| Objective: To uncover the effect and mechanism of lnc ROPM on regulating phospholipid metabolism and enhancing breast cancer stem cells stemness and chemoresistance.Methods:(1)Lnc RNA microarray analysis was used to compare differentially expressed lnc RNA between MCF7/BCSCs and MCF7/non-BCSCs with a cut-off threshold of fold change > 2.0 and P <0.05.Bioinformatics analysis was employed to predict interesting potential metabolic pathways-related lnc RNAs.q RT-PCR analysis was applied to detect lnc ROPM expression in BCSCs and non-BCSCs from various breast cancer cells.The location of lnc ROPM in human chromosome and its protein-coding capability were queried from database.RNA fluorescence in situ hybridization and subcellular fractionation experiments were conducted to identify the subcellular localization of lnc ROPM in BCSCs.(2)TCGA database was utilized to validate the prognostic significance of lnc ROPM in patients with breast cancer.q RT-PCR assays were conducted to examine lnc ROPM expression in breast cancer tissues and adjacent normal tissues,as well as different pathological types tissues from patients with breast cancer.RNA fluorescence in situ hybridization was employed to detect lnc ROPM expression in representative tissues.Pearson correlation analysis was carried out to evaluate the relationship between lnc ROPM and Sox2 or Oct4 in breast cancer tissues.q RT-PCR analysis was applied to detect lnc ROPM expression in non-CSCs and CSCs from breast cancer samples.(3)Two pairs of lentivirus-mediated short hairpin RNAs(sh RNAs)were transfected into BCSCs to establish the stable lnc ROPM knockdown cell lines,and the lentivirus-carried lnc ROPM was transfected into non-BCSCs to establish the stable lnc ROPM overexpression cell lines.q RT-PCR analysis and western blot were performed to evaluate the expression of stemness-related genes(Sox2,Nanog,Oct4 and Klf4)in BCSCs with lnc ROPM knockdown or in non-BCSCs with lnc ROPM overexpression.The mammosphere forming efficiency and sphere size were counted in BCSCs with lnc ROPM deficiency or in non-BCSCs with lnc ROPM overexpression.Limiting dilution analysis was performed to determine the frequency of CSCs in BCSCs with lnc ROPM knockdown or in non-BCSCs with lnc ROPM overexpression both in vitro and in vivo.(4)Bioinformatic analysis was used to predict the potential target gene of lnc ROPM and q RT-PCR was further applied to confirm the results.q RT-PCR and western blot analysis were conducted to test PLA2G16 expression in cells with lnc ROPM knockdown or overexpression.q RT-PCR assays were performed to detect PLA2G16 expression in different pathological types tissues from patients with breast cancer,and immunohistochemical staining was used to examine PLA2G16 expression in representative tissues.Pearson correlation analysis was carried out to evaluate the relationship between PLA2G16 and lnc ROPM in breast cancer tissues.GEO database was employed to analyze PLA2G16 expression in tumor tissues from breast cancer patients.(5)q RT-PCR analysis was conducted to measure the levels of PLA2G16 pre-m RNA and mature m RNA in lnc ROPM deficient BCSCs.Sequence alignment analysis was performed to examine whether lnc ROPM directly binds to PLA2G16 m RNA.RNA pull-down assays were used to verify the interaction between lnc ROPM and PLA2G16 3’-UTR region,CDS region,or 5’-UTR region.Luciferase reporter assays were carried out to further verify the binding relationship between lnc ROPM and PLA2G16.Cells in lnc ROPM knockdown or overexpression group and their control groups were treated with actinomycin D(RNA synthesis inhibitor)in a time-dependent manner,respectively.And the levels of PLA2G16 m RNA at indicated time point were examined by q RT-PCR.(6)q RT-PCR analysis was applied to detect PLA2G16 expression in CSCs and non-CSCs from various breast cancer cells and tissues.Pearson correlation analysis was carried out to evaluate the relationship between PLA2G16 and Sox2 or Oct4 in breast cancer tissues.Two pairs of different lentivirus-mediated short hairpin RNAs(sh RNAs)were transfected into BCSCs to establish the stable PLA2G16 knockdown cell lines,and Giripladib which was an inhibitor of cytoplasmic phopholipase A2 was also used.Meanwhile,PLA2G16 was overexpressed in non-BCSCs using lentiviral vectors.Subsequently,western blot assays were conducted to verify the expression of stemness-related genes(Sox2,Oct4,Klf4)in PLA2G16 knockdown group,Giripladib treated group,PLA2G16 overexpression group,and their corresponding control group.The mammosphere forming efficiency and sphere size were counted in above groups.Then PLA2G16 was overexpressed in lnc ROPM-deficient BCSCs and silenced or inhibited in lnc ROPM-overexpressing non-BCSCs.Western blot analysis was performed to detect the expression of stemness-related genes(Sox2,Oct4,Klf4)in above groups and the mammosphere forming ability was also detected.(7)Lipidomic analysis was performed to examine changes in lipid metabolites after lnc ROPM knockdown or overexpression.Statistical analysis was applied to identify significantly differentially expressed metabolites.Lipidomic analysis was conducted to detect the content of arachidonic acid(AA)in PLA2G16 knockdown group,Giripladib treated group,PLA2G16 overexpression group,and their corresponding control group cells.At the same time,AA levels were also measured in BCSCs and non-BCSCs.Next,the effect of exogenous AA on the mammosphere forming ability,the expression levels of stemness-related genes(Sox2,Oct4,Klf4),and the key proteins of classical stemness-related pathways(Wnt/β-catenin,Notch,Hedgehog,Hippo/YAP,PI3K/AKT)was detected in lnc ROPM or PLA2G16 deficient BCSCs.(8)CCK-8 assays were performed to assess the chemosensitivity of lnc ROPM or PLA2G16 knockdown group,Giripladib treated group,lnc ROPM or PLA2G16 overexpression group,and their corresponding control group cells to chemotherapeutic drugs(dxorubicin,cisplatin,tamoxifen).q RT-PCR analysis was utilized to examine the expression of lnc ROPM,PLA2G16,Oct4,and Sox2 in BCSCs from tamoxifen-resistant MCF7 cells,cisplatin-resistant MDA-MB-231 cells,doxorubicin-resistant BT549 cells and their parental cells.The effect of exogenous AA on the chemosensitivity was detected by CCK-8 in lnc ROPM knockdown group,PLA2G16 knockdown group,Giripladib treated group,and their corresponding control group cells.Next,the mammosphere forming ability was detected in BCSCs treated with single chemotherapy agent(dxorubicin,cisplatin,or tamoxifen),Giripladib,chemotherapy agent combined with Giripladib,or DMSO(control group).And orthotopic xenografts were established in nude mice to detect the tumorigenicity of BCSCs from the four groups mentioned above.Then the volume of tumors in each group was calculated and the expression of KLF4 protein was examined by immunohistochemical staining in each group.Results:(1)The potential metabolism-related lnc ROPM was highly expressed in BCSCs compared with non-BCSCs and lnc ROPM was mainly present in the cytoplasm of BCSCs.Moreover,lnc ROPM was located at human chromosome 11q12.3-q13.1 with no protein coding ability.(2)Kaplan-Meier survival analysis revealed that the expression of lnc ROPM in patients with breast cancer was negatively correlated with 3-year disease-free survival rate and poor progression-free survival rate.And high lnc ROPM expression in ER-PR-Her2-breast cancer patients who received treatment was significantly correlated with poor prognosis.Furthermore,lnc ROPM was highly expressed in breast cancer tissues in comparison to adjacent normal tissues and associated with tumor size,TNM stage,histology grade,lymph node metastasis,tumor recurrence and chemoresistance.Pearson correlation analysis showed that lnc ROPM was positively correlated with Sox2 and Oct4 expression in breast cancer tissues.And compared with non-BCSCs,lnc ROPM was obviously increased in BCSCs derived from primary breast cancer samples,especially in high tumor grade group.(3)Efficiently knockdown of lnc ROPM in BCSCs significantly reduced stemness-related genes(Sox2,Nanog,Oct4,Klf4)expression,attenuated mammospheres forming efficiency,sphere sizes,and the frequency of CSCs.While overexpression of lnc ROPM in non-BCSCs dramatically promoted stemness-related genes(Sox2,Nanog,Oct4,Klf4)expression,enhanced mammospheres forming efficiency,sphere sizes,and the frequency of CSCs.(4)PLA2G16 was a target gene of lnc ROPM.Knockdown or overexpression of lnc ROPM decreased or increased the expression of PLA2G16 at the m RNA and protein levels.And the elevated PLA2G16 levels in breast tumor tissues were closely associated with advanced TNM stage,high histology grade,lymph node metastasis,tumor recurrence and chemoresistance.Pearson correlation analysis indicated a positive correlation between lnc ROPM and PLA2G16 expression in breast cancer samples.Besides,GSE22513 data showed that PLA2G16 was much higher in tumor tissues from breast cancer patients with a partial response to neoadjuvant paclitaxel/radiation treatment than patients with pathologic complete response to neoadjuvant treatment.(5)The levels of PLA2G16 mature m RNA(including 3’-UTR,CDS,and5’-UTR)were significantly decreased in lnc ROPM-silenced BCSCs,while the levels of PLA2G16 pre-m RNA containing three intrnic regions(intron-1,intron-2,and intron-3)were remain unchanged.Using sequence alignment analysis,we discovered that lnc ROPM might interact with the3’-UTR of PLA2G16 m RNA(energy =-17.86 kcal/mol).RNA pull-down and luciferase reporter assays showed that lnc ROPM could specifically bind to the 3’-UTR region of PLA2G16 m RNA.Knockdown or overexpression of lnc ROPM weakened or enhanced the stability of PLA2G16 m RNA.(6)PLA2G16 was highly expressed in BCSCs compared with non-CSCs.Pearson correlation analysis showed that PLA2G16 was positively correlated with Sox2 and Oct4 expression in breast cancer tissues.Knockdown of PLA2G16 or Giripladib treatment in BCSCs significantly reduced stemness-related genes(Sox2,Oct4,Klf4)expression and attenuated mammospheres forming ability.While overexpression of PLA2G16 in non-BCSCs dramatically promoted stemness-related genes(Sox2,Oct4,Klf4)expression and enhanced mammospheres forming ability.Moreover,overexpression of PLA2G16 in lnc ROPM-silenced BCSCs could partially rescue mammospheres forming ability and Sox2,Oct4,Klf4,Nanog expression caused by lose of lnc ROPM in BCSCs,while these enhanced CSCs phenotypes in non-BCSCs induced by lnc ROPM overexpression were obviously reversed by PLA2G16 knockdown or Giripladib treatment.(7)PC (Phosphatidylcholines)and PG(Phosphatidylglycerols),two of metabolic substrates of PLA2G16,were significantly increased or decreased in lnc ROPM-knocked down or overexpressing cells;whereas Cer(Ceramides)and FFA(Free fatty acids),two of representative metabolites of PLA2G16,were dramatically reduced or elevated in lnc ROPM-knocked down or overexpressing cells.Among these metabolites,FFA exhibited the highest fold change and arachidonic acid(AA)was the most significantly changed metabolite in FFA.Moreover,AA contents were significantly decreased in PLA2G16-knocked down and Giripladib treated MCF7 CSCs,while increased in PLA2G16-overexpressing MCF7 non-CSCs.And AA was markedly enriched in BCSCs compared with non-BCSCs.Furthermore,exogenous AA treatment could rescue the repressed stemness-related genes(Sox2,Oct4,Klf4)expression,attenuated mammospheres forming ability,and downregulated PI3K/AKT,Wnt/β-catenin,and Hippo/YAP signaling pathways in sh-lnc ROPM or sh-PLA2G16 BCSCs.(8)Knockdown of lnc ROPM or PLA2G16 in BCSCs could increase BCSCs sensitivity to chemotherapeutic drugs(dxorubicin,cisplatin,tamoxifen),and treating BCSCs with Giripladib had similar results.While overexpression of lnc ROPM or PLA2G16 in non-BCSCs decreased cells sensitivity to chemotherapeutic drugs.Furthermore,the enhanced lnc ROPM and PLA2G16 were detected in chemotherapy drugs resistant BCSCs,which possessed strong stemness properties,in comparison to their parental BCSCs.And exogenous AA treatment could rescue the increased sensitivity to chemotherapeutic drugs in BCSCs from sh-lnc ROPM group,sh-PLA2G16,and Giripladib treated group.Additionally,Giripladib combined with chemotherapeutic drugs(doxorubicin,cisplatin,or tamoxifen)could efficiently decrease BCSCs numbers and tumourigenicity.Conclusions:(1)The metabolism-related lnc ROPM is highlyexpressed in BCSCs,located at human chromosome 11q12.3-q13.1,and mainly present in the cytoplasm of BCSCs.(2)Lnc ROPM is highly expressed in breast cancer tissues and chemoresistant breast cancer tissues.And the expression of lnc ROPM is significantly correlated with clinicopathological features,stemness phenotypes,and poor prognosis in breast cancer patients.(3)Lnc ROPM contributes to BCSCs properties and is required for the maintenance of BCSCs properties.(4)PLA2G16 is an important target of lnc ROPM and associates with breast cancer development and chemoresistance.(5)Lnc ROPM binds to the 3’UTR of PLA2G16 m RNA to enhance PLA2G16 m RNA stability,thereby enhancing PLA2G16 levels.(6)PLA2G16 is aberrantly highly expressed in BCSCs and promotes BCSCs properties.Moreover,PLA2G16 is crucial for lnc ROPM-driven BCSCs properties.(7)Lnc ROPM-PLA2G16 signaling axis regulates phospholipid metabolism,promotes the production of arachidonic acid,thereby activating PI3K/AKT,Wnt/β-catenin and Hippo/YAP pathways and facilitating BCSCs stemness features.(8)Lnc ROPM,PLA2G16 and AA promotes BCSCs resistance to chemotherapeutic drugs and Giripladib combining with chemotherapeutic drugs can efficiently eliminate BCSCs. |
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