Role of microRNA-130b in placental PGC-1α/TFAM mitochondrial biogenesis pathway

Abstract

Diabetes during pregnancy is associated with abnormal placenta mitochondrial function and increased oxidative stress, which affect fetal development and offspring long-term health. Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) is a master regulator of mitochondrial biogenesis and energy metabolism. The molecular mechanisms underlying the regulation of PGC-1α in placenta in the context of diabetes remain unclear. The present study examined the role of microRNA 130b (miR-130b-3p) in regulating PGC-1α expression and oxidative stress in a placental trophoblastic cell line (BeWo). Prolonged exposure of BeWo cells to high glucose mimicking hyperglycemia resulted in decreased protein abundance of PGC-1α and its downstream factor, mitochondrial transcription factor A (TFAM). High glucose treatment increased the expression of miR-130b-3p in BeWo cells, as well as exosomal secretion of miR-130b-3p. Transfection of BeWo cells with miR-130b-3p mimic reduced the abundance of PGC-1α, whereas inhibition of miR-130b-3p increased PGC-1α expression in response to high glucose, suggesting a role for miR-130b-3p in mediating high glucose-induced down regulation of PGC-1α expression. In addition, miR-130b-3p anti-sense inhibitor increased TFAM expression and reduced 4-hydroxynonenal (4-HNE)-induced production of reactive oxygen species (ROS). Taken together, these findings reveal that miR-130b-3p down-regulates PGC-1α expression in placental trophoblasts, and inhibition of miR-130b-3p appears to improve mitochondrial biogenesis signaling and protect placental trophoblast cells from oxidative stress.

Keywords: Hyperglycemia; Oxidative stress; PGC-1α; Placental trophoblast; miR-130b.

Figure 1

Regulation of PGC-1α/TFAM in BeWo cells by glucose. BeWo cells were cultured in normal medium (N, 10mM glucose, for 6 days), medium with high glucose (HG, 42mM, for 4 or 6 days), or osmotic control mannitol (M, 10mM glucose+32 mM mannitol, for 6 days). For HG 4-days treatment group, cells were incubated in osmotic control medium for 2 days followed by high glucose treatment for 4 days. Representative Western blots, and bar graphs of PGC-1α and TFAM quantification are shown. Mean ± SD, n=3, * P<0.05 compared to mannitol group.

Figure 2

Regulation of miR-130b-3p expression in BeWo cells. a: BeWo cells were cultured in medium with high glucose (42mM) or mannitol (M) for 24 hours. The miRNA abundance from cell extracts was quantified by qPCR, n=4. b: BeWo cells were cultured in high glucose (42mM) or mannitol (M) for 72 hours, after which exosomes were isolated from medium (verified by Western blot analysis with exosome-specific antibodies for HSP70 and CD81, left panel). Exosome-associated miR-130b-3p quantified by qPCR (right panel, n=5). c, d and e: BeWo cells were treated with 4-HNE (10 µM) (c, n=4), TNFα (0, 10, 30 ng/ml) (d, n=2–3) or palmitate (0, 0.3, 0.6, and 1 mM) (e, n=2) for 16 hours, and miR-130b-3p levels in cells were quantified by qPCR. * P<0.05.

Figure 3

Regulation of PGC-1α expression by miR-130b-3p. a: BeWo cells were transfected with 100 nM of miR-130b-3p mimic or negative control. PGC-1α protein expression was determined after 2 days. Left panel: representative Western blot. Right panel: Quantification of expression (mean ± SD, n=6, *** P < 0.001). b: BeWo cells were transfected with anti-sense miR-130b-3p (anti-miR-130b, 100 nM) or negative control (100 nM), then incubated in medium containing high glucose (42 mM) or mannitol for 3 days. Representative Western blots (left panel) and quantification of PGC-1α protein abundance (right panel) are shown. Mean ± SD, n=3 – 4, * P < 0.05.

Figure 4

Effects of miR-130b-3p inhibition on TFAM expression and ROS production in response to 4-HNE in BeWo cells. BeWo cells were transfected with miR-130b mimic or negative control (100 nM), maintained in culture for 48 h, and then exposed to 4-HNE (0, 3, or 10 µM) for 6 hours in serum free medium. a and b: Cell protein extracts were subjected to Western blot analysis. Representative blots (a) and quantification (b) of PGC-1α and TFAM protein levels were shown. Mean ± SD, n=4–5, * P < 0.05 compared to negative control at each dose of 4-HNE treatment. c and d: Production of ROS was determined with probe DHR-123. Representative images (c) and intensity of green fluorescence (d) were shown. Mean ± SD, n=3–5, * P < 0.05 compared to negative control at each dose of 4-HNE treatment.