Enhanced functional properties of human limbal stem cells by inhibition of the miR-31/FIH-1/P21 axis
Authors
Zhiping Liu, Weijiao Zhan, Minzhi Zeng, Jinghong Chen, Huyong Zou and Zhiqun Min
Institution
TheDepartment ofOphthalmology, The SecondAffiliatedHospital ofGuangzhouMedical University,Guangzhou, Guangdong
Country
China
Year
2017
Journal
Acta Ophthalmologica
Abstract
Objective: On the basis of the functional roles of the embryonic stem cell niche
(ESCN) in the human limbal stem cells (LSCs), we proposed to explore the
potential roles of microRNAs in regulating the self-renewal and differentiation of
LSCs cultured in the ESCN.
Methods: The LSCs were cultured in different media, either in CnT-20
media or in CnT-20 + 20% ES culture supernatant (ESC-CM). The LSCs
cultured in ESC-CM were then transfected with microRNA-31 (miR-31)
mimic or antago-31. The colony-forming efficiency (CFE) was analysed. Cell
cycle, apoptosis, mitochondrial potential and reactive oxygen species were
analysed by flow cytometry, and quantitative real-time PCR was used to
determine the expression levels of FIH-1, P21, P63, ABCG2, CK3,
microRNA-31, microRNA-143, microRNA-145 and microRNA-184. Indirect
immunostaining was employed to detect the expression of P63, ABCG2,
survivin, connexin-43 and CK3. Western blot was employed to detect the
expression of FIH-1, P63, P21, CK3, caspase 3, Tcf4, b-catenin, survivin,
GSK3b and pGSK3b.
Results: Compared with cells grown in CnT-20, the level of miR-31 in cells
grown in ESC-CM was lower. We investigated the roles that miR-31 and FIH-1
play in regulating the functional properties of LSCs. We used antagomirs
(antago) to reduce the level of miR-31 in LSCs. Antago-31 increased FIH-1
levels and significantly reduced P21 expressional level in LSCs compared to
irrelevant-antago (Ir-antago) treatment. The downregulation of miR-31 in LSCs
promotes the maintenance of stemness.
Conclusion: ES culture supernatant (ESC-CM) regulates the fate of LSCs in
part by inhibiting the miR-31/FIH-1/P21 axis. This study may have a high
impact on the expansion of LSCs in regenerative medicine, especially for ocular
surface reconstruction.