| Background:Breast cancer ranks first in cancer incidence and mortality among women.However,the mechanism of occurrence and progression of BC is still unknow.Exploring the mechanism of energy metabolism in the initiation and progression of BC is expected to provide scientific basis for the drug exploring,the individualized therapy and the improvement of prognosis for the patients.Long noncoding RNA(lncRNA)is involved in the regulation of gene expression in many diseases by forming complexes with DNA,RNA or proteins,acting as catalysts,molecular guides,decoys,scaffolds and other roles at the transcription,translation and epigenetic levels.However,the regulatory mechanism of lncRNA in the reprogramming of energy metabolism in BC remains to be elucidated.Methods:First,lncRNA microarray and lncRNA-seq were used to screen indicators.lncRNA HOXC-AS3,which was significantly related to BC occurrence and metastasis,was finally selected.Databases and RT-qPCR of BC tissues were used to analyze the relationship between HOXC-AS3 and the pathological parameters of patients.Nuclear and cytoplasmic separation of RNA and fluorescence in situ hybridization(FISH)experiments were used to clarify the nuclear and cytoplasmic distribution of HOXC-AS3.Actinomycin D experiment was used to detect the expression stability of HOXC-AS3.mRNA-seq was used to predict the downstream pathways and biological processes regulated by HOXC-AS3.glucose absorption assay,Lactate production assay,ECAR,OCR,CCK8,EDU,and transwell assays revealed that HOXC-AS3 enhanced the energy metabolism reprogramming,cell proliferation,migration and invasion capabilities of BC.The effect of HOXC-AS3 on tumor size,weight and infiltration ability was detected after subcutaneously inoculating BC cells overexpressed with HOXC-AS3 in nude mice.At the same time,liquid chromatograph-mass spectrometer(LC-MS)of metabolomics was used to detect the changes of energy metabolites in tumors to verify the effects of HOXC-AS3 on various biological behaviors of BC in vivo.Next,we used dual luciferase assay,RT-qPCR and chromatin immunoprecipitation(ChIP)assays determined SP1 as the transcription factor of HOXC-AS3.Subsequently,chromatin isolation by RNA purification(ChIRP),ChIP,RNA immunoprecipitation(RIP),dual luciferase assays and western blotting assays were used to verify the endogenous competitive RNA(ceRNA)regulatory mechanism of HOXC-AS3/miR-1224-5p/SP1.The protein bound with HOXC-AS3 was identified through ChIRP-mass spectrometry(ChIRP-MS),ChIRP-western blotting,FISH and RIP experiments.Western blotting,co-immunoprecipitation(CO-IP)and xenograft tumor model in mouse were used to further verify the downstream regulatory mechanism of HOXC-AS3/SIRT6 complex.We designed and synthesized oligo-anti-HOXC-AS3,which was modified by locked nucleic acid(LNA)targeting the binding sites of HOXC-AS3 with SIRT6.The miRNA pulldown,RIP and FISH assays confirmed that oligo-anti-HOXC-AS3 can bind to HOXC-AS3,and inhibit the binding of HOXC-AS3 to SIRT6 by blocking the binding sites.Glucose uptake assays,lactate production assays and xenograft tumor model in mouse experiments confirmed that oligo-anti-HOXC-AS3 could reverse the tumor-promoting effect of HOXC-AS3.Results:(1)The expression of HOXC-AS3 in BCThe expression of HOXC-AS3 was significantly upregulated in lymph node metastasis(LNM)compared with non LNM groups.HOXC-AS3 expressed higher in patients with higher tumor stage.In addition,patients with higher expression of HOXC-AS3 have a shorter overall survival.Receiver operating characteristic curve(ROC curve)analysis showed that HOXC-AS3 can be used to distinguish patients with or without LNM and tumor stage.(2)Biological characteristics of HOXC-AS3Software prediction and RNA pulldown-western blotting assays showed that HOXC-AS3 did not have coding potential.Nuclear and cytoplasmic separation of RNA and FISH assays showed that HOXC-AS3 was distributed both in the nucleus and cytoplasm.ActD chase assay showed that HOXC-AS3 was stably expressed in BC cells.(3)HOXC-AS3 regulates BC energy metabolism in vitro and in vivoHOXC-AS3 could promote the glucose absorption and the lactate accumulation of BC cells.The extracellular acidification rate(ECAR)and oxygen consumption rate(OCR)detection assays showed that HOXC-AS3 could increase the concentration of extracellular acid,while inhibiting oxygen consumption.LC-MS of metabonomics in mouse xenograft tumor model showed that HOXC-AS3 could enrich glycolysis-related metabolites,while inhibiting the accumulation of metabolites in the tricarboxylic acid cycle.(4)The transcription factor SP1 activates the transcription of HOXC-AS3SP1 bound to the-195 bp to-186 bp and-137 bp to-128 bp promoter regions of HOXC-AS3,promoting the expression of HOXC-AS3 in BC.The expression of SP1 and HOXC-AS3 were positively correlated in BC tissues.(5)SP1,HOXC-AS3 and miR1224-5p form a positive feedback loopHOXC-AS3 in the cytoplasm competitively bound to miR-1224-5p in a ceRNA manner to promote SP1 expression.Reversal experiments confirmed that SP1,HOXC-AS3 and miR1224-5p formed a positive feedback loop,which promoted the upregulation of HOXC-AS3 in BC.(6)HOXC-AS3 is bound to SIRT6The P1 region(600 nt at the 5’end of HOXC-AS3)of HOXC-AS3 binds to SIRT6.The sequences 106CTGCTGCT113 and 344TTGTGG349 of HOXC-AS3 was the binding sequences with amino acid sequence-113QNVD116 of SIRT6.(7)HOXC-AS3 antagonizes SIRT6-mediated deacetylation of histone H3 lysine 9(H3K9)in the promoter regions of PFK1,PDK4 and LDHAThe expression of SIRT6 was not affected by HOXC-AS3.However,the location of SIRT6 on chromatin was inversely proportional to the level of HOXC-AS3.HOXC-AS3 promotes the acetylation level of H3K9ac and the expression of PFK1,PDK4 and LDHA by regulating SIRT6.HOXC-AS3 inhibited the enrichment of SIRT6 in the promoter regions of PFK1,PDK4,and LDHA,and promoted the level of H3K9ac acetylation in the promoter regions of these three genes.(8)HOXC-AS3 promotes BC progression by regulating SIRT6HOXC-AS3 could promote the proliferation,migration and infiltration ability of BC cells.The tumor volume and weight of the HOXC-AS3 overexpression group were larger,and showed stronger local infiltration ability,while the tumor growth and infiltration were significantly inhibited after intratumoral injection of SIRT6.SIRT6 reversed the effects of HOXC-AS3 on lactate,glucose 6 phosphate(G6P)and citrate production and the promotion of the expression of genes PFK1,PDK4 and LDHA.(9)HOXC-AS3 is a potential therapeutic target for BCWe designed and synthesized oligo-anti-HOXC-AS3,labeled with LNA,targeting the binding sequences to block the interaction between HOXC-AS3 and SIRT6.Oligo-anti-HOXC-AS3 could reverse the promoting effect of HOXC-AS3 on glucose uptake and lactate production.Tumor burden and invasion of mice treated with the oligo-anti-HOXC-AS3 were significantly reduced.And it also reversed the regulation effect of HOXC-AS3 on energy metabolites.Conclusion:SP1,HOXC-AS3,and miR-1224-5p formed a feedback loop to upregulate the expression of HOXC-AS3.HOXC-AS3 could selectively antagonize SIRT6-mediated H3K9ac level of energy metabolism-related genes PFK1,PDK4 and LDHA,which further leads to reprogramming of energy metabolism in BC.Oligo-anti-HOXC-AS3 could specifically block the binding of HOXC-AS3 to SIRT6,effectively reversing the promoting effect of HOXC-AS3 on BC.These results revealed the important roles of HOXC-AS3 in the regulation of energy metabolism reprogramming in BC cells,revealing that HOXC-AS3 is a potential therapeutic target. |
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