不良心理应激通过上调乳酸脱氧酶A水平促进胶质瘤增殖和侵袭

Psychological distress promotes proliferation and invasion of glioma by upregulating lactate deoxygenase A

目的探讨乳酸脱氢酶A(LDHA)表达在不良心理应激促进脑胶质瘤细胞增殖及侵袭中的作用。方法将胶质瘤荷瘤裸鼠分为荷瘤组、荷瘤应激组、阴性对照组、短发夹RNA(shRNA)-乳酸脱氧酶A(LDHA)组、shRNA-LDHA应激组。束缚应激4周后,测量各组裸鼠肿瘤大小;利用ELISA检测血清中去甲肾上腺素(NE)、肾上腺素(EPI)的含量;通过比色法检测瘤组织中乳酸的量;Western blot法检测各组瘤组织LDHA蛋白表达。采用CCK-8法和平板克隆形成实验检测NE对胶质瘤LN229细胞增殖的影响;TranswellTM法检测其侵袭能力;实时定量PCR检测LN229细胞LDHA mRNA表达水平;Western blot法检测LDHA、胞外信号调节激酶1/2(ERK1/2)、缺氧诱导因子1α(HIF-1α)蛋白表达;利用荧光素酶报告基因活性检测实验研究NE调节LDHA表达的分子机制。结果与荷瘤组比较,荷瘤应激组肿瘤的大小、EPI、NE、乳酸的含量及LDHA蛋白表达水平均明显增加;而沉默LDHA后,肿瘤的增殖速度、乳酸的含量显著降低;与对照组比较,NE促进LN229细胞LDHA mRNA水平,上调LDHA蛋白表达、ERK1/2和HIF-1α蛋白磷酸化水平,且细胞增殖速度、侵袭能力均增强;荧光素酶报告基因实验证实NE通过HIF-1α上调LDHA的表达。结论不良心理应激通过上调LDHA表达促进胶质瘤增殖和侵袭。

Objective To investigate the role of lactate deoxygenase A （LDHA） expression in the proliferation and invasion of gUoma promoted by the psychological distress. Methods The glioma-bearing nude mice were divided into tumor bearing group, stress tumor bearing group, negative control, short hairpin RNA （shRNA）-1actate dehydrogenase A （LDHA） group and shRNA-LDHA stress group. Four weeks after stress, the tumor size of each group was measured; meanwhile, norepinephrine （NE） andadrenaline （EPI） in the blood were detected by ELISA; lactic acid in the tumor tissue was determined by colorimetry. And the expression level of LDHA was detected by Western blotting. The proliferation of glioma LN229 cells stimulated by NE was detected by CCK-8 assay and plate clone formation assay; TranswellTM assay was used to test the invasive ability of LN229 cells. The level of LDHA mRNA was detected by real-time quantitative PCR. The expressions of LDHA, ERK1/2 and hypoxia-induced factor-1α （HIF-1α） were observed using Western blotting. By constructing the reporter gene, we studied the mechanism of LDHA expression regulated by NE. Results The tumor size and the levels of EPI, NE, lactic acid and LDHA in the stress group significantly increased as compared with the tumor bearing group. After silencing LDHA, the proliferation rate and lactate content decreased. Compared with the control group, the mRNA and protein levels of LDHA in NE group was significantly elevated; the phosphorylation levels of HIF-1α and ERK1/2 was significantly raised; and cell proliferation and invasion ability were also enhanced. Promoter luciferase reporter assay confirmed that NE up-regulated the expression of LDHA by HIF-1α. Conclusion Adverse psychological stress can promote the proliferation and invasion of glioma cells by upregulating LDHA expression.