The miRNA-34a/Sirt1/p53 pathway in a rat model of lens regeneration

Abstract

Background: There are many molecular factors involved in Wolffian and corneal lens regeneration, but few in lens regeneration by lens epithelial cells (LECs) in mammals. Silent information regulator 1 (Sirt1) has a variety of physiological functions, such as a transport hub, and is involved in pathological conditions. We studied the expression of the microRNA (miRNA)-34a/Sirt1/tumor protein p53 (p53) pathway in a rat model of lens regeneration.

Methods: We performed extracapsular lens extraction in 42 healthy female Sprague-Dawley rats. Slit lamp observation was performed at 3, 7, 14, 21, 30, 60 and 90 days postoperatively, and the rats were killed humanely by cervical dislocation at 30, 60 and 90 days postoperatively to remove the eyeballs. We performed semiquantitative immunofluorescence analysis of Sirt1, p53, alpha-smooth muscle actin (α-SMA) and fibronectin (fn), and real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) to detect the relative expressions of miRNA-34a, Sirt1, p53, aquaporin 0 (AQP 0), γA-crystallin, and beaded filament structural protein 1 (BFSP1) mRNA in the lens and posterior capsule.

Results: The posterior capsule wrinkled at 3 days and it increased at 7 days. At 14 days, pearl-like opacification appeared under the capsule, with increasing shrinkage. Greater mass-like proliferators in size and number accumulated under the capsule and at the equator after 21 days. A regenerated lens developed in the central depression of the capsule at 30 days, slightly protruding from it. Despite being thickened at 60 days, the central depression persisted, with a smaller change at 90 days than at 60 days. Although the relative mRNA expression of miRNA-34a and p53 in the lens and posterior capsule decreased over time (P=0.000), that of Sirt1 increased (P<0.01). α-SMA was uniformly expressed in the crystals and gradually decreased, while fn expression gradually increased.

Conclusions: miRNA-34a expression decreased and Sirt1 expression increased during lens regeneration. Furthermore, p53 expression decreased, thus reducing apoptosis. Therefore, Sirt1 acted as a key factor in the pathway, and played a protective role in lens regeneration.

Keywords: Lens regeneration; miRNA-34a/Sirt1/p53 pathway; ophthalmology; rat model.

Figure 1

Model-making process. After the incision was enlarged, the lens nucleus and cortex were delivered, and the incision was sutured with 10/0 sutures. At the end of the operation, the anterior chamber had formed, with little bleeding, no obvious iris incarceration, and a clear posterior capsule.

Figure 2

Slit-lamp image at each time point. The posterior capsule was clear (0 days) at the end of the extracapsular lens extraction. The shrinkage of the posterior capsule gradually increased (3 days, 7 days). Pearl corpuscles gradually appear in the posterior capsule (14 days, blue arrow), the shrinkage further increases, and the greater mass-like proliferators (21 days, blue arrow) appeared. After 30 days, the equatorial area had completely proliferated, with an irregular central area. At 60 days, the shape of the regenerated lens is similar to that of the primary lens. At 90 days, the regenerated lens depicts no obvious swelling, with uniform depth of the anterior chamber.

Figure 3

HE staining of a normal lens, and a regenerated lens at 30, 60, and 90 days. The normal lens fibers are arranged in order, and the single layer of LECs of the anterior capsule membrane are arranged in order to the equator. At 30 days, there was a fusiform appearance, and residual epithelial cells in the anterior sac were slightly loose. The layers of the anterior capsule at 60 days are denser, and lens fibers are arranged in order. At 90 days, the anterior capsule LECs are arranged in a monolayer similar to that of a normal lens. LECs, lens epithelial cells.

Figure 4

Sirt1 immunofluorescence. Sirt1 expression was higher in the lens epithelial cells close to the capsule. It was non-uniform in crystal fibroblasts at 30 days. Other groups are expressed as homogenized. (A) 30 days; (B) 60 days; (C) 90 days; (D) normal. Sirt1, sirtuin 1.

Figure 5

p53 immunofluorescence. p53 was more expressed near the capsule and the equator. Its expression decreased over time; the normal group had lower p53 levels. (A) 30 days; (B) 60 days; (C) 90 days; (D) normal. p53, tumor protein p53.

Figure 6

α-SMA immunofluorescence. α-SMA is uniformly expressed in the crystal. However, it decreases gradually over time and tends to be expressed in the lens epithelial cells close to the capsule, but at a significantly lower level than in the normal group at 90 days. (A) 30 days; (B) 60 days; (C) 90 days; (D) normal. α-SMA, alpha-smooth muscle actin.

Figure 7

Fn immunofluorescence. Fn is expressed in crystal fibroblasts, with a stripe-like appearance. The expression in the normal group is higher, but that in the experimental group gradually increased over time. (A) 30 days; (B) 60 days; (C) 90 days; (D) normal. fn, fibronectin.

Figure 8

Immunofluorescence semiquantitative expression analysis demonstrated a gradual and significant increase in Sirt1 expression over time. Other expressions decreased gradually. However, p53 levels were still higher than normal. Although α-SMA expression was lower than normal at 90 days, it was higher than normal at 30 and 60 days. fn was always lower than normal. AU, arbitrary units; Sirt1, sirtuin 1; p53, p53 tumor protein; α-SMA, alpha-smooth muscle actin; fn, fibronectin.

Figure 9

RT-qPCR expression of components of the miRNA-34a/Sirt1/p53 pathway. miRNA-34a negatively correlated with Sirt1, and Sirt1 negatively correlated with tumor protein p53. All factors in the pathway were significantly different at each time point between and within groups. *, P<0.01. Sirt1, sirtuin 1; p53, p53 tumor protein; RT-qPCR, real-time fluorescence quantitative polymerase chain reaction.

Figure 10

RT-qPCR expression of crystal markers. AQP 0 and γA-crystallin expression were significantly different at each time point between and within groups. Beaded filament structural protein 1 was significantly different at each time point between groups. However, there was no significant difference within groups. AQP 0, aquaporin 0; BFSP1, beaded filament structural protein 1; RT-qPCR, real-time fluorescence quantitative polymerase chain reaction.