南方医科大学学报

南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (2): 193-199.doi: 10.12122/j.issn.1673-4254.2021.02.05

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长链非编码RNA UPK1A-AS1通过稳定HIF-1α的表达促进肝癌细胞的糖酵解

张冬艳, 邹雪晶, 宋 旸, 吴德华   

  • 发布日期:2021-02-05

Long non-coding RNA UPK1A-AS1 promotes glycolysis in hepatocellular carcinoma cells via stabilization of HIF-1α

  • Published:2021-02-05

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摘要：

目的 探讨长链非编码RNA UPK1A-AS1对肝癌细胞糖酵解的影响及其分子机制。方法 通过慢病毒过表达系统构建UPK1A-AS1稳定过表达组（UPK1A-AS1）和慢病毒阴性对照组肝癌细胞株，两组细胞均分别置于常氧（21% O2）及乏氧（1% O2）条件下培养24 h，获得不同氧气条件下的UPK1A-AS1过表达组和阴性对照组肝癌细胞，利用葡萄糖及乳酸试剂盒检测过表达UPK1A-AS1对肝癌细胞糖酵解的影响。利用qRT-PCR法检测糖酵解相关基因HIF1A、GLUT1、HK1、HK2及PGK1的表达，采用双荧光素酶报告实验检测过表达UPK1A-AS1对HRE活性的影响。用放线菌酮处理肝癌细胞，检测过表达UPK1A-AS1对HIF-1α蛋白质稳定性的影响。利用泛素化蛋白质免疫共沉淀法明确UPK1A-AS1对HIF-1α蛋白泛素化修饰的影响。将肝癌细胞分为对照组、UPK1A-AS1稳定过表达组（UPK1A-AS1）、HIF-1α敲除组（si-HIF-1α）、UPK1A-AS1稳定过表达联合HIF-1α敲除组（UPK1A-AS1+si-HIF-1α），检测在过表达UPK1A-AS1的基础上干扰HIF-1α的表达后，肝癌细胞葡萄糖消耗及乳酸生成，HRE活性和糖酵解相关基因HK1、HK2及PGK1的表达情况，明确UPK1A-AS1是否通过上调HIF-1α的表达促进肝癌细胞的糖酵解水平。结果 与对照组相比，不论在常氧还是乏氧的条件下，过表达UPK1A-AS1均能促进肝癌细胞葡萄糖的消耗水平，并促进肝癌细胞生成乳酸，差异具有统计学意义（P<0.05）；过表达UPK1A-AS1能明显上调糖酵解相关基因HIF1A、GLUT1、HK1、HK2及PGK1的表达，且过表达UPK1A-AS1能促进HRE的转录活性（P<0.05）；Western blot显示，过表达UPK1A-AS1能增加HIF-1α蛋白的稳定性，并显著减少HIF-1α的泛素化修饰。葡萄糖消耗及乳酸生成实验表明，敲除HIF-1α能逆转过表达UPK1A-AS1对葡萄糖消耗及乳酸生成的促进作用（P<0.05）；干扰HIF-1α能恢复过表达UPK1A-AS1对HRE活性的上调作用。结论 长链非编码RNA UPK1A-AS1通过稳定HIF-1α的表达上调糖酵解相关基因的表达，进而促进肝癌细胞的糖酵解水平。

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Abstract:

Objective To investigate the effect of long non-coding RNA UPK1A-AS1 on glycolysis of hepatocellular carcinoma cells and its underlying molecular mechanisms. Methods A hepatocellular carcinoma (HCC) cell line with lentivirus-mediated stable UPK1A-AS1 overexpression and the cells infected with a negative control lentiviral vector were cultured under normoxic (21% O2) or hypoxic (1% O2) conditions for 24 h. The effect of UPK1A-AS1 overexpression on glycolysis of the HCC cells was examined. The expressions of glycolysis-related genes HIF1A, GLUT1, HK1, HK2 and PGK1 were detected by qRT-PCR, and the effect of UPK1A-AS1 overexpression on HRE activity was detected by dual luciferase report assay. The HCC cells were treated with cycloheximide to detect the effect of UPK1A-AS1 overexpression on the stability of HIF-1α protein. Immunoprecipitation assay was used to analyze the changes in ubiquitin modification of HIF-1α protein in response to UPK1A-AS1 overexpression. The effects of UPK1A-AS1 overexpression and RNA interference of HIF-1α expression on glucose consumption, lactate production and expressions of HRE activity and glycolysis- related genes (HK1, HK2 and PGK1) were examined in the HCC cells. Results Compared with the control group, overexpression of UPK1A-AS1 significantly promoted glucose consumption and lactate production in HCC cells under both normoxic and hypoxic conditions (P<0.05). Overexpression of UPK1A-AS1 significantly increased the expression of glycolysis-related genes including HIF1A, GLUT1, HK1, HK2 and PGK1. Upregulation of UPK1A-AS1 obviously promoted the transcriptional activity of HRE (P<0.05). Western blotting showed that UPK1A-AS1 overexpression obviously increased the stability of HIF-1α protein and significantly reduced ubiquitin modification of HIF-1α. Downregulation of HIF-1α obviously reversed the effect of UPK1A-AS1 overexpression in promoting glucose consumption, lactate production and HRE luciferase activity. Silencing HIF-1α also suppressed the upregulation of glycolysis-related gene expressions induced by UPK1A-AS1 overexpression (P<0.05). Conclusion The long non-coding RNA UPK1A-AS1 upregulates the expression of glycolysis-related genes by stabilizing the expression of HIF-1α, thereby promoting glycolysis level in HCC cells.

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