Circular RNA LPAR3通过miR-143-5p/USP14通路调控食管癌细胞糖酵解的研究  

Circular RNA LPAR3 Regulates Glycolysis in Esophageal Cancer Cells through miR-143-5p/USP14 Pathway

作　　者：查建栋 徐志渊 陈文琦 ZHA Jiandong;XU Zhiyuan;CHEN Wenqi(Shenzhen Hospital,University of Hong Kong,Shenzhen,Guangdong,518053,China)

机构地区：[1]香港大学深圳医院,广东省深圳市518053

出　　处：《医学分子生物学杂志》2023年第4期332-338,共7页Journal of Medical Molecular Biology

基　　金：深圳市医学重点学科建设经费(No.SZXK014)。

摘　　要：目的探究调控食管癌细胞糖酵解的机制。方法逆转录实时定量PCR(quantitative real-time,qRT-PCR)法分别检测环状RNA溶血磷脂酸受体(circular RNA lysophosphatidic acid receptor 3,CircLPAR3),微小RNA-143-5(microRNA-143-5p,miR-143-5p)和泛素特异性蛋白酶14(ubiquitin-specific protease 14,USP14)的表达水平;使用Seahorse XF 24细胞外通量分析仪测量细胞外酸化率(extracellular acidification rate,ECAR)和耗氧率(oxygen comsumpition rate,OCR);Western印迹法检测糖酵解途径关键酶HK2的表达水平;使用Starbase数据库预测CircLPAR3与miR-143-5p以及miR-143-5p与USP14的靶向结合位点,双荧光素酶试验验证CircLPAR3和miR-143-5p以及miR-143-5p与USP14的靶向关系。结果与正常人食管上皮细胞(normal human esophageal epithelial cells,Het-1A)组比较,食管癌细胞系中CircLPAR3高表达,且KYSE450细胞系的表达最高,同时miR-143-5p低表达而USP14高表达。与Het-1A组比较,KYSE450组HK2的蛋白表达水平增加,同时细胞ECAR增加,整体糖酵解OCR减少;敲低CircLPAR3的表达导致细胞HK2的蛋白表达水平减少,ECAR减少,整体糖酵解OCR增加;在抑制CircLPAR3的表达情况下,抑制miR-143-5p的表达能够部分逆转细胞的糖酵解途径;双荧光素酶实验验证了CircLPAR3和miR-143-5p的靶向关系,以及miR-143-5p和USP14的靶向关系。结论CircLPAR3能够通过调控miR-143-5p/USP14通路调节食管癌细胞糖酵解途径。Objective Esophageal cancer is a serious threat to human health,and the metabolic pattern of tumor cells is dominated by glycolysis.This study analyzed the mechanisms regulating glycolysis in esophageal cancer cells.Methods The expression levels of Circular RNA lysophosphatidic acid receptor 3(CircLPAR3),microRNA-143-5p(miR-143-5p)and ubiquitin-specific protease 14(USP14)were measured using quantitative real-time(qRT-PCR).The extracellular acidification rate(ECAR)and oxygen consumption rate(OCR)were measured using Seahorse XF 24 Extracellular Flux Analyzer.The expression levels of HK2(a key enzyme of glycolytic pathway)was measured by Western blotting.The target binding site of CircLPAR3 to miR-143-5p and that of miR-143-5p to USP14 were all predicted by the Starbase database and verified through dual luciferase gene reporter assay.Results CircLPAR3 was highly expressed in the esophageal cancer cell lines,especially in the KYSE450 cell lines.At the same time,KYSE450 cells had lower miR-143-5p expression level and higher USP14 expression level.Compared to the Het-1A group,the KYSE450 group showed increased protein expression level of HK2,along with an increased ECAR and a reduced overall glycolytic OCR.Knockdown of CircLPAR3 expression resulted in reduced protein expression levels of cellular HK2,reduced ECAR and increased overall glycolytic OCR.Inhibition of miR-143-5p expression was able to partially reverse the suppression of cellular glycolysis induced by the inhibition of CircLPAR3 expression.The dual luciferase gene reporter assay verified the targeting relationship between CircLPAR3 and miR-143-5p,as well as the targeting relationship between miR-143-5p and USP14.Conclusion Circular RNA LPAR3 regulates the glycolytic pathway in esophageal cancer cells by regulating the miR-143-5p/USP14 pathway.

关 键 词：环状RNA溶血磷脂酸受体 微小RNA-143-5 泛素特异性蛋白酶14 食管癌 糖酵解 

分 类 号：R730.3[医药卫生—肿瘤]