中药黄芪蛋白抗肝癌细胞HepG2作用机制

The mechanism of anti-hepatocellular carcinoma cell line HepG2 by Chinese medicine Astragalus protein

本文考察了黄芪蛋白对肝癌细胞HepG2的增殖抑制作用,结合转录组学探讨黄芪蛋白抗肿瘤作用机制。黄芪干燥根部经硫酸铵沉淀,得到分子质量大小不一的黄芪蛋白(Huang Qi protein,HQP)。通过血球计数法检测黄芪蛋白对肿瘤细胞HepG2的影响及其毒性作用;结合流式细胞术和Hoechst/propidium iodide(PI)双染测定细胞死亡情况;Western blot测定坏死标志蛋白受体相互作用的丝氨酸/苏氨酸激酶1(RIP1);将对照组与加药组RNA进行转录组测序,对RNA测序(RNA-seq)结果进行差异表达基因分析;qRT-PCR验证候选基因mRNA相对表达量。结果表明,随着HQP浓度增加,对肝癌细胞HepG2增殖抑制作用愈加明显,当HQP质量浓度为100μg·mL^-1时,细胞坏死率增加到18.78%,同时在显微镜下观察到PI单染的红色坏死细胞增多,Western blot结果显示RIP1蛋白水平增加。RNA-seq结果分析得到2.6万个相关基因受HQP调控,其中979个基因受调控较明显。KEGG分析发现部分差异表达基因与p53信号通路相关,qRT-PCR验证测序结果可靠。黄芪蛋白使HepG2细胞发生程序性坏死可能与p53信号通路有关。

To detect the inhibitory effect of Astragalus protein on the proliferation of hepatocellular carcinoma cell line HepG2,transcriptomics was used to explore the anti-tumor mechanism of Astragalus protein.The dried roots of Astragalus was precipitated by ammonium sulfate to obtain Huang Qi protein(HQP)with different molecular weights.The effect of HQP on HepG2 and its toxic effect were detected by hemocytometry.Cell necrosis was detected by flow cytometry and Hoechst/propidium iodide(PI)double staining.The necrotic marker protein receptor interacting serine/threonine kinase 1(RIP1)was determined by Western blot.Transcriptome sequencing was performed on the control group and dosing group RNA,and differential expression genes were analyzed for RNA-seq results.qRT-PCR was used to verified the relative mRNA expression levels of candidate genes.The results showed that the inhibition of HepG2 proliferation was more obvious with the increase of HQP concentration.When the concentration of HQP was 100μg·mL^-1,the necrosis rate increased to 18.78%,and the number of red necrotic cells stained with PI was observed under the microscope.The Western blot results showed an increase in RIP1 protein levels.The results of RNA-seq analysis showed that 26000 related genes were regulated by HQP,and 979 genes were more regulated.KEGG analysis found that some differentially expressed genes were associated with p53 signaling pathway,and qRT-PCR further verified that the sequencing results were reliable.HQP may cause programmed necrosis of HepG2 cells and may be involved in the p53 signaling pathway.