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

芮一奇; 邓飞; 王文文; 许华; 李晓伟; 丁永斌; 范姝琳

doi:10.3971/j.issn.1000-8578.2022.22.0057

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

摘要

摘要:
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。

Abstract:
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.

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