ZNRF3-AS1/let-7a-5p/STAT3/hnRNPA1 Loop Regulates PKM2-Mediated Aerobic Glycolysis And Proliferation Of Breast Cancer Cells

Objective:Breast cancer is a common malignant tumor that occurs mostly in women.A growing body of evidence suggests that aerobic glycolysis in tumors is associated with tumor expansion.Tumor cells metabolize more glucose to lactate in aerobic or hypoxic conditions than normal cells.PKM2 is crucial for tumor cell aerobic glycolysis.We established a role for ZNRF3-AS1 / let-7a-5p / STAT3 / hnRNP-A1 /PKM2 signaling in breast cancer cell glucose metabolism.Methods:The expression plasmids of STAT3 and hnRNP-A1 and the luciferase reporter plasmids pGL3-hnRNPA1-WT,pGL3-hnRNPA1-MUT,pGL3-ZNRF3-AS1-WT,pGL3-ZNRF3-AS1-MUT,pmirGLO-STAT3-WT,pmirGLO-STAT3-MUT,pmirGLO-ZNRF3-AS1-WT and pmirGLO-ZNRF3-AS1-MUT were constructed by molecular biology techniques.Using luciferase reporter gene activity assay and chromatin immunoprecipitation to demonstrate the binding of STAT3 to the promoters of hnRNP-A1,the luciferase reporter gene activity assay was used to demonstrate the binding of the promoters of STAT3 and ZNRF3-AS1 and the binding of let-7a-5p and the 3'UTR of STAT3.Western Blot was used to detect the expression of STAT3,hnRNP-A1,PKM2 and other genes at the protein level.Real-time quantitative fluorescent PCR was used to detect the expression of ZNRF3-AS1,let-7a-5p,STAT3,hnRNP-A1,PKM1 and PKM2 at the RNA level.Glucose and lactate assay kits were used to detect the glycolysis activity of breast cancer cells.Cell count kit-8(CCK-8)and Edu kit was used to monitor the proliferation of breast cancer cells.Results:According to the above experimental protocol,the following results were obtained: interference of PKM2 inhibited cell proliferation and aerobic glycolysis of breast cancer cells.STAT3 promotes the up-regulation of hnRNP-A1 expression,and the binding of hnRNP-A1 to PKM precursor RNA promotes the formation of PKM2.However,let-7a-5p targets the 3'UTR of STAT3 to inhibit its expression.HnRNPA1 can inhibit the maturation of let-7a-5p and promote the expression of STAT3,forming a positive feedback loop.On the other hand,STAT3 can promote ZNRF3-AS1.ZNRF3-AS1 can adsorb let-7a-5p and then release the inhibitory effect of let-7a-5p on STAT3 to form another positive feedback loop.Conclusion:These results indicate that let-7a-5p,ZNRF3-AS1,STAT3 and hnRNP-A1 form two positive feedback loops that regulate PKM2 expression to regulate glucose metabolism in breast cancer cells.These findings shed light on new ways to mediate aerobic glycolysis in breast cancer and offer potential targets for breast cancer treatment.