[Puerarin Inhibits the Proliferation,Invasion,and Migration of Non-small Cell Lung Cancer Cells through Regulating miR-490/Denticleless E3 Ubiquitin Protein Ligase].

Objective To explore the mechanism of puerarin inhibiting the proliferation,invasion,and migration of non-small cell lung cancer cells. Methods A549 cells were cultured and treated with different concentrations of puerarin.The inhibition rate (IR) on cell proliferation was detected by CCK-8,and qRT-PCR was performed to detect the mRNA levels of miR-490 and denticleless E3 ubiquitin protein ligase(DTL).Double luciferase reporter assay was employed to identify the targets of miR-490 and DTL based on the establishment of NC mimic group,miR-490 mimic group,NC inhibitor group,and miR-490 inhibitor group.The cells treated by 20 μmol/L puerarin were classified into six groups:DMSO,puerarin,puerarin+NC inhibitor,puerarin+miR-490 inhibitor,puerarin+miR-490 inhibitor+Si-NC,and puerarin+miR-490 inhibitor+Si-DTL.Transwell was used to detect cell migration and invasion.Western blotting was performed to detect the protein levels of epithelial-mesenchymal transition-related markers E-cadherin,N-cadherin,and Vimentin. Results With the increase in puerarin concentration,the IR gradually elevated (=105.375,<0.001),miR-490 expression gradually increased (=32.919,<0.001),and DTL expression gradually decreased (=116.120,<0.001).Compared with NC mimic group,miR-490 mimic group had decreased luciferase activity (=7.762,=0.016),raised miR-490 mRNA level (=13.319,<0.001),and declined DTL mRNA level (=7.415,=0.002).Compared with those in NC inhibitor group,miR-490 demonstrated decreased mRNA level (=9.523,=0.001) and DTL presented increased mRNA level (=11.305,<0.001) in miR-490 inhibitor group.Western blotting showed that the protein level of DTL was higher in NC mimic group (=7.953,=0.001) than in miR-490 mimic group and higher in miR-490 inhibitor group than in NC inhibitor group (=10.552,<0.001).Compared with DMSO group,puerarin group showed up-regulated mRNA level of miR-490 (=10.255,=0.001) while down-regulated mRNA level of DTL (=6.682,=0.003).Compared with those in puerarin+NC inhibitor group,the mRNA level of miR-490 declined (=10.995,<0.001) while that of DTL raised (=12.478,<0.001) in puerarin+miR-490 inhibitor group.The mRNA level of miR-490 had no significant difference between puerarin+miR-490 inhibitor+Si-NC group and puerarin+miR-490 inhibitor+Si-DTL group (=1.081,=0.341),and that of DTL was lower in the latter group (=14.321,<0.001).The protein level of DTL was higher in puerarin+miR-490 inhibitor group than in puerarin+NC inhibitor group (=11.423,<0.001),and lower in puerarin+miR-490 inhibitor+Si-DTL group than in puerarin+miR-490 inhibitor+Si-NC group (=12.080,<0.001).Compared with DMSO group,puerarin group showed inhibited cell proliferation (=129.27,<0.001).The activity of cell proliferation was higher in puerarin+miR-490 inhibitor group than in puerarin+NC inhibitor group (=75.12,<0.001),and higher in puerarin+miR-490 inhibitor+Si-NC group than in puerarin+miR-490 inhibitor+Si-DTL group (=52.59,<0.001).Compared with DMSO group,puerarin group had suppressed cell migration (=8.963,=0.001).The cell migration ability was higher in puerarin+miR-490 inhibitor group than in puerarin+NC inhibitor group (=12.117,<0.001) and higher in puerarin+miR-490 inhibitor+Si-NC group than in puerarin+miR-490 inhibitor+Si-DTL group (=12.934,<0.001).Puerarin group showed weakened cell invasion ability compared with DMSO group (=4.710,=0.009).The cell invasion ability was higher in puerarin+miR-490 inhibitor group than in puerarin+NC inhibitor group (=13.264,<0.001) and lower in puerarin+miR-490 inhibitor+Si-DTL group than in puerarin+miR-490 inhibitor+Si-NC group (=13.476,<0.001).Compared with DMSO group,puerarin group showed up-regulated protein level of E-cadherin (=7.137,=0.002) while down-regulated protein levels of N-cadherin (=8.828,=0.001) and vimentin (=6.594,=0.003).Compared with those in puerarin+NC inhibitor group,the protein level of E-cadherin (=12.376,<0.001) decreased while those of N-cadherin (=13.436,<0.001) and vimentin (=11.467,<0.001) increased in puerarin+miR-490 inhibitor group.Compared with puerarin+miR-490 inhibitor+Si-NC group,puerarin+miR-490 inhibitor+Si-DTL group up-regulated the protein level of E-cadherin (=13.081,<0.001) while down-regulated the protein levels of N-cadherin (=10.835,<0.001) and vimentin (=11.862,<0.001). Conclusion Puerarin could inhibit the proliferation,invasion,and migration of non-small cell lung cancer cells by up-regulating miR-490 and down-regulating DTL.