“三棱-莪术”单体成分对丙肝病毒基因组3’-UTR介导的肝细胞恶性的研究

Influence of the Active Ingredients in Rhizoma Sparganii-Radices Zedoariae on Malignant Transformation of Hepatocyte Caused by the 3’-UTR of Hepatitis C Virus

目的:探索丙型肝炎病毒(Hepatitis C Virus, HCV)基因组3’非编码区(3’ Untranslated Region, 3’UTR)对肝细胞的恶性转化作用,在此基础上初步筛查"三棱-莪术"配伍单体成分对肝细胞恶性转化的影响。方法:(1)将HCV 3’UTR的cDNA克隆到pcDNA3.1(+)质粒中,构建表达HCV 3’UTR的真核表达载体pcDNA-3’UTR;用pcDNA-3’UTR质粒转染L02细胞,构建稳定表达HCV 3’UTR的模型对照组L02-3’UTR细胞。(2)通过检测模型对照组的 Eya1和Hk2基因的表达、细胞增殖速度、软琼脂克隆形成能力、糖代谢能力,判断模型对照组是否发生恶性转化。(3)以不同浓度(0、1、10、50、100和200μg/mL)的"三棱-莪术"中药单体分别处理模型对照组L02-3’UTR,计算出药物的干预浓度;采用干预浓度的药物单体处理L02-3’UTR,检测Eya1和Hk2基因的表达、细胞增殖速度、糖代谢能力,判断药物对模型对照组恶性特征的影响。结果:(1)构建了pcDNA-3’UTR质粒,并筛选出表达HCV 3’UTR的模型对照组L02-3’UTR。(2)与空白对照组L02-NC比较,模型对照组L02-3’UTR细胞在增殖速度、软琼脂克隆形成能力、糖代谢能力及基因Eya1和Hk2表达上均显著升高(P<0.01)。(3)与模型对照组L02-3’UTR相比,三棱-莪术的单体成分(10μg/mL的姜黄素和50μg/mL的山柰酚)可明显降低模型对照细胞的增殖速度、糖代谢能力及Eya1和Hk2基因的表达(P<0.05或P<0.01)。结论:正常肝细胞中HCV 3’UTR对应的lncRNA的高表达,使细胞中肝癌相关基因Eya1和Hk2表达增高,细胞过度增殖、接触抑制消失、糖代谢紊乱等典型的恶性转化特征;中药单体姜黄素和山柰酚能有效防止这些恶性特征的发展。该项工作揭示了HCV 3’UTR导致肝细胞恶性转化的一种全新、清晰的途径,这种早期恶性转化细胞模型可用于大规模筛选中药成分用于阻止细胞恶性进展甚至逆转恶性特征,对三棱-莪术有效成份的筛选则为此类工作提供了范例。

Objective:To explore the malignant transformation of hepatocytes by the 3’-untranslated region(UTR) of hepatitis C virus(HCV), and to screen the monomer of Sparganii Rhizoma-Curcumae Rhizoma with effective regulation on the malignant transformation of hepatocytes. Methods:(1) The double-strand complementary DNAs(dscDNAs) of HCV 3’UTR was cloned into pcDNA3.1(+) vector, to construct the eukaryotic plasmid pcDNA-3’UTR expressing HCV 3’UTR. Then pcDNA-3’UTR was used to transfect L02 cells, to establish L02-3’UTR model cell which can stably express HCV 3’UTR RNA.(2) The malignant transformation state of L02-3’UTR was evaluated by detecting the expressions of Eya1 and Hk2 genes, the proliferation speed, clone formation ability in soft agar and glucose metabolism.(3) The intervention concentration of the medicines was calculated by treating L02-3’UTR cells with different concentrations(0,1,10,50,100 and 200 μg/mL) of monomer of Sparganii Rhizoma-Curcumae Rhizoma, then the influences of the medicines on cell malignant characteristics were evaluated by measuring the expressions of Eya1 and Hk2 genes, the proliferation speed, glucose metabolism in L02-3’UTR cells after treatment with the intervention concentration of the medicines. Results:(1) The pcDNA-3’UTR plasmid was constructed and the L02-3’UTR model cell expressing HCV-3’UTR was screened.(2) Compared with the control group L02-NC,The expressions of Eya1 and Hk2 in the model group were significantly up-regulated, the levels of sugar metabolism, the cell proliferation rate and soft AGAR cloning formation ability were significantly increased(P<0.01).(3) Compared with the model group L02-3’UTR,after treatment with Sparganii Rhizoma-Curcumae Rhizoma(10 μg/mL curcumin or 50 μg/mL kaempferol),the proliferation rate, the capacity of glucose metabolism and the expression levels of Eya1 and Hk2 genes were significantly decreased(P<0.05 or P<0.01). Conclusion:The high expression of the lncRNA of HCV 3’UTR in normal hepatocytes is able to induce the typical malignant transformation such as the increasing expression of Eya1 and Hk2 genes, the excessive proliferation of cells, the disappearance of contact inhibition, the disordered glucose metabolism. Monomers curcumin and kaempferol show the capability to efficiently reduce these malignant characteristics. The study revealed a novel method of HCV 3’UTR-induced the malignant transformation of liver cells. This early stage of malignant transformation of liver cells provides an ideal model for large-scale screening of effective components from traditional Chinese herbs to prevent or reverse the progress. The screening of active components from Sparganii Rhizoma-Curcumae Rhizoma herein provids an example for future work.