Linc00460通过海绵吸附影响miR-320a对乳腺癌细胞的有氧糖酵解作用

Effects of Linc00460 on Aerobic Glycolysis in Breast Cancer Cells via Sponge Adsorption of miR-320a

目的探讨Linc00460通过海绵吸附miR-320a对乳腺癌(BC)细胞有氧糖酵解作用的影响。方法qRT-PCR法检测正常乳腺上皮细胞系MCF-10A及5种BC细胞系中Linc00460和miR-320a表达水平。qRT-PCR检测干扰Linc00460对miR-320a表达的影响,双荧光素酶报告基因实验检测miR-320a和Linc00460的靶向关系。将si-Linc00460和miR-320a inhibitor共转染至MDA-MB-231细胞,qRT-PCR检测细胞中miR-320a表达水平;MTT法检测细胞增殖能力;2-NBDG法检测细胞葡萄糖摄取率;比色法检测细胞上清液中乳酸含量;酶活性试剂盒检测糖酵解关键酶的活性;Western blot检测酵解途径中关键蛋白表达水平。结果与MCF-10A细胞比较,5种BC细胞系中Linc00460高表达,而miR-320a低表达。干扰Linc00460后,MDA-MB-231细胞中miR-320a表达显著升高。双荧光素酶报告基因实验证实,miR-320a和Linc00460可靶向结合。干扰Linc00460表达后MDA-MB-231细胞增殖能力受到明显抑制(P=0.000),细胞葡萄糖摄取率和细胞上清液中乳酸含量降低(均P=0.000),PFK、PK和LDH酶活性受到抑制(均P=0.000),PFKM、GLUT1和LDHA蛋白表达水平下调(均P=0.000)。抑制miR-320a可明显逆转si-Linc00460对MDA-MB-231细胞增殖和糖酵解的抑制作用(均P=0.000或0.001)。结论Linc00460可能通过海绵吸附miR-320a,上调PFKM表达,从而促进BC细胞有氧糖酵解作用。

Objective To explore the effect of Linc00460 on the aerobic glycolysis of breast cancer(BC)cells through sponge adsorption of miR-320a.Methods The qRT-PCR method was used to detect Linc00460 and miR-320a expression levels in normal breast epithelial cell line MCF-10A and five BC cell lines.The effect of interfering Linc00460 on miR-320a expression was detected by qRT-PCR.The double luciferase reporter gene experiment was used to analyze the targeting relationship between miR-320a and Linc00460.In addition,the si-Linc00460 and miR-320a inhibitor were co-transfected into MDA-MB-231 cells,and the expression level of miR-320a in the cells was detected by qRT-PCR;cell proliferation ability was measured by the MTT method;glucose uptake rate was detected by 2-NBDG method;the content of lactic acid in the cell supernatant was detected by colorimetric method;the key enzymes of glycolysis was detected by the enzyme activity kit;and the expression levels of the key proteins in the glycolysis pathway were detected by Western blot.Results Linc00460 was highly expressed in five BC cell lines,while miR-320a was lowly expressed as compared with MCF-10A cells.The expression of miR-320a in MDA-MB-231 cells significantly increased after interfering with Linc00460.The double luciferase reporter gene experiment confirmed that miR-320a and Linc00460 could target binding.Interfering with the expression of Linc00460 could inhibit MDA-MB-231 cells proliferation(all P=0.000),reduce the rate of cellular glucose uptake and the content of lactic acid in the cell supernatant(all P=0.000),inhibit the activities of PFK,PK,and LDH enzymes(all P=0.000),and downregulate the protein expression levels of PFKM,GLUT1,and LDHA(all P=0.000).Meanwhile,inhibition of miR-320a could reverse the inhibitory effect of si-Linc00460 on the proliferation and glycolysis of MDA-MB-231 cells(all P=0.000 or 0.001).Conclusion Linc00460 might adsorb miR-320a,consequently leading to upregulation of PFKM expression,thereby promoting aerobic glycolysis in BC cells.