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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 different regulation of UBE2D2 expression in rat hepatocarcinoma induced by different traditional Chinese medicine and the role of UBE2D2 in the proliferation of human hepatoma cells. Methods: DEN-induced hepatocellular carcinoma (HCC) was induced in rats. Affy-metrix rat 230A GeneChip arrays was used to detect the expression of UBE2D2 in HCC tissues. SiRNA targeting UBE2D2 The recombinant plasmids were transfected into SMMC-7721 human hepatocellular carcinoma cell lines by lipofectamine. The proliferation and proliferation of SMMC-7721 cells were detected by MTT assay. Realtime-PCR was used to detect the gene expression difference. Results: The results of the chip showed that the expression of UBE2D2 gene was significantly increased after the formation of hepatocarcinoma in rats. Different TCM treatment regulates the expression of UBE2D2 in different degree. The down-regulating effect of Jianpiqiqi method is obvious. One UBE2D2 gene was screened by MTT assay After 6 days of transfection, the cell proliferation was significantly inhibited (compared with the negative control group, P <0.01). Real-time fluorescent quantitative PCR showed that the expression of the gene was significantly increased 6 days after transfection reduce. Conclusion: The high expression of UBE2D2 gene is related to the malignant proliferation of hepatocellular carcinoma cells.