Enhanced functional properties of human limbal stem cells by inhibition of the miR-31/FIH-1/P21 axis.

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, β-catenin, survivin, GSK3β and pGSK3β.

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.