沉默DNA甲基转移酶上调N-myc下游调节基因1表达对前列腺癌DU145细胞生物学行为的影响

Upregulation of N - myc downstram regulated gene 1 expression by silencing DNA methyltrans- ferases influences the biological behaviors of prostate cancer DU145 cells

目的观察小干扰RNA（siRNAs）沉默DNA甲基转移酶（DNMTs）上调N-myc下游调节基因1（NDRG1）表达对前列腺癌DU145细胞生物学行为的影响。方法使用siRNAs分别抑制DU145细胞中DNMT1、DNMT3b及DNMT1＋DNMT3b的表达；使用Westernblot、实时定量反转录聚合酶链反应（RT—qPCR）检测细胞中DNMT1、DNMT3b及NDRG1的表达改变；通过细胞计数试剂盒（CCK-8）、Muse凋亡检测仪、划痕实验和Transwell侵袭实验，检测细胞增殖、凋亡、迁移及侵袭能力的改变；筛选上述改变最显著组DU145细胞，抑制细胞中NDRG1表达，再次检测上述细胞生物学行为。结果Westernblot及RT-qPCR结果显示，与对照组比较，DNMT1抑制组、DNMT3b抑制组及联合抑制组的NDRG1mRNA（1．59±0．03，1．23±0．01，1．36±0．11比0．90±0．15，1．00±0．03）和蛋白（1．07±0．16，0．49±0．01，0．92±0．09比0．23±0．04，0．30±0．07）表达显著增加（P〈0．05）。CCK-8结果显示，在24、48、72h，DNMT1抑制组吸光度（A）值为0．189±0．030、0．266±0．048、0．419±0．058；DNMT3b抑制组为0．221±0．023、0．318±0．072、0．498±0．064；联合抑制组为0．199±0．021、0．284±0．062、0．488±0，091，与对照组比较，显著减少（P〈0．05）。凋亡检测结果显示，DNMT1抑制组凋亡率为（22．40±1．36）％、DNMT3b抑制组为（14．20±0．80）％、联合抑制组为（17．60±0．74）％，与对照组比较，显著增加（P〈0．05）。划痕实验结果显示，DNMT1抑制组划痕面积愈合率为（53．01±3．22）％、DNMT3b抑制组为（70．97±2．12）％、联合抑制组为（63．93±4．42）％，与对照组比较显著减少（P〈0．05）。Transwell实验结果显示，DNMT1抑制组侵袭细胞数为（16±3）个、DNMT3b抑制组为（32±5）个、联合抑制组为（20±4）个，与对照组比较，显著减少（P〈0．05）。其中，DNMT1抑制组效果最显著（P〈0．05），联合抑制未见显著协同效应。与对照组比较，NDRG1-siRNA可显著逆转由DNMT1-siRNA引起的DU145细胞生物学行为改变（P〈0．05）。结论通过抑制DNMT1、DNMT3b在前列腺癌DU145细胞中的表达，可部分恢复NDRG1的表达，并可显著抑制肿瘤细胞增殖，促进细胞凋亡，减弱细胞迁移及侵袭能力，其中单独抑制DNMT1效果最显著．联合抑制无明显协同效应．提示DNMT1可以成为前列腺癌去甲基化治疗的一个有效靶点。

Objective To investigate the influence of the upregulation of N -myc downstram regu- lated gene 1 （NDRG1） expression by silencing DNA methyltransferases （DNMTs） using sInall interfering RNAs （siRNAs） on the biological behaviors of prostate cancer DU145 cells. Methods SiRNAs were used to inhibit the expression of DNMT1, DNMT3b of DU145 cells ; Western blotting and quantitative real - timereverse transcriptase - polymerase chain reaction （ RT - qPCR） were used to detect the expression levels of DNMT1, DNMT3b and NDRGI of DU145 cells ; cell counting kit - 8 （ CCK - 8） assay, muse cell analy- zer, wound healing assay and Transwell migration assay were used to detect the proliferation, apoptosis, migration and invasion of DUlg5 cells; then we inhibited the expression of NDRG1 of DU145 cells which had the most significant changes of cell biological behaviors; and we detected the expression level of NDRG1 and the changes of cell biological behaviors. Results Western blotting and RT - qPCR showed that the mRNA （ 1.59 ± 0. 03, 1.23 ±0. 01, 1.36±0. 11 vs. 0. 90 ± 0. 15, 1.00 ±0. 03 ） and protein （ 1. 07 ±0. 16, 0. 49±0. 01,0. 92 ±0. 09 vs. 0. 23 ±0. 04, 0. 30 ±0. 07） expression levels of NDRG1 of DNMT1 inhibition group, DNM33b inhibition group and double inhibition group were significantly higher than these in control groups （P 〈 0. 05）. CCK - 8 assay showed that, at 24, 48 and 72 h, the optical den- sity （A） values of DNMT1 inhibition group （0. 189 ±0. 030, 0. 266 ±0. 048, 0. 419 ±0. 058）, DNMT3b inhibition group （0. 221 ±0. 023,0. 318 ±0. 072, 0. 498 ±0.064） and double inhibition group （0. 199 ±0. 021,0. 284 ±0. 062, 0. 488 ±0. 091 ） were significantly lower than control groups （P 〈0. 05）. Apopto- sis results showed that the apoptosis rates of DNMT1 inhibition group （22. 40 ＋ 1.36） % , DNMT3b inhibi- tion group （ 14. 20 ± 0. 80） % and double inhibition group （ 17. 60± 0. 74） % were significantly higher than control groups （ P 〈 0. 05）. Wound healing assay showed that the healing rates of DNMT1 inhibition group （53.01 ±3.22）%, DNMT3b inhibition group （70.97 ±2. 12）% and double inhibition group （63.93± 4. 42） % were significantly lower than control groups （ P 〈 0. 05 ）. Transwell migration assay showed that the amount of invasion cells in DNMT1 inhibition group [ （ 16 ± 3） cells], in DNMT3b inhibition group [ （32± 5 ） cells] and in double inhibition group [ （20 ± 4） cells] were significantly less than control groups （P 〈 0.05 ）. Among these groups, DNMT1 inhibition group had the most significant effect （P 〈 0. 05）, but the co - transfection had no significant synergistic effect. Compared with control groups, NDRG1 - siRNA could significantly reverse the changes of cell biological behaviors of DU145 cells （ P 〈 0. 05）. Conclusion Inhibition of DNMT1, DNMT3b of DU145 cells could partially restore NDRG1 ex- pression, which could inhibit the proliferation, apoptosis, migration and invasion of cancer cells. Moreo- ver, DNMT1 inhibition group had the most significant effect, but the co - transfection had no significant synergistic effect, suggesting that DNMT1 could become an effective target for demethylation treatment of prostate cancer.