姜黄素介导微小RNA-199a-3p基因对前列腺癌细胞的影响

Effects of curcumin mediating miR-199a-3p gene on prostate cancer C4-2 cells

目的探讨姜黄素调控微小RNA-199a-3p(miR-199a-3p)的表达对前列腺癌C4-2细胞增殖、迁移和侵袭的影响。方法(1)将体外培养的前列腺癌C4-2细胞分为4组:空白组(无药物干预)和低、中、高3个剂量实验组(20,40,80μmol·L^-1姜黄素干预),选取对C4-2细胞的增殖抑制最明显姜黄素浓度用于以下实验。(2)将体外培养的前列腺癌C4-2细胞分为3组:空白对照组(仅加入脂质体)、第1转染组和第2转染组,第1转染组、第2转染组分别转染阴性对照物与miR-199a-3p抑制物后再加入姜黄素40μmol·L^-1处理。用溴化噻唑蓝四氮唑法检测细胞增殖水平,以Transwell法检测细胞迁移和侵袭水平,以实时荧光定量-PCR法检测miR-199a-3p基因表达(2﹣△△Ct值),以蛋白质印迹法检测基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶-9(MMP-9)、Wnt/β-catenin通路β-catenin、Cyclin D1和c-Myc蛋白表达(灰度值的比值)。结果(1)空白组和低、中、高3个剂量实验组干预72 h的细胞增殖率分别为(1.76±0.31)%,(1.52±0.27)%,(0.78±0.16)%和(0.95±0.19)%,低、中、高3个剂量实验组与空白组相比,差异均有统计学意义(均P<0.05),其中40μmol·L^-1姜黄素对C4-2细胞的增殖抑制最明显,故选用该浓度做后续实验。空白组和中剂量实验组的细胞迁移数目分别为(165.48±27.83)和(68.42±16.67)个;这2组的细胞增殖数目分别为(104.62±21.64)和(43.58±10.91)个;这2组的miR-199a-3p基因表达量分别为1.03±0.12和3.91±0.29,中剂量实验组与空白组比较,以上指标的差异均有统计学意义(均P<0.05)。(2)空白对照组、第1转染组和第2转染组在转染72 h的细胞增殖率分别为(0.95±0.19)%,(0.95±0.27)%和(1.66±0.41)%;这3组的细胞迁移数目分别为(62.45±12.11),(71.52±15.12)和(138.46±39.49)个;这3组的细胞侵袭数目分别为(34.49±8.43),(39.34±9.46)和(81.49±24.16)个;这3组的MMP-2表达分别为0.46±0.11,0.45±0.13和0.76±0.17;这3组的MMP-9表达分别为0.36±0.11,0.38±0.12和0.59±0.15;这3组的β-catenin表达分别为0.53±0.13,0.49±0.16和0.71±0.17;这3组的Cyclin D1表达分别为0.26±0.09,0.29±0.11和0.69±0.15;这3组的c-Myc表达分别为0.21±0.06,0.22±0.07和0.56±0.12。以上指标:第2转染组与空白对照组比较,或第2转染组与第1转染组比较,差异均有统计学意义(均P<0.05)。结论姜黄素可上调miR-199a-3p基因的表达,从而抑制前列腺癌C4-2细胞的增殖、迁移和侵袭。

Objective To explore the effect of curcumin regulating the expression of miR-199 a-3 p on the proliferation,migration and invasion of prostate cancer C4-2 cells.Methods(1)Prostate cancer C4-2 cells were cultured in vitro and divided into 4 groups:blank group(not intervention)and experimental-L,-M,-H groups,which were intervened with 20,40,80μmol·L^-1 curcumin,respectively.The concentration of curcumin that inhibits the proliferation of C4-2 cells the most obviously was selected to be used in the experiment.(2)Prostate cancer C4-2 cells were cultured in vitro and divided into 3 groups:blank control group,the first transfection group,and the second transfection group.The blank control group only was added liposomes;the first transfection group and the second transfection group were transfected with negative control substance and miR-199 a-3 p inhibitor respectively,and curcumin 40μmol·L^-1 was added.Methyl thiazolyl tetrazoliun was used to detect cell proliferation.Transwell method was used to detect cell migration and invasion.Fluorescence real time quantitative-PCR was used to detect miR-199 a-3 p gene expression(2﹣△△Ctvalue).Western blot method was used to detect matrix metalloproteinase-2(MMP-2),matrix metalloproteinase-9(MMP-9)and Wnt/β-catenin pathwayβ-catenin,Cyclin D1 and c-Myc protein expression(ratios of gray value).Results(1)The cell proliferation rates for 72 h in blank group,experimental-L group,experimental-M group,and experimental-H group were(1.76±0.31)%,(1.52±0.27)%,(0.78±0.16)%and(0.95±0.19)%,with statistically significant difference between experimental-L group,experimental-M,experimental-H group and blank group(all P<0.05).Among them,40μmol·L^-1 curcumin inhibited the proliferation of C4-2 cells most obviously.Therefore,the concentration was selected for subsequent experiments.The number of cell migration in blank group and experimental-M group were(165.48±27.83)and(68.42±16.67),respectively;the number of cell proliferation were(104.62±21.64)and(43.58±10.91),respectively;the expression levels of miR-199 a-3 p gene were 1.03±0.12 and 3.91±0.29,respectively;with statistically significant difference among above indicators between experimental-M group and the blank group(all P<0.05).(2)The cell proliferation rates in blank control group,first transfection group and,second transfection group were(0.95±0.19)%,(0.95±0.27)%and(1.66±0.41)%after 72 h of transfection;the number of cell migration in the three groups were(62.45±12.11),(71.52±15.12)and(138.46±39.49)cells;the number of cell invasion in the three groups were(34.49±8.43),(39.34±9.46)and(81.49±24.16)cells;the expression of MMP-2 protein in the three groups were 0.46±0.11,0.45±0.13 and 0.76±0.17;the expression of MMP-9 protein in the three groups were0.36±0.11,0.38±0.12 and 0.59±0.15;the expression ofβ-catenin protein in the three groups were0.53±0.13,0.49±0.16 and 0.71±0.17;the expression of Cyclin D1 protein in the three groups were0.26±0.09,0.29±0.11 and 0.69±0.15;the expression of c-Myc protein in the three groups were expressions were 0.21±0.06,0.22±0.07 and 0.56±0.12,with statistically significant difference among above indicators between second transfection group and blank control group,or between second transfection group and first transfection group(all P<0.05).Conclusion Curcumin can up-regulate the expression of miR-199 a-3 p gene,thereby inhibiting the proliferation,migration and invasion of C4-2 cells in prostate cancer.