| Purpose: Breast Cancer(BC)is a malignant tumor that endangers women’s health.Triple-negative Breast Cancer(TNBC)constitutes the worst prognosis subtype among breast cancers,metastasis mostly contributes the death of patients diagnosed with TNBC.Hypoxia Induced Factor-1α(HIF-1α)is an important factor which promotes the metastasis of TNBC,deemed to be the most important molecule in the process of tumorigenesis under hypoxia.HIF-1α promotes tumor metabolism,angiogenesis and metastasis by transcriptional activation of downstream genes.Many lncRNAs are involved in this process mediated by HIF-1αunder hypoxia,but the specific mechanisms have not been fully elucidated.We aimed to uncover the mechanism of lncRNA participating the HIF signaling to regulation the metastasis of TNBC.Methods: 1)Screening of hypoxia induced lncRNA according to bioinformatic analysis and prognosis of breast cancer patients.2)Transwell assay detected metastasis of triple-negative breast cancer cells under hypoxia after knocking down,overexpressing of full-length DARSAS1 and overexpression of DARS-AS1-encoded peptides.3)Ch IP-q PCR and Luciferase Reporter Assay proved DARS-AS1 is promoted by HIF-1α.4)RNA pulldown assay identified DARS-AS1 binding RBP and uncovered the mechanism of DARS-AS1 regulating triple-negative breast cancer metastasis through RNA-binding protein.5)Identification of DARS-AS1 encoding small peptide by detecting exogenous vector’s fusion tag protein.Results: 1)DARS-AS1 was highly expressed in hypoxia and related to the bad prognosis of patients with TNBC.Differentially expressed lncRNAs under hypoxia were obtained through bioinformatics analysis.To narrow the candidates,we combined HIF-1α Ch IP-seq data and clinical prognosis analysis,DARS-AS1 was the best candidates.2)DARS-AS1 promoted the metastasis of triple-negative breast cancer.Transwell assay showed significant inhibitions in migration and invasion of MDA-MB-231 and BT-549 cells after silencing DARS-AS1 with si RNA.The most significantly upregulated transcript of DARS-AS1 under hypoxia was cloned into the vector,and DARS-AS1 was overexpressed in TNBC cells by lentivirus vector.Transwell assay showed significant promotions in migration and invasion in two TNBC cells after overexpressing DARS-AS1.3)HIF-1α mediated DARS-AS1 to promote the metastasis of triple-negative breast cancer.To confirm that HIF-1αinduced upregulation of DARS-AS1.We obtained the Ch IP-Seq data of HIF-1α and found a HIF-1α binding peak on the promoter of DARS-AS1,then,a Ch IP-q PCR primer was designed according to the HIF-1α binding region,result of Ch IP-q PCR showed 135 foldchanges relative to negative control.In addition,the expression of DARS-AS1 was decreased by HIF-1α si RNA under hypoxia.Eventually,we cloned HIF-1α binding site(HRE)from the DARS-AS1 promoter into the p GL3 vector.MDA-MB-231 was transfected with p GL3 vector and treated with hypoxia,inserting the HRE motif into p GL3 vector showed a higher enrichment relative to empty vector according to luciferase reporter assay.Hence,HIF-1α binds to the DARS-AS1 promoter region to promote the transcription of DARS-AS1 under hypoxia.4)DARS-AS1 mediates the nuclear trans-location of PKM2 under hypoxia to promote the transcriptional activity of HIF-1α.Relying on RNA pulldown and mass spectrometry,we confirmed that DARS-AS1 binds to PKM2,a RIP-q PCR was used to identify that PKM2 could also binds to DARS-AS1.After silencing DARS-AS1 with si RNA,many HIF-1α-induced glycolytic genes were downregulated,while the expression of these genes was increased by overexpression of DARS-AS1,indicating that DARS-AS1 could promote the transcriptional activity of HIF-1α.PKM2 binds and promotes the transcriptional regulation of HIF-1α.Silencing DARS-AS1 by si RNA decreased the interaction of PKM2 and DARS-AS1.Cell fraction and immunofluorescence assay showed a decreased distribution of PKM2 in nucleus.The above results indicated that DARS-AS1 promoted the nuclear translocation of PKM2,increased the interaction of PKM2 to HIF-1α in the nucleus,and enhanced the transcriptional regulation of HIF-1α.5)DARS-AS1 inhibits the metastasis of triple-negative breast cancer by encoding a micropeptide.We analyzed the Ribosome Profile Sequencing data according to the online website GWIPS-viz,it was revealed that DARS-AS1 has the potential to bind to ribosome and encode protein.Bioinformatics analysis showed that there are two ORFs in DARS-AS1.The ORF fusion tag protein sequence was inserted into the PCDH vector.By detecting the exogenous tag expression,it was determined that the ORF1 of DARS-AS1 could encode a 58aa-length micropeptide.A further Transwell assay was used to detect the regulation of overexpressed micropeptide,and it was found that the migration and invasion of TNBC was inhibited,dramatically,its regulation was contradictory to that of overexpressed full length lncRNA DARS-AS1,which indicated that lncRNA DARS-AS1 may act bifunction in regulation the metastasis of TNBC,that is,the coding micropeptide and non-coding RNA respectively.Conclusion: We found the highly expressed lncRNA DARS-AS1 under hypoxia has dual regulation on the metastasis of TNBC.On the one hand,DARS-AS1 promoted the metastasis of TNBC by promoting the PKM2 nuclear translocation and enhancing the transcriptional regulation of HIF-1α;on the other hand,DARS-AS1 inhibits the metastasis of TNBC by encoding micropeptide,which acted as a "brake"-like regulation.Our study discovered a lncRNA-mediated bifunction in metastasis of triplenegative breast cancer,providing a new potential target for the diagnosis and treatment of triple-negative breast cancer. |
PDF Full Text Request