季德胜蛇药通过调控miR-335抑制肝癌Huh-7细胞增殖作用机制探讨

Mechanism discussion of JiDeSheng Sheyao inhibiting liver cancer cell Huh-7 proliferation through regulating miR-335

目的探讨季德胜蛇药对肝癌Huh-7细胞增殖作用的影响并进一步从miRNA层面探讨其抑制增殖的调控机制。方法以肝癌Huh-7细胞为模型,制备不同浓度的兔含药血清后培养Huh-7细胞,并同时设置相应浓度的对照组。CCK-8实验检测转染miR-335入Huh-7细胞后对该细胞增殖能力的影响;采用实时定量聚合酶链式反应qRT-PCR检测季德胜蛇药含药血清对Huh-7细胞中miR-335的调控作用;CCK-8实验检测季德胜蛇药含药血清对Huh-7细胞增殖能力的影响;Western-blot检测miR-335潜在靶向Bcl-w表达水平的变化。结果 miR-335过表达后CCK-8实验组与空白对照组及阴性对照组相比,其OD值显著降低(P<0.01)。含药血清干预下,与对照组相比,同等浓度的蛇药组miR-335表达水平较高,且在一定浓度范围内含药血清浓度越高,miR-335在Huh-7细胞中的表达也相继增高;此外,CCK-8实验下季德胜蛇药含药血清组都较正常血清组OD值低,且浓度越高,其OD值越低,都有较明显的统计学差异(P<0.01)。上调miR-335后,Huh-7细胞Bcl-w蛋白表达水平降低。结论季德胜蛇药通过调控miR-335能抑制肝癌Huh-7细胞增殖。

Objective To discuss the mechanism of JiDeSheng Sheyao inhibiting liver cancer cell Huh-7 proliferation through regulating miRNA. Methods Cell Huh-7 were used as the model for study . Rabbits were given Jidesheng Sheyao by gastrogavage to prepare medicated serum. Then Huh-7 was cultured with medicated serum by different concentration. At the same time, the corresponding concentrations of the control group were set up. The effect of miR-335 on Huh-7 ceils proliferation was tested by CCK-8 assay. QRT-PCR was used to detect the regulation function of miR - 335 on human liver cancer Huh-7 cell. The effect of Jidesheng Sheyao medicated serum on Huh-7 cells proliferation was tested by CCK-8 assay. The effect of miR-335 on potential target protein Bcl-w was tested by Western-blot. Results The OD value of the experimental group was decreased significantly compared with blank control group and the negative control group by CCK-8 assay after up-regulation of miR-335 （ P〈0.01 ）. The miR-335 expression level was higher compared with the control group in the same concentration of snake medicine group. In a certain concentration range, the expression of miR-335 in Huh-7 ceils increased with increasing serum concentration; In addition, the OD value of JiDeSheng Sheyao medicated serum group were lower compared with normal serum, and the concentration were higher , its OD value were lower by CCK 8 experimental. They had also a significant statistical difference （ P〈0. 01, P〈0. 001 ）. After raising miR-335, the Bcl-w protein expression levels in Huh-7 cells were lower. Conclusion JiDeSheng Sheyao can inhibite liver cancer Huh-7 cell proliferation through regulating miR-335.