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Differential miRNA Expression in Cells and Matrix Vesicles in Vascular Smooth Muscle Cells from Rats with Kidney Disease.

Chaturvedi P, Chen NX, O'Neill K, McClintick JN, Moe SM, Janga SC - PLoS ONE (2015)

Bottom Line: The percentage of miRNA to total RNA was increased in MV compared to VSMC.We found several processes including vascular smooth muscle contraction, response to hypoxia and regulation of muscle cell differentiation to be enriched.In conclusion, our results demonstrate that miRs are concentrated in MV from calcifying VSMC, and that important functions and pathways are affected by the miRs dysregulation between calcifying VSMC and the MV they produce.

View Article: PubMed Central - PubMed

Affiliation: Department of Biohealth Informatics, School of Informatics and Computing, Indiana University Purdue University, Indianapolis, Indiana, United States of America.

ABSTRACT
Vascular calcification is a complex process and has been associated with aging, diabetes, chronic kidney disease (CKD). Although there have been several studies that examine the role of miRNAs (miRs) in bone osteogenesis, little is known about the role of miRs in vascular calcification and their role in the pathogenesis of vascular abnormalities. Matrix vesicles (MV) are known to play in important role in initiating vascular smooth muscle cell (VSMC) calcification. In the present study, we performed miRNA microarray analysis to identify the dysregulated miRs between MV and VSMC derived from CKD rats to understand the role of post-transcriptional regulatory networks governed by these miRNAs in vascular calcification and to uncover the differential miRNA content of MV. The percentage of miRNA to total RNA was increased in MV compared to VSMC. Comparison of expression profiles of miRNA by microarray demonstrated 33 miRs to be differentially expressed with the majority (~ 57%) of them down-regulated. Target genes controlled by differentially expressed miRNAs were identified utilizing two different complementary computational approaches Miranda and Targetscan to understand the functions and pathways that may be affected due to the production of MV from calcifying VSMC thereby contributing to the regulation of genes by miRs. We found several processes including vascular smooth muscle contraction, response to hypoxia and regulation of muscle cell differentiation to be enriched. Signaling pathways identified included MAP-kinase and wnt signaling that have previously been shown to be important in vascular calcification. In conclusion, our results demonstrate that miRs are concentrated in MV from calcifying VSMC, and that important functions and pathways are affected by the miRs dysregulation between calcifying VSMC and the MV they produce. This suggests that miRs may play a very important regulatory role in vascular calcification in CKD by controlling an extensive network of post-transcriptional targets.

No MeSH data available.


Related in: MedlinePlus

RT-PCR validation of selected miRNAs n MV and VSMC.To validate the miRNA identified by the arrays that regulate multiple genes, we performed Real time PCR on VSMc and MV to determine the expression of miR-667, miR-702, miR-3562, mir-3568 and miR-3584 and normalized by U6. Each sample (n = 3 with MV and VSMC isolated from 3 CKD rats, same samples as arrays) was assayed in triplicate. The results demonstrated increased expression in MV compared to VSMC for each of these miRNAs, confirming the array results. Data were expressed as mean ± SEM. * p<0.05, MV vs. VSMC.
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pone.0131589.g007: RT-PCR validation of selected miRNAs n MV and VSMC.To validate the miRNA identified by the arrays that regulate multiple genes, we performed Real time PCR on VSMc and MV to determine the expression of miR-667, miR-702, miR-3562, mir-3568 and miR-3584 and normalized by U6. Each sample (n = 3 with MV and VSMC isolated from 3 CKD rats, same samples as arrays) was assayed in triplicate. The results demonstrated increased expression in MV compared to VSMC for each of these miRNAs, confirming the array results. Data were expressed as mean ± SEM. * p<0.05, MV vs. VSMC.

Mentions: Our microarray analysis and target prediction using computational methods for the 33 miRNAs revealed that the representative set of miRs: miR-667, miR-702, miR-3562, miR-3568 and miR-3584 either controlled genes of interest in the formation of MV, or were predicted to target many genes, with significantly higher expression in MV compared to VSMC from microarray data analysis (Table 1 & Fig 3). Hence, to confirm the expression of these miRNA in MV and VSMC, we performed the real time PCR. The results demonstrated that miR-667, miR-702, miR-3562, miR-3568 and miR-3584 are all expressed in VMSC from CKD rats and are highly concentrated in MV, confirming directional changes from microarray analysis (Fig 7).


Differential miRNA Expression in Cells and Matrix Vesicles in Vascular Smooth Muscle Cells from Rats with Kidney Disease.

Chaturvedi P, Chen NX, O'Neill K, McClintick JN, Moe SM, Janga SC - PLoS ONE (2015)

RT-PCR validation of selected miRNAs n MV and VSMC.To validate the miRNA identified by the arrays that regulate multiple genes, we performed Real time PCR on VSMc and MV to determine the expression of miR-667, miR-702, miR-3562, mir-3568 and miR-3584 and normalized by U6. Each sample (n = 3 with MV and VSMC isolated from 3 CKD rats, same samples as arrays) was assayed in triplicate. The results demonstrated increased expression in MV compared to VSMC for each of these miRNAs, confirming the array results. Data were expressed as mean ± SEM. * p<0.05, MV vs. VSMC.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482652&req=5

pone.0131589.g007: RT-PCR validation of selected miRNAs n MV and VSMC.To validate the miRNA identified by the arrays that regulate multiple genes, we performed Real time PCR on VSMc and MV to determine the expression of miR-667, miR-702, miR-3562, mir-3568 and miR-3584 and normalized by U6. Each sample (n = 3 with MV and VSMC isolated from 3 CKD rats, same samples as arrays) was assayed in triplicate. The results demonstrated increased expression in MV compared to VSMC for each of these miRNAs, confirming the array results. Data were expressed as mean ± SEM. * p<0.05, MV vs. VSMC.
Mentions: Our microarray analysis and target prediction using computational methods for the 33 miRNAs revealed that the representative set of miRs: miR-667, miR-702, miR-3562, miR-3568 and miR-3584 either controlled genes of interest in the formation of MV, or were predicted to target many genes, with significantly higher expression in MV compared to VSMC from microarray data analysis (Table 1 & Fig 3). Hence, to confirm the expression of these miRNA in MV and VSMC, we performed the real time PCR. The results demonstrated that miR-667, miR-702, miR-3562, miR-3568 and miR-3584 are all expressed in VMSC from CKD rats and are highly concentrated in MV, confirming directional changes from microarray analysis (Fig 7).

Bottom Line: The percentage of miRNA to total RNA was increased in MV compared to VSMC.We found several processes including vascular smooth muscle contraction, response to hypoxia and regulation of muscle cell differentiation to be enriched.In conclusion, our results demonstrate that miRs are concentrated in MV from calcifying VSMC, and that important functions and pathways are affected by the miRs dysregulation between calcifying VSMC and the MV they produce.

View Article: PubMed Central - PubMed

Affiliation: Department of Biohealth Informatics, School of Informatics and Computing, Indiana University Purdue University, Indianapolis, Indiana, United States of America.

ABSTRACT
Vascular calcification is a complex process and has been associated with aging, diabetes, chronic kidney disease (CKD). Although there have been several studies that examine the role of miRNAs (miRs) in bone osteogenesis, little is known about the role of miRs in vascular calcification and their role in the pathogenesis of vascular abnormalities. Matrix vesicles (MV) are known to play in important role in initiating vascular smooth muscle cell (VSMC) calcification. In the present study, we performed miRNA microarray analysis to identify the dysregulated miRs between MV and VSMC derived from CKD rats to understand the role of post-transcriptional regulatory networks governed by these miRNAs in vascular calcification and to uncover the differential miRNA content of MV. The percentage of miRNA to total RNA was increased in MV compared to VSMC. Comparison of expression profiles of miRNA by microarray demonstrated 33 miRs to be differentially expressed with the majority (~ 57%) of them down-regulated. Target genes controlled by differentially expressed miRNAs were identified utilizing two different complementary computational approaches Miranda and Targetscan to understand the functions and pathways that may be affected due to the production of MV from calcifying VSMC thereby contributing to the regulation of genes by miRs. We found several processes including vascular smooth muscle contraction, response to hypoxia and regulation of muscle cell differentiation to be enriched. Signaling pathways identified included MAP-kinase and wnt signaling that have previously been shown to be important in vascular calcification. In conclusion, our results demonstrate that miRs are concentrated in MV from calcifying VSMC, and that important functions and pathways are affected by the miRs dysregulation between calcifying VSMC and the MV they produce. This suggests that miRs may play a very important regulatory role in vascular calcification in CKD by controlling an extensive network of post-transcriptional targets.

No MeSH data available.


Related in: MedlinePlus