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. 2015 Mar 15;7(3):489-501.
eCollection 2015.

Evaluation of miR-29c inhibits endotheliocyte migration and angiogenesis of human endothelial cells by suppressing the insulin like growth factor 1

Yun Hu  1 , Feng Deng  1 , Jinlin Song  1 , Juhong Lin  1 , Xue Li  2 , Yuying Tang  1 , Jie Zhou  1 , Tian Tang  3 , Leilei Zheng  1
Affiliations

Evaluation of miR-29c inhibits endotheliocyte migration and angiogenesis of human endothelial cells by suppressing the insulin like growth factor 1

Yun Hu et al. Am J Transl Res. .

Abstract

MicroRNAs, a class of 22-nucleotide non-coding RNAs, modulate gene expression by associating with the 3'-untranslated regions (3'-UTRs) of messenger RNAs (mRNAs). Although multiple miRNAs are known to be regulated during angiogenesis, their individual roles in blood vessel development are still not fully understood. Herein, we investigate the role of miR-29c in regulating cell cycle and angiogenic phenotype of endothelial cells. The results showed that IGF-1 is highly expressed and down-regulated by miR-29c in human umbilical vein endothelial cells (HUVEC). Consistent with this preliminary finding, introduction of exogenous miR-29c or miR-29c inhibitor alters cell cycle progression, proliferation and tube formation of HUVEC, respectively. Furthermore, by using luciferase reporter assay, we find that the expression of IGF-1, a suppressor transcription factor, is directly regulated by miR-29c through 3'-UTR. In addition, we show that the selective inhibition of PI3K/AKT pathway prior to miR-29c stimulation prevents the expression of angiogenesis suppressor miRNAs that are family and cluster specific. As a conclusion, we find that miR-29c plays a significant role in regulating cell cycle, proliferation and angiogenic properties of HUVECs. This function is likely mediated through IGF-1 proteins at the post-transcriptional level. As a novel molecular target, miR-29c may have a potential value in the treatment of angiogenesis-associated diseases, such as cardiovascular diseases and cancers.

Keywords: IGF-1; angiogenesis; endothelial cell; miRNA-29c.

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Figures

Figure 1
Figure 1
(A) HUVECs were incubated with IGF-1 (40 ng/ml) for 72 h. The expression levels of miR-29c was descend significantly after IGF-1 treatment compared to control. (B) HUVECs were incubated with different concentrations of IGF-1 for 24 h, the expression levels of miR-29c was detected by qRT-PCR. Control cells were untreated. (C) Immunocytochemical assay shown the IGF1expression levels after treatment. (D) miR-29c directly down-regulates IGF-1 expression. (E) Predict IGF-1 was potential targets for miR-29c and its validation. Luciference reporter assay was performed to detect the effect of miR-29c and anti-sense miR-29c (F) on the luciference intensity controlled by 3’UTR of IGF-1. Mean ± SD (n = 3). *p < 0.05, **p < 0.01.
Figure 2
Figure 2
Overexpression of miR-29c reduces HUVECs proliferation in vitro. (A) CCK-8 assays revealed that upregulation of miR-29c reduced cell proliferation of HUVECs and 293T cells (B), compared to negative (NC) transfected cells. (C, D) EDU incorporation assay. In the image overlay, the purple nuclei are EDU stained and indicate proliferating cells, while the blue nuclei are DAPI stained. (E, F) Effect of miR-29c on cell cycle of HUVECs. HUVECs were subjected to Propidium iodide (PI) Flow Cytometry analysis. Overexpression of miR-29c by miR-29c mimic accelerated S to G1 cell cycle transition, while inhibition of miR-29c increase G2 population and S phase population. Data represents Mean ± SE of three independent experiments, *p < 0.05, **p < 0.01.
Figure 3
Figure 3
Effect of miR-29c on tube network formation in HUVECs. HUVECs were transfected with miR-29c mimic or miR-29c mimic NC or miR-29c inhibitor /miR-29c inhibitor NC. After 48 hrs, cells in suspension were seeded on wells that precoated with Matrigel. Tube network formations were measured after 6~8 h. A. Representative images of three independent experiments. B. Total tube length was measured with image analysis software and normalized to that of NC group. *p < 0.05, **p < 0.01.
Figure 4
Figure 4
miR-29c overexpression can downregulate IGF-1 mRNA expression. Quantitative RT-PCR results revealed that miR-29c delivery markedly reduced the IGF-1-PI3K/AKT expression. (A) IGF-1, (B) PI3K, (C) AKT, and (D) VEGF mRNA expression. The upregulation of IGF-1 mRNA following miR-29c transfection in HUVECs was analyzed by RT-PCR. (*p < 0.05, **p < 0.01).
Figure 5
Figure 5
Effect of miR-29c overexpression suppress HUVECs migration and regulate IGF-1 and VEGF protein expression. A, B. Reintroduction of miR-29c depressed cells migration ability by cell migration assay. C, D. The down-regulated expression of miR-29c promoted the migration of HUVECs in a wound-healing assay. E, F. Immunocytochemistry assay to compare the activity of VEGF in miR-29c overexpressing and control cells. G, H. HUVECs that were transfected with miR-29c inhibitors and miR-29c mimics, respectively, were subjected to western blot for IGF-1, p-PI3K, p-AKT and VEGF protein expression. Representative WB showed that up-regulation of miR-29c obviously depressed protein expression of IGF1, p-PI3K, p-AKT and VEGF expression in HUVECs. (n = 3 repeats with similar results. *p < 0.05, **p < 0.01).
Figure 6
Figure 6
Abridged general view for the interplay among miR-29-PI3K/AKT-VEGF pathway in HUVEC. miR-29c as a angiogenesis suppressor by targeting IGF-1, which decreased the angiogenesis of HUVECs through the modulation of the PI3K/AKT pathway. Overexpression of miR-29c, which suppresses the expression of IGF1, activates the PI3K/AKT-VEGF pathway by increasing the phosphorylation of p-PI3K, p-AKT and VEGF expression, which inhibits cell proliferation and angiogenesis in HUVECs.
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References

    1. Zhao Y, Ransom JF, Li A, Vedantham V, von Drehle M, Muth AN, Tsuchihashi T, McManus MT, Schwartz RJ, Srivastava D. Dysregulation of cardiogenesis, cardiac conduction, and cell cycle in mice lacking miRNA-1-2. Cell. 2007;129:303–317. - PubMed
    1. Li W, Liu M, Feng Y, Xu YF, Huang YF, Che JP, Wang GC, Yao XD, Zheng JH. Downregulated miR-646 in clear cell renal carcinoma correlated with tumour metastasis by targeting the nin one binding protein (NOB1) Br J Cancer. 2014;111:1188–1200. - PMC - PubMed
    1. Wu YH, Hu TF, Chen YC, Tsai YN, Tsai YH, Cheng CC, Wang HW. The manipulation of miRNA-gene regulatory networks by KSHV induces endothelial cell motility. Blood. 2011;118:2896–2905. - PubMed
    1. Pencheva N, Tran H, Buss C, Huh D, Drobnjak M, Busam K, Tavazoie SF. Convergent multi-miRNA targeting of ApoE drives LRP1/LRP8-dependent melanoma metastasis and angiogenesis. Cell. 2012;151:1068–1082. - PMC - PubMed
    1. Kriegel AJ, Liu Y, Fang Y, Ding X, Liang M. The miR-29 family: genomics, cell biology, and relevance to renal and cardiovascular injury. Physiol Genomics. 2012;44:237–244. - PMC - PubMed

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