微小RNA-106a-5p/E2F3/β-catenin轴增加NK细胞对喉癌细胞的杀伤作用

miR-106a-5p/E2F3/β-catenin axis increases the killing effect of NK cells on laryngeal cancer cells

目的 分析微小RNA-106a-5p(miR-106a-5p)/E2F3/β-catenin轴对喉癌细胞恶性发展的影响及其可能的作用机制。方法 通过实时荧光定量PCR(qRT-PCR)技术检测喉癌组织和癌旁组织中miR-106a-5p和E2F3的基因表达量。生物信息学软件及其双荧光素酶实验检测miR-106a-5p和E2F3之间的关系。下调miR-106a-5p和E2F3表达,CCK-8试剂盒检测喉癌Hep-2细胞的增殖能力,Western blot实验检测Hep-2细胞上皮间质转化(epithelial mesenchymal transformation,EMT)能力,检测NK细胞对Hep-2细胞的杀伤活性,酶联免疫吸附实验(ELISA)检测γ干扰素(interferon-γ,IFN-γ)和肿瘤坏死因子α(tumor necrosis factor α,TNF-α)的分泌。Western blot分析miR-106a-5p通过E2F3对β-catenin通路的影响。进一步检测抑制β-catenin通路对Hep-2细胞恶性发展及NK细胞杀伤作用的影响。结果 喉癌组织中miR-106a-5p表达(0.62±0.08)低于癌旁组织(1.53±0.24),E2F3表达(2.65±0.86)高于癌旁组织(1.48±0.74)。miR-106a-5p与E2F3特异性结合。下调miR-106a-5p促进Hep-2细胞增殖和EMT,抑制NK细胞对Hep-2细胞的杀伤活性,而下调E2F3表达抑制Hep-2细胞增殖和EMT,上调NK细胞对Hep-2细胞的杀伤活性。下调miR-106a-5p通过E2F3激活β-catenin通路。XAV939干扰β-catenin通路后显著逆转了下调miR-106a-5p通对Hep-2细胞恶性发展以及NK细胞杀伤作用的影响。结论miR-106a-5p靶向E2F3激活β-catenin通路调控喉癌细胞的恶性发展。

OBJECTIVE To study the effect of miR-106a-5p/E2F3/β-catenin axis on the development of laryngeal carcinoma cells and its possible mechanism.METHODSThe expression levels of miR-106a-5p and E2F3 in laryngeal carcinoma and adjacent tissues were detected by qRT-PCR.Bioinformatics software and dual luciferase assay were used to detect the relationship between miR-106a-5p and E2F3.After the miR-106a-5p and E2F3 expressions of the laryngeal carcinoma Hep-2 cells were down-regulated,the proliferation ability of the Hep-2 cells was detected by CCK-8 kit,the EMT ability was detected by Western blot,and killing activity of NK cells on Hep-2 cells was detected.IFN-γ and TNF-α were detected by ELISA.Western blot was used to study the effect of miR-106a-5p on β-catenin pathway via E2F3.The effect of β-catenin pathway inhibition on development of Hep-2 cell and NK cell killing activity was furtherinvestigated.RESULTS The expression of miR-106a-5Pin laryngeal cancer tissues(0.62±0.08) was lower than that in adjacent tissues(1.53±0.24),and the expression of E2F3(2.65±0.86) was higher than that in adjacent tissues (1.48±0.74).MiR-106a-5p specifically bound to E2F3.Down-regulation of miR-106a-5p promoted the proliferation and EMT of Hep-2 cells,and inhibited the killing activity of NK cells to Hep-2 cells.The down-regulation of E2F3 expression inhibited the proliferation and EMT of Hep-2 cells,and up-regulated the killing activity of NK cell to Hep-2 cells.Down-regulation of miR-106a-5p activated the β-catenin pathway via E2F3.XAV939 interfered with β-catenin pathway and significantly reversed the effect of down-regulated miR-106a-5p on Hep-2 cell development and NK cell killing activity.CONCLUSION MiR-106a-5p regulates the development of laryngeal carcinoma cells vie activation of the β-catenin pathway by targeting the E2F3.