基于LDHA基因和Wnt/β-catenin信号通路探讨自噬在卵巢癌细胞侵袭和迁移及糖酵解中的作用

Role of autophagy in invasion and migration and glycolysis of ovarian cancer cells investigated based on LDHA gene and Wnt/β-catenin signaling pathway

摘要

目的 探讨自噬通过调控乳酸脱氢酶A(LDHA)和Wnt/β-连环蛋白(β-catenin)信号通路对卵巢癌细胞侵袭、迁移能力和糖酵解的影响。方法 2021年1月至2022年12月在空军军医大学西京医院使用厄尔平衡盐溶液(EBSS)饥饿处理诱导人卵巢腺癌细胞系SKOV3细胞诱导自噬.细胞实验分组1为:Control组、EBSS组和EBSS+3-MA组;细胞实验分组2为:EBSS+NC-shRNA组、EBSS+LDHA-shRNA1组和EBSS+LDHA-shRNA2组;细胞实验分组3为:EBSS+NC-shRNA组、EBSS+β-catenin-shRNA1组和EBSS+β-catenin-shRNA2组.采用实时荧光定量PCR(RT-qPCR)法、Western blotting法、Transwell实验、细胞免疫荧光染色等检测并统计分析有关表达量的情况。结果 Control组和EBSS组及EBSS+3-MA组间细胞的微管相关蛋白1轻链3(LC3)-Ⅱ/LC3-Ⅰ表达量相对比值比较,差异有统计学意义(F=113.900,P<0.01).Control组和EBSS组及EBSS+3-MA组间细胞的侵袭和迁移细胞数及基质金属蛋白酶(MMP)2和MMP9蛋白相对表达量比较,差异均有统计学意义(F值分别为2448.000、1405.000、111.000、132.200,P<0.01).Control组和EBSS组及EBSS+3-MA组间细胞的LDHA mRNA、GLUT1 mRNA、LDHA蛋白相对表达量比较,差异均有统计学意义(F值分别为311.400、66.940、154.200,P<0.01).NC-shRNA组和LDHA-shRNA1组及LDHA-shRNA2组间细胞的LDHA蛋白相对表达量比较,差异有统计学意义(F=123.200,P<0.01).Control组和EBSS组及EBSS+3-MA组间细胞的β-catenin、p-β-catenin磷酸化相对表达量比较,差异均有统计学意义(F值分别为206.500、222.200,P<0.01).NC-shRNA组和β-catenin-shRNA1组及β-catenin-shRNA2组间细胞的β-catenin相对表达量比较,差异有统计学意义(F=127.000,P<0.01)。结论 饥饿诱导的自噬激活可促进卵巢癌的侵袭、迁移能力及糖酵解活性,其机制可能与自噬激活Wnt/β-catenin信号通路并上调LDHA表达有关.

Abstract

Objective To investigate the effects of autophagy on ovarian cancer cell invasion, migration and glycolysis by regulating lactate dehydrogenase A (LDHA) and Wnt/β-catenin signaling pathways. Methods From January 2021 to December 2022, autophagy was induced by starvation treatment with Earl balanced salt solution (EBSS) in Xijing Hospital of Air Force Medical University, human ovarian adenocarcinoma cell line SKOV3 cells. Cell experiment group 1 was divided into control group, EBSS group and EBSS+3-MA group. Cell experimental group 2 was as follows: EBSS+NC-shRNA group, EBSS+LDHA-shRNA1 group and EBSS+LDHA-shRNA2 group. Group 3 of the cell experiment was EBSS+NC-shRNA group, EBSS+β-catenin-shRNA1 group, and EBSS+β-catenin-shRNA2 group. Real-time quantitative PCR (RT-qPCR), Western blotting, Transwell assay, and cell immunofluorescence staining were used to detect and statistically analyze the expression level. Results There was a significant difference in the relative ratio of LC3-Ⅱ/LC3-Ⅰ expression between control group, EBSS group and EBSS+3-MA group (F=113.900, P<0.01). There were significant differences in the number of invasive and migrating cells and the relative expression levels of MMP2 and MMP9 proteins between the Control group, the EBSS group and the EBSS+3-MA group (F values were 2448.000, 1405.000, 111.000 and 132.200, respectively, P<0.01). There were significant differences in the relative expressions of LDHA mRNA, GLUT1 mRNA and LDHA protein between the control group, the EBSS group and the EBSS+3-MA group (F values were 311.400, 66.940 and 154.200, P<0.01, respectively). There was a significant difference in the relative expression of LDHA protein between the NC-shRNA group, LDHA-shRNA1 group and LDHA-shRNA2 group (F=123.200, P<0.01). There were significant differences in the relative expressions of β-catenin and phosphorylated p-β-catenin between the control group, the EBSS group and the EBSS+3-MA group (F-values were 206.500 and 222.200, P<0.01, respectively). There was a significant difference in the relative expression of β-catenin between the NC-shRNA group, the β-catenin-shRNA1 group and the β-catenin-shRNA2 group (F=127.000, P<0.01). Conclusion Starvation-induced autophagy activation can promote the invasion, migration and glycolytic activity of ovarian cancer, which may be related to the activation of Wnt/β-catenin signaling pathway and up-regulation of LDHA expression by autophagy.

关键词

卵巢癌 / 自噬 / 乳酸脱氢酶A / 糖酵解 / β-连环蛋白

Key words

ovarian cancer / autophagy / lactate dehydrogenase A / glycolysis / β-catenin

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基金资助

国家自然科学基金(81172458)

陕西省自然科学基础研究计划重点项目(2024JC-GZDXM-G52)