MiR-375 mitigates retinal angiogenesis by depressing the JAK2/STAT3 pathway

Abstract

Aberrant neovascularization in the retina is an important threat to vision and closely related to several retinal diseases, such as wet form of age-related macular degeneration, diabetic retinopathy, and retinopathy of prematurity. However, the pathogenesis remains largely unknown. MicroRNAs (miRNAs) have been demonstrated to play critical regulatory roles in angiogenesis. Therefore, we aimed to identify the key miRNAs that regulate retinal neovascularization and elucidate the potential underlying mechanisms. In the present study, we performed RNA sequencing of microRNAs in the retina and found that miR-375 was significantly downregulated in the retina of oxygen-induced retinopathy mice. In retinal microvascular endothelial cells (RMECs), overexpression of miR-375 inhibited cell proliferation and angiogenesis. Conversely, inhibition of miR-375 had the opposite effects. Moreover, our results showed that miR-375 negatively regulated the protein expression of JAK2 by inhibiting its translation. The promoting effects of anti-miR-375 on cell proliferation and angiogenesis were attenuated by an inhibitor of STAT3. These results indicate that miR-375 mitigates cell proliferation and angiogenesis, at least in part, through the JAK2/STAT3 pathway in RMECs, which implies an important underlying mechanism of retinal angiogenesis and provides potential therapeutic targets for retinal microangiopathy.

Keywords: JAK2; angiogenesis; miR-375; proliferation; retina.

Figure 1

Expression of miR-375 is decreased in the retina of OIR. (A, B) A heat map (A) of the significantly differentially expressed microRNAs and the volcano plots (B) between groups. (C) A heat map of the differentially expressed microRNAs, which were consistent with our results, in the retina of OIR from GSE84303. (D, E) miR-375 was significantly downregulated in the retina of OIR group compared with that of the control group both in our samples (D) and in GSE84303 (E). (F) Expression of miR-375 was down-regulated in the retina of CNV. *P < 0.05.

Figure 2

Inhibition of miR-375 promotes cell proliferation and angiogenesis in RMECs. (A) Cell viability was increased by the inhibition of miR-375 in RMECs. (B, C) The incorporation of BrdU (B) and the protein levels of PCNA (C) were facilitated by the treatment with anti-miR-375. (D, E) The inhibition of miR-375 enhanced cell migration (D) and tube formation (E) in RMECs. Scale bar: 100 μm (D) and 500 μm (E), respectively. All values are represented as the mean ± standard error of the mean. n = 3 per group. *P < 0.05.

Figure 3

MiR-375 mitigates cell proliferation and angiogenesis. (A) Expression of miR-375 was significantly increased after the treatment with miR-375 in RMECs. (B) Cell viability was decreased by treatment with miR-375. (C, D) miR-375 inhibited the incorporation of BrdU (C) and the protein levels of PCNA (D). (E, F) Cell migration (E) and tube formation (F) were repressed by miR-375 in RMECs. Scale bar: 100 μm (E) and 500 μm (F), respectively. All values are represented as the mean ± standard error of the mean. n = 3 per group. *P < 0.05.

Figure 4

MiR-375 negatively regulates the expression of JAK2 by inhibiting its translation. (A) The potential binding site for miR-375 in the 3’UTR of JAK2. (B) miR-375 significantly decreased the luciferase activity of the JAK2 WT plasmid, while no detectable effects were observed on the luciferase activity of the MT plasmid. (C, D) miR-375 had no detectable effects on the mRNA expression of JAK2 (C), but significantly depressed its protein expression (D). (E) The mRNA expression of JAK2 was not affected by anti-miR-375. (F) Treatment with anti-miR-375 induced the protein expression of JAK2. All values are represented as the mean ± standard error of the mean. n = 3 per group. *P < 0.05.

Figure 5

Effects of miR-375 on cell proliferation are mediated by JAK2. (A) Expression of JAK2 was significantly decreased by siJAK2. (B) The increased cell viability induced by anti-miR-375 was mitigated by Jak2 knockdown. * P < 0.05 vs. anti-miR-375+siJAK2 group. (C, D) Knockdown of JAK2 attenuated the promoting effects of anti-miR-375 on BrdU incorporation (C) and PCNA expression (D). (E) Cell migration enhanced by anti-miR-375 was inhibited by JAK2 knockdown. Scale bar: 100 μm. (F) Expression of JAK2 was increased by the transfection with a recombinant plasmid. (G, H) miR-375-mitigated cell viability (G) and BrdU incorporation (H) was reversed by the reintroduction of JAK2. * P < 0.05 vs. miR-375+JAK2 group. (I, J) The inhibitory effects of miR-375 on PCNA expression (I) and cell migration (J) were attenuated by the restoration of JAK2 expression. Scale bar: 100 μm. All values are represented as the mean ± standard error of the mean. n = 3 per group. *P < 0.05.

Figure 6

STAT3 is involved in miR-375-regulated cell proliferation in RMECs. (A) The phosphorylation of STAT3 was induced by anti-miR-375, whereas miR-375 repressed the phosphorylation of STAT3. There were no significant changes on the expression of total STAT3 after the treatment with miR-375 or anti-miR-375. The expression of p-STAT3 was quantified by the ratio of p-STAT3 to STAT3 and the expression of STAT3 was quantified by the ratio of STAT3 to β-actin. (B, C) The increase in cell viability (B) and BrdU incorporation (C) induced by anti-miR-375 was mitigated by the inhibition of STAT3. * P < 0.05 vs. anti-miR-375+Stattic group. (D, E) Anti-miR-375-increased PCNA expression (D) and cell migration (E) was attenuated by blocking the STAT3 pathway. Scale bar: 100 μm. All values are represented as the mean ± standard error of the mean. n = 3 per group. *P < 0.05.