买麻藤下调LINC00673表达对肝癌细胞增殖、迁移和细胞周期的影响

Effects of Gnetum montanum down-regulating LINC00673 on proliferation,metastasis and cell cycle of hepatoma cells

目的探讨买麻藤是否通过下调LINC00673表达影响肝癌细胞增殖、迁移和细胞周期。方法取肝癌细胞Huh-7,分别使用低、中、高剂量(20、40、60 ng/mL)的买麻藤处理;或在Huh-7细胞中转染LINC00673过表达质粒pcDNA-LINC00673、过表达空载质粒pcDNA,并使用高剂量的买麻藤处理。采用MTT法测算细胞存活率,克隆形成实验检测细胞克隆形成数,流式细胞术检测细胞周期,实时荧光定量PCR检测LINC00673,Transwell检测细胞迁移,Western blotting法检测细胞中细胞核相关抗原Ki-67(Ki-67)、E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)。结果低、中、高剂量的买麻藤降低Huh-7细胞的存活率、克隆形成数、S期细胞比例、迁移细胞数及LINC00673、Ki-67蛋白、N-cadherin蛋白表达,提高G0+G1期细胞比例、E-cadherin蛋白表达(P均<0.05),且买麻藤浓度越高,作用效果越显著(P均<0.05)。LINC00673过表达后,买麻藤高剂量作用的Huh-7细胞的存活率、克隆形成数、迁移细胞数、S期细胞比例及LINC00673、Ki-67、N-cadherin表达增加,G0+G1期细胞比例与E-cadherin表达减少(P均<0.05)。结论买麻藤通过下调LINC00673的表达,抑制肝癌细胞的增殖、迁移,并诱导细胞周期阻滞。

Objective To investigate whether Gnetum montanum can affect the proliferation,migration and cell cy⁃cle of hepatocellular carcinoma cells by down-regulating long intergenic non-coding RNA 00673(LINC00673).Meth⁃ods Hepatocellular carcinoma cells Huh-7 were treated with low-,medium-,and high-dose Gnetum montanum(20,40,and 60 ng/mL),respectively.Huh-7 cells were transfected with PCDNA-LINC00673 or pcDNA,followed by highdose Gnetum montanum treatment.Cell viability,clonal formation number,and cell cycle were detected by MTT assay,clonal formation assay and flow cytometry,respectively.The real-time fluorescent quantitative PCR was applied to analyze the expression of LINC00673,Transwell method to detect the cell migration,and Western blotting to detect the expression levels of nuclear-associated antigens(Ki-67),E-cadherin,and N-cadherin.Results The low-,medium-and high-dose Gnetum montanum significantly decreased the survival rate,clonal formation,proportion of cells in the S phase,and the number of migrating cells of Huh-7 cells,and down-regulated the expression of LINC00673,Ki-67 and N-cadherin,and increased the proportion of cells in the G0-G1 phase,as well as up-regulated the expression of E-cadherin protein(all P<0.05);the higher the Gnetum montanum concentration,the more significant the effect became(P<0.05).After over-ex⁃pression of LINC00673,the survival rate,clonal formation,S-phase cell proportion,migrating cells,and expression of LINC00673,Ki67 and N-cadherin increased in the Huh-7 cells treated with the high-dose Gnetum montanum,while the proportion of cells in the G0-G1 phase and E-cadherin expression decreased(all P<0.05).Conclusion Gnetum monta⁃num can down-regulate the expression of LINC00673,inhibit the proliferation and migration of liver cancer cells,and in⁃duce cell cycle arrest.