Moreover, miRNA-548ah-5p might involve in the regulation of cell cycle-related gene expressions according to the bioinformatics analysis. Open in a separate window FIGURE 4 The disturbation of cell cycle-related pathways contributed to the cell apoptosis. signal pathway associated with the antiproliferative property of curcumin. Finally, miRNAs significantly changed, as well as the regulatory roles Kenpaullone of those miRNAs in cell apoptosis were determined. Result The apoptosis rate of H446 cells increased under the elevated concentration of curcumin treatment. And cell cycle-related genes downregulated in the curcumin-treated cells. Besides, miRNA-548ah-5p of a high level acted as a negative role in the anticarcinogenic activity of curcumin. Conclusion Our findings not only enriched the understanding of anti-tumor activity initiated by curcumin through figuring out the downregulated cell cycle-related pathways but also shed light on its novel therapeutic application. < 0.05. The bioinformatic analysis further analyzed expression levels of microRNA, including the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, Gene Ontology (GO) enrichment analysis, and heat map. KEGG pathway enrichment analysis could find the significant pathways1, and the criteria were count 3 and false discovery rate (FDR) < 0.05. GO functional enrichment analysis further assessed the biological process2, and the criteria were gene count 3 and < 0.05. Quantitative Real Time-PCR Analysis The total RNA was extracted from H446 cells by Buffer MZ (Tiangen) after incubation with or without curcumin at room temperature. The RNA was reversely transcribed to cDNA by MiRcute Plus miRNA First-Strand cDNA Synthesis kit (Tiangen). A quantitative real-time PCR was performed by MiRcute Plus miRNA qPCR kit (Tiangen). The primers of miR-548-5p, miR-4725-3p, miR-195-5p, miR-10394-5p, and small nuclear RNA-U6 (RNU6) were provided by Genecopoeia. The expression level of each microRNA was determined by miRNA sequence-specific primers and normalized to the expression level of RNU6. Transfection of MicroRNA Inhibitor Cells were cultured in the growth medium for 24 h. Transfection of inhibitors of miR-548-5p, Akt1 miR-4725-3p, miR-195-5p, miR-10394-5p, and control inhibitor was performed using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufactures protocol. After 24 h of transfection, cells were cultured in fresh medium with or without curcumin. And cells were collected to detect the cell proliferation and apoptosis using CCK-8 Kenpaullone assay or FITC-annexin V/PI assay, respectively. Statistical Analysis All data were presented as mean standard error (SE). The unpaired Students < 0.05 was considered to indicate a statistical difference. Results Curcumin Reduced Cell Viability and Induced Cell Apoptosis in H446 Human SCLS Cell Line To assess the anticarcinogenic property of curcumin, different cell lines (HCT116, Hela, MB231, PC-9, A549, H446) were cultured with curcumin. As shown in Supplementary Figure S1, the cell viability of these cell lines all decreased in a dose-dependent manner after curcumin treatment. Moreover, H466 cell lines were confirmed to be most sensitive to curcumin due to the most significant decline in cell viability than other cell types. Therefore, H446 cell lines were adopted for a subsequent experiment. And we obtained the cell viability of H446 cells under a lower concentration gradient after 24 h (Figure 1A). H446 cells were examined by Annexin-V/PI Kenpaullone apoptosis detection kit after curcumin treatment at different time points to explore its effect of curcumin on cell apoptosis. As demonstrated in Figure 1B, the curcumin-induced cell apoptosis rate significantly increased from 3.03 to 12.82% or 46.82% at time points 24 or 48 h, respectively. Furthermore, the TUNEL assay indicated the apoptotic cells by marking DNA fragmentation (Figure 1C). As a result, TUNEL positive cells were detected in the curcumin-treated group. Besides, we examined the apoptosis-related protein expression after curcumin incubation (Figure 1D). The result showed that Curcumin treatment reduced the expression of Bcl-2 and increased the expression of Bax and cytochrome-C, which are involved in crucial regulation of cell growth (Ow et al., 2008). These results showed that curcumin effectively promoted the apoptosis of H446 cells. Open in a separate window FIGURE 1 Curcumin reduced cell viability and induced cell apoptosis in H446 human SCLS cell line. (A) The relative proliferation of H446 cells was determined after 24 h curcumin treatment at different concentration (0, 5, 10, 15, 20 M). (B) The apoptosis rate of H446 cells was evaluated by Annexin-V/PI apoptosis detection kit after.