UBE2D2基因在肝癌细胞中的作用及不同中医治法对其调控作用的影响

Function of UBE2D2 Gene in Hepatocarcinoma Cells and Effects of Different Therapies of Traditional Chinese Medicine on Expression of UBE2D2

目的:观察不同中医治法对大鼠肝癌UBE2D2表达的调控差异,以及UBE2D2在人肝癌细胞增殖中的作用。方法:采用DEN诱发大鼠肝癌,分别经中药健脾益气、清热解毒、活血化瘀等治疗后,Affy-metrix rat 230A GeneChip arrays检测肝癌组织UBE2D2表达的差异;设计2个人UBE2D2基因siRNA靶点,将重组质粒经脂质体转入SMMC-7721人肝癌细胞株,MTT法检测细胞生长增殖情况、Realtime-PCR检测该基因的表达差异。结果:芯片结果显示,UBE2D2基因在大鼠肝癌形成后表达显著增加,不同中医治法对其具有不同程度的下调作用,其中健脾理气法下调作用较明显;采用MTT法筛选到1个UBE2D2基因RNA干扰有效靶序列,转染6 d后细胞的生长增殖得到了明显的抑制(与阴性对照组相比,P<0.01);实时荧光定量PCR检测表明,转染6 d后该基因的表达明显降低。结论:UBE2D2基因的高表达与肝癌细胞恶性增殖有关,而中药健脾理气治法对其具有下调作用。

Objective： To observe the effects of different therapies of traditional Chinese medicine （TCM） on expression of UBE2D2 in rats with hepatocarcinoma and the function of UBE2D2 on proliferation of hepatocarcinoma cells. Methods： Rat hepatoceltular carcinoma （ HCC ） models were induced by diethylnitrosamine （ DEN ）, and were treated by herbs of strengthening spleen and replenishing qi, clearing away heat and removing toxin, and activating blood to resolve stagnation. After treatment, the expressions of UBE2D2 gene in hepatocarcinoma cells were determined by Affymetrix rat 230A GeneChip arrays. Two siRNA targets toward UBE2D2 gene were designed, recombinant plasmids were transfected to human hepatocellular carcinoma cell line SMMC-7721, the proliferation of cells and the expression of UBE2D2 gene were detected by MTr assay and real-time quantitative PCR method respectively. Results： According to the gene chip results, the expression of UBE2D2 increased significantly after formation of hepatocellular carcinoma, different therapies of TCM had down-regulating function especially the herbs of strengthening spleen and replenishing qi was more obvious. An effective siRNA target sequence in UBE2D2 was obtained by MTT assay, the cell growth and proliferation were inhibited obviously when interferential target effectivly was transfected for 6 days （ compared with negative control group, P 〈 0.01 ）. The results of real-time quantitative PCR method showed that the geue expression decreased after transfection of 6 days. Conclusion ： The high-expression of UBE2D2 has relation with the malignant proliferation of hepatocarcinoma cells and the herbs of strengthening spleen and replenishing qi can down-regulate the high-expression.