姜黄素抑制肺癌细胞A549上皮间质转化的机制研究

Mechanism of curcumin inhibiting epithelial mesenchymal transformation of lung cancer cell line A549

摘要:

目的 探讨姜黄素对肺癌细胞A549上皮间质转化的影响。方法 姜黄素诱导肺癌细胞A549 24 h，CCK-8法检测A549细胞活性，Transwell实验检测A549细胞迁移、侵袭的能力，Western blotting检测上皮间质转化标志蛋白的相对表达量，实时荧光定量聚合酶链反应（qRT-PCR）检测HOTAIR、E-cadherin、N-cadherin及Snail mRNA的相对表达量，测序方法检测姜黄素诱导后lncRNA表达的变化，实验验证介导姜黄素发挥作用的具体非编码RNA，分析HOTAIR与临床病理参数之间的关系。结果 CCK-8法检测结果显示，姜黄素诱导肺癌细胞A549 24 h后，不同浓度组肺癌细胞A549的OD值比较，差异有统计学意义（P <0.05），随着姜黄素浓度的升高，OD值降低。Transwell结果显示，姜黄素诱导肺癌细胞A549 24 h后，姜黄素诱导组抑制肺癌细胞A549的侵袭、迁移，与对照组比较，差异有统计学意义（P <0.05）。Western blotting和qRT-PCR检测结果显示，姜黄素诱导组的间质表型基因和蛋白（E-cadherin、N-cadherin、Snail）与对照组比较，差异有统计学意义（P <0.05），N-cadherin、Snail相对表达量下降，E-cadherin相对表达量升高。癌旁正常组和肺癌组中HOTAIR的相对表达量比较，差异有统计学意义（P <0.05），肺癌组高于癌旁正常组。测序检测和qRT-PCR结果显示，姜黄素抑制HOTAIR的表达。分析HOTAIR在肺癌组织中的表达及其与临床病理参数之间的关系显示，HOTAIR的表达与TNM分期、分化程度、淋巴结转移密切相关。对照组、姜黄素诱导组、姜黄素诱导+HOTAIR过表达组A549细胞OD值比较，差异有统计学意义（P <0.05）。结论 姜黄素有效抑制肺癌细胞A549的增殖、侵袭及迁移，姜黄素抑制lncRNA HOTAIR表达，姜黄素抑制肺癌细胞A549上皮间质转化是通过HOTAIR实现的。

关键词:肺肿瘤;姜黄素;上皮间质转化;HOTAIR

Abstract:

Objective To investigate the effect of curcumin on epithelial-mesenchymal transition of A549.Methods The inhibitory effect of curcumin on lung cancer cell line A549 was detected by Transwell assay; the change of epithelial-mesenchymal transformation marker protein was detected by Western blotting; the relative expression of HOTAIR was detected by real-time reverse transcriptase polymerase chain reaction (Real-time RT-PCR); and the change of lncRNA expression after curcumin induction was detected by sequencing. The specific non-coding RNA mediating the role of curcumin was verified, and the relationship between HOTAIR and clinicopathological parameters was analyzed.Results The results of CCK-8 assay showed that after 24 hours of curcumin-induced lung cancer cell line A549, the OD value of lung cancer cell A549 in different concentration groups was significantly different (P < 0.05), and the OD value decreased with the increase of curcumin concentration. Transwell results showed that 24 hours after curcumin-induced lung cancer cell A549, the curcumin-induced group inhibited the invasion and migration of lung cancer cell line A549, and there was significant difference between the curcumin-induced group and the control group (P < 0.05). The results of Western blotting and Real-time RT-PCR showed that the interstitial phenotypic genes and proteins (E-ca, N-ca, and Snail) in the curcumin-induced group were significantly different from those in the control group. The relative expression of N-ca and Snail decreased and the relative expression of E-ca increased in the curcumin-induced group. The relative expression of HOTAIR in lung cancer group was higher than that in normal group. The results of sequencing and Real-timeRT-PCR showed that curcumin inhibited the expression of HOTAIR. The expression of HOTAIR in lung cancer and its relationship with clinicopathological parameters were analyzed. The results showed that the expression of HOTAIR was closely related to stage, differentiation, and lymph node metastasis.Conclusion Curcumin effectively inhibits the proliferation, invasion, and migration of lung cancer cell line A549. Curcumin inhibits the expression of lncRNA HOTAIR. Curcumin inhibits epithelial-mesenchymal transformation of lung cancer cell line A549 through HOTAIR.

Key words:lung neoplasms;curcumin;epithelial-mesenchymal transition;HOTAIR long non-coding RNA