Abstract
Purpose:
It is well known that Ciliary Epithelial (CE) cells of mammals are retinal progenitor/stem cells capable to reenter the cell cycle, loose some epithelial properties and generate retinal neurons and glia after proper stimulation. Despite this regenerative potential, the efficiency is still low due to the inefficient reprogramming and/or the presence of inhibitory factors such as microRNAs (miRNAs), small non-coding RNAs that regulate a variety of pluripotent genes and proteins expression. Here, we investigated the expression of let-7 family of miRNAs and its regulatory protein Lin28 in CE cells from newborn and adult rodents.
Methods:
Adults Wistar rats (45 days-old) and newborns (1 day-old) were euthanized and the eyeballs removed. CE cells were carefully separated from retina, sclera and iris, and further manually dissected into outer and inner CE. Let-7 miRNAs expression, pluripotent genes (Sox2, Nanog, Klf4, Oct-4) and retinal progenitor genes (Pax6, Chx10) were analyzed by Real-Time PCR. The Lin28A protein expression was analyzed by western blotting assay. Statistical analysis between adult and newborn groups were evaluated by Student’s t-test.
Results:
Our results indicated that Pax6 and Chx10 progenitor genes were highly expressed in newborn CE cells, where Pax6 presents higher expression (35%) in the inner and Chx10 in the outer (180%) CE cells. In adults, the expression of progenitor genes were lower than the newborn. Similarly, Nanog, Oct4, Sox2 and Klf-4 pluripotent genes were about 70% more expressed in newborn compared to adults. The higher expression of Nanog, Oct4 and Sox2 were detected in the inner cells (2-, 6- and 30- fold, respectively) and Klf-4 in the outer cells (1.3-fold) in newborns. The let-7s miRNAs expression were detected in adult CE cells, at higher levels when compared to the expression in the retina. In average, let-7 miRNAs were expressed more than twofold in adult inner cells compared to the outer, whereas Lin28 protein expression was lower.
Conclusions:
The adult CE cells express progenitor and pluripotent genes; however, the regenerative capacity of the mammalian retina does not develop efficiently. The high expression of let-7 family members in adult CE cells, especially in the inner cells, which can’t form neurospheres, suggests that miRNAs might be an important negative regulators for adult CE regenerative capacity.