二氯乙酸对人肾癌细胞株A498侵袭及迁移的抑制作用研究

目的探讨二氯乙酸(DCA)对人肾癌细胞株A498侵袭及迁移的影响,并分析其机制。方法取人肾癌细胞株A498培养,分为阴性对照(等容积无菌生理盐水)组、DCA低、中、高(5、10、20 mmol/L)剂量组,培养48 h。Transwell小室实验检测侵袭能力,细胞划痕实验检测迁移能力,实时荧光定量聚合酶链反应(qRT-PCR)检测c-Jun氨基末端激酶(JNK)、钙粘附蛋白E(E-cadherin)、N-钙粘蛋白(N-cadherin)基因表达,Western blot检测蛋白表达及磷酸化JNK(p-JNK)。多组间比较采用单因素方差分析,组间两两比较采用SNK-q检验。结果与DCA高剂量组相比,DCA中、低剂量组、阴性对照组侵袭活性(75.33±10.09)比(167.89±47.12)、(372.74±50.06)、(530.26±81.22)个/视野]、E-cadherin mRNA相对表达量(1.52±0.12比1.35±0.11、1.02±0.11、0.85±0.12)、E-cadherin蛋白相对表达量(1.32±0.19比0.89±0.14、0.37±0.08、0.13±0.03)均降低(P均<0.001);而迁移率(10.05±2.31)﹪比(23.95±4.20)﹪、(36.26±5.01)﹪、(53.59±6.71)﹪]、N-cadherin mRNA蛋白相对表达量(0.42±0.06比0.58±0.07、0.80±0.10、0.95±0.11),N-cadherin蛋白相对表达量(0.15±0.03比0.25±0.04、0.74±0.07、0.95±0.11)、p-JNK水平(0.14±0.03比0.29±0.05、0.68±0.07、0.95±0.10)均升高(P均<0.001)。不同剂量和阴性对照组的JNK mRNA与蛋白表达差异均无统计学意义(P>0.05)。结论DCA可抑制人肾癌细胞株A498侵袭及迁移,且一定范围内呈剂量依赖性,推测与调控侵袭及迁移相关基因与蛋白表达有关。

Inhibitory effect of dichloroacetic acid on invasion and migration of human renal cell carcinoma cell line A498

Objective To investigate the effects and mechanisms of dichloroacetic acid(DCA)on the invasion and migration of the renal cell carcinoma cell line A498.Methods Human renal cell carcinoma cell line A498 were cultured and divided into negative a control(NC)group,a low dose DCA group,a medium dose DCA group and a high dose DCA group.The 3 dose groups of DCA were treated with 5,10,20 mmol/L DCA,and the NC group was administered isovolumetric sterile saline for 48 hours.Transwell chamber test was used to detect invasive ability and the cell scratch test to detect migration ability.Real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)was used to detect the expression of c-Jun amino-terminal kinase(JNK),E-cadherin and N-cadherin genes.Western blot was used to detect the expressions of the above proteins and phosphorylated JNK(p-JNK).The differences in the data of these groups were analyzed by one-way ANOVA in SPSS Statistics 25.0,and SNK-q test was used for comparison between each two groups.Results Comparing with high dose DCA group,the invasive activity decreased in the middle dose DCA group,the low dose DCA group and the negative control group(75.33±10.09 vs 167.89±47.12,372.74±50.06 and 530.26±81.22/field),and the relative expression of E-cadherin mRNA decreased(1.52±0.12 vs 1.35±0.11,1.02±0.11 and 0.85±0.12,respectively),as well as the relative expression of E-cadherin protein decreased(1.32±0.19 vs 0.89±0.14,0.37±0.08 and 0.13±0.03,respectively)(all P<0.001),while the mobility(10.05±2.31)﹪vs(23.95±4.20)﹪,(36.26±5.01)﹪and(53.59±6.71)﹪,respectively],the relative expression of N-cadherin mRNA(0.42±0.06 vs 0.58±0.07,0.80±0.10 and 0.95±0.11,respectively),the relative expression of N-cadherin protein(0.15±0.03 vs 0.25±0.04,0.74±0.07 and 0.95±0.11,respectively),and level of p-JNK(0.14±0.03 vs 0.29±0.05,0.68±0.07 and 0.95±0.10,respectively)were increased(all P<0.001).There was no significant difference in JNK mRNA and protein expression among four groups(P>0.05).Conclusion DCA mayinhibit the invasion and migration of human renal cell carcinoma cell line A498 in a dose-dependent manner within a certain range,which is presumed to be related to the expressions of genes and proteins related to invasion and migration.