姜黄素调控LncRNA-UCA1/miR-582-5p通路抑制膀胱癌细胞增殖的机制研究

Mechanism of Curcumin in the Inhibition of Bladder Cancer Cell Proliferation via LncRNA-UCA1/miR-582-5p Pathway

目的:探讨姜黄素调控LncRNA-UCA1/miR-582-5p通路抑制膀胱癌细胞增殖的机制。方法:以不同浓度的姜黄素干预人膀胱癌T24细胞,MTT法检测姜黄素对T24细胞的增殖抑制率,并绘制抑制率曲线图,筛选姜黄素最佳作用浓度及时间进行后续试验;双荧光素酶报告基因试验验证UCA1与miR-582-5p的靶向关系;RT-PCR法检测姜黄素对T24细胞中UCA1和miR-582-5p表达水平的影响;流式细胞术检测姜黄素对T24细胞周期以及凋亡的影响;划痕试验检测姜黄素对T24细胞迁移能力的影响;Transwell侵袭试验检测姜黄素对T24细胞侵袭能力的影响。结果:姜黄素能够显著抑制人膀胱癌T24细胞增殖,并呈浓度时间依赖性,姜黄素0、5、10、20、30、40、50μmol/L作用T24细胞后,增殖抑制率随浓度增高以及时间的延长显著增高(P<0.01);UCA1上存在miR-582-5p的结合位点,进一步研究发现miR-582 mimic可显著降低UCA1野生型质粒荧光素酶活性(P<0.01),而对UCA1突变型质粒荧光素酶活性无调控作用,表明UCA1上存在miR-582-5p的结合位点,两者可以直接相互作用,且呈负相关关系;姜黄素作用T24细胞24 h后,UCA1表达水平随姜黄素浓度增高而下调,miR-582-5p表达上调(P<0.05或P<0.01);姜黄素10、20、40μmol/L显著诱导T24细胞凋亡,抑制T24细胞迁移(P<0.01),将细胞周期阻滞于S期(P<0.01),明显降低侵袭细胞数(P<0.05或者P<0.01)。结论:姜黄素能明显抑制人膀胱癌T24细胞增殖、迁移、侵袭、阻滞细胞周期、诱导细胞凋亡,表明姜黄素可能通过LncRNA-UCA1/miR-582-5p通路抑制人膀胱癌T24细胞增殖。

Objective:To investigate the mechanism of curcumin in inhibiting bladder cancer cell proliferation by regulating the LncRNA-urothelial carcinoma-associated 1(UCA1)/miR-582-5 p pathway.Methods:Human bladder cancer T24 cells were treated with curcumin at different concentrations.The inhibition rate of curcumin on the proliferation of T24 cells was detected by MTT assay,and the inhibition rate curve was plotted to screen the optimal effect concentration of curcumin and time for subsequent experiments.Dual-luciferase reporter assay was performed to verify the relationship between UCA1 and miR-582-5 p.The effect of curcumin on the expression of UCA1 and miR-582-5 p in T24 cells was determined by RT-PCR.Flow cytometry was employed to detect the effect of curcumin on the cell cycle and apoptosis of T24 cells.The effects of curcumin on the migration and invasion of T24 cells were detected by wound healing assay and Transwell assay,respectively.Results:Curcumin significantly inhibited the proliferation of human bladder cancer T24 cells in a concentration-and time-dependent manner.After treatment with curcumin at different concentrations(0,5,10,20,30,40,and 50μmol/L),the inhibition rates on the proliferation of T24 cells were increased with the concentration and time(P<0.01).The binding site of miR-582-5 p was observed on UCA1.Further investigation revealed that miR-582 mimic could reduce the activity of luciferase reporter plasmids containing wild-type UCA1(P<0.01),but showed no regulatory effect on the activity of those containing mutant UCA1,indicating that there was a binding site for miR-582-5 p on UCA1 and they directly interacted with each other in a negative relationship.After curcumin treatment on T24 cells for 24 hours,UCA1 expression level was down-regulated with the increase in the curcumin concentration,while the expression of miR-582-5 p was up-regulated(P<0.05 or P<0.01).Curcumin at 10,20,and 40μmol/L significantly induced the apoptosis,inhibited the migration(P<0.01),arrested the cell cycle of T24 cells at the S phase(P<0.01),and decreased the invaded cells(P<0.05 or P<0.01).Conclusion:Curcumin can significantly inhibit the proliferation,migration,and invasion,arrest cell cycle,and induce the apoptosis of human bladder cancer T24 cells,which is presumedly achieved via the LncRNA-UCA1/miR-582-5 p pathway.