MicroRNA-30 inhibits neointimal hyperplasia by targeting Ca(2+)/calmodulin-dependent protein kinase IIδ (CaMKIIδ)

Abstract

The multifunctional Ca(2+)/calmodulin-dependent protein kinase II δ-isoform (CaMKIIδ) promotes vascular smooth muscle (VSM) proliferation, migration, and injury-induced vascular wall neointima formation. The objective of this study was to test if microRNA-30 (miR-30) family members are endogenous regulators of CaMKIIδ expression following vascular injury and whether ectopic expression of miR-30 can inhibit CaMKIIδ-dependent VSM cell function and neointimal VSM hyperplasia induced by vascular injury. The CaMKIIδ 3'UTR contains a consensus miR-30 binding sequence that is highly conserved across species. A significant decrease in miR-30 family members and increase in CaMKIIδ2 protein expression, with no change in CaMKIIδ mRNA expression, was observed in medial layers of VSM 7 days post-injury. In vitro, overexpression of miR-30c or miR-30e inhibited CaMKIIδ2 protein expression by ~50% in cultured rat aortic VSM cells, and inhibited VSM cell proliferation and migration. In vivo, lenti-viral delivery of miR-30c into injured rat carotid arteries prevented the injury-induced increase in CaMKIIδ2. Furthermore, neointima formation was dramatically inhibited by lenti-viral delivery of miR-30c in the injured medial smooth muscle. These studies define a novel mechanism for regulating CaMKIIδ expression in VSM and provide a new potential therapeutic strategy to reduce progression of vascular proliferative diseases, including atherosclerosis and restenosis.

Figure 1. Vascular injury induces reciprocal regulation of miR-30 family members and CaMKIIδ protein in rat carotid artery.

(a) Rat carotid artery was intraluminally injured and the protein levels of CaMKIIδ, Calponin, SM-MHC and β-actin (as loading reference) were measured by SDS-PAGE and immnuno bloting using specific antibodies. Histogram shows the quantification of protein levels normalized over β-actin. (b) Quantitative reverse transcriptase polymerase chain reaction (qPCR) results show mRNA levels of smooth muscle myosin heavy chain (SM-MHC), smooth muscle 22α, Klf4 and CaMKIIδ in the contra uninjured and injured carotid arteries 7 days after injury. (c) Complimentary sequences between rat CaMKIIδ 3′UTR and rat miR-30 family members. The expressions of miR-30 family members and miR-145 were compared between injured and uninjured carotid arteries and U6 expression was measured and used for normalization. Values shown are mean±S.E.M., n = 3 pairs and *p < 0.05 **p < 0.01 analyzed by paired t-test.

Figure 2. MiR-30 inhibits CaMKIIδ expression by targeting CaMKIIδ 3′UTR in primarily cultured smooth muscle cells.

Rat aorta medial layers were enzymatically dispersed and primarily cultured for 3–7 passages. (a) The miRNA and mRNA levels of miR-30a, miR-30b, miR-30c, miR-30d, miR-30e, SM-MHC, SM-22α and Klf4 were measured by qPCR in aorta and cultured smooth muscle cells. Data were normalized over U6 (miRNA) or GAPDH (mRNA) (n = 3 aorta and 5 cultured SM cells). (b) miR-30c mimic or miR-30e (Invitrogen) (0.2pmol) was eletroporated into primarily cultured SM cells (1 million) and the expression of CaMKIIδ and GAPDH was analyzed by Western blot 3 days post-electroporation. (c) Full length of CaMKIIδ 3′UTR or truncated CaMKIIδ 3′UTR was introduced into pmiR reporter vector (Invitrogen). Full length CaMKIIδ 3′UTR reporter or truncated CaMKIIδ 3′UTR reporter as well as miR-30c and renilla were transfected into HEK293 cells and luciferase activity was measured 3 days after transfection. Values are shown as mean +/- S.E. M., n ≥ 4 and analyzed by two-way ANOVA or t-test. *p < 0.05 **p < 0.01 and *** p < 0.001.

Figure 3. MiR-30 inhibits vascular smooth muscle cell proliferation.

miR-30e mimic (0.2 pmol) was eletroporated into cultured vascular smooth muscle cells (50,000). (a) Cell numbers were counted at 24 h, 48 h and 72 h post eletroporation to monitor cell growth and proliferation. (b) The level of miR-30 family members was tested using qPCR 72 h after eletroporation. (c) Cell lysates were immnuoblotting for PCNA and GAPDH and quantified data were normalized over GAPDH. Values shown are mean±S.E.M., n = 3 pairs and *p < 0.05 ***p < 0.001 analyzed by two-way ANOVA.

Figure 4. Overexpression of CaMKIIδ partially rescues VSM proliferation inhibition by miR-30e.

Cultured VSM cells were eletroporated with scrambled miRNA or miR-30e mimic (0.2 pmol) and after 24 h, adeno-virus encoding CaMKIIδ2HA or GFP (2.5 MOI) was utilized to infect electroporated VSM cells for CaMKIIδ rescue. (a) Cell lysates were analyzed at 48 h and 72 h post eletroporation by western blot using specific antibodies for CaMKIIδ and GAPDH. Quantified data were normalized over GAPDH. (b) VSM Cell growth were examined by counting cell number 24 h, 48 h, and 72 h post electroporation. Values are shown as mean ± S.E.M., n = 3 and *p < 0.05, **p < 0.01 ***p < 0.001 analyzed by two-way ANOVA.

Figure 5. Overexpression of miR-30c prevents injury induced increase of CaMKIIδ and attenuates neointima formation.

Rat carotid artery was balloon injured and locally infected with lentivirus encoding GFP and miR-30c. After 14 days, immunoblotting and histochemistry were employed for protein and morphology analysis. (a) The protein level of CaMKIIδ, calponin, GFP and β-actin was tested in uninjured and injured carotid arteries. Quantified data were normalized over β-actin and shown as mean ± S.E.M., n = 3. *p < 0.05 by paired t-test comparing CaMKIIδ expression between injured (IN) and uninjured (UN) arteries. (b) Injured carotid artery was fixed followed with sectioning (8μm) and staining with hematoxylin and eosin. The areas of medial layer and neointima were measured and quantified. Data shown are the ratio of neointima over medial layer, and presented as mean ± S.E.M., n = 3. ** indicates p < 0.01 by unpaired t-test.