Mir-301a-5p Affects The Biological Performance And Glycolysis Mechanism Of Triple-Negative Breast Cancer Through The PTEN/AKT/mTOR Signaling Pathway

Backgroud:Breast Cancer(BC)is the leading cause of Cancer and Cancer-related death among women worldwide[1].However,Triple-negtive breast cancer has the worst prognosis in the classification of breast cancer because of its early onset age,predisposition to early local recurrence and distant metastasis,and strong invasiveness[52].Early diagnosis and treatment are very important to improve the quality of life and reduce the related mortality.Therefore,it is urgent to explore new strategies and techniques for early screening,diagnosis and treatment of TNBC.Objective:1.To analyze the expression and clinical significance of miR-301a-5p in TNBC.2.To investigate the molecular mechanism by which MiR-301 a-5p affects glycolysis in triple-negative breast cancer cells through PTEN/AKT/mTOR signaling pathway.3.To investigate the expression of CD24 in TNBC and the potential of potential targeting molecular vectors.Methods:1.Download miRNA transcriptome data and clinicopathological data of breast cancer patients from the Cancer Genome Atlas(TCGA)database,analyze differentially expressed miRNA genes by R language,and analyze miR-301a-5p expression with clinicopathological characteristics,prognosis.2.Mononuclear cells from peripheral blood of patients with clinically diagnosed breast cancer were collected,and the expression levels of miR-301a-5p in peripheral mononuclear cells of TNBC patients and their controls were detected by real-time PCR.3.Transient transfection technique was used to inhibit and increase the expression of miR-301a-5p,RT-PCR technique was used to detect whether the expression was successful,and CCK-8 and cell cloning experiments were used to verify the effect of miR-301a-5p on the proliferation of triple-negative breast cancer cells4.Transient transfection technique inhibited and increased the expression of miR-301a-5p,glucose uptake was detected by glucose assay kit;lactate production was detected by using lactate assay kit.5.Western-blot assay was performed to verify the expression levels of PTEN gene and PI3K/AKT/mTOR genes and glycolysis-related genes(PKM2,LDHA).6.The expression level of CD24 on the surface of breast cancer cells was detected by immunohistochemistry and flow cytometry,and its effect as a targeting molecule was assessed by CD24 aptamer.Results:1.The expression of miR-301a-5p was found to be significantly elevated in breast cancer by TCGA analysis(P<0.05).In breast cancer subjects the area under the ROC curve(AUC)was 0.776,indicating that miR-301a-5p could be a breast cancer diagnostic marker.High miR-301a-5p expression was also found to be associated with poor prognosis in breast cancer patients(P<0.05).The expression of miR-301a-5p was significantly correlated with prognostic survival analysis,tumor size,age,molecular typing,menopause,but not with pathological stage and lymph node metastasis.The expression level of miR-301a-5p was higher in the peripheral blood of triple-negative breast cancer patients than in normal controls.2.The expression of miR-301a-5p in PBMCs of TNBC patients and their controls was detected by real-time PCR,and the expression level of miR-301a-5p in PBMCs of TNBC was found to be significantly increased compared with their controls,and the difference was statistically significant(P<0.01).3.Real-time PCR assay experiments showed that the expression levels of miR-301 a-5p were significantly different after transient transfection of miR-301a-5p(P<0.05),suggesting that transient transfection was successfully constructed.The proliferation and colony formation ability of MDA-MB-231 and MDA-MB-468 cells were significantly enhanced after overexpression of miR-301a-5p(mimic);the proliferation and colony formation ability of MDA-MB-231 and MDA-MB-468 cells were significantly inhibited by inhibition of expression of miR-301a-5p(inhibitor).4.After transfection with miR-3 01a-5p mimic,glucose uptake and lactate production were significantly increased in MDA-MB-231 and MDA-MB-468 cells.Transfection with miR-301a-5p inhibitor,glucose uptake and lactate production were significantly lower in MDA-MB-231 and MDA-MB-468 cells.5.After transfection with miR-301a-5p mimic,the expression of p-AKT p-STAT3 p-mTOR,PKM2 and LDHA was increased in MDA-MB-231 and MDA-MB-468 compared with the mimic-NC group.Transfection with miR-301a-5p inhibitor,the expression of p-AKT、p-STAT3、p-mTOR,PKM2 and LDHA was decreased in MDA-MB-231 and MDA-MB-468 compared with mimic-NC group.Among them,the expression of AKT,STAT3 and mTOR protein did not change significantly.6.The protein expression levels of p-AKT、p-STAT3、p-mTOR、PKM2 and LDHA in the miR-301a-5p mimic group after NVP-BEZ235 treatment were lower than those in the mimic-NC group.However,miR-301a-5p inhibitor p-AKT、p-STAT3、p-mTOR、PKM2 and LDHA protein expression was higher than inhibitor-NC group.7.The expression level of CD24 in triple-negative breast cancer was significantly higher than that in tissue.As demonstrated by CD24 aptamer binding,the CD24 molecule can act as a brand-new targeting molecule.Conclusions:MiR-301a-5p is highly expressed in triple-negative breast cancer and is associated with survival rate and clinicopathological characteristics,and miR-301a-5p can regulate the PI3K/AKT/mTOR signaling pathway through the PTEN gene,participate in the glycolytic process of triple-negative breast cancer,and affect the biological function of triple-negative breast cancer cells.CD24 may serve as a potential therapeutic target and drug delivery system.