| [Objectives]Global Cancer Statistics 2018 has recently published by the International Agency for Research on Cancer(IARC),which shows that the incidence and mortality of lung cancer have become the first in the world,accounting for 11.6%of the total cases and 18.4%of the total deaths,respectively.Lung adenocarcinoma(LAC)is the main subtype of lung cancer,invasion and metastasis are the main reasons for its poor prognosis.Cholesterol metabolism has been implicated in tumor proliferation and metastasis.Because of the complexity of cholesterol metabolism,its role is also controversial.The inconsistentfunctions of cholesterol metabolites may explain the uncertainty in the association between high cholesterol and poor prognosis in lung adenocarcinoma.Therefore,it is necessary to investigate the role of cholesterol in the proliferation and metastasis of lung adenocarcinoma.27-HC is the most common circulating oxidized cholesterol.It has been reported that 27-HC links hypercholesterolemia and breast cancer proliferation and metastasis.Previous studies have confirmed that 27-HC promoted osteoclast formation in the microenvironment of lung adenocarcinoma,but it is still unclear whether 27-HC is involved in lung adenocarcinoma metastasis or whether 27-HC links high cholesterol and lung adenocarcinoma metastasis.[Methods and Results]1、27-hydroxycholesterol promoted the proliferation,migration and invasion of lung adenocarcinoma cells(1)The CCK8 assay demonstrated that 27-HC promoted the proliferation of LAC at a dose-dependent manner.(2)The scratch assay confirmed that 1μM of 27-HC accelerated the cell migration,especially in a coculture system with THP-1-derived macrophage.(3)In addition,1 μM of 27-HC also elevated the invasion abilities of LAC.2、27-HC was required for cholesterol-induced proliferation,migration and invasion of LAC(1)Knockdown of CYP27A1 and CYP7B1 were transfected into lung adenocarcinoma cells with targeting shRNA by lentivirus.(2)27-HC significantly promoted the proliferation of lung adenocarcinoma cells,and cholesterol stimulation partly promoted the proliferation of lung adenocarcinoma cells,which was inhibited by CYP27A1 knockdown.CYP7B1 knockdown did not affect 27-HC effect,but enhanced cholesterol effect at 120 h,and had not obvious effect before 96 h,which might be due to the inhibition of proliferation of lung adenocarcinoma cells by CYP7B1 knockdown alone.(3)In the co-culture system,27-HC and cholesterol obviously promoted the proliferation of lung adenocarcinoma cells.CYP27A1 knockdown significantly inhibited the effects of cholesterol and 27-HC,while CYP7B1 knockdown significantly enhanced the effects of cholesterol and 27-HC.(4)In the monoculture system,both cholesterol and 27-HC promoted the migration and invasion of lung adenocarcinoma cells,which was significantly enhanced in the co-culture system.CYP27A1 knockdown inhibited the migration and invasion of lung adenocarcinoma cells,while CYP7B1 knockdown accelerated the migration and invasion of lung adenocarcinoma cells.3、27-HC was required for cholesterol-induced release of FGF-2 and IL-6.(1)The cytokines stimulated by cholesterol and 27-HCwere determined using the Milliplex Map Multiple cytokines detection kit.The results showed that both cholesterol and 27-HC induced the release of FGF-2 and IL-6,especially in coculture system.(2)CYP27A1 knockdown significantly blocked the effect of cholesterol,but did not affect the effect of 27-HC.(3)CYP7B1 knockdown notably enhanced the effects of cholesterol and 27-HC.4、27-HC was key for cholesterol-induced PPIB expression(1)iTRAQ analysis revealed thatPPIB expression was increased in both monoculture and coculture system exposed to 27-HC,and 27-HC moderately induced PPIB expression in monoculture system,which was significantly enhanced in coculture system.(2)CYP7B1 knockdown intensified 27-HC-induced PPIB expression in both monoculture and coculture system.Cholesterol treatment did not significantly alter PPIB expression in monoculture system,but remarkably elevated PPIB expression in coculture system.CYP27A1 knockdown almost blocked cholesterol-induce PPIB expression in both monoculture and coculture system.(3)In coculture system,both cholesterol and 27-HC promoted LXR expression and phosphorylation of AKT andNFKB p65(pAKT and pNFκB p65),which were attenuated by CYP27A1 knockdown and enhanced by CYP7B1 knockdown,respectively.5、27-HC induced PPIB expression by activatingNFκB(1)27-HC induced the expression of LXR,PPIB,Snail and vimentin,and activated AKT and NFκB p65.LXR knockdown partly reduced 27-HC-induced expression of Snail and vimentin,but did not significantly affect the expression of PPIB,pAKT and pNFκB p65.(2)FGF2 stimulated the expression of PPIB and pNFκB p65,moderately promoted the expression of Snail and Vimentin,but did not increase the expression of LXR and pAKT.(3)IL-6 exposure elevated the expression of pAKT,pNFκB p65,PPIB,Snail and Vimentin,but did not affect LXR expression.(4)SN50 was used to inhibit the activation of NFκB p65.The results showed that SN50 pretreatment simultaneously reduced the expression of LXR,PPIB,pAKT,pNFrcB p65,Snail and Vimentin,despite the presence of 27-HC.6、High cholesterol diet promoted LAC metastasis in vivo(1)In vivo imaging,high cholesterol diet significantly increased LAC metastasis in vivo compared to normal diet,which was attenuated by CYP27A1 knockdown and enhanced by CYP7B1 knockdown.(2)HE stains demonstrated that high cholesterol diet mainly promoted colonization of tumor cells in lung tissue,which was restrained by CYP27A1 knockdown and strengthened by CYP7B1 knockdown.(3)High cholesterol diet elevated PPIB expression,which was inhibited by CYP27A1 knockdown and reinforced by CYP7B1 knockdown.(4)The oxycholesterol levels in mice serum were determined using LC-MS method.The results showed that high cholesterol diet elevated serum 27-HC level in CYP7B1 knockdown group.Furthemore,high cholesterol diet increased the levels of serum 4p-hydroxycholesterol(4β-HC)and 25-hydroxycholesterol(25-HC)in control and CYP7B1 knockdown groups.Conversely,high cholesterol diet reduced serum 4-cholesten-3-one level in control and CYP27A1 knockdown groups7、CYP27A1 deficiency reduced high cholesterol diet-induced LAC metastasis in vivo(1)We created CYP27A1-deficient mice(CYP27Ar-/-),after intravenous injection of LAC via tail vein,CYP27A+/+and CYP27A-/-mice were divided into two group,and fed with normal diet and high cholesterol diet,respectively.The results showed that high cholesterol diet increased lung metastasis of LAC,which was significantly blocked by CYP27AI deficiency.(2)HE stains showed that CYP27A1 deficiency reduced lung nodules induced by high cholesterol diet.(3)Immunohistochemistry analysis revealed that high cholesterol diet increased expression of PPIB,LXR,IL-6 and FGF2,and phosphorylation of NFκB p65 and AKT,which conformably inhibited by CYP27A1 deficiency.(4)Serological analysis showed that high cholesterol diet elevated serum 27-HC level,which was remarkably blocked by CYP27A1 deficiency.[Conclusion]PPIB played a key role in cholesterol-induced and 27-HC-induced signal transduction as well as cells migration and invasion.In addition,27-HC stimulated the secretion of FGF2 and IL-6,which also contributed to activate NFκB and increased the expression of PPIB,snail and Vimentin.On the one hand,27-HC promoted the expression of snail and Vimentin by activating LXR,facilitating migration and invasion of lung adenocarcinoma cells;on the other hand,27-HC induced the expression of PPIB,snail and Vimentin by activating NFκB and increasing secretion of FGF2 and IL-6,accelerating cells migration and invasion.[Objectives]Lung cancer is one of the most common tumors in the world,accounting for about 11.6%of cancer cases and 18.4%of cancer-related deaths worldwide.Lung cancer contains many types,and is mainly divided into small cell lung cancer and non-small cell lung cancer(NSCLC).Despite significant advances in diagnosis and treatment,the 5-year survival rate for NSCLC remains relatively low.Recently,development in high-throughput sequencing technology have shown that most eukaryotic genomes are widely transcribed.Only about 2%of genes with protein-coding activity has been found,and the remaining 98%are non-coding RNAs,among which long non-coding RNAs(lncRNAs)accounted for the largest proportion and had important functions.Multiple studies have found that IncRNA molecules are involved in a variety of biological processes.In this study,the expression,function and mechanism of LINC01089 in NSCLC were analyzed,which explored whether LINC01089 is a potential target for the treatment of NSCLC,with a view to improve the overall survival of patients.[Methods and Results]1、Collect clinical tissue samples.A total of 60 pairs of NSCLC tumorand adjacent normal tissues were collected from patients at the Department of thoracic surgery,Cancer Hospital,Chinese Academy of Medical Sciences and Peking Union Medical College,between November 2018 and January 2019.LINC01089 expression was determined by RT-qPCR.Results showed that LINC01089 expression in NSCLC was significantly lower than that in adjacent normal tissues.Furthermore,Kaplan-Meier curves and Log-rank test,based on the GEPIA database,revealed that lower LINC01089 expression NSCLC was positively associated with a poor overall survival(OS)(Hazard Ratio(HR)=0.68,P=0.0067).Besides,RT-qPCR revealed that the expression of LINC01089 was downregulated in NSCLC cell lines(A549,H1299,H460,PC9).2.LINC01089 inhibits the development and progression of NSCLC cells(1)A549 and H1299 cells were transfected with shRNAtargeting LINC01089,whereas PC9 and H460 cells were transfected with pLV-LINC01089 plasmid.CCK-8 assay revealed that inhibition of LINC01089 significantly promoted A549 and H1299 cells proliferation.(2)The colony formation assay verified that LINC01089 knockdown,using shLINC01089 transfection,significantly increased the colony-forming efficiency of A549 cells.(3)Subcutaneous inj ection of the pLV-LINC01089-A549 cells into nude mice significantly inhibited tumors growth compared to the pLV-Empty-A549 cells.Conversely,subcutaneous injection of shLENC0 1089-A549 cells into nude mice promoted tumorigenesis.(4)Wound-healing assay revealed that knockdown of LINC0 1089 significantly increased cell migration ability of A549 cells.Reversely,overexpression of LINC0 1089 significantly restrained migration ability of PC9 cells.(5)Results from transwell assays showed that LINC0 1089 knockdown significantly increased the migratory abilities of A549 cells,whereas its overexpression dramatically inhibited the migratory activities of PC9 cells.(6)Tumor metastasis model was constructed by intravenously injected the A549 cells into nude mice.In vivo imaging data showed that LINC01089 knockdown induced more metastasis in the lungs.(7)Flow cytometry was used to determine the effect of LINC01089 on cell cycle and apoptosis in NSCLC cells.Results indicated that downregulation of LINC0 1089 promoted the transition from G1 to S phases of the cell cycle,Conversely,LINCO 1089 overexpression reduced the number of cells in the S phase and generated an unstable number of cells in the G2 phase.Flow cytometry analysis revealed no significant differences in the proportion of apoptotic cells between the experimental and control groupsafter 48 h incubation.3、LINC01089 functions as a ceRNA for miR-27a in NSCLC(1)We analyzed subcellular localization of LINC0 1089in NSCLC cells by the nucleocytoplasmic separation assay.The results showed that LINC01089 was mainly localized in the cytoplasm.(2)We predicted the potential targets of LINC01089 using StarBase V3.0.The results suggested that LINC0 1089 contained complementary sequences binding miR-27a.(3)To further verify the direct binding between miR-27a and LINC01089 endogenously,we performed RNA immunoprecipitation(RIP)experiments in the A549 cells.The results revealed significant enrichment of LINC01089 immunoprecipitated by anti-Ago2 antibody in miR-27a overexpressing cells.Results from a biotin-labeled system,revealed significantly high levels of LINC01089 in the pull-down product isolated from A549 and H1299 cells transfected with biotin-labeled miR-27a.(4)We further performed a dual-luciferase reporter assay by co-transfecting LINC01089-wt or LINC01089-mut containing target sequences and miR-27a in PC9 cells.The results revealed a significantly lower luciferase activity in LINC01089-wt group compared to the LINC01089-mut group.(5)LINC01089 expression was negatively correlated with miR-27a expression in clinical samples.(6)RT-qPCR analysis indicated that knockdown of LINC01089 in A549 and H1299 cells promoted the expression of miR-27a mRNA.4、miR-27a promoted EMT of NSCLC via the SFRP1-Wnt/β-catenin pathway(1)RT-qPCR revealed significantly higher level of miR-27a in NSCLC than that in adjacent normal tissues.(2)A549 cells were transfected with miR-27a inhibitor or mimic.CCK-8 assay revealed that miR-27a overexpression significantly promoted cells proliferation,whereas miR-27a inhibition significantly suppressed cells proliferation.Transwell assay showed that miR-27a overexpression significantly enhanced cell migration ability,which was significantly inhibited by its knockdown.(3)Spearman’s correlation analysis showed that miR-27a expression was inversely associated with SFRP1 expression in NSCLC tissue samples.(4)We co-transfected mimics or inhibitors of miR-27a with siSFRP1 in A549 and H1299 cells.Results indicated that miR-27a overexpression increased the expression of p-GSK-3β and β-catenin,whereas miR-27a inhibition decreased the expression of p-GSK-3 β andβ-catenin.Furthermore,knockdown of SFRP1 enhanced p-GSK-3β expression,which was attenuated by miR-27a inhibitor.(5)RT-qPCR revealed that overexpression of miR-27a increased the expression of Slug,Snail,and N-cadherin,whereas decreased the expression of SFRP1 and E-cadherin.5、LINC01089 suppressed EMT in NSCLC via the miR-27a-SFRPl-Wnt/β-catenin axis(1)Throughout rescue experiments,LINC01089 knockdown significantly promoted the expression of miR-27a,and miR-27a inhibitor significantly reversed the cell proliferation and migration mediated by LINC01089 knockdown.(2)The mRNA expression of LINC01089 was positively correlated with SFRP1 expression.(3)LINC01089 knockdown mediated an increase in mRNA levels of N-cadherin,Snail,Slug,and SFRP1,and simultaneously elevated the protein levels of p-GSK-3βand β-catenin.(4)miR-27a inhibitor reversedthe mRNA expression of N-cadherin,E-cadherin,Snail,Slug,and SFRP1 mediated by LINC0 1089 knockdown.Furthermore,miR-27a inhibitor reversed the protein expression of p-GSK-3β and β-catenin mediated by LINC0 1089 knockdown.Overexpressing LINC01089 reduced mRNA levels ofN-cadherin,Snail,Slug,and SFRP1,and also decreased protein expression of p-GSK-3βand β-catenin.[Conclusion]In this study,the expression,function and mechanism of LINC0 1089 in NSCLC were analyzed.The results showed that the expression of LINC0 1089 was downregulated in NSCLC tissues and cells,and the reduced expression of LINC01089 was closely related to the prognosis of NSCLC.LINC01089 inhibited the development of NSCLC by regulating miR-27a-SFRP1-Wnt/β-catenin-EMT signaling pathway.These results identified LINC01089 as a potential biomarker in NSCLC diagnosis and therapy. |
PDF Full Text Request