石上柏乙酸乙酯萃取物下调LINC02474抑制结直肠癌细胞增殖、迁移和侵袭

Effects of Selaginella doederleinii ethyl acetate extract on the proliferation,migration and invasion of colorectal cancer cells

目的研究石上柏乙酸乙酯萃取物对结直肠癌细胞(Caco-2)增殖、迁移和侵袭的影响,并探讨其机制。方法集落形成实验检测石上柏乙酸乙酯萃取物对Caco-2细胞增殖的影响。流式细胞数检测周期分布。Transwell和划痕愈合实验检测细胞侵袭和迁移。实时荧光定量PCR(RT-qPCR)检测LINC02474表达水平。蛋白质印记(Western blot)检测上皮细胞钙粘蛋白(E-cadherin)、神经钙黏素(N-cadherin)蛋白表达水平。转染LINC02474过表达质粒至Caco-2细胞,采用上述方法检测上调LINC02474对石上柏乙酸乙酯萃取物处理的Caco-2细胞增殖、迁移和侵袭的影响。结果石上柏乙酸乙酯萃取物处理后Caco-2细胞克隆形成数、S期细胞比例、侵袭细胞数、划痕愈合率、LINC02474和N-cadherin蛋白表达显著降低,G0-G1期细胞比例和E-cadherin蛋白表达显著升高(P<0.05),并呈剂量依赖效应。上调LINC02474表达后,石上柏乙酸乙酯萃取物处理的Caco-2细胞克隆形成数、S期细胞比例、侵袭细胞数、划痕愈合率、N-cadherin蛋白表达显著升高,G0-G1期细胞比例和E-cadherin蛋白表达显著降低(P<0.05)。结论石上柏乙酸乙酯萃取物抑制结直肠癌细胞的增殖、迁移和侵袭,其机制可能与下调LINC02474表达有关。

Objective To investigate the effects of Selaginella doederleinii ethyl acetate extract on the proliferation,migration and invasion of colorectal cancer cells(Caco-2),and to explore its action mechanism.Methods The colony formation experiment was used to detect the effects of Selaginella doederleinii ethyl acetate extract on the proliferation of Caco-2 cells.Flow cytometry was used to detect the cycle distribution,and Transwell and scratch healing test were used to detect cell invasion and migration.Real-time quantitative PCR(RT-qPCR)detected the expression levels of LINC02474,and Western Blot measured the expression levels of E-cadherin and N-cadherin.Moreover the LINC02474 over-expression plasmid was transfected into Caco-2 cells,and the above methods were used to detect the effects of up-regulating LINC02474 on the proliferation,migration,and invasion of Caco-2 cells treated by Selaginella doederleinii ethyl acetate extract.Results After treatment by Selaginella doederleinii ethyl acetate extract,the cell clone formation number,S-phase cell ratio,invasive cell number,scratch healing rate,LINC02474 and N-cadherin protein expression levels in Caco-2 cells were significantly decreased,however,the G0-G1 phase cell ratio and E-cadherin protein expression levels were significantly increased(P<0.05),with a dose-dependent manner.After up-regulating the expression levels of LINC02474,the number of clone formation,the proportion of S-phase cells,the number of invasive cells,the scratch healing rate,the expression levels of N-cadherin protein in Caco-2 cells were significantly increased,but,the cell proportion in G0-G1 phase and the expression levels of E-cadherin protein were significantly decreased(P<0.05).Conclusion Selaginella doederleinii ethyl acetate extract can inhibit the proliferation,migration and invasion of colorectal cancer cells,and its action mechanism may be related to the down-regulation of LINC02474 expression.