microRNA regulatory mechanism by which PLLA aligned nanofibers influence PC12 cell differentiation

Aligned nanofibers (AFs) are regarded as promising biomaterials in nerve tissue engineering. However, a full understanding of the biocompatibility of AFs at the molecular level is still challenging. Therefore, the present study focused on identifying the microRNA (miRNA)-mediated regulatory mechanism by which poly-L-lactic acid (PLLA) AFs influence PC12 cell differentiation.

Firstly, the effects of PLLA random nanofibers (RFs)/AFs and PLLA films (control) on the biological responses of PC12 cells that are associated with neuronal differentiation were examined. Then, SOLiD sequencing and cDNA microarray were employed to profile the expressions of miRNAs and mRNAs. The target genes of the misregulated miRNAs were predicted and compared with the mRNA profile data. Functions of the matched target genes (the intersection between the predicted target genes and the experimentally-determined, misregulated genes) were analyzed.

The results revealed that neurites spread in various directions in control and RF groups. In the AF group, most neurites extended in parallel with each other. The glucose consumption and lactic acid production in the RF and AF groups were higher than those in the control group. Compared with the control group, 42 and 94 miRNAs were significantly dysregulated in the RF and AF groups, respectively. By comparing the predicted target genes with the mRNA profile data, five and 87 matched target genes were found in the RF and AF groups, respectively. Three of the matched target genes in the AF group were found to be associated with neuronal differentiation, whereas none had this association in the RF group. The PLLA AFs induced the dysregulation of miRNAs that regulate many biological functions, including axonal guidance, lipid metabolism and long-term potentiation. In particular, two miRNA-matched target gene-biological function modules associated with neuronal differentiation were identified as follows: (1) miR-23b, miR-18a, miR-107 and miR-103 regulate the Rras2 and Nf1 gene and thereby, affect cytoskeleton regulation and MAPK pathway; (2) miR-92a, miR-339-5p, miR-25, miR-125a-5p, miR-351 and miR-19b co-regulate the Pafah1b1 gene, affecting PC12 cell migration and differentiation.

This work demonstrates a bioinformatic approach to accomplish miRNA-mRNA profile integrative analysis and provides more insights for understanding the regulatory mechanism of miRNA in AFs affecting neuronal differentiation. These findings will be greatly beneficial for the application and design of AFs in nerve tissue engineering.