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目的:探讨NDRG2在热疗诱导的热应激抗肝癌细胞侵袭中所发挥的作用和机制研究。方法:构建NDRG2过表达和干涉表达的HepG-2细胞稳转细胞株,通过Transwell和Western-blot方法检测了和细胞侵袭力和细胞内NDRG2、MMP-2和MMP-9的表达量变化;构建荷瘤鼠模型,通过HE染色及免疫组化方法检测并对比了热对肿瘤细胞向周围肌肉组织的侵袭抑制作用。结果:给予NDRG2过表达的HepG-2细胞45℃、30min热处理后,细胞内NDRG2的表达明显增高,同时伴随细胞侵袭力、MMP-2和MMP-9的表达明显降低(P<0.05)。与对照组相比,45℃的局部热作用能有效抑制肿瘤细胞对周围肌肉组织的侵袭,而干涉细胞内NDRG2的表达则降低了热对肿瘤细胞侵袭的抑制。对其机制的研究中发现,给予对照和NDRG2过表达的HepG-2细胞45℃,30min热刺激后,HSP70在热后6h表达量开始升高,而在两个组之间没有显著差异;对照组的HepG-2细胞在给予热处理后ERK1/2的磷酸化水平降低;NDRG2的过表达不仅降低了细胞中ERK1/2的本底水平,还降低了热作用早期对ERK1/2的诱导;进一步分别应用ERK1/2,p38MAPK和JNK三个激酶的抑制剂作用于NDRG2被敲除的HepG-2细胞,经过热处理后ERK1/2抑制剂组可以明显抑制HepG-2细胞的侵袭。结论:热应激所诱导NDRG2的表达量与肝癌细胞的侵袭力呈现一种负相关性;在热应激抗肝癌细胞侵袭的作用中,是通过影响NDRG2-ERK1/2通路而实现的。
Objective: To investigate the role and mechanism of NDRG2 in hyperthermia-induced hepatocellular carcinoma cell invasion. METHODS: The HepG-2 cells with NDRG2 overexpression and interference expression were stably transfected into the cell line. The changes of invasiveness and intracellular levels of NDRG2, MMP-2 and MMP-9 were detected by Transwell and Western-blot. Tumor-bearing mice model was established by HE staining and immunohistochemistry to detect and compare the heat on tumor cells to the surrounding muscle invasion inhibition. Results: The NDRG2 expression in HepG-2 cells overexpressing NDRG2 was significantly increased at 45 ℃ for 30min, while the expression of MMP-2 and MMP-9 was significantly decreased with cell invasion (P <0.05). Compared with the control group, the local thermal effect at 45 ℃ can effectively inhibit the invasion of tumor cells to the surrounding muscle tissue, while the expression of NDRG2 in the interference cells reduced the inhibition of heat on tumor cell invasion. The mechanism of the study found that the control and NDRG2 over-expression of HepG-2 cells at 45 ℃, 30min thermal stimulation, HSP70 6h after heat began to increase the expression level, but no significant difference between the two groups; control The phosphorylation level of ERK1 / 2 in HepG-2 cells decreased after heat-treatment. Overexpression of NDRG2 not only reduced the level of ERK1 / 2 in cells, but also decreased the induction of ERK1 / 2 in the early stage of heat. The HepG-2 cells knocked out by NDRG2 were treated with inhibitors of ERK1 / 2, p38MAPK and JNK, respectively. After heat-treatment, the ERK1 / 2 inhibitor group could significantly inhibit the invasion of HepG-2 cells. CONCLUSION: The expression of NDRG2 induced by heat stress has a negative correlation with the invasiveness of hepatocellular carcinoma cells. In the effect of heat stress on invasion of hepatocellular carcinoma cells, the effect of NDRG2-ERK1 / 2 pathway can be achieved.