| Objective: To investigate the effect and molecular mechanism of LncRNA408 on facilitating breast cancer stem cells(BCSCs)enrichment and stemness maintenance.Methods:(1)the expression of stemness-related lncRNA after EMT transformation was analyzed by bioinformatics analysis,the microarray data were verified by qRT-PCR,and the stemness characteristic(CD44+/CD24-)population in BT549 and Hs578 T cells were sorted by flow cytometry.Western blotting was used to detect the expression of stemness markers(SOX2,KLF4,c-Myc)in CSCs and non-CSCs of BT549 and Hs578 T cells,and qRT-PCR was used to detect the expression of lncRNA408 in CSCs and non-CSCs.The expression of lncRNA408 in non-CSC sphere and serial passaged CSC sphere was detected by qRT-PCR.The expression of lncRNA408 in breast tumor and paracancerous tissues was detected by qRT-PCR,the expression of lncRNA408 in different grades of clinical breast cancer samples was detected by qRT-PCR.(2)shRNA was used to knockdown lncRNA408 in BT549 and Hs578 T cells,and qRT-PCR was used to verify the knockdown efficiency,the mammosphere formation efficiency(MFE)and mammosphere size were counted,the expression of SOX2,KLF4 and c-Myc in knockdown models of CSCs were detected by qRT-PCR,Western blot and immunofluorescence,respectively.lncRNA408 was overexpressed using lentivirus packaged plasmids in BT549 and Hs578 T cells,qRT-PCR was used to verify the overexpression efficiency.The population of stemness characteristic cells in overexpressed cells was detected by flow cytometry,the mammosphere formation efficiency and mammosphere size were detected,qRT-PCR and Western blot were used to detect the expression of SOX2,KLF4 and c-Myc in CSCs.(3)The localization of lncRNA408 on chromosome was analyzed by bioinformatics,CBY1 was confirmed to be its adjacent target gene,the protein coding potential of lncRNA408 was predicted,and the subcellular localization was detected by in situ hybridization.qRT-PCR and Western blot were used to detect the expression of CBY1 in lncRNA408 knockout and overexpression models,respectively.Kaplan-meier survival analysis was used to analyze the relationship between CBY1 expression and the prognosis of breast cancer.(4)shRNA was used to knock down the expression of CBY1 in BT549 and Hs578 T cells,the knockdown efficiency was detected by qRT-PCR and Western blot.The mammosphere formation efficiency and mammosphere size were detected,the expression of SOX2,KLF4 and c-Myc in CSC were determined using qRT-PCR and Western blot,respectively.Knockdown the CBY1 expression in the lncRNA408 knockdown models simultaneously,the mammosphere formation efficiency and mammosphere size were detected,the expression of KLF4 and c-Myc in "shLnc408+shCBY1" CSC were detected by qRT-PCR and Western blot,respectively.(5)The protein interaction network of CBY1 was analyzed by bioinformatics,the interaction among CBY1,14-3-3 and ?-catenin proteins was detected by immunoprecipitation in CBY1 knockdown models,and the CBY1,14-3-3 and ?-catenin interaction was performed after CBY1 was mutated.Western blot and immunofluorescence were used to detect the subcellular localization of ?-catenin in CBY1 knockdown and mutant cell models,respectively.CBY1 knockdown cells were treated with PRI-724(?-catenin inhibitor),the mammosphere formation efficiency and mammosphere size were detected,and the expression of KLF4 and c-Myc were detected by qRT-PCR and Western blot,respectively.(6)The effect of LncRNA408/CBY1 axis on BCSC tumorigenesis was determined using ectopic tumorigenesis in nude mice,the volume and weight of tumors in each group was measured,and the expression of c-Myc in each group was detected by immunohistochemistry.Results:(1)LncRNA408 was highly expressed in EMT transformed breast cancer cells.The expression of lncRNA408 in CSCs(CD44+/CD24-)was higher than that in non-CSC(CD44+/CD24+,CD44-/CD24+,CD44-/CD24-),and the expression of lncRNA408 increased gradually with the CSCs were serial passaged.The expression of lncRNA408 in clinical breast cancer samples was higher than that in paracancerous tissues,and lncRNA408 expression was gradually increased with the progression of breast cancer grade.(2)The mammosphere formation efficiency and mammosphere size of BCSCs were decreased after lncRNA408 knockdown and the expression of stemness markers SOX2,KLF4 and c-Myc decreased significantly;After lncRNA408 overexpressed,the subpopulation of stemness characteristic cells were increased and the mammosphere formation efficiency and mammosphere size of BCSCs were enhanced,the expression of stemness markers SOX2,KLF4 and c-Myc were significantly increased.(3)Bioinformatics analysis determines that lncRNA408 is an antisense lncRNA located on chromosome 22,without the ability to encode protein,the nearest coding gene is CBY1;in situ hybridization shows that lncRNA408 was located in the nucleus.The mRNA and protein expression of CBY1 were increased after lncRNA408 knockdown,whereas the mRNA and protein expression of CBY1 attenuated after lncRNA408 was overexpressed.Kaplan-meier survival analysis showed that CBY1 was a good prognostic factor.(4)The mammosphere formation efficiency and mammosphere size of BCSCs were significantly increased after CBY1 knockdown,and the expression of stemness markers SOX2,KLF4 and c-Myc were apparently increased.However,there was no significant change of mammosphere formation efficiency and mammosphere size in BCSCs of "shLnc408+shCBY1" models,so was KLF4 and c-Myc expression.(5)The protein interaction network of CBY1 displayed that CBY1 mainly bound to 14-3-3 and ?-catenin proteins.No matter CBY1 knockdown or mutant,it did not interact with 14-3-3 or ?-catenin,and ?-catenin transferred from cytoplasm to nucleus.The mammosphere formation efficiency and mammosphere size of BCSCs were significantly increased after CBY1 knockdown,but it obviously decreased after ?-catenin was inhibited,so were the stemness markers.(6)LncRNA408 promoted the tumorigenesis of BCSCs and the expression of c-Myc in the tumor,knocked down of CBY1 enhanced the tumorigenesis of BCSC and the expression of c-Myc in the tumor.Conclusions:(1)lncRNA408 is highly expressed in breast cancer stem cells.(2)lncRNA408 promoted the enrichment and stemness maintenance of BCSC.(3)lncRNA408 is an antisense lncRNA located on chromosome 22 without the ability to encode protein.It is located in the nucleus and could inhibit its adjacent gene CBY1 mRNA and protein expression.(4)CBY1 per se repress the enrichment and stemness maintenance of BCSCs,and lncRNA408 promoted the enrichment and stemness maintenance of BCSCs through inhibiting the expression of CBY1.(5)CBY1 binds to 14-3-3 and ?-catenin protein in the nucleus,then pumps ?-catenin out of the nucleus and inactivates the WNT signaling pathway.(6)lncRNA408/CBY1 axis promoted the tumorigenesis of BCSC in vivo. |
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