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Identification of retinopathy of prematurity related miRNAs in hyperoxia-induced neonatal rats by deep sequencing.

Zhao R, Qian L, Jiang L - Int J Mol Sci (2014)

Bottom Line: Some of the differentially expressed miRNAs were confirmed by quantitative reverse transcription-PCR (qRT-PCR).A total of 594 target genes of the differentially expressed microRNAs were identified using a bioinformatics approach.This study provides some insights into the molecular mechanisms that underlie ROP development, thereby aiding the diagnosis and treatment of this disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Zhongda Hospital, Southeast University, Nanjing 210096, China. tczhaoruibin@163.com.

ABSTRACT
Retinopathy of prematurity (ROP) remains a major problem for many preterm infants. MicroRNAs (miRNAs) are a class of small noncoding RNAs that regulate gene expression at the posttranscriptional level and have been studied in many diseases. To understand the roles of miRNAs in ROP model rats, we constructed two small RNA libraries from the plasma of hyperoxia-induced rats and normal controls. Sequencing data revealed that 44 down-regulated microRNAs and 22 up-regulated microRNAs from the hyperoxia-induced rats were identified by deep sequencing technology. Some of the differentially expressed miRNAs were confirmed by quantitative reverse transcription-PCR (qRT-PCR). A total of 594 target genes of the differentially expressed microRNAs were identified using a bioinformatics approach. Functional annotation analysis indicated that a number of pathways might be involved in angiogenesis, cell proliferation and cell differentiation, which might be involved in the genesis and development of ROP. The elevated expression level of the vascular endothelial growth factor (VEGF) protein in the hyperoxia-induced neonatal rats was also confirmed by enzyme linked immunosorbent assay (ELISA). This study provides some insights into the molecular mechanisms that underlie ROP development, thereby aiding the diagnosis and treatment of this disease.

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Comparison of the high-throughput sequencing and quantitative real-time PCR results. (a) The differentially expressed miRNAs, including rno-miR-183-5p, rno-miR-9a-5p, rno-miR-199a-5p, rno-miR-351-5p, rno-miR-200b-3p, rno-miR-191a-3p, rno-miR-181c-3p, rno-miR-330-5p rno-miR-126a-5p and rno-miR-351-3p were used to perform expression analyses by qPCR. Comparisons of the high-throughput sequencing and qRT-PCR results indicated that the two plarforms exhibited good correlation; and (b) The 5p/3p ratio validation results for miR-126a, miR-330 and miR-351 by qPCR also indicated good correlation between the two platforms.
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ijms-16-00840-f003: Comparison of the high-throughput sequencing and quantitative real-time PCR results. (a) The differentially expressed miRNAs, including rno-miR-183-5p, rno-miR-9a-5p, rno-miR-199a-5p, rno-miR-351-5p, rno-miR-200b-3p, rno-miR-191a-3p, rno-miR-181c-3p, rno-miR-330-5p rno-miR-126a-5p and rno-miR-351-3p were used to perform expression analyses by qPCR. Comparisons of the high-throughput sequencing and qRT-PCR results indicated that the two plarforms exhibited good correlation; and (b) The 5p/3p ratio validation results for miR-126a, miR-330 and miR-351 by qPCR also indicated good correlation between the two platforms.

Mentions: To confirm the deep sequencing results, we used qRT-PCR to assess the expressions of 10 of the miRNAs (miR-183-5p, miR-9a-5p, miR-199a-5p, miR-351-5p, miR200b-3p, miR-191a-3p, miR-181c-3p, miR-330-5p, miR-126a-5p and miR-351-3p) in the 12-pair plasma samples from the hyperoxia rats and controls. Among these miRNAs, the 5p/3p ratios of miR-351, miR-330 and miR-126a were also measured for validation. These 10 selected miRNAs covered both highly expressed miRNAs and lowly expressed miRNAs that exhibited dysregulations in the hyperoxia rats compared to the controls. The expression levels of these 10 miRNAs were calculated using the comparative Ct method in which the Ct value of each miRNA is normalized to U6 snRNA using the comparative Ct (ΔCt). All 10 of the miRNAs were expressed at significantly different levels in the plasma samples from the hyperoxia rats and controls. As shown in Figure 3, the qPCR results demonstrated a very good correspondence between the two analysis methods.


Identification of retinopathy of prematurity related miRNAs in hyperoxia-induced neonatal rats by deep sequencing.

Zhao R, Qian L, Jiang L - Int J Mol Sci (2014)

Comparison of the high-throughput sequencing and quantitative real-time PCR results. (a) The differentially expressed miRNAs, including rno-miR-183-5p, rno-miR-9a-5p, rno-miR-199a-5p, rno-miR-351-5p, rno-miR-200b-3p, rno-miR-191a-3p, rno-miR-181c-3p, rno-miR-330-5p rno-miR-126a-5p and rno-miR-351-3p were used to perform expression analyses by qPCR. Comparisons of the high-throughput sequencing and qRT-PCR results indicated that the two plarforms exhibited good correlation; and (b) The 5p/3p ratio validation results for miR-126a, miR-330 and miR-351 by qPCR also indicated good correlation between the two platforms.
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Related In: Results  -  Collection

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ijms-16-00840-f003: Comparison of the high-throughput sequencing and quantitative real-time PCR results. (a) The differentially expressed miRNAs, including rno-miR-183-5p, rno-miR-9a-5p, rno-miR-199a-5p, rno-miR-351-5p, rno-miR-200b-3p, rno-miR-191a-3p, rno-miR-181c-3p, rno-miR-330-5p rno-miR-126a-5p and rno-miR-351-3p were used to perform expression analyses by qPCR. Comparisons of the high-throughput sequencing and qRT-PCR results indicated that the two plarforms exhibited good correlation; and (b) The 5p/3p ratio validation results for miR-126a, miR-330 and miR-351 by qPCR also indicated good correlation between the two platforms.
Mentions: To confirm the deep sequencing results, we used qRT-PCR to assess the expressions of 10 of the miRNAs (miR-183-5p, miR-9a-5p, miR-199a-5p, miR-351-5p, miR200b-3p, miR-191a-3p, miR-181c-3p, miR-330-5p, miR-126a-5p and miR-351-3p) in the 12-pair plasma samples from the hyperoxia rats and controls. Among these miRNAs, the 5p/3p ratios of miR-351, miR-330 and miR-126a were also measured for validation. These 10 selected miRNAs covered both highly expressed miRNAs and lowly expressed miRNAs that exhibited dysregulations in the hyperoxia rats compared to the controls. The expression levels of these 10 miRNAs were calculated using the comparative Ct method in which the Ct value of each miRNA is normalized to U6 snRNA using the comparative Ct (ΔCt). All 10 of the miRNAs were expressed at significantly different levels in the plasma samples from the hyperoxia rats and controls. As shown in Figure 3, the qPCR results demonstrated a very good correspondence between the two analysis methods.

Bottom Line: Some of the differentially expressed miRNAs were confirmed by quantitative reverse transcription-PCR (qRT-PCR).A total of 594 target genes of the differentially expressed microRNAs were identified using a bioinformatics approach.This study provides some insights into the molecular mechanisms that underlie ROP development, thereby aiding the diagnosis and treatment of this disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Zhongda Hospital, Southeast University, Nanjing 210096, China. tczhaoruibin@163.com.

ABSTRACT
Retinopathy of prematurity (ROP) remains a major problem for many preterm infants. MicroRNAs (miRNAs) are a class of small noncoding RNAs that regulate gene expression at the posttranscriptional level and have been studied in many diseases. To understand the roles of miRNAs in ROP model rats, we constructed two small RNA libraries from the plasma of hyperoxia-induced rats and normal controls. Sequencing data revealed that 44 down-regulated microRNAs and 22 up-regulated microRNAs from the hyperoxia-induced rats were identified by deep sequencing technology. Some of the differentially expressed miRNAs were confirmed by quantitative reverse transcription-PCR (qRT-PCR). A total of 594 target genes of the differentially expressed microRNAs were identified using a bioinformatics approach. Functional annotation analysis indicated that a number of pathways might be involved in angiogenesis, cell proliferation and cell differentiation, which might be involved in the genesis and development of ROP. The elevated expression level of the vascular endothelial growth factor (VEGF) protein in the hyperoxia-induced neonatal rats was also confirmed by enzyme linked immunosorbent assay (ELISA). This study provides some insights into the molecular mechanisms that underlie ROP development, thereby aiding the diagnosis and treatment of this disease.

Show MeSH
Related in: MedlinePlus