Abstract: BACKGROUND: Studies demonstrated that miR-135a-5p was highly expressed in mouse embryonic palatal mesenchymal cells with cleft palate induced by dexamethasone. The primary cilium and its mediated Shh signaling pathway were involved in the autophagy of mouse embryonic palatal mesenchymal cells. It is speculated that miR-135a-5p may regulate autophagy in mouse embryonic palatal mesenchymal cells through primary cilia and its mediated Shh signaling pathway.
OBJECTIVE: To investigate the regulatory effect of miR-135a-5p on autophagy of mouse embryonic palatal mesenchymal cells.
METHODS: In vitro, palatal mesenchymal cells from C57BL/6J mouse embryos were extracted and cultured. Cell transfections were set up as follows: (1) the cells were divided into control group, miR-135a-5p negative control group and miR-135a-5p mimic group; (2) NC+miR-NC group, KIF3B overexpression group, and miR-135a-5p+KIF3B group: qRT-PCR was performed to verify transfection efficiency of miR-135a-5p and KIF3B. A transmission electron microscope was used to observe the number of autophagosome/autophagolysosome in the cells of each group. The degree of fluorescence expression of autophagy marker LC3B was determined by the immunofluorescence technique. The protein expression of KIF3B, LC3 and P62 was determined by western blot assay. (3) The cells were divided into miR-135a-5p negative control group, and SAG treated group, and SAG+miR-135a-5p group. qRT-PCR was used to detect the mRNA expression levels of Gli3, a key transcription factor downstream of Shh signaling. The protein expressions of autophagy-related proteins LC3 and P62 were detected by western blot assay. 
RESULTS AND CONCLUSION: (1) After overexpression of miR-135a-5p, the number of autophagosome/autophagolysosome was significantly increased (P < 0.01). The fluorescence density of LC3B increased significantly (P < 0.01); the protein expression of KIF3B and P62 decreased (P < 0.01), and the protein expression of LC3 increased. (2) After overexpression of KIF3B, the number of autophagosome/autophagolysosome was significantly decreased (P < 0.01); the fluorescence density of LC3B was decreased (P < 0.01); the protein expression of P62 was increased (P < 0.01), and the protein expression of LC3 was decreased (P < 0.01). Targeted expression of KIF3B was inhibited by miR-135a-5p (P < 0.01); the number of autophagosome/autophagolysosome, the fluorescence intensity of LC3B as well as the protein expression of LC3 were reversed (P < 0.01) and the protein expression of P62 was decreased (P < 0.01). (3) SAG significantly increased the mRNA expression of Gli3 (P < 0.01), increased the protein expression of P62 (P < 0.01), and decreased the protein expression of LC3 (P < 0.01). When miR-135a-5p was added, Gli3 mRNA expression was significantly decreased (P < 0.01); P62 protein expression was decreased (P < 0.01), and LC3 protein expression was reversed (P < 0.01). (4) These results indicate that miR-135a-5p targets the inhibition of KIF3B and promotes autophagy in mouse embryonic mesenchymal cells possibly by negatively regulating the Shh signaling pathway.

Key words: autophagy, embryonic palatal mesenchymal cell, miRNA, KIF3B, Shh signaling pathway, cleft palate