| الوصف: |
This study aimed to explore the regulatory molecular mechanism of microRNA (miRNA) in poly(L-lactic acid) (PLLA) nanofiber-induced PC12 cell differentiation by combining transcriptomics, proteomics and miRNA sequencing technology. Primarily, PLLA random/aligned nanofibers were fabricated by electrospinning, and their morphology, chemical composition and mechanical properties were measured. Next, PC12 cells were planted on the PLLA film (control), PLLA random nanofibers (RF) and PLLA aligned nanofibers (AFs), and the morphology and synapse length of the PC12 cells were observed. Furthermore, isobaric tags for relative and absolute quantitation (iTRAQ)-labeled proteomics qualification technologies were applied to identify protein expression information of PC12 cells, and differentially expressed proteins were determined. To elucidate the regulatory relationship among mRNA, miRNA and protein, bioinformatics methods were adopted to analyze the proteomics data using early mRNA profile data and miRNA sequencing data, from which collective gene ontologies, biological pathways and the matched miRNA mRNA/protein target pairs were filtered. The experiment yielded the following results: 1. PLLA random/aligned nanofibers were composed of PLLA, and all the materials were uniform. 2. The modulus elasticity of PLLA aligned nanofibers was dramatically higher than that of PLLA random nanofibers. 3. PC12 cells were in good condition in all three groups. 4. Cells were nearly round in the control group and extended synapses in the RF and AF groups, yet they had shorter and randomly oriented synapses in the RF group. However, the length of the synapses was significantly increased in the AF group compared with that of the control and RF groups. More specifically, there were 235 and 281 differentially expressed proteins in the RF and AF groups, respectively, compared with the control group. Comparing the integrated analysis of the proteomics experimental data with the miRNA and mRNA profiles, collective differentially expressed data were involved in 2 and 5 gene ontologies and 4 and 7 biological pathways in the RF and AF groups, respectively, which were related to cell differentiation. Through data comparison, 0 and 5 matched miRNA genes/proteins pairs were filtered in the RF and AF groups, respectively. Further discussion revealed that PLLA aligned nanofibers could regulate 5 mRNA/protein target pairs (Dnajb12, Rhob, Wbp11, Hmgcs1 and Hmgn2) by 8 miRNAs (miR-24, miR-25, miR-92a, miR-19b, miR-183, miR-29a, miR-29c and miR-23b) and further regulate 'focal adhesion' pathways and many functions (e.g., cell adhesion, transport, gene transcription, biosynthesis, and DNA assembly), which could finally influence the migration and differentiation of PC12 cells. The results showed that the focal adhesion pathway, integrin-mediated cell adhesion pathway, MAPK pathway and TGF-β pathway play key functions in the process of PLLA-aligned, nanofiber-induced PC12 cell differentiation. |
