The Molecular Mechanism Of LncRNA KB-1980E6.3 In Facilitating The Stemness Of Breast Cancer Stem Cells

Objective: To investigate the molecular mechanism of lncRNA KB-1980E6.3 in facilitating the stemness of breast cancer stem cells.Methods:(1)RNA-seq analysis was used to detect the Hypoxia-responsive lncRNAs(HRLs)in breast cancer cells,the microarray data were verified by q RT-PCR,and the candidate HRL was selected.q RT-PCR was used to detect the expression of lncRNA KB-1980E6.3 in breast cancer cells cultured under normoxia and hypoxia conditions.q RT-PCR was used to detect the expression of lncRNA KB-1980E6.3 in breast cancer cells cultured under hypoxia conditions at designed time points.Subcellular fractionation assay was used to detect the localization of lncRNA KB-1980E6.3 in breast cancer cells cultured under hypoxia conditions.The expression of lncRNA KB-1980E6.3 in breast cancer tissues and adjacent non-cancerous tissues was detected by q RT-PCR.The expression of lncRNA KB-1980E6.3 in breast tumors with different clinical stages were measured by q RT-PCR.(2)shRNA was used to knockdown HIF-1? and HIF-2? in BT549 and Hs578 T cells,q RT-PCR and Western Blot was used to verify the knockdown efficiency.q RT-PCR was used to detect the lncRNA KB-1980E6.3 expression in HIF-1? or HIF-2? knockdown BT549 and Hs578 T cells under normoxia or hypoxia conditions.Luciferase reporter assay and Chromatin Immunoprecipitation(Ch IP)assay were conducted to evaluate HIF's involvement in the regulation of lncRNA KB-1980E6.3expression.(3)shRNA was used to knockdown lncRNA KB-1980E6.3 in BT549 and Hs578T cells,lncRNA KB-1980E6.3 was overexpressed using lentivirus packaged plasmids in BT549 and Hs578 T cells,q RT-PCR used to verify the knockdown and overexpression efficiency.KEGG pathway analysis was used to detect the signaling pathways that related to lncRNA KB-1980E6.3.Mammosphere formation efficiency was determined by mammosphere assay in lncRNA KB-1980E6.3 knockout and overexpression cell models.Colony formations were measured in lncRNA KB-1980E6.3 knockout and overexpression cell models.The effect of lncRNA KB-1980E6.3 on breast cancer stem cells(BCSCs)tumorigenesis was detected by using ectopic tumorigenesis in nude mice.(4)qRT-PCR and Western Blot was used to detect the expression of Nanog,OCT4,KLF4,SOX2 and c-Myc in lncRNA KB-1980E6.3knockout and overexpression cell models.The expression of c-Myc in breast cancer tissues and adjacent non-cancerous tissues was detected by q RT-PCR.The expression of c-Myc in breast tumors with different clinical stages were measured by q RT-PCR.The correlation between c-Myc mRNA and lncRNA KB-1980E6.3 expression levels in breast cancer patients based on the data from TCGA database.Immunohistochemistry(IHC)was used to detect the expression of c-Myc in breast cancer tissues.The correlation between lncRNA KB-1980E6.3RNA level and c-Myc protein IHC scores was analyzed by correlation analysis.q RT-PCR was used to determine c-Myc mRNA levels in lncRNA KB-1980E6.3 knockout and overexpression cells that treated with Actinomycin D for the indicated time.(5)The probability of the interaction between lncRNA KB-1980E6.3and insulin-like growth factor II mRNA-binding proteins 1(IGF2BP1)was predicted by RPISeq.RNA Binding Protein Immunoprecipitation(RIP)assay was performed using cell lysates from normoxic or hypoxic breast cancer cells using anti-IGF2BP1 antibody,the lncRNA KB-1980E6.3 and c-Myc enrichment in the RIP precipitates was analyzed by q RT-PCR.Lysates from hypoxic breast cancer cells were subjected toRNA-pulldown with biotin labeled full-length or truncated lncRNA KB-1980E6.3 probe,followed by Western Blot with anti-IGF2BP1 antibody.RIP and q RT-PCR was used to identify the lncRNA KB-1980E6.3 binding domain in IGF2BP1 using full length or truncated IGF2BP1.q RT-PCR was used to determine c-Myc mRNA levels in "sh KB-1980E6.3+oe IGF2BP1" cell models that treated with Actinomycin D for the indicated time.q RT-PCR and Western Blot was used to detect the expression of c-Myc in "sh KB-1980E6.3+oe IGF2BP1" cell models.q RT-PCR and Western Blot was used to detect the expression of IGF2BP1 in lncRNA KB-1980E6.3knockout cell models.Using a biotin-labeled antisense DNA probe specifically against lncRNA KB-1980E6.3 in pulldown assay,q RT-PCR and Western Blot to verify the relationship among lncRNA KB-1980E6.3/IGF2BP1/ c-myc coding region instability determinant(CRD)mRNA complexes.(6)The effect of lncRNA KB-1980E6.3/IGF2BP1/c-Myc axis on mammosphere formation efficiency was determined by mammosphere assay.The effect of lncRNA KB-1980E6.3/IGF2BP1/c-Myc axis on colony formation was determined by colony formation assay.The effect of lncRNA KB-1980E6.3/IGF2BP1/c-Myc axis on BCSCs tumorigenesis was detected by using ectopic tumorigenesis in nude mice.Results:(1)LncRNA KB-1980E6.3 was the most increased lncRNA under hypoxia conditions when compared with normoxia.The expression of lncRNA KB-1980E6.3 was hypoxia-dependence in breast cancer cells.LncRNA KB-1980E6.3 was mainly located in the cytoplasm of breast cancer cells cultured under hypoxia conditions.The expression of lncRNA KB-1980E6.3 in clinical breast tumor tissues was higher than that in adjacent normal tissues.The expression of lncRNA KB-1980E6.3 in breast cancer patients with advanced clinical stages was higher than that in patients with early clinical stages.(2)Hypoxic lncRNA KB-1980E6.3 was significantly decreased in HIF-1? knockdown breast cancer cells rather than in HIF-2? silenced cells under hypoxia conditions.HIF-1? could bind to the Hypoxia response element(HRE)site of lncRNA KB-1980E6.3 promoter,and activate the transcription of lncRNA k B-1980E6.3.(3)Hypoxia could notably stimulate spheroid formation of breast cancer cells in suspended culture and loss of lncRNA KB-1980E6.3significantly abrogated the spheroid formation abilities of breast cancer cells.Ectopic lncRNA KB-1980E6.3 overexpressing breast cancer cells acquired strong spheroid formation potentials.Clone survival of breast cancer cells was strikingly increased under hypoxia conditions,while the loss of lncRNA KB-1980E6.3 reduced clone survival.Ectopic lncRNA KB-1980E6.3 notably increased clone survival of breast cancer cells.LncRNA KB-1980E6.3 is essential for BCSCs tumorigenesis in vivo.(4)LncRNA KB-1980E6.3 knockdown decreased the expression levels of cancer stem cells(CSCs)-associated genes,ectopic lncRNA KB-1980E6.3 increase the expression levels of CSCs-associated genes,and the effect of lncRNA KB-1980E6.3 on c-Myc was more apparent.The expression of c-Myc in clinical breast tumor tissues was higher than that in adjacent normal tissues.The expression of c-Myc in breast cancer patients with advanced clinical stages was higher than that in patients with early clinical stages.There was a positive correlation between lncRNA KB-1980E6.3 and c-Myc in clinical breast cancer tissues.Knockdown of lncRNA KB-1980E6.3 resulted in a reduced half-life of c-Myc mRNA.Ectopic lncRNA KB-1980E6.3 increase the half-life of c-Myc mRNA.(5)LncRNA KB-1980E6.3 and c-Myc mRNA was clearly enriched in IGF2BP1 immunoprecipitates.IGF2BP1 was co-precipitated with synthesized sense lncRNA KB-1980E6.3 rather than antisense lncRNA KB-1980E6.3.The 201–400 nt of lncRNA KB-1980E6.3 was required for its interaction with IGF2BP1.The KH1/2 domain of IGF2BP1 was required for association with lncRNA KB-1980E6.3.The mRNA stabilities,mRNA and protein of c-Myc caused by lncRNAKB-1980E6.3 knockdown were partially restored in "sh KB-1980E6.3+oe IGF2BP1" cell models.Knockdown of lncRNA KB-1980E6.3 did not affect the expression of IGF2BP1.The recruited IGF2BP1 by lncRNA KB-1980E6.3 could bind significantly more with c-Myc CRD WT than c-Myc CRD MUT in breast cancer cells cultured under hypoxia conditions.Knockdown of lncRNA KB-1980E6.3 or IGF2BP1 markedly reduced the recognition between IGF2BP1 and c-Myc CRD mRNA in breast cancer cells cultured under hypoxia conditions.Significantly stable c-Myc mRNA was detected in breast cancer cells with ectopic WT c-Myc CRD rather than mutant c-Myc CRD under hypoxia conditions.Knockdown of lncRNA KB-1980E6.3 or IGF2BP1 dramatically decreased the stability of c-Myc mRNA in breast cancer cells cultured under hypoxia conditions.(6)LncRNA KB-1980E6.3 or IGF2BP1 knockdown decreased the spheroid formation capacity and cell colony formation,and could be partially rescued by ectopic c-Myc expression.LncRNA KB-1980E6.3 or IGF2BP1 knockdown attenuated tumor growth of xenograft mice,and could be effectively rescued by ectopic c-Myc expression.LncRNA KB-1980E6.3/IGF2BP1/c-myc axis is essential for BCSCs tumorigenesis in vivo.Conclusions:(1)LncRNA KB-1980E6.3 is a hypoxia-responsive lncRNA and upregulated in breast cancer tissues.(2)LncRNA KB-1980E6.3 is upregulated by HIF-1?.(3)LncRNA KB-1980E6.3 promotes stemness maintenance of BCSCs.(4)LncRNA KB-1980E6.3 enhances the expression levels c-Myc by increasing c-Myc mRNA stability.(5)LncRNA KB-1980E6.3 increases c-Myc mRNAs stability via binding with IGF2BP1.(6)LncRNA KB-1980E6.3/IGF2BP1/c-myc axis is essential for BCSCs stemness and tumorigenesis in vivo.