[In vitro study of effects and mechanism of rapamycin-induced autophagy in keloid fibroblasts].

Objective: To investigate the effect of rapamycin on biological characteristics and autophagy of keloid fibroblasts, and the regulation of rapamycin in mTOR (mammalian target of rapamycin) signaling pathway and autophagy-related non-coding RNAs in keloid fibroblasts.

Methods: After Keloid fibroblasts were treated with rapamycin (10、50、100 nmol/L), and MTS assay was used to test the cell proliferation. The apoptosis of cells was tested by the flow cytometry analysis. The formation of autophagy was observed by TEM, and the Western Blot was used to detect the expression of autophagy-related protein LC3.Real-time PCR was used to detect the expression of genes of involued in mTOR pathway and autophagy-related non-coding RNAs. Statistical significance was determined using Paired-Samples t Test,P value less than 0.05 was considered statistically significant.

Results: The ratio of 490nm was decreased significantly in rapamycin-treated keloid fibroblasts compared with that in untreated cells (P ＜ 0.05).Meanwhile the mRNA expressions of extracellular matrix (ECM) genes, including collagen-1 、α-SMA and Fibronectin, were inhibited by rapamycin (P ＜ 0.05).The flow cytometry analysis showed that the percent of apoptosis cells was not increased in rapamycin-induced cells (P ＞ O.05). The double-layer membrane structure of autophagosomes could be observed under the TEM in rapamycin-treated fibroblasts, accompanied by the increased expression of autophagy-related protein LC3.The mRNA expressions of downstream genes of mTOR pathway,4EBP1 and p70S6K,were down-regulated in rapamycin-treated fibroblasts, while the expressions of autophagy-related miRNAs, including miR-885-3p,miR-204,miR-101,miR-376b and lncRNA FLJ11812 were enhanced, and miR-30a,lncRNA HULC5 was decreased in rapamycin-treated fibroblasts (P ＜ 0.05).

Conclusions: Rapamycin could inhibit the proliferation of keloid fibroblasts, and could not affect the apoptosis of cells.However, rapamycin induced the autophagy of keloid fibroblasts through regulating the expression of autophagy-related non-coding RNAs and genes in the mTOR signaling pathway.