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Enhanced levels of microRNA-125b in vascular smooth muscle cells of diabetic db/db mice lead to increased inflammatory gene expression by targeting the histone methyltransferase Suv39h1.

Villeneuve LM, Kato M, Reddy MA, Wang M, Lanting L, Natarajan R - Diabetes (2010)

Bottom Line: Conversely, miR-125b inhibitors showed opposite effects.Furthermore, miR-125b mimics increased expression of inflammatory genes, monocyte chemoattractant protein-1, and interleukin-6, and reduced H3K9me3 at their promoters in nondiabetic cells.In addition, we found that the increase in miR-125b in db/db VSMC is caused by increased transcription of miR-125b-2.

View Article: PubMed Central - PubMed

Affiliation: Department of Diabetes, Beckman Research Institute of City of Hope, Duarte, California, USA.

ABSTRACT

Objective: Diabetes remains a major risk factor for vascular complications that seem to persist even after achieving glycemic control, possibly due to "metabolic memory." Using cultured vascular smooth muscle cells (MVSMC) from type 2 diabetic db/db mice, we recently showed that decreased promoter occupancy of the chromatin histone H3 lysine-9 methyltransferase Suv39h1 and the associated repressive epigenetic mark histone H3 lysine-9 trimethylation (H3K9me3) play key roles in sustained inflammatory gene expression. Here we examined the role of microRNAs (miRs) in Suv39h1 regulation and function in MVSMC from diabetic mice.

Research design and methods: We used luciferase assays with Suv39h1 3'untranslated region (UTR) reporter constructs and Western blotting of endogenous protein to verify that miR-125b targets Suv39h1. We examined the effects of Suv39h1 targeting on inflammatory gene expression by quantitative real time polymerase chain reaction (RT-qPCR), and H3K9me3 levels at their promoters by chromatin immunoprecipitation assays.

Results: We observed significant upregulation of miR-125b with parallel downregulation of Suv39h1 protein (predicted miR-125b target) in MVSMC cultured from diabetic db/db mice relative to control db/+. miR-125b mimics inhibited both Suv39h1 3'UTR luciferase reporter activity and endogenous Suv39h1 protein levels. Conversely, miR-125b inhibitors showed opposite effects. Furthermore, miR-125b mimics increased expression of inflammatory genes, monocyte chemoattractant protein-1, and interleukin-6, and reduced H3K9me3 at their promoters in nondiabetic cells. Interestingly, miR-125b mimics increased monocyte binding to db/+ MVSMC toward that in db/db MVSMC, further imitating the proinflammatory diabetic phenotype. In addition, we found that the increase in miR-125b in db/db VSMC is caused by increased transcription of miR-125b-2.

Conclusions: These results demonstrate a novel upstream role for miR-125b in the epigenetic regulation of inflammatory genes in MVSMC of db/db mice through downregulation of Suv39h1.

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Related in: MedlinePlus

Increased transcriptional regulation of miR-125b-2 in db/db MVSMC. A: Chromosomal map of the genomic locations of miR-125b isoforms, as well as two RIKEN cDNA clones that overlap miR-125b-2 along with primer locations designated as numbered arrowheads. B–G: Total RNA was extracted from MVSMC followed by RT-PCRs. B: RT-qPCRs using primers p1–p2 for 2610203C20Rik-201 (Rik-201) or primers p3–p4 flanking the premiR-125b-2 sequence. Results expressed as fold over db/+ (*P < 0.05 vs. db/+, n = 6). C and D: Using conventional PCR and gel electrophoresis with primers flanking the premiR-125b-2 on chromosome 16 (primers p3–p4), an unprocessed premiR-125b-2 would result in a 240 bp fragment. D: Primers p3–p4 reveal a 240-bp fragment which is increased in db/db relative to db/+ VSMC. As a negative control, an RNA sample with no reverse transcriptase (no RT) was used for conventional PCR to demonstrate the absence of contamination of genomic DNA in the RNA used for RT-PCR. As an internal control in conventional PCR, 18S primers were used. Contrast of the gel was adjusted uniformly across the whole gel to better visualize the bands. E: RT-qPCR of transcript levels downstream of premiR-125b-2 overlapping RIKEN cDNA clones using primers p5–p6. Results are expressed as fold over db/+ (*P < 0.05 vs. db/+, n = 5). F: Representative results of overall cDNA transcript levels relative to db/+ amplified with primers p3–p4 (*P < 0.05 vs. db/+ within each primer set, primers p3–p4, n = 6, and primers p5–p6, n = 5). G: RT-qPCR amplification of transcript containing both miR-125b-2 and the downstream cDNA using primers p3–p6. Results from one experiment performed in triplicate.
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Figure 6: Increased transcriptional regulation of miR-125b-2 in db/db MVSMC. A: Chromosomal map of the genomic locations of miR-125b isoforms, as well as two RIKEN cDNA clones that overlap miR-125b-2 along with primer locations designated as numbered arrowheads. B–G: Total RNA was extracted from MVSMC followed by RT-PCRs. B: RT-qPCRs using primers p1–p2 for 2610203C20Rik-201 (Rik-201) or primers p3–p4 flanking the premiR-125b-2 sequence. Results expressed as fold over db/+ (*P < 0.05 vs. db/+, n = 6). C and D: Using conventional PCR and gel electrophoresis with primers flanking the premiR-125b-2 on chromosome 16 (primers p3–p4), an unprocessed premiR-125b-2 would result in a 240 bp fragment. D: Primers p3–p4 reveal a 240-bp fragment which is increased in db/db relative to db/+ VSMC. As a negative control, an RNA sample with no reverse transcriptase (no RT) was used for conventional PCR to demonstrate the absence of contamination of genomic DNA in the RNA used for RT-PCR. As an internal control in conventional PCR, 18S primers were used. Contrast of the gel was adjusted uniformly across the whole gel to better visualize the bands. E: RT-qPCR of transcript levels downstream of premiR-125b-2 overlapping RIKEN cDNA clones using primers p5–p6. Results are expressed as fold over db/+ (*P < 0.05 vs. db/+, n = 5). F: Representative results of overall cDNA transcript levels relative to db/+ amplified with primers p3–p4 (*P < 0.05 vs. db/+ within each primer set, primers p3–p4, n = 6, and primers p5–p6, n = 5). G: RT-qPCR amplification of transcript containing both miR-125b-2 and the downstream cDNA using primers p3–p6. Results from one experiment performed in triplicate.

Mentions: We next examined potential regulatory mechanisms for miR-125b in db/db MVSMC. The mouse genome has two copies of miR-125b, namely125b-1 and 125b-2, with both processed into the same mature miR (http://microrna.sanger.ac.uk/sequences) by enzymes such as Drosha and Dicer (26–27). The miR-125b-1 is located in the intron of a known protein coding transcript (2610203C20Rik-201) within chromosome 9, whereas miR-125b-2 is intergenic within chromosome 16 (Fig. 6A). Primers flanking the premiR-125b-1 sequence did not pick up any transcripts (not shown), suggesting that it is processed from the intron of 2610203C20Rik-201. Yet, RT-qPCR to amplify 2610203C20Rik-201 itself (primers p1-p2) indicated no change in its transcription in db/db MVSMC relative to db/+ (Fig. 6B, left two bars). On the other hand, interestingly, RT-qPCR with primers flanking the premiR-125b-2 sequence (primers p3-p4) showed increased transcription of miR-125b-2 in db/db cells relative to db/+ (Fig. 6B, right two bars).


Enhanced levels of microRNA-125b in vascular smooth muscle cells of diabetic db/db mice lead to increased inflammatory gene expression by targeting the histone methyltransferase Suv39h1.

Villeneuve LM, Kato M, Reddy MA, Wang M, Lanting L, Natarajan R - Diabetes (2010)

Increased transcriptional regulation of miR-125b-2 in db/db MVSMC. A: Chromosomal map of the genomic locations of miR-125b isoforms, as well as two RIKEN cDNA clones that overlap miR-125b-2 along with primer locations designated as numbered arrowheads. B–G: Total RNA was extracted from MVSMC followed by RT-PCRs. B: RT-qPCRs using primers p1–p2 for 2610203C20Rik-201 (Rik-201) or primers p3–p4 flanking the premiR-125b-2 sequence. Results expressed as fold over db/+ (*P < 0.05 vs. db/+, n = 6). C and D: Using conventional PCR and gel electrophoresis with primers flanking the premiR-125b-2 on chromosome 16 (primers p3–p4), an unprocessed premiR-125b-2 would result in a 240 bp fragment. D: Primers p3–p4 reveal a 240-bp fragment which is increased in db/db relative to db/+ VSMC. As a negative control, an RNA sample with no reverse transcriptase (no RT) was used for conventional PCR to demonstrate the absence of contamination of genomic DNA in the RNA used for RT-PCR. As an internal control in conventional PCR, 18S primers were used. Contrast of the gel was adjusted uniformly across the whole gel to better visualize the bands. E: RT-qPCR of transcript levels downstream of premiR-125b-2 overlapping RIKEN cDNA clones using primers p5–p6. Results are expressed as fold over db/+ (*P < 0.05 vs. db/+, n = 5). F: Representative results of overall cDNA transcript levels relative to db/+ amplified with primers p3–p4 (*P < 0.05 vs. db/+ within each primer set, primers p3–p4, n = 6, and primers p5–p6, n = 5). G: RT-qPCR amplification of transcript containing both miR-125b-2 and the downstream cDNA using primers p3–p6. Results from one experiment performed in triplicate.
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Figure 6: Increased transcriptional regulation of miR-125b-2 in db/db MVSMC. A: Chromosomal map of the genomic locations of miR-125b isoforms, as well as two RIKEN cDNA clones that overlap miR-125b-2 along with primer locations designated as numbered arrowheads. B–G: Total RNA was extracted from MVSMC followed by RT-PCRs. B: RT-qPCRs using primers p1–p2 for 2610203C20Rik-201 (Rik-201) or primers p3–p4 flanking the premiR-125b-2 sequence. Results expressed as fold over db/+ (*P < 0.05 vs. db/+, n = 6). C and D: Using conventional PCR and gel electrophoresis with primers flanking the premiR-125b-2 on chromosome 16 (primers p3–p4), an unprocessed premiR-125b-2 would result in a 240 bp fragment. D: Primers p3–p4 reveal a 240-bp fragment which is increased in db/db relative to db/+ VSMC. As a negative control, an RNA sample with no reverse transcriptase (no RT) was used for conventional PCR to demonstrate the absence of contamination of genomic DNA in the RNA used for RT-PCR. As an internal control in conventional PCR, 18S primers were used. Contrast of the gel was adjusted uniformly across the whole gel to better visualize the bands. E: RT-qPCR of transcript levels downstream of premiR-125b-2 overlapping RIKEN cDNA clones using primers p5–p6. Results are expressed as fold over db/+ (*P < 0.05 vs. db/+, n = 5). F: Representative results of overall cDNA transcript levels relative to db/+ amplified with primers p3–p4 (*P < 0.05 vs. db/+ within each primer set, primers p3–p4, n = 6, and primers p5–p6, n = 5). G: RT-qPCR amplification of transcript containing both miR-125b-2 and the downstream cDNA using primers p3–p6. Results from one experiment performed in triplicate.
Mentions: We next examined potential regulatory mechanisms for miR-125b in db/db MVSMC. The mouse genome has two copies of miR-125b, namely125b-1 and 125b-2, with both processed into the same mature miR (http://microrna.sanger.ac.uk/sequences) by enzymes such as Drosha and Dicer (26–27). The miR-125b-1 is located in the intron of a known protein coding transcript (2610203C20Rik-201) within chromosome 9, whereas miR-125b-2 is intergenic within chromosome 16 (Fig. 6A). Primers flanking the premiR-125b-1 sequence did not pick up any transcripts (not shown), suggesting that it is processed from the intron of 2610203C20Rik-201. Yet, RT-qPCR to amplify 2610203C20Rik-201 itself (primers p1-p2) indicated no change in its transcription in db/db MVSMC relative to db/+ (Fig. 6B, left two bars). On the other hand, interestingly, RT-qPCR with primers flanking the premiR-125b-2 sequence (primers p3-p4) showed increased transcription of miR-125b-2 in db/db cells relative to db/+ (Fig. 6B, right two bars).

Bottom Line: Conversely, miR-125b inhibitors showed opposite effects.Furthermore, miR-125b mimics increased expression of inflammatory genes, monocyte chemoattractant protein-1, and interleukin-6, and reduced H3K9me3 at their promoters in nondiabetic cells.In addition, we found that the increase in miR-125b in db/db VSMC is caused by increased transcription of miR-125b-2.

View Article: PubMed Central - PubMed

Affiliation: Department of Diabetes, Beckman Research Institute of City of Hope, Duarte, California, USA.

ABSTRACT

Objective: Diabetes remains a major risk factor for vascular complications that seem to persist even after achieving glycemic control, possibly due to "metabolic memory." Using cultured vascular smooth muscle cells (MVSMC) from type 2 diabetic db/db mice, we recently showed that decreased promoter occupancy of the chromatin histone H3 lysine-9 methyltransferase Suv39h1 and the associated repressive epigenetic mark histone H3 lysine-9 trimethylation (H3K9me3) play key roles in sustained inflammatory gene expression. Here we examined the role of microRNAs (miRs) in Suv39h1 regulation and function in MVSMC from diabetic mice.

Research design and methods: We used luciferase assays with Suv39h1 3'untranslated region (UTR) reporter constructs and Western blotting of endogenous protein to verify that miR-125b targets Suv39h1. We examined the effects of Suv39h1 targeting on inflammatory gene expression by quantitative real time polymerase chain reaction (RT-qPCR), and H3K9me3 levels at their promoters by chromatin immunoprecipitation assays.

Results: We observed significant upregulation of miR-125b with parallel downregulation of Suv39h1 protein (predicted miR-125b target) in MVSMC cultured from diabetic db/db mice relative to control db/+. miR-125b mimics inhibited both Suv39h1 3'UTR luciferase reporter activity and endogenous Suv39h1 protein levels. Conversely, miR-125b inhibitors showed opposite effects. Furthermore, miR-125b mimics increased expression of inflammatory genes, monocyte chemoattractant protein-1, and interleukin-6, and reduced H3K9me3 at their promoters in nondiabetic cells. Interestingly, miR-125b mimics increased monocyte binding to db/+ MVSMC toward that in db/db MVSMC, further imitating the proinflammatory diabetic phenotype. In addition, we found that the increase in miR-125b in db/db VSMC is caused by increased transcription of miR-125b-2.

Conclusions: These results demonstrate a novel upstream role for miR-125b in the epigenetic regulation of inflammatory genes in MVSMC of db/db mice through downregulation of Suv39h1.

Show MeSH
Related in: MedlinePlus