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MicroRNA modulation induced by AICA ribonucleotide in J1 mouse ES cells.

Shi X - PLoS ONE (2014)

Bottom Line: The result showed that AICAR can significantly modulate the expression of multiple miRNAs, including those have crucial functions in ES cell development.For the differently expressed miRNAs identified, further study was conducted regarding the pluripotency and differentiation associated miRNAs with their targets.Moreover, miR-134 was significantly down-regulated after AICAR treatment, and this was suggested to be directly associated with the up-regulated pluripotency markers, Nanog and Sox2.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, Northwest A&F University, Yangling, Shaanxi, China; Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling, Shaanxi, China.

ABSTRACT
ES cells can propagate indefinitely, maintain self-renewal, and differentiate into almost any cell type of the body. These properties make them valuable in the research of embryonic development, regenerative medicine, and organ transplantation. MicroRNAs (miRNAs) are considered to have essential functions in the maintenance and differentiation of embryonic stem cells (ES cells). It was reported that, strong external stimuli, such as a transient low-pH and hypoxia stress, were conducive to the formation of induced pluripotent stem cells (iPS cells). AICA ribonucleotide (AICAR) is an AMP-activated protein kinase activator, which can let cells in the state of energy stress. We have demonstrated that AICAR can maintain the pluripotency of J1 mouse ES cells through modulating protein expression in our previous research, but its effects on ES cell miRNA expression remain unknown. In this study, we conducted small RNA high-throughput sequencing to investigate AICAR influence on J1 mouse ES cells by comparing the miRNA expression patterns of the AICAR-treated cells and those without treatment. The result showed that AICAR can significantly modulate the expression of multiple miRNAs, including those have crucial functions in ES cell development. Some differentially expressed miRNAs were selected and confirmed by real-time PCR. For the differently expressed miRNAs identified, further study was conducted regarding the pluripotency and differentiation associated miRNAs with their targets. Moreover, miR-134 was significantly down-regulated after AICAR treatment, and this was suggested to be directly associated with the up-regulated pluripotency markers, Nanog and Sox2. Lastly, Myc was significantly down-regulated after AICAR treatment; therefore, we predicted miRNAs that may target Myc and identified that AICAR induced up-regulation of miR-34a, 34b, and 34c can repress Myc expression in J1 mouse ES cells. Taken together, our study provide a new mechanism for AICAR in ES cells pluripotency maintenance and give insight for its usage in iPS cells generation.

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Several miRNAs targeted Myc and down-regulated its expression.(A) The expression of Myc and the predicted miRNAs was validated by real-time PCR in the presence of 1 mM AICAR. (B) Western blot analysis of Myc expression in J1 ES cells in the presence of 1,000 U/ml LIF and with or without 1 mM AICAR for 24 h. Cell lysates were extracted and analyzed by western blot. Relative expression level were comparing to Gapdh. (C/D/E/F/G) MiR-34a/34b/34c/340/135b expression vector pCDH-mir34a/34b/34c/340/135b and their negative control were transfected into J1 ES cells, and miR-34a/34b/34c/340/135b expression was detected by real-time PCR. (H) The schematic representation for construction of the firefly luciferase reporter vector. Myc 3′-UTR was inserted into psiCHECK-2 vector by XbaI and EcoRI sites. (I) Brief description of predicted miRNAs and their target sites on Myc 3′-UTR. (J) The luciferase reporters were co-transfected with miRNA expression vectors, and empty vector pCDH-GFP without insertion was used as control. 24 and 48 h after transfection, luciferase activity was detected using dual-luciferase reporter assay. (K) Brief description of mutated sites on Myc 3′-UTR. (L) The mutated luciferase reporters were co-transfected with miRNA expression vectors, and empty vector pCDH-GFP without insertion was used as control. 24 h after transfection, luciferase activity was detected using dual-luciferase reporter assay. (M) The mutated luciferase reporters were co-transfected with miRNA expression vectors, and empty vector pCDH-GFP without insertion was used as control. 48 h after transfection, luciferase activity was detected using dual-luciferase reporter assay. (N) The indicated miRNA expression vectors were transfected into J1 ES cells. 48 h after transfection, Myc expression was detected by real-time PCR. Gapdh was used to normalize template levels. (O) The indicated miRNA expression vectors were transfected into J1 ES cells. 48 h after transfection, cell lysates were extracted and analyzed by western blot. Relative expression level were comparing to Gapdh. (WB: western blot; *: p<0.05; **: p<0.01).
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pone-0103724-g004: Several miRNAs targeted Myc and down-regulated its expression.(A) The expression of Myc and the predicted miRNAs was validated by real-time PCR in the presence of 1 mM AICAR. (B) Western blot analysis of Myc expression in J1 ES cells in the presence of 1,000 U/ml LIF and with or without 1 mM AICAR for 24 h. Cell lysates were extracted and analyzed by western blot. Relative expression level were comparing to Gapdh. (C/D/E/F/G) MiR-34a/34b/34c/340/135b expression vector pCDH-mir34a/34b/34c/340/135b and their negative control were transfected into J1 ES cells, and miR-34a/34b/34c/340/135b expression was detected by real-time PCR. (H) The schematic representation for construction of the firefly luciferase reporter vector. Myc 3′-UTR was inserted into psiCHECK-2 vector by XbaI and EcoRI sites. (I) Brief description of predicted miRNAs and their target sites on Myc 3′-UTR. (J) The luciferase reporters were co-transfected with miRNA expression vectors, and empty vector pCDH-GFP without insertion was used as control. 24 and 48 h after transfection, luciferase activity was detected using dual-luciferase reporter assay. (K) Brief description of mutated sites on Myc 3′-UTR. (L) The mutated luciferase reporters were co-transfected with miRNA expression vectors, and empty vector pCDH-GFP without insertion was used as control. 24 h after transfection, luciferase activity was detected using dual-luciferase reporter assay. (M) The mutated luciferase reporters were co-transfected with miRNA expression vectors, and empty vector pCDH-GFP without insertion was used as control. 48 h after transfection, luciferase activity was detected using dual-luciferase reporter assay. (N) The indicated miRNA expression vectors were transfected into J1 ES cells. 48 h after transfection, Myc expression was detected by real-time PCR. Gapdh was used to normalize template levels. (O) The indicated miRNA expression vectors were transfected into J1 ES cells. 48 h after transfection, cell lysates were extracted and analyzed by western blot. Relative expression level were comparing to Gapdh. (WB: western blot; *: p<0.05; **: p<0.01).

Mentions: First, we predicted miRNAs that may target the 3′-UTR of Myc using TargetScan and miRanda database. By combining our sequencing data with the predicted result, we selected miR-34a, miR-34b, miR-34c, miR-340, and miR-135b to conduct further research. We performed real-time PCR to detect the expression level of Myc and of those predicted miRNAs. Real-time PCR results showed that Myc expression was significantly down-regulated, whereas the expression of miRNAs that may target Myc was increased (Figure 4A). The expression of Myc was also verified by western blot analysis (Figure 4B).


MicroRNA modulation induced by AICA ribonucleotide in J1 mouse ES cells.

Shi X - PLoS ONE (2014)

Several miRNAs targeted Myc and down-regulated its expression.(A) The expression of Myc and the predicted miRNAs was validated by real-time PCR in the presence of 1 mM AICAR. (B) Western blot analysis of Myc expression in J1 ES cells in the presence of 1,000 U/ml LIF and with or without 1 mM AICAR for 24 h. Cell lysates were extracted and analyzed by western blot. Relative expression level were comparing to Gapdh. (C/D/E/F/G) MiR-34a/34b/34c/340/135b expression vector pCDH-mir34a/34b/34c/340/135b and their negative control were transfected into J1 ES cells, and miR-34a/34b/34c/340/135b expression was detected by real-time PCR. (H) The schematic representation for construction of the firefly luciferase reporter vector. Myc 3′-UTR was inserted into psiCHECK-2 vector by XbaI and EcoRI sites. (I) Brief description of predicted miRNAs and their target sites on Myc 3′-UTR. (J) The luciferase reporters were co-transfected with miRNA expression vectors, and empty vector pCDH-GFP without insertion was used as control. 24 and 48 h after transfection, luciferase activity was detected using dual-luciferase reporter assay. (K) Brief description of mutated sites on Myc 3′-UTR. (L) The mutated luciferase reporters were co-transfected with miRNA expression vectors, and empty vector pCDH-GFP without insertion was used as control. 24 h after transfection, luciferase activity was detected using dual-luciferase reporter assay. (M) The mutated luciferase reporters were co-transfected with miRNA expression vectors, and empty vector pCDH-GFP without insertion was used as control. 48 h after transfection, luciferase activity was detected using dual-luciferase reporter assay. (N) The indicated miRNA expression vectors were transfected into J1 ES cells. 48 h after transfection, Myc expression was detected by real-time PCR. Gapdh was used to normalize template levels. (O) The indicated miRNA expression vectors were transfected into J1 ES cells. 48 h after transfection, cell lysates were extracted and analyzed by western blot. Relative expression level were comparing to Gapdh. (WB: western blot; *: p<0.05; **: p<0.01).
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pone-0103724-g004: Several miRNAs targeted Myc and down-regulated its expression.(A) The expression of Myc and the predicted miRNAs was validated by real-time PCR in the presence of 1 mM AICAR. (B) Western blot analysis of Myc expression in J1 ES cells in the presence of 1,000 U/ml LIF and with or without 1 mM AICAR for 24 h. Cell lysates were extracted and analyzed by western blot. Relative expression level were comparing to Gapdh. (C/D/E/F/G) MiR-34a/34b/34c/340/135b expression vector pCDH-mir34a/34b/34c/340/135b and their negative control were transfected into J1 ES cells, and miR-34a/34b/34c/340/135b expression was detected by real-time PCR. (H) The schematic representation for construction of the firefly luciferase reporter vector. Myc 3′-UTR was inserted into psiCHECK-2 vector by XbaI and EcoRI sites. (I) Brief description of predicted miRNAs and their target sites on Myc 3′-UTR. (J) The luciferase reporters were co-transfected with miRNA expression vectors, and empty vector pCDH-GFP without insertion was used as control. 24 and 48 h after transfection, luciferase activity was detected using dual-luciferase reporter assay. (K) Brief description of mutated sites on Myc 3′-UTR. (L) The mutated luciferase reporters were co-transfected with miRNA expression vectors, and empty vector pCDH-GFP without insertion was used as control. 24 h after transfection, luciferase activity was detected using dual-luciferase reporter assay. (M) The mutated luciferase reporters were co-transfected with miRNA expression vectors, and empty vector pCDH-GFP without insertion was used as control. 48 h after transfection, luciferase activity was detected using dual-luciferase reporter assay. (N) The indicated miRNA expression vectors were transfected into J1 ES cells. 48 h after transfection, Myc expression was detected by real-time PCR. Gapdh was used to normalize template levels. (O) The indicated miRNA expression vectors were transfected into J1 ES cells. 48 h after transfection, cell lysates were extracted and analyzed by western blot. Relative expression level were comparing to Gapdh. (WB: western blot; *: p<0.05; **: p<0.01).
Mentions: First, we predicted miRNAs that may target the 3′-UTR of Myc using TargetScan and miRanda database. By combining our sequencing data with the predicted result, we selected miR-34a, miR-34b, miR-34c, miR-340, and miR-135b to conduct further research. We performed real-time PCR to detect the expression level of Myc and of those predicted miRNAs. Real-time PCR results showed that Myc expression was significantly down-regulated, whereas the expression of miRNAs that may target Myc was increased (Figure 4A). The expression of Myc was also verified by western blot analysis (Figure 4B).

Bottom Line: The result showed that AICAR can significantly modulate the expression of multiple miRNAs, including those have crucial functions in ES cell development.For the differently expressed miRNAs identified, further study was conducted regarding the pluripotency and differentiation associated miRNAs with their targets.Moreover, miR-134 was significantly down-regulated after AICAR treatment, and this was suggested to be directly associated with the up-regulated pluripotency markers, Nanog and Sox2.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, Northwest A&F University, Yangling, Shaanxi, China; Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling, Shaanxi, China.

ABSTRACT
ES cells can propagate indefinitely, maintain self-renewal, and differentiate into almost any cell type of the body. These properties make them valuable in the research of embryonic development, regenerative medicine, and organ transplantation. MicroRNAs (miRNAs) are considered to have essential functions in the maintenance and differentiation of embryonic stem cells (ES cells). It was reported that, strong external stimuli, such as a transient low-pH and hypoxia stress, were conducive to the formation of induced pluripotent stem cells (iPS cells). AICA ribonucleotide (AICAR) is an AMP-activated protein kinase activator, which can let cells in the state of energy stress. We have demonstrated that AICAR can maintain the pluripotency of J1 mouse ES cells through modulating protein expression in our previous research, but its effects on ES cell miRNA expression remain unknown. In this study, we conducted small RNA high-throughput sequencing to investigate AICAR influence on J1 mouse ES cells by comparing the miRNA expression patterns of the AICAR-treated cells and those without treatment. The result showed that AICAR can significantly modulate the expression of multiple miRNAs, including those have crucial functions in ES cell development. Some differentially expressed miRNAs were selected and confirmed by real-time PCR. For the differently expressed miRNAs identified, further study was conducted regarding the pluripotency and differentiation associated miRNAs with their targets. Moreover, miR-134 was significantly down-regulated after AICAR treatment, and this was suggested to be directly associated with the up-regulated pluripotency markers, Nanog and Sox2. Lastly, Myc was significantly down-regulated after AICAR treatment; therefore, we predicted miRNAs that may target Myc and identified that AICAR induced up-regulation of miR-34a, 34b, and 34c can repress Myc expression in J1 mouse ES cells. Taken together, our study provide a new mechanism for AICAR in ES cells pluripotency maintenance and give insight for its usage in iPS cells generation.

Show MeSH
Related in: MedlinePlus