摘要
目的探讨沉默半胱氨酸蛋白酶抑制剂C(Cys C)对非小细胞肺癌(NSCLC)A549细胞增殖、凋亡、侵袭等生物学行为的影响。方法体外培养人NSCLC A549细胞,并以脂质体转染法分别将Cys C特异性siRNA(si-Cys C组)、si-NC(阴性对照组)及空脂质体(Blank组)转染A549细胞,并以RT-PCR和Western blot法检测转染效率;使用CCK-8实验、流式细胞术、Transwell等方法检测沉默Cys C表达对A549细胞增殖、凋亡、侵袭等生物学行为的影响;使用组织蛋白酶L荧光定量检测试剂盒检测组织蛋白酶L活性。结果转染后,si-Cys C组的Cys C miRNA和蛋白水平均显著下调(均P<0.05);CCK-8实验显示沉默Cys C后,与Blank组和si-NC组比较,si-Cys C组在48、72h时A549细胞增殖倍数显著增加(P<0.05);流式细胞术检测结果提示,与Blank组和si-NC组比较,si-Cys C组A549细胞凋亡水平显著下降(P<0.05);Transwell实验提示与Blank组和si-NC组比较,si-Cys C组细胞侵袭数明显增加(P<0.05);与Blank组和si-NC组比较,沉默Cys C表达后si-Cys C组A549细胞组织蛋白酶L活性显著增加(P<0.05);Blank组和si-NC组上述各指标比较,差异均无统计学意义(均P>0.05)。结论脂质体转染构建稳定低表达Cys C的A549细胞模型可靠,Cys C表达下调可以诱导A549细胞组织蛋白酶L水平升高,减少A549细胞凋亡,并促进其增殖和侵袭能力。
Objective To explore the effects of silencing cysteine protease inhibitor C(Cys C) on biological behaviors(proliferation, apoptosis, invasion) of non-small cell lung cancer(NSCLC) A549 cells.Methods Human NSCLC A549 cells were cultured in vitro.A549 cells were transfected with Cys C specific siRNA(si-Cys C group),si-NC(negative control group) and empty liposome(Blank group) by liposome transfection method.The transfection efficiency was detected by RT-PCR and Western blot.The effects of silencing Cys C expression on biological behaviors(proliferation, apoptosis, invasion) of A549 cells were detected by CCK-8,flow cytometry and Transwell assay.The activity of cathepsin L was detected by cathepsin L fluorescence quantitative detection kits.Results After transfection, levels of Cys C miRNA and protein were significantly down-regulated in si-Cys C group(P<0.05).CCK-8 showed that after silencing Cys C,compared with Blank group and si-NC group, proliferation rate of A549 cells at 48 h and 72 h was significantly increased in si-Cys C group(P<0.05).The results of flow cytometry showed that compared with Blank group and si-NC group, apoptosis level of A549 cells was significantly decreased in si-Cys C group(P<0.05).Transwell assay indicated that compared with Blank group and si-NC group, number of invasion cells was significantly increased in si-Cys C group(P<0.05).Compared with Blank group and si-NC group, after silencing Cys C expression, activity of cathepsin L was significantly increased in si-Cys C group(P<0.05).There was no significant difference in any index between Blank group and si-NC group(P>0.05).Conclusion It is reliable to construct A549 cell model with stable and low expression of Cys C by liposome transfection.Down-regulation of Cys C expression can induce the increase of cathepsin L level in A549 cells, reduce apoptosis of A549 cells, and promote their proliferation and invasion abilities.
作者
段松
黄春
DUAN Song;HUANG Chun(Three Gorges Hospital Affiliated to Chongqing University,Wanzhou 404000,China)
出处
《中国煤炭工业医学杂志》
2021年第2期127-131,共5页
Chinese Journal of Coal Industry Medicine
基金
重庆市自然科学基金资助项目(编号:cstc2018jcyjAX0469)。
关键词
非小细胞肺癌
半胱氨酸蛋白酶抑制剂C
增殖
凋亡
侵袭
Non-small cell lung cancer
Cysteine protease inhibitor C
Proliferation
Apoptosis
Invasion
