DNA甲基转移酶1抑制对涎腺腺样囊性癌细胞生长、侵袭及转移的影响

Effect of DNA methyltransferases 1 inhibition on proliferation, invasion, and metastasis in ACC-M line

目的研究DNA甲基转移酶1（DNA methyltransferases 1，DNMT-1）表达抑制对涎腺腺样囊性癌（salivary adenoid cystic carcinoma，SACC）细胞体内、体外的生物学影响，探讨DNMT-1在SACC发生、侵袭及转移中的作用。方法对前期实验建立的DNMT-1表达稳定抑制的ACC-M细胞（实验干扰组）、空载对照ACC—M细胞系（空载对照组）及ACC—M细胞系（空白对照组），在体外分别应用甲基噻唑基四唑（MTT）法生长曲线分析、流式细胞周期分析、平板克隆实验、体外侵袭能力分析等方法，体内对裸鼠皮下、尾静脉注射肿瘤细胞，综合分析评估DNMT-1表达抑制对ACC—M细胞体内增殖、侵袭及转移的影响。结果实验干扰组细胞倍增时间延长[（34．7±2．1）h]、细胞周期S期比例[（17．4±1．7）％]、克隆形成率[（43．0±1．3）％]降低，与空白对照组[分别为（26．2±3．1）h、（31．5±2．0）％、（71．0±4．7）％]及空载对照组[分别为（28．4±3．9）h、（39．0±2．0）％、（66．0±5．2）％]差异有统计学意义（P〈0．05）；各组体外侵袭能力的差异无统计学意义（P〉0．05）；体内DNMT-1表达稳定抑制的ACC—M细胞，其皮下成瘤率（6／10）、瘤体体积[（2．18±0．83）mm^3]、重量[（O．0156±0．0046）g]均显著低于空白对照组[分别为10／10，（155．44±1．67）mm^3、（0．0724±0．0157）g]及空载对照组[分别为10／10、（147．46±1．73）mm^3、（0．0729±0．0177）g]，差异均有统计学意义（P〈0．05）；各组的肺转移率差异无统计学意义（P〉0．05）；DNMT-1表达稳定抑制的ACC-M细胞，其肺转移瘤的个数[（2．0±0．5）个]、直径[（70．0±20．3）μm]均显著低于空白[（28．0±5．5）个、（195．0±25．4）μm]及空载（27．0±4．5）个、（190．0±19．9）μm]对照组，P〈0．05。结论抑制DNMT-1的表达能有效降低ACC细胞的生长、增殖及转移能力。

Objective To investigate the effect of DNA methyltransferases 1 （DNMT-1） inhibition on the ACC-M ceils in vitro and in vivo and discuss the role of DNMT-1 in the development, invasion and metastasis of salivary adenoid cystic carcinoma （SACC）. Methods ACC-M cells of stable DNMT-1 inhibition were established in a previous research. In vitro, the growth and invasion of ACC-M cells which stably inhibited DNMT-1 were detected and analyzed by methyl thiazolyl tetrazolium （MTT） growth curve, flow cytometry, plating efficiency and invasion assay. In vivo, the growth and metastasis of ACC-M cells which persistently inhibited DNMT-1 were observed and analyzed by subcutaneous injection and tail vein injection into the nude mice. Results In vitro, the doubling time [ （34. 7 ± 2. 1 ） h ], S phase fraction [ （ 17.4 ± 1.7 ） % ], plating efficiency [ （ 43.0 ± 1.3 ） % ] of ACC-M cells was significantly different from those of blank [ （ 26. 2 ± 3. 1 ） h, （31.5 ± 2. 0） %, （71.0 ± 4. 7 ） % ], empty load control [ （28.4 ± 3.9） h, （39. 0 ± 2. 0 ） %, （ 66. 0 ± 5.2 ） % ], P 〈 0.05, and the invasion ability was not significantly different among these groups（ P 〉 0. 05 ）. In vivo, the subcutaneous tumor forming rate （ 6/10 ）, volume [ （ 2. 18 ± 0. 83 ） mm^3 ], weight [ （0. 0156 ± 0. 0046） g] of ACC-M cells was also significantly lower than that of blank [ 10/10, （ 155.44 ± 1.67） mm^3, （0. 0724 ± 0. 0157 ） g], empty load control [ 10/10, （ 147.46 ± 1.73 ） mm^3, （0. 0729 ± 0. 0177） g], P 〈 0. 05, but the rate of lung metastasis was not significantly different among these groups （ P 〉 0.05 ）, and the masses （ 2. 0 ± 0. 5 ）, diameter （ 70. 0 ± 20. 3 ） μm of ACC-M cells was significantly lower than that of blank [ （28. 0 ± 5.5 ）, （ 195 ± 25.4）μm ], empty load control [ （27.0 ± 4. 5）, （ 190. 0 ± 19. 9 ） μm ], P 〈 0. 05. Conclusions Inhibition of DNMT-1 is able to inhibit the proliferation and metastasis of ACC-M cells in vitro and in vivo.