MicroRNA-93 controls perfusion recovery after hindlimb ischemia by modulating expression of multiple genes in the cell cycle pathway

Abstract

Background: MicroRNAs are key regulators of gene expression in response to injury, but there is limited knowledge of their role in ischemia-induced angiogenesis, such as in peripheral arterial disease. Here, we used an unbiased strategy and took advantage of different phenotypic outcomes that follow surgically induced hindlimb ischemia between inbred mouse strains to identify key microRNAs involved in perfusion recovery from hindlimb ischemia.

Methods and results: From comparative microRNA profiling between inbred mouse strains that display profound differences in their extent of perfusion recovery after hindlimb ischemia, we found that the mouse strain with higher levels of microRNA-93 (miR-93) in hindlimb muscle before ischemia and the greater ability to upregulate miR-93 in response to ischemia had better perfusion recovery. In vitro, overexpression of miR-93 attenuated hypoxia-induced apoptosis in both endothelial and skeletal muscle cells and enhanced proliferation in both cell types. In addition, miR-93 overexpression enhanced endothelial cell tube formation. In vivo, miR-93 overexpression enhanced capillary density and perfusion recovery from hindlimb ischemia, and antagomirs to miR-93 attenuated perfusion recovery. Both in vitro and in vivo modulation of miR-93 resulted in alterations in the expression of >1 cell cycle pathway gene in 2 different cell types.

Conclusions: Our data indicate that miR-93 enhances perfusion recovery from hindlimb ischemia by modulation of multiple genes that coordinate the functional pathways of cell proliferation and apoptosis. Thus, miR-93 is a strong potential target for pharmacological modulation to promote angiogenesis in ischemic tissue.

Figure 1

Micro-RNA-93 is differentially expressed between C57Bl/6J and BALB/cJ mice. C57BL/6J (B6) mice have greater levels of miR-93 compared to BALB/cJ(BC). (a) Relative expression of miR-93 in non-ischemic gastrocnemius (GA) muscle from C57BL/6J mice is higher compared to that in BALB/cJ mice. (b) Relative expression of miR-106b in non-ischemic GA muscle from C57Bl/6J and BALB/cJ mice was not significantly different.(c) Following induction of hind-limb ischemia, ischemic GA from C57BL/6J mice showed up-regulation of miR-93 at 5.6 ±1.4 fold at day 3 and 3.3 ± 0.5 fold at day-7 compared to non-ischemic GA. BALB/cJ mice did not show this up-regulation of miR-93 (1.4 ± 0.5 fold at day 3, 1.1 ± 0.3 fold at day 7-post hind-limb ischemia). Data represent mean ± SEM; n=5/group); Non-isch=Non-ischemic GA muscle.

Figure 2

Effects of miR-93 modulation in cellular apoptosis and proliferation. (a) In both endothelial cells (HUVEC) and skeletal muscle cells (C2C12), miR-93 is up-regulated after exposure to 6-hours of hypoxia and serum starvation (6h-HSS). Data are representative of three separate experiments, n=3–5 wells/group. Bars represent mean ± SEM. (b) In both HUVECs and C2C12 cells, knockdown of miR-93 resulted in up-regulation of the apoptotic gene caspase-9 even in the absence of any external injury. (c) In HUVECs, over-expression of miR-93 (PremiR-93) attenuates, and knockdown of miR-93 (AntimiR-93) increases hypoxia and serum starvation (HSS, 48- hours exposure)-induced apoptosis compared to scramble treated controls. (d) Similarly, in C2C12 cells, over-expression of miR-93 (PremiR-93) attenuates, while knockdown of miR-93 (AntimiR-93) increases HSS-induced (3-hours exposure) apoptosis. (e) Over-expression of miR-93 increased cell proliferation in both HUVEC and C2C12 cells 48-hours after transfection. Data are mean ± SEM, n=6–8wells/group, data representative of three separate experiments. (f) Forty-eight hours after transfection with Scramble miR-mimic or PremiR-93, HUVECs were plated in matrigel with reduced growth factor, and incubated for 6-hours in basal medium without or with 5% low serum growth supplement (LSGS). PremiR-93 treated HUVECs showed enhanced tube formation, which was quantitated as the number of full tubes per area (represented by bar graph). (g). Similarly, forty-eight hours after transfection with AntimiR-93 or Scramble siRNA sequences, HUVECs were plated in growth factor enriched matrigel and incubated for 6-hours in basal medium with or without a mixture of Endothelial Cell Growth Factors (GF). Tube formation was quantified as number of full tubes per area. As represented by the bar graph, tube formation was reduced in antimiR-93 treated HUVECs. Data are mean ± SEM, n=4wells/group, data representative of two separate experiments.

Figure 2

Effects of miR-93 modulation in cellular apoptosis and proliferation. (a) In both endothelial cells (HUVEC) and skeletal muscle cells (C2C12), miR-93 is up-regulated after exposure to 6-hours of hypoxia and serum starvation (6h-HSS). Data are representative of three separate experiments, n=3–5 wells/group. Bars represent mean ± SEM. (b) In both HUVECs and C2C12 cells, knockdown of miR-93 resulted in up-regulation of the apoptotic gene caspase-9 even in the absence of any external injury. (c) In HUVECs, over-expression of miR-93 (PremiR-93) attenuates, and knockdown of miR-93 (AntimiR-93) increases hypoxia and serum starvation (HSS, 48- hours exposure)-induced apoptosis compared to scramble treated controls. (d) Similarly, in C2C12 cells, over-expression of miR-93 (PremiR-93) attenuates, while knockdown of miR-93 (AntimiR-93) increases HSS-induced (3-hours exposure) apoptosis. (e) Over-expression of miR-93 increased cell proliferation in both HUVEC and C2C12 cells 48-hours after transfection. Data are mean ± SEM, n=6–8wells/group, data representative of three separate experiments. (f) Forty-eight hours after transfection with Scramble miR-mimic or PremiR-93, HUVECs were plated in matrigel with reduced growth factor, and incubated for 6-hours in basal medium without or with 5% low serum growth supplement (LSGS). PremiR-93 treated HUVECs showed enhanced tube formation, which was quantitated as the number of full tubes per area (represented by bar graph). (g). Similarly, forty-eight hours after transfection with AntimiR-93 or Scramble siRNA sequences, HUVECs were plated in growth factor enriched matrigel and incubated for 6-hours in basal medium with or without a mixture of Endothelial Cell Growth Factors (GF). Tube formation was quantified as number of full tubes per area. As represented by the bar graph, tube formation was reduced in antimiR-93 treated HUVECs. Data are mean ± SEM, n=4wells/group, data representative of two separate experiments.

Figure 2

Effects of miR-93 modulation in cellular apoptosis and proliferation. (a) In both endothelial cells (HUVEC) and skeletal muscle cells (C2C12), miR-93 is up-regulated after exposure to 6-hours of hypoxia and serum starvation (6h-HSS). Data are representative of three separate experiments, n=3–5 wells/group. Bars represent mean ± SEM. (b) In both HUVECs and C2C12 cells, knockdown of miR-93 resulted in up-regulation of the apoptotic gene caspase-9 even in the absence of any external injury. (c) In HUVECs, over-expression of miR-93 (PremiR-93) attenuates, and knockdown of miR-93 (AntimiR-93) increases hypoxia and serum starvation (HSS, 48- hours exposure)-induced apoptosis compared to scramble treated controls. (d) Similarly, in C2C12 cells, over-expression of miR-93 (PremiR-93) attenuates, while knockdown of miR-93 (AntimiR-93) increases HSS-induced (3-hours exposure) apoptosis. (e) Over-expression of miR-93 increased cell proliferation in both HUVEC and C2C12 cells 48-hours after transfection. Data are mean ± SEM, n=6–8wells/group, data representative of three separate experiments. (f) Forty-eight hours after transfection with Scramble miR-mimic or PremiR-93, HUVECs were plated in matrigel with reduced growth factor, and incubated for 6-hours in basal medium without or with 5% low serum growth supplement (LSGS). PremiR-93 treated HUVECs showed enhanced tube formation, which was quantitated as the number of full tubes per area (represented by bar graph). (g). Similarly, forty-eight hours after transfection with AntimiR-93 or Scramble siRNA sequences, HUVECs were plated in growth factor enriched matrigel and incubated for 6-hours in basal medium with or without a mixture of Endothelial Cell Growth Factors (GF). Tube formation was quantified as number of full tubes per area. As represented by the bar graph, tube formation was reduced in antimiR-93 treated HUVECs. Data are mean ± SEM, n=4wells/group, data representative of two separate experiments.

Figure 2

Effects of miR-93 modulation in cellular apoptosis and proliferation. (a) In both endothelial cells (HUVEC) and skeletal muscle cells (C2C12), miR-93 is up-regulated after exposure to 6-hours of hypoxia and serum starvation (6h-HSS). Data are representative of three separate experiments, n=3–5 wells/group. Bars represent mean ± SEM. (b) In both HUVECs and C2C12 cells, knockdown of miR-93 resulted in up-regulation of the apoptotic gene caspase-9 even in the absence of any external injury. (c) In HUVECs, over-expression of miR-93 (PremiR-93) attenuates, and knockdown of miR-93 (AntimiR-93) increases hypoxia and serum starvation (HSS, 48- hours exposure)-induced apoptosis compared to scramble treated controls. (d) Similarly, in C2C12 cells, over-expression of miR-93 (PremiR-93) attenuates, while knockdown of miR-93 (AntimiR-93) increases HSS-induced (3-hours exposure) apoptosis. (e) Over-expression of miR-93 increased cell proliferation in both HUVEC and C2C12 cells 48-hours after transfection. Data are mean ± SEM, n=6–8wells/group, data representative of three separate experiments. (f) Forty-eight hours after transfection with Scramble miR-mimic or PremiR-93, HUVECs were plated in matrigel with reduced growth factor, and incubated for 6-hours in basal medium without or with 5% low serum growth supplement (LSGS). PremiR-93 treated HUVECs showed enhanced tube formation, which was quantitated as the number of full tubes per area (represented by bar graph). (g). Similarly, forty-eight hours after transfection with AntimiR-93 or Scramble siRNA sequences, HUVECs were plated in growth factor enriched matrigel and incubated for 6-hours in basal medium with or without a mixture of Endothelial Cell Growth Factors (GF). Tube formation was quantified as number of full tubes per area. As represented by the bar graph, tube formation was reduced in antimiR-93 treated HUVECs. Data are mean ± SEM, n=4wells/group, data representative of two separate experiments.

Figure 2

Effects of miR-93 modulation in cellular apoptosis and proliferation. (a) In both endothelial cells (HUVEC) and skeletal muscle cells (C2C12), miR-93 is up-regulated after exposure to 6-hours of hypoxia and serum starvation (6h-HSS). Data are representative of three separate experiments, n=3–5 wells/group. Bars represent mean ± SEM. (b) In both HUVECs and C2C12 cells, knockdown of miR-93 resulted in up-regulation of the apoptotic gene caspase-9 even in the absence of any external injury. (c) In HUVECs, over-expression of miR-93 (PremiR-93) attenuates, and knockdown of miR-93 (AntimiR-93) increases hypoxia and serum starvation (HSS, 48- hours exposure)-induced apoptosis compared to scramble treated controls. (d) Similarly, in C2C12 cells, over-expression of miR-93 (PremiR-93) attenuates, while knockdown of miR-93 (AntimiR-93) increases HSS-induced (3-hours exposure) apoptosis. (e) Over-expression of miR-93 increased cell proliferation in both HUVEC and C2C12 cells 48-hours after transfection. Data are mean ± SEM, n=6–8wells/group, data representative of three separate experiments. (f) Forty-eight hours after transfection with Scramble miR-mimic or PremiR-93, HUVECs were plated in matrigel with reduced growth factor, and incubated for 6-hours in basal medium without or with 5% low serum growth supplement (LSGS). PremiR-93 treated HUVECs showed enhanced tube formation, which was quantitated as the number of full tubes per area (represented by bar graph). (g). Similarly, forty-eight hours after transfection with AntimiR-93 or Scramble siRNA sequences, HUVECs were plated in growth factor enriched matrigel and incubated for 6-hours in basal medium with or without a mixture of Endothelial Cell Growth Factors (GF). Tube formation was quantified as number of full tubes per area. As represented by the bar graph, tube formation was reduced in antimiR-93 treated HUVECs. Data are mean ± SEM, n=4wells/group, data representative of two separate experiments.

Figure 2

Effects of miR-93 modulation in cellular apoptosis and proliferation. (a) In both endothelial cells (HUVEC) and skeletal muscle cells (C2C12), miR-93 is up-regulated after exposure to 6-hours of hypoxia and serum starvation (6h-HSS). Data are representative of three separate experiments, n=3–5 wells/group. Bars represent mean ± SEM. (b) In both HUVECs and C2C12 cells, knockdown of miR-93 resulted in up-regulation of the apoptotic gene caspase-9 even in the absence of any external injury. (c) In HUVECs, over-expression of miR-93 (PremiR-93) attenuates, and knockdown of miR-93 (AntimiR-93) increases hypoxia and serum starvation (HSS, 48- hours exposure)-induced apoptosis compared to scramble treated controls. (d) Similarly, in C2C12 cells, over-expression of miR-93 (PremiR-93) attenuates, while knockdown of miR-93 (AntimiR-93) increases HSS-induced (3-hours exposure) apoptosis. (e) Over-expression of miR-93 increased cell proliferation in both HUVEC and C2C12 cells 48-hours after transfection. Data are mean ± SEM, n=6–8wells/group, data representative of three separate experiments. (f) Forty-eight hours after transfection with Scramble miR-mimic or PremiR-93, HUVECs were plated in matrigel with reduced growth factor, and incubated for 6-hours in basal medium without or with 5% low serum growth supplement (LSGS). PremiR-93 treated HUVECs showed enhanced tube formation, which was quantitated as the number of full tubes per area (represented by bar graph). (g). Similarly, forty-eight hours after transfection with AntimiR-93 or Scramble siRNA sequences, HUVECs were plated in growth factor enriched matrigel and incubated for 6-hours in basal medium with or without a mixture of Endothelial Cell Growth Factors (GF). Tube formation was quantified as number of full tubes per area. As represented by the bar graph, tube formation was reduced in antimiR-93 treated HUVECs. Data are mean ± SEM, n=4wells/group, data representative of two separate experiments.

Figure 3

Modulation of miR-93 regulates perfusion recovery in C57BL/6J (B6) and BALB/cJ (BC) mice. (a) A single intravenous dose of antagomir-93 (8 mg/kg bw) given prior to hind-limb ischemia (HLI) effectively knocked down miR-93 in the ischemic GA muscle compared to scramble treated mice. This effect was seen as early as day 1, and was persistent until at least day 7 post-HLI. Data are mean ± SEM, n=3/group at days 1 and 7, n=6 at day 3; *p<0.01 at all time points (b) C57Bl/6J mice received three intravenous doses of antagomir-93 or scramble (8 mg/kg bw prior to HLI and repeated at day 7 and 14 post-HLI). Perfusion recovery in the hind-limb was monitored using Doppler imaging. Antagomir-93 treated mice showed significantly impaired perfusion recovery compared to scramble treated mice at day 14 and day 21 post-HLI (n=8–12/group, data represents mean ± SEM). (c) Local intramuscular injections of premiR-93 or miR-mimic negative control (100 nM in 25μl in two sites in the GA and 100nM in 25μl in one site in TA) were done prior to induction of HLI. Following HLI, ischemic tissue of premiR-93 treated mice showed significant up-regulation of miR-93 at day 4, and this effect was persistent until day 10-post-HLI. (n=3/group/time point; data are mean ±SEM). (d) BALB/cJ mice received a single intramuscular injection of premiR-93 prior to induction of HLI, and post-HLI perfusion recovery was monitored using Doppler imaging. PremiR-93 treated mice showed significantly improved perfusion recovery compared to scramble treated mice at day 14 and day 21 post-HLI (n=9–12/group, data represents mean ± SEM).(e)At day 21 following HLI, ischemic gastrocnemius muscle from premiR-93 treated mice showed significantly higher capillary density compared to scramble treated mice (Average capillaries/muscle fiber, Scramble vs. PremiR: 1.2 ±0.1 vs. 1.8 ±01, p=0.004, data represents mean ±SEM. IGA=21-day post-HLI ischemic gastrocnemius muscle).

Figure 3

Modulation of miR-93 regulates perfusion recovery in C57BL/6J (B6) and BALB/cJ (BC) mice. (a) A single intravenous dose of antagomir-93 (8 mg/kg bw) given prior to hind-limb ischemia (HLI) effectively knocked down miR-93 in the ischemic GA muscle compared to scramble treated mice. This effect was seen as early as day 1, and was persistent until at least day 7 post-HLI. Data are mean ± SEM, n=3/group at days 1 and 7, n=6 at day 3; *p<0.01 at all time points (b) C57Bl/6J mice received three intravenous doses of antagomir-93 or scramble (8 mg/kg bw prior to HLI and repeated at day 7 and 14 post-HLI). Perfusion recovery in the hind-limb was monitored using Doppler imaging. Antagomir-93 treated mice showed significantly impaired perfusion recovery compared to scramble treated mice at day 14 and day 21 post-HLI (n=8–12/group, data represents mean ± SEM). (c) Local intramuscular injections of premiR-93 or miR-mimic negative control (100 nM in 25μl in two sites in the GA and 100nM in 25μl in one site in TA) were done prior to induction of HLI. Following HLI, ischemic tissue of premiR-93 treated mice showed significant up-regulation of miR-93 at day 4, and this effect was persistent until day 10-post-HLI. (n=3/group/time point; data are mean ±SEM). (d) BALB/cJ mice received a single intramuscular injection of premiR-93 prior to induction of HLI, and post-HLI perfusion recovery was monitored using Doppler imaging. PremiR-93 treated mice showed significantly improved perfusion recovery compared to scramble treated mice at day 14 and day 21 post-HLI (n=9–12/group, data represents mean ± SEM).(e)At day 21 following HLI, ischemic gastrocnemius muscle from premiR-93 treated mice showed significantly higher capillary density compared to scramble treated mice (Average capillaries/muscle fiber, Scramble vs. PremiR: 1.2 ±0.1 vs. 1.8 ±01, p=0.004, data represents mean ±SEM. IGA=21-day post-HLI ischemic gastrocnemius muscle).

Figure 3

Modulation of miR-93 regulates perfusion recovery in C57BL/6J (B6) and BALB/cJ (BC) mice. (a) A single intravenous dose of antagomir-93 (8 mg/kg bw) given prior to hind-limb ischemia (HLI) effectively knocked down miR-93 in the ischemic GA muscle compared to scramble treated mice. This effect was seen as early as day 1, and was persistent until at least day 7 post-HLI. Data are mean ± SEM, n=3/group at days 1 and 7, n=6 at day 3; *p<0.01 at all time points (b) C57Bl/6J mice received three intravenous doses of antagomir-93 or scramble (8 mg/kg bw prior to HLI and repeated at day 7 and 14 post-HLI). Perfusion recovery in the hind-limb was monitored using Doppler imaging. Antagomir-93 treated mice showed significantly impaired perfusion recovery compared to scramble treated mice at day 14 and day 21 post-HLI (n=8–12/group, data represents mean ± SEM). (c) Local intramuscular injections of premiR-93 or miR-mimic negative control (100 nM in 25μl in two sites in the GA and 100nM in 25μl in one site in TA) were done prior to induction of HLI. Following HLI, ischemic tissue of premiR-93 treated mice showed significant up-regulation of miR-93 at day 4, and this effect was persistent until day 10-post-HLI. (n=3/group/time point; data are mean ±SEM). (d) BALB/cJ mice received a single intramuscular injection of premiR-93 prior to induction of HLI, and post-HLI perfusion recovery was monitored using Doppler imaging. PremiR-93 treated mice showed significantly improved perfusion recovery compared to scramble treated mice at day 14 and day 21 post-HLI (n=9–12/group, data represents mean ± SEM).(e)At day 21 following HLI, ischemic gastrocnemius muscle from premiR-93 treated mice showed significantly higher capillary density compared to scramble treated mice (Average capillaries/muscle fiber, Scramble vs. PremiR: 1.2 ±0.1 vs. 1.8 ±01, p=0.004, data represents mean ±SEM. IGA=21-day post-HLI ischemic gastrocnemius muscle).

Figure 3

Modulation of miR-93 regulates perfusion recovery in C57BL/6J (B6) and BALB/cJ (BC) mice. (a) A single intravenous dose of antagomir-93 (8 mg/kg bw) given prior to hind-limb ischemia (HLI) effectively knocked down miR-93 in the ischemic GA muscle compared to scramble treated mice. This effect was seen as early as day 1, and was persistent until at least day 7 post-HLI. Data are mean ± SEM, n=3/group at days 1 and 7, n=6 at day 3; *p<0.01 at all time points (b) C57Bl/6J mice received three intravenous doses of antagomir-93 or scramble (8 mg/kg bw prior to HLI and repeated at day 7 and 14 post-HLI). Perfusion recovery in the hind-limb was monitored using Doppler imaging. Antagomir-93 treated mice showed significantly impaired perfusion recovery compared to scramble treated mice at day 14 and day 21 post-HLI (n=8–12/group, data represents mean ± SEM). (c) Local intramuscular injections of premiR-93 or miR-mimic negative control (100 nM in 25μl in two sites in the GA and 100nM in 25μl in one site in TA) were done prior to induction of HLI. Following HLI, ischemic tissue of premiR-93 treated mice showed significant up-regulation of miR-93 at day 4, and this effect was persistent until day 10-post-HLI. (n=3/group/time point; data are mean ±SEM). (d) BALB/cJ mice received a single intramuscular injection of premiR-93 prior to induction of HLI, and post-HLI perfusion recovery was monitored using Doppler imaging. PremiR-93 treated mice showed significantly improved perfusion recovery compared to scramble treated mice at day 14 and day 21 post-HLI (n=9–12/group, data represents mean ± SEM).(e)At day 21 following HLI, ischemic gastrocnemius muscle from premiR-93 treated mice showed significantly higher capillary density compared to scramble treated mice (Average capillaries/muscle fiber, Scramble vs. PremiR: 1.2 ±0.1 vs. 1.8 ±01, p=0.004, data represents mean ±SEM. IGA=21-day post-HLI ischemic gastrocnemius muscle).

Figure 3

Modulation of miR-93 regulates perfusion recovery in C57BL/6J (B6) and BALB/cJ (BC) mice. (a) A single intravenous dose of antagomir-93 (8 mg/kg bw) given prior to hind-limb ischemia (HLI) effectively knocked down miR-93 in the ischemic GA muscle compared to scramble treated mice. This effect was seen as early as day 1, and was persistent until at least day 7 post-HLI. Data are mean ± SEM, n=3/group at days 1 and 7, n=6 at day 3; *p<0.01 at all time points (b) C57Bl/6J mice received three intravenous doses of antagomir-93 or scramble (8 mg/kg bw prior to HLI and repeated at day 7 and 14 post-HLI). Perfusion recovery in the hind-limb was monitored using Doppler imaging. Antagomir-93 treated mice showed significantly impaired perfusion recovery compared to scramble treated mice at day 14 and day 21 post-HLI (n=8–12/group, data represents mean ± SEM). (c) Local intramuscular injections of premiR-93 or miR-mimic negative control (100 nM in 25μl in two sites in the GA and 100nM in 25μl in one site in TA) were done prior to induction of HLI. Following HLI, ischemic tissue of premiR-93 treated mice showed significant up-regulation of miR-93 at day 4, and this effect was persistent until day 10-post-HLI. (n=3/group/time point; data are mean ±SEM). (d) BALB/cJ mice received a single intramuscular injection of premiR-93 prior to induction of HLI, and post-HLI perfusion recovery was monitored using Doppler imaging. PremiR-93 treated mice showed significantly improved perfusion recovery compared to scramble treated mice at day 14 and day 21 post-HLI (n=9–12/group, data represents mean ± SEM).(e)At day 21 following HLI, ischemic gastrocnemius muscle from premiR-93 treated mice showed significantly higher capillary density compared to scramble treated mice (Average capillaries/muscle fiber, Scramble vs. PremiR: 1.2 ±0.1 vs. 1.8 ±01, p=0.004, data represents mean ±SEM. IGA=21-day post-HLI ischemic gastrocnemius muscle).

Figure 4

Gene changes in ischemic muscles with miR-93 modulation in-vivo in C57Bl/6J and BALB/cJ mice. (a) In gastrocnemius (GA) muscle from C57BL/6J mice at day 3 post-HLI, miR-93 knockdown resulted in increased mRNA levels of p21 and p53 compared to scramble treated mice, while E2F-1 levels were not different at the mRNA level (b) In GA muscle from C57BL/6J mice at day 3 post-HLI, miR-93 knockdown resulted in increased protein levels of p21, E2F-1 and P53 compared to scramble treated mice as assessed by western blot. (n=6/group; data represent mean ± SEM). At day 3 post-hind-limb ischemia, ischemic GA from BALB/cJ mice with miR-93 over-expression showed down-regulation of p21, E2F-1 and p53 at both mRNA (c) and protein (d) levels compared to scramble treated mice (n=6/group; data represent mean ± SEM; western blot quantitation done using densitometry, expression levels normalized to actin). (e). heat map of the genes in the cell cycle pathway in ischemic (IGA) vs. non-ischemic (NGA) gastrocnemius muscle from untreated BALB/cJ mice at day 3 post-hind-limb ischemia. P21, E2F-1 and p53, all three genes downregulated by miR-93 over-expression in BALB/cJ mice were upregulated in ischemic muscles from untreated BALB/cJ mice. Other highlighted genes are known and/or predicted targets of miR-93 based on literature and computational predictions, but were not found to be changed with miR-93 modulation in our experiments.

Figure 4

Gene changes in ischemic muscles with miR-93 modulation in-vivo in C57Bl/6J and BALB/cJ mice. (a) In gastrocnemius (GA) muscle from C57BL/6J mice at day 3 post-HLI, miR-93 knockdown resulted in increased mRNA levels of p21 and p53 compared to scramble treated mice, while E2F-1 levels were not different at the mRNA level (b) In GA muscle from C57BL/6J mice at day 3 post-HLI, miR-93 knockdown resulted in increased protein levels of p21, E2F-1 and P53 compared to scramble treated mice as assessed by western blot. (n=6/group; data represent mean ± SEM). At day 3 post-hind-limb ischemia, ischemic GA from BALB/cJ mice with miR-93 over-expression showed down-regulation of p21, E2F-1 and p53 at both mRNA (c) and protein (d) levels compared to scramble treated mice (n=6/group; data represent mean ± SEM; western blot quantitation done using densitometry, expression levels normalized to actin). (e). heat map of the genes in the cell cycle pathway in ischemic (IGA) vs. non-ischemic (NGA) gastrocnemius muscle from untreated BALB/cJ mice at day 3 post-hind-limb ischemia. P21, E2F-1 and p53, all three genes downregulated by miR-93 over-expression in BALB/cJ mice were upregulated in ischemic muscles from untreated BALB/cJ mice. Other highlighted genes are known and/or predicted targets of miR-93 based on literature and computational predictions, but were not found to be changed with miR-93 modulation in our experiments.

Figure 4

Gene changes in ischemic muscles with miR-93 modulation in-vivo in C57Bl/6J and BALB/cJ mice. (a) In gastrocnemius (GA) muscle from C57BL/6J mice at day 3 post-HLI, miR-93 knockdown resulted in increased mRNA levels of p21 and p53 compared to scramble treated mice, while E2F-1 levels were not different at the mRNA level (b) In GA muscle from C57BL/6J mice at day 3 post-HLI, miR-93 knockdown resulted in increased protein levels of p21, E2F-1 and P53 compared to scramble treated mice as assessed by western blot. (n=6/group; data represent mean ± SEM). At day 3 post-hind-limb ischemia, ischemic GA from BALB/cJ mice with miR-93 over-expression showed down-regulation of p21, E2F-1 and p53 at both mRNA (c) and protein (d) levels compared to scramble treated mice (n=6/group; data represent mean ± SEM; western blot quantitation done using densitometry, expression levels normalized to actin). (e). heat map of the genes in the cell cycle pathway in ischemic (IGA) vs. non-ischemic (NGA) gastrocnemius muscle from untreated BALB/cJ mice at day 3 post-hind-limb ischemia. P21, E2F-1 and p53, all three genes downregulated by miR-93 over-expression in BALB/cJ mice were upregulated in ischemic muscles from untreated BALB/cJ mice. Other highlighted genes are known and/or predicted targets of miR-93 based on literature and computational predictions, but were not found to be changed with miR-93 modulation in our experiments.

Figure 4

Gene changes in ischemic muscles with miR-93 modulation in-vivo in C57Bl/6J and BALB/cJ mice. (a) In gastrocnemius (GA) muscle from C57BL/6J mice at day 3 post-HLI, miR-93 knockdown resulted in increased mRNA levels of p21 and p53 compared to scramble treated mice, while E2F-1 levels were not different at the mRNA level (b) In GA muscle from C57BL/6J mice at day 3 post-HLI, miR-93 knockdown resulted in increased protein levels of p21, E2F-1 and P53 compared to scramble treated mice as assessed by western blot. (n=6/group; data represent mean ± SEM). At day 3 post-hind-limb ischemia, ischemic GA from BALB/cJ mice with miR-93 over-expression showed down-regulation of p21, E2F-1 and p53 at both mRNA (c) and protein (d) levels compared to scramble treated mice (n=6/group; data represent mean ± SEM; western blot quantitation done using densitometry, expression levels normalized to actin). (e). heat map of the genes in the cell cycle pathway in ischemic (IGA) vs. non-ischemic (NGA) gastrocnemius muscle from untreated BALB/cJ mice at day 3 post-hind-limb ischemia. P21, E2F-1 and p53, all three genes downregulated by miR-93 over-expression in BALB/cJ mice were upregulated in ischemic muscles from untreated BALB/cJ mice. Other highlighted genes are known and/or predicted targets of miR-93 based on literature and computational predictions, but were not found to be changed with miR-93 modulation in our experiments.

Figure 4

Gene changes in ischemic muscles with miR-93 modulation in-vivo in C57Bl/6J and BALB/cJ mice. (a) In gastrocnemius (GA) muscle from C57BL/6J mice at day 3 post-HLI, miR-93 knockdown resulted in increased mRNA levels of p21 and p53 compared to scramble treated mice, while E2F-1 levels were not different at the mRNA level (b) In GA muscle from C57BL/6J mice at day 3 post-HLI, miR-93 knockdown resulted in increased protein levels of p21, E2F-1 and P53 compared to scramble treated mice as assessed by western blot. (n=6/group; data represent mean ± SEM). At day 3 post-hind-limb ischemia, ischemic GA from BALB/cJ mice with miR-93 over-expression showed down-regulation of p21, E2F-1 and p53 at both mRNA (c) and protein (d) levels compared to scramble treated mice (n=6/group; data represent mean ± SEM; western blot quantitation done using densitometry, expression levels normalized to actin). (e). heat map of the genes in the cell cycle pathway in ischemic (IGA) vs. non-ischemic (NGA) gastrocnemius muscle from untreated BALB/cJ mice at day 3 post-hind-limb ischemia. P21, E2F-1 and p53, all three genes downregulated by miR-93 over-expression in BALB/cJ mice were upregulated in ischemic muscles from untreated BALB/cJ mice. Other highlighted genes are known and/or predicted targets of miR-93 based on literature and computational predictions, but were not found to be changed with miR-93 modulation in our experiments.