Finally, precursors were identified by assessing the expression of clusters of miRNA members. These approaches were used to distinguish miR-128-1 and miR-128-2 as well as miR-194-1 and miR-194-2. The mechanism of transcription related to the differential expression of miR-194-1 and miR-194-2 was also investigated.ConclusionThis is the first report to distinguish paralogous miRNA copies by analyzing the expression of major-minor pairs, the host gene, and miRNA clusters. Discriminating paralogous precursors can provide useful information for investigating the mechanisms that regulate miRNA gene expression under different physiological and pathological conditions.
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