RNA干扰技术下调营养缺乏自噬因子1表达对肝癌细胞侵袭转移能力的影响

Effect of down-regulation of nutrient-deprivation autophagy factor-1 expression by RNA interference on the invasion and metastasis of hepatocellular cancer cells

目的采用RNA干扰手段抑制营养缺乏自噬因子1(NAF1)在肝细胞癌MHCC-97H细胞中的表达,观察其对肝癌细胞侵袭转移能力的影响。方法采用NAF1-si RNA和空白对照NC-si RNA转染肝细胞癌MHCC-97H细胞,设为NAF组和NC组,同时设立未转染空白对照组(Blank组)。荧光实时定量PCR法检测MHCC-97H细胞NAF1m RNA表达。Western blotting法检测MHCC-97H细胞NAF蛋白和基质金属蛋白酶9(MMP9)蛋白表达。细胞划痕实验检测MHCC-97H细胞迁移能力。Transwell实验检测MHCC-97H细胞侵袭能力。荧光素酶报告基因实验检测MHCC-97H细胞核因子κB(NF-κB)信号通路的相对活性。多组间数据的比较用单因素方差分析,两组之间数据的比较采用t检验。结果 NAF组MHCC-97H细胞NAF1-m RNA的相对表达量为(6.50±0.23)﹪,低于Blank组的(100.80±0.60)﹪,且差异具有统计学意义(t=14.17,P=0.02)。NAF组MHCC-97H细胞NAF1蛋白和MMP9蛋白的相对表达量分别为0.12±0.01、0.07±0.01,均低于Blank组的0.79±0.05、0.81±0.02,且差异均有统计学意义(t=5.21、7.45,P=0.00、0.02)。NAF组MHCC-97H细胞的迁移距离明显小于Blank组。NAF组穿膜细胞的数量为(11.28±1.56)个/视野,少于Blank组的(197.87±3.18)个/视野,且差异有统计学意义(t=7.97,P=0.00),NAF组NF-κB信号通路的相对活性值3.25±0.53明显低于Blank组的26.69±7.41,差异具有统计学意义(t=13.33,P=0.01)。结论采用RNA干扰手段抑制NAF1的表达可能成为抑制肝细胞癌侵袭转移的有效手段。

Objective RNA interference was used to inhibit the expression of nutrient- deprivation autophagy factor-1（NAF1） in MHCC-97H hepatocellular carcinoma （HCC） cells, and its effect on the invasion and metastasis of HCC cells was observed. Methods MHCC- 97H cells were transfected with NAF 1-siRNA and blank control NC-siRNA. To detect the expression of NAF1 mRNA in MHCC-97H cells, real time fluorescence quantitative PCR was used. Expression of NAF1 protein and MMP9 protein of MHCC-97H cells were detected by western blotting assay. MHCC-97H cell migration and invasion ability was evaluated using cell scratch assay and transwell assay respectively. Luciferase reporter gene assay was used to evaluated the activity of nuclear factor κB （NF-κB） signaling pathway. Data among multiple groups were compared with one-way ANOVA, and data between two groups with LSD-t test. Results The relative expression of NAFI-mRNA in NAF group was （6.50± 0.23） %, as compared with blank control group （100.80 ± 0.60） % （t = 14.17, P = 0.02）. The relative expression of NAF1 protein and MMP9 protein of MHCC-97H cells, which were 0,12 ± 0.01 and 0.07 ± 0.01 respectively in the NAF group, were lower than those of the blank group （0.79 ± 0.05 and 0.81 ± 0.02; t = 5.21 and 7.45; P = 0.00 and 0.02, respectively）. Cell migration in the NAF group was significantly inhibited as compared with the blank group. The number of penetrating cells in the NAF group （ 11.28 ± 1.56） / visual field was significantly less than that in the blank group （ 197.87± 3.18） / visual field, （t = 7.97, P = 0.00）. The relative activity value of NF-ld3 signaling pathway of the NAF group was 3.25 ± 0.53, significantly lower than that of the blank group （26.69 ± 7.41, t = 13.33, P = 0.01）. Conclusion RNA interference to inhibit the expression of NAF 1 may be an effective means of suppressing liver cancer cell invasion and metastasis.