鞣酸通过下调TMEM16A的表达抑制人结肠癌细胞SW620细胞的增殖

Tannic acid inhibits the proliferation of SW620 cell lines by downregulating TMEM16A expression

目的：探讨鞣酸对人结肠癌SW620细胞增殖及TMEM16AmRNA与蛋白水平的影响。方法将对数生长期的人结肠癌细胞SW620分为3组，分别是鞣酸低剂量组（50μmol/L）及高剂量组（100μmol/L），以及二甲基亚砜（DMSO）作为空白对照组，培养48、72h。MTT法检测细胞的增殖情况，流式细胞仪测定细胞周期的分布及凋亡情况，RT-PCR和Westernblot分别检测TMEM16A的mRNA水平和蛋白水平。计量资料采用x±s表示，多组间比较采用单因素方差分析，两两比较采用SNK检验。结果MTT结果显示：鞣酸处理能显著抑制SW620的增殖。高剂量组对SW620细胞的抑制作用比低剂量大。处理48h，高剂量组比低剂量组抑制作用明显（t＝15．35，P＜0．01）；处理72h，高剂量组同样比低剂量组抑制作用明显（t＝22．32，P＜0．01）。流式细胞检测结果显示：与对照组相比，各个剂量组都能显著将SW620细胞抑制在G0/G1期和S期（F＝6782．1、1509．3，P＜0．01），G2/M期的细胞比例差异无统计学意义（F＝1．37，P＞0．05）。各个剂量组处理后，凋亡率差异有统计学意义（F＝545．3，P＜0．01）。与对照组比较，鞣酸高剂量组的3H-TdR、3H-Leucine掺入量在48、72h均明显低于对照组（P＜0．05）。鞣酸低剂量组药物干预48、72h后TMEM16AmRNA相对表达量分别为0．63±0．01和0．62±0．01；鞣酸高剂量组药物干预48、72hTMEM16AmRNA相对表达量分别为0．58±0．01和0．50±0．01，均低于空白对照组的0．85±0．01（F＝7．645，P＜0．05）。鞣酸低剂量组药物干预48、72h后TMEM16A蛋白相对表达量分别为0．68±0．14和0．65±0．12；鞣酸高剂量组药物干预48、72hTMEM16蛋白相对表达量分别为0．64±0．15和0．63±0．11，低于空白对照组的1．28±0．06（F＝4．508，P＜0．05）。结论鞣酸能明显抑制人结肠癌细胞SW620细胞的增殖，将其细胞周期阻滞于G1～S期，并下调TMEM16A的mRNA及蛋白水平。

Objective To investigate the effects of Tannic acid on the proliferation of human colon cancer SW 620 cell line and the mRNA and protein levels of TMEM16A .Methods Human colon cancer cell line SW620 were divided into the low dose（50 μmol/L） ,high dose（100 μmol/L） ,they were cultured for 48 h or 72 h separately .Control groups were cultured in the medium with DMSO .The proliferation of SW620 cell line was detected by the MTT assay at different time points （48 h or 72 h） .The cell cycle and apoptosis in the Tannic acid-treated groups were detected by flow cytometry .RT-PCR and Western blotting were used to de-termine the mRNA and protein levels of TMEM16A separately .All data were analyzed using the one-way analysis of variance （ANOVA） ,SNK test by the SPSS software .Results Compared with the control group ,the proliferation of SW620 cell line was significantly inhibited after the treatment by Tannic acid at the concentration of 50 μmol/L and 100 μmol/L for 48 h or 72 h（t=15 .35 ,P〈0 .01 ;t=22 .32 ,P〈0 .01） .Determined by flow cytometry ,after treated with Tannic acid ,the numbers of SW620 cells were inhibited in the G0/G1 phase and the S phase（F=6 782 .1 ,1 509 .3 ,P〈0 .01） ,and the numbers of SW620 cells in G2/M phase were not varied in each group（F=1 .37 ,P〉0 .05） ,and increased apoptotic rate when compared with control group （F=545 .3 ,P〈0 .01） .The value of 3H-TdR and 3H-Leucine incorporation of SW620 cells treated with Tannic acid（100 μmol/L） 48 h and 72 h separately ,were obviously decreased as compared with that of control group （P〈0 .05） .In the low dose treated groups （50 μmol/L） ,the mRNA levels in 48 h group and 72 h group were（0 .633 ± 0 .009） and（0 .621 ± 0 .011） ,and in the high dose treated groups （100 μmol/L） ,the mRNA levels in 48 h group（0 .64 ± 0 .15） and 72 h group（0 .63 ± 0 .11） ,were lower than the control group（F=7 .645 ,P〈 0 .05） .After treating SW620 with Tannic acid for 48 h and 72 h ,in the low dose groups ,the protein expression of TMEM16A were（0 .68 ± 0 .14） and（0 .65 ± 0 .12） ,and in the high dose groups ,the protein expression of TMEM16A were（0 .64 ± 0 .15） and（0 .63 ± 0 .11） were decreased when compared with the control group （1 .28 ± 0 .06）（F=4 .508 ,P〈0 .05） .Conclusion Tannic acid arrested SW620 at G1-S phase and decrease the mRNA and protein expression of TMEM16A .