STK4-AS1 Delivered By Breast Cancer Derived Exosomes Promotes Tumor Proliferation Through The Glycolytic Pathway

Objective: Breast cancer is the most prevalent malignancy among women,and its incidence has been steadily rising each year.Despite substantial advancements in the development of personalized treatment strategies for molecular subtypes of breast cancer,the results of treatment often fall short of expectations.Consequently,an investigation into the gene expression patterns in breast cancer and an understanding of its pathogenesis are crucial for early diagnosis and improvement in the survival rates of breast cancer patients.Long non-coding RNAs(lncRNAs)are capable of being specifically expressed in different tissues and cells,making them a valuable resource for use as diagnostic biomarkers,prognostic factors,and specific therapeutic targets.Thus,an in-depth understanding of the regulatory mechanisms of lncRNAs in breast cancer is imperative for the development of novel therapeutic strategies.Intercellular communication between tumor cells and their surrounding and distant microenvironments is vital for tumor survival,progression,and metastasis.Exosomes are crucial mediators of cell-to-cell communication,transferring proteins,lipids,and nucleic acids(mRNA,non-coding RNA,and DNA).In recent years,research has increasingly focused on the functionalities of the various contents of exosomes,which can contribute to tumor progression by promoting tumor cell growth,invasion,metastasis,and even tumorigensis.In the present study,we analyzed the differential gene expression in exosomes derived from breast cancer cells and mammary epithelial cells,and found that tumorderived exosomes carrying the lncRNA STK4-AS1 could enhance the proliferative capacity of cancer cells and tumor growth.Furthermore,this study also demonstrated that STK4-AS1 promotes tumor proliferation by influencing the glycolytic mechanism.In conclusion,our study provides empirical evidence that STK4-AS1,a lncRNA present in breast cancer-derived exosomes,contributes to the development and progression of breast cancer by regulating glycolysis,and offers a theoretical basis for predicting a poor prognosis of breast cancer and the development of new therapeutic targets.Methods: Part Ⅰ: In this study,gene microarray analysis was conducted to identify differentially expressed genes in three pairs of primary cultured breast cancer cell exosomes and normal epithelial cell exosomes.qRT-PCR was employed to quantify the relative expression of STK4-AS1 in tissues,cells,and cell exosomes.The GenotypeTissue Expression project(GTEx),The Cancer Genome Atlas(TCGA),and Cancer Cell Line Encyclopedia(CCLE)databases were analyzed to evaluate the expression of STK4-AS1 in various tissues or cancer tissues.The TCGA Breast Cancer(BRCA)database was specifically utilized to examine the expression of STK4-AS1 in different subtypes of breast cancer and its prognostic significance.The subcellular localization and distribution of STK4-AS1 in breast cancer cell lines MCF-7 and T-47 D was investigated using a lncRNA STK4-AS1 probe with a fluorescent dye.To further explore the function of STK4-AS1,tumor-derived exosomes containing STK4-AS1 were extracted from MCF-7cells,co-incubated with MCF-7 cells,and the effects on cell proliferation were assessed through CCK8 and plate cloning assays.Additionally,the impact on tumor growth was evaluated in a nude mouse tumorigenicity model.Gene Set Enrichment Analysis(GSEA)was performed to uncover the functional pathways that STK4-AS1 may modulate.Lentiviral transfection was employed to generate STK4-AS1-differentially expressed cell models,and cellular glycolysis was assessed through glucose consumption assay,lactate production assay,and extracellular acidification rate(ECAR)assay.Part Ⅱ: The dual luciferase reporter assay was utilized to determine the binding of STK4-AS1 to the ZNF483 3’UTR Alu element.Western blot assay was performed to assess the expression of ZNF483 protein.Cellular glycolysis was further evaluated through glucose consumption assay,lactate production assay,and ECAR assay.Part Ⅲ: The expression of ZNF483 was examined in tissues and cells using qRTPCR and Western blot assays.The TCGA BRCA data was analyzed to evaluate the expression of ZNF483.Lentiviral transfection was used to generate cell models with differential expressions of ZNF483.The expression of HK2 was quantified through qRTPCR and Western blot assays in different subgroups.Chromatin immunoprecipitation(CHIP)assay was performed to confirm the binding of ZNF483 to the HK2 promoter region.Results: Part Ⅰ: A total of 817 differentially expressed genes were identified through a comparison of breast cancer cell exosomes and normal breast epithelial cell exosomes.315 of these genes were upregulated and 502 were downregulated.One of the genes that showed high expression in breast cancer cell exosomes was STK4-AS1,which was expressed approximately 1.79 times more in breast cancer cell exosomes than in breast epithelial cell exosomes.Further,STK4-AS1 expression was significantly increased in breast cancer tissues,cancer cells,and cancer cell exosomes compared to breast epithelial tissues,cells,and cellular exosomes.The GTEx,TCGA,and CCLE databases were searched for STK4-AS1 expression in all tissues,revealing that this gene was expressed in the majority of cancer tissues and cells compared to normal tissues and cells.In the TCGA BRCA dataset,STK4-AS1 was highly expressed in HR+/Her2-and Her2+ breast cancer patients,compared to the corresponding normal tissues,with statistically significant differences.However,in TNBC patients,STK4-AS1 expression was not found to be significantly different.Breast cancer patients were divided into high and low expression groups based on their STK4-AS1 expression,which was ordered from highest to lowest using the median value.Results showed that overall survival(OS)was lower in the high expression group of HR+/Her2-breast cancer patients compared to the low expression group,with a statistically significant difference.However,there were no significant differences in OS observed between the high and low expression groups in Her2+ patients or TNBC patients.An STK4-AS1 probe with fluorescent dye was used to detect the gene’s expression,which was found to be primarily in the cytoplasm of MCF-7and T-47 D cells.Laser confocal microscopy indicated that STK4-AS1-positive cellular exosomes were taken up by MCF-7 cells,resulting in an increase in STK4-AS1 expression and enhanced cell proliferation,as well as an increase in tumor size and weight in nude mice.The GSEA revealed that STK4-AS1 was enriched in the glycolytic pathway in multiple datasets.The glucose consumption assay,lactate production assay,and ECAR assay showed that the knockdown of STK4-AS1 led to reduced glycolysis,while its overexpression resulted in increased glycolysis.Part Ⅱ: A dual luciferase reporter assay was performed to investigate the interaction between the Alu element of STK4-AS1 and the Alu element in the 3’UTR of ZNF483.The results of the RNA-IP assay confirmed the binding of STAU1 to both STK4-AS1 and ZNF483 mRNAs.To further validate these findings,the expression of ZNF483 was examined using nascent RNA and RNA half-life assays.In comparison to the control group with the empty plasmid,the knockdown of STK4-AS1 did not result in any significant change in the nascent RNA of ZNF483,but did result in an increase in ZNF483 mRNA half-life.On the other hand,high expression of STK4-AS1 led to no significant change in ZNF483 nascent RNA but a decrease in ZNF483 mRNA half-life.Knockdown of either STAU1 or UPF1 similarly had no impact on the nascent RNA of ZNF483,but increased ZNF483 mRNA half-life.The results of the Western blot confirmed the impact of STK4-AS1 on ZNF483 protein expression,with increased expression observed in the STK4-AS1 knockdown group and decreased expression in the STK4-AS1 high expression group.In terms of glycolysis,the results of glucose consumption,lactate production,and ECAR assays revealed that the knockdown of STK4-AS1 resulted in reduced glycolysis,which was restored after concomitant knockdown of ZNF483.Conversely,high expression of STK4-AS1 resulted in increased glycolysis,which was restored after concomitant overexpression of ZNF483.Part Ⅲ: The results obtained from the TCGA database indicated that the expression of ZNF483 was lower in breast cancer tissues compared to normal breast tissues.This was further confirmed by qRT-PCR and Western blot analyses,which revealed lower expression of ZNF483 in both breast cancer tissues and cells in comparison to normal breast tissues and cells as well as to the null plasmid group and the negative control group.CHIP experiments showed that ZNF483 had the ability to bind to the upstream region of the HK2 transcription start site(TSS),specifically between positions 1917 bp to1908bp.Conclusion: 1.High expression of STK4-AS1 has been observed in cancer tissues,cells,and exosomes.2.Evidence suggests that breast cancer exosomes can transfer STK4-AS1 to recipient cells,thereby influencing proliferation and tumor growth.3.Studies have revealed that STK4-AS1 can increase glycolysis in cancer cells.4.The SMD pathway has been implicated in the degradation of ZNF483 mRNA by STK4-AS1.5.It is believed that the reduction in ZNF483 expression by STK4-AS1 contributes to the enhancement of cellular glycolysis.6.Compared to normal breast tissues,ZNF483 is expressed at low levels in breast cancer tissues.7.ZNF483 bind to the HK2 promoter region and regulate HK2 expression.