| Cancer seriously harms human health.Lung cancer is the most common cancer and is the leading cause of cancer death.Despite rapid advances in lung cancer treatment,the prognosis of lung cancer patients remains less than optimal.The 5-year survival percentage of lung cancer patients is 4–17% depending on different tumor stages and regions.It is urgent to explore the mechanisms of its occurrence and development and design effective therapy strategy.Tumor is composed of multiple subpopulations of cells.Cancer stem cells(CSCs),a small population of cancer cells that retain stem cell properties,play an essential role in tumor initiation,progression and therapy.The self-renewal and differentiation of CSCs contribute to the tumor initiation.CSCs also act as tumorigenic cells in metastatic tumor tissues.As tumors growing,the survival of tumor cells is becoming more and more difficult,and the pressure of survival drives the evolution of tumor cells.The differentiation ability confers CSC a greate evolution advantage.CSCs undergoing epithelial-mesenchymal transition(EMT)get stronger migration and invation ability to metastasize from the primary site of the tumor to other organs of the body.CSCs differentiate into cells that can adapt to the new environment and eventually form metastatic tumor tissues.Drug resistance,a major problem for NSCLC treatment,is another essential property of CSCs,which resist therapy by enhancing membrane transporter activity and activating anti-apoptotic pathways.Conventional anti-cancer drugs kill most tumor cells but not CSCs,and surviving CSCs re-establish tumors.Because of the conspicuous role of CSCs in tumor initiation,metastasis,drug resistance and recurrence,inhibiting CSCs by targeting signaling pathways that regulate these cells is an effective anti-lung cancer strategy.Forkhead box(FOX)proteins,characterized by a winged helix DNA-binding domain,are important transcription factors affecting lung cancer progression.Several FOX family members,such as FOXA1,FOXM1,and FOXQ1,have been shown to regulate CSC-like propertie.Forkhead box C1(FOXC1),a member of the FOX family,acts as an oncogene in many types of cancers such as breast cancer,liver cancer,and esophageal cancer.Recently,several studies have shown that FOXC1 is closely related to stemness.FOXC1 play an essential role in regurateing the hair follicle stem cells and hematopoietic stem cells.Hedgehog and Notch signaling pathways are essential pathways in regulating CSCs.FOXC1 activates Notch signaling pathway by directly regulating Dll4 expression in endothelial cells,and enhances Hedgehog signaling pathway activity by directly binding with Gli2 in basal-like breast cancer cells.We analyzed FOXC1 expression in NSCLC based on information in the Cancer Genome Atlas(TCGA)database on UALCANC and found elevated FOXC1 expression in NSCLC tissues compared to normal lung tissues.However,few studies have reported the role of FOXC1 in the regulation of CSCs in NSCLC.Therefore,we investigate whether FOXC1 has an effect on the CSC-like properties in NSCLC.Part 1 The expression of FOXC1 in NSCLC tissues and its effect on NSCLC patients’ survival probabilityObjective: To explore the FOXC1 expression in NSCLC tissues and the relationship between FOXC1 expression and NSCLC patients’ survival probability.Methods: 1.We analyzed the FOXC1 expression data of the NSCLC patients in TCGA database on UALCANC.2.The survival analysis of NSCLC patients was performed on the Human Protein Atlas.Results: 1.FOXC1 expression was upregulated in lung adenocarcinoma(LUAD)and lung squamous cell carcinoma(LUSC)tissues compared with normal lung tissues(P<0.01,P<0.01).2.Compared with patients with low FOXC1 levels,LUAD and LUSC patients with high FOXC1 levels had lower survival probability(P<0.05,P<0.05).Conclusions: 1.FOXC1 expression is significantly upregulated in NSCLC tissues.2.FOXC1 expression is negatively correlated with NSCLC patients’ survival probabilityPart 2 The effects of FOXC1 on the cancer stem cell-like properties in NSCLCObjective: To explore the effects of FOXC1 on cancer stem cell-like properties in NSCLC.Methods: 1.Lentivirus was used to establish stable FOXC1-knockdown and FOXC1-overexpression cell lines.2.Q-PCR was used to detected the m RNA level.3.Western blot was used to detecte the protenin level.4.The percentage of CD133+ cells was analyzed on flow cytometer using CD133-PE antibody.5.The sphere formation assay was used to detect the self-renewal ability of CSCs.6.Xenograft assay was used to detcct the NSCLC cell tumorigenicity.7.Transwell was used to detcct the migration and invasion ability of NSCLC cells.8.The cell viability was measured using CCK-8.9.The proportion of apoptotic cells was measured by flow cytometry.Results: 1.FOXC1 enhanced the stemness of NSCLC cells:(1)FOXC1 knockdown inhibited the stemness of NSCLC cells.1)Compared with A549-LV-sh Control cells,the m RNA and protein levels of FOXC1 were stably downregulated in A549-LV-sh FOXC1 cells(P < 0.01,P < 0.01).2)FOXC1 knockdown decreased the percentage of CD133+ cells(P < 0.01),3)reduced the number of spheres(diameter > 100 μm)(P < 0.01),4)downregulated the m RNA levels of SOX2,Oct4,NANOG,and ABCG2(P < 0.01,P < 0.01,P < 0.01,P < 0.01)and the protein levels of SOX2,Oct4,NANOG,and ABCG2(P < 0.01,P < 0.01,P < 0.01,P < 0.01).(2)FOXC1 overexpression enhanced the stemness of NSCLC cells.1)Comp ared with NCI-H1299-LV-Control cells,the m RNA and protein levels of FOXC1 were stably upregulated in NCI-H1299-LV-FOXC1 cells(P < 0.01,P < 0.01).2)FOXC1 overexpression increased the percentage of CD133+ cells(P < 0.05),3)increased the number of spheres(diameter > 100 μm)(P < 0.01),4)upregulated the m RNA levels of SOX2,Oct4,NANOG,and ABCG2(P < 0.05,P < 0.01,P < 0.05,P < 0.01)and the protein levels of SOX2,Oct4,NANOG,and ABCG2(P < 0.01,P < 0.05,P < 0.01,P < 0.01).2.FOXC1 enhances the tumorigenicity of NSCLC cells:(1)FOXC1 knockdown inhibited the tumorigenicity of NSCLC cells.1)In the 5×103,5×104,5×105 groups,the tumor incidence rates of A549-LV-sh FOXC1 cells(1/8,6/8,7/8) were lower than these of A549-LV-sh Control cells(4/8,7/8,8/8).2)In the 5×103,5×104,5×105 groups,the tumor volume of A549-LV-sh FOXC1 cells was smaller than these of A549-LV-sh Control cells.3)In 5 × 105 group,the tumor weight of A549-LV-sh FOXC1 cells was significantly less than this of A549-LV-sh Control cells(P <0.01).In 5 × 103 and 5 × 104 groups,the tumor weights of A549-LV-sh FOXC1 cells were less than that these of A549-LV-sh Control cells,but there were no statistically significant difference(P >0.05,P > 0.05).(2)FOXC1 overexpression enhanced the tumorigenicity of NSCLC cells.1)In the 5×103,5×104,5×105 groups,the tumor incidence rates of NCI-H1299-LV-FOXC1 cells(3/8,6/8,8/8)were higher than these of NCI-H1299-LV-Control cells(1/8,4/8,7/8).2)In the 5×103,5×104,5×105 groups,the tumor volume of NCI-H1299-LV-FOXC1 cells was larger than these of NCI-H1299-LV-Control cells.3)In 5 × 104 group,the tumor weight of NCI-H1299-LV-FOXC1 cells was significantly greater than this of NCI-H1299-LV-Control cells(P <0.01).In 5 × 103 and 5 × 104 groups,the tumor weights of NCI-H1299-LV-FOXC1 cells were greater than these of NCI-H1299-LV-Control cells,but there were no statistically significant difference(P >0.05,P > 0.05).3.FOXC1 promotes the migration and invasion of NSCLC cells:(1)FOXC1 knockdown inhibited the migration and invasion of NSCLC cells.1)FOXC1 knockdown inhibited the migration ability(P<0.01)and 2)the invasion ability(P<0.01)of NSCLC cells.3)FOXC1 knockdown reduced the protein levels of Vimentin,fibronectin,N-cadherin(P<0.01,P<0.05,P<0.01)and did not affect the protein levels of E-Cadherin(P >0.05).(2)FOXC1 overexpression enhanced the migration and invasion of NSCLC cells.1)FOXC1 overexpression promoted the migration ability(P<0.01)and 2)the invasion ability(P<0.01)of NSCLC cells.3)FOXC1 overexpression increased the protein levels of Vimentin,fibronectin,N-cadherin(P<0.05,P<0.01,P<0.05),The E-Cadherin protein was not detected in NCI-H1299 cells.4.FOXC1 confers drug resistance in NSCLC cells:(1)FOXC1 knockdown enhanced the sensitivity of NSCLC cells to chemotherapy.1) FOXC1 knockdown enhanced the inhibitory effects of cisplatin and docetaxel on the viability of A549 cells,2)increased the percentage of apoptotic cells induced by cisplatin or docetaxel(P<0.01,P<0.01),3)upregulated the protein levels of cleaved caspase-3 and BIM(P<0.01,P<0.05).(2)FOXC1 overexpression inhibited the sensitivity of NSCLC cells to chemotherapy.1)FOXC1 overexpression attenuated the inhibitory effects of cisplatin and docetaxel on the viability of NCI-H1299 cells,2)decreased the percentage of apoptotic cells induced by cisplatin or docetaxel(P<0.01,P<0.01),3)downregulated the protein levels of cleaved caspase-3 and BIM(P<0.01,P<0.05).Conclusions: FOXC1 contributes to CSC-like properties in NSCLC,including enhanced stemness,self-renewal ability,migration and invation and induction of drug resistance.Part 3 The molecular mechanism underlying the promoting effects of FOXC1 on CSC-like propertiesObjective: To investigate the molecular mechanism underlying the promoting effects of FOXC1 on CSC-like properties.Methods: 1.Q-PCR was used to detect the m RNA level.2.Western Blot was used to detect the protenin level.3.Immunohistochemical staining was used to detect the protenin level in the xenograft tumors.4.Luciferase reporter assay was used to investigate the effect of FOXC1 on beta-catenin promoter activity.5.Chromatin immunoprecipitation(Ch IP)assay was used to investigate the binding of FOXC1 to beta-catenin promoter.6.Lentivirus was used to establish stable beta-catenin-knockdown and beta-catenin-overexpressing cell lines.7.The percentage of CD133+ cells was analyzed on flow cytometer using CD133-PE antibody.8.The sphere formation assay was used to detect the self-renewal ability of CSCs.9.Transwell without matrigel was used to detcct the migration ability of NSCLC cells,and transwell with matrigel was used to detect the invasion ability of NSCLC cells.10.The cell viability was measured using CCK-8.Results: 1.FOXC1 promotes beta-catenin expression by enhancing beta-catenin promoter activity:(1)FOXC1 knockdown downregulated the beta-catenin m RNA level,the total and nuclear beta-catenin protein levels of A549 cells(P < 0.01,P < 0.01,P < 0.01).(2)Immunohistochemical staining showed that FOXC1 knockdown downregulated beta-catenin protein level in the xenograft tumors.(3)FOXC1 overexpression upregulated the beta-catenin m RNA level,the total and nuclear beta-catenin protein levels of NCI-H1299 cells(P < 0.01,P < 0.05,P < 0.05).(4)Immunohistochemical staining showed that FOXC1 overexpression upregulated beta-catenin protein level in the xenograft tumors.5)Luciferase reporter assay showed that FOXC1 significantly increased the beta-catenin promoter activity(P < 0.01).6)After analyzing the sequence of the beta-catenin promoter on the TRANSFAC and JASPARJ databases,we found three putative FOXC1 binding sites(BSs)in beta-catenin promoter: BS1,GTTCGTTTGTT(-1268/-1258)beta-catenin;BS2,TCTATAAACAT(-997/-987)beta-catenin;BS3,TTATTTGTTCA(-821/-811)beta-catenin.The BS1 and BS3 mutation did not significantly affect the luciferase activity(P > 0.05),while the BS2 mutation decreased the FOXC1-induced luciferase activity(P < 0.01).7)Ch IP-PCR assays confirmed the binding of FOXC1 to BS2.2.Beta-catenin mediates FOXC1-induced CSC-like properties in NSCLC:(1)Beta-catenin overexpression reversed the inhibitory effects of FOXC1 knockdown on the CSC-like properties.1)LV-beta-catenin lentivirus was used to upregulate total and nuclear beta-catenin protein levels in A549-LV-sh FOXC1 cells(P < 0.01,P < 0.05);2)Beta-catenin overexpression reversed the inhibitory effects of FOXC1 knockdown on the stemness of NSCLC cells.Beta-catenin overexpression increased the percentage of CD133+ cells(P < 0.01)and the number of spheres(diameter > 100 μm)(P < 0.01),upregulated the protein levels of SOX2,Oct4,NANOG,and ABCG2(P < 0.05,P < 0.05,P < 0.01,P < 0.01).3)Beta-catenin overexpression reversed the inhibitory effects of FOXC1 knockdown on the migration and invasion.Beta-catenin overexpression promoted the migration and invasion of FOXC1-knockdown cells(P<0.01,P<0.05).4)Beta-catenin overexpression reversed chemo-sensitization enhanced by FOXC1 knockdown.Beta-catenin overexpression attenuated the inhibitory effects of cisplatin and docetaxel on the viability of FOXC1-knockdown cells.(2)Beta-catenin knockdown reversed the enhancement of CSC-like properties induced by FOXC1 overexpression.1)LV-shbeta-catenin lentivirus was used to downregulate the total and nuclear beta-catenin protein levels in NCI-H1299-LV-FOXC1 cells(P < 0.01,P < 0.05).2)Beta-catenin knockdown reversed the enhancement of the stemness induced by FOXC1 overexpression.Beta-catenin knockdown reduced the percentage of CD133+ cells(P < 0.01)and the number of spheres(diameter > 100 μm)(P < 0.01),downregulated the protein levels of SOX2,Oct4,NANOG,and ABCG2(P < 0.01,P < 0.01,P < 0.01,P < 0.01).3)Beta-catenin knockdown reversed the enhancement of the migration and invasion induced by FOXC1 overexpression.Beta-catenin knockdown inhibited the migration and invasion of FOXC1-overexpression cells(P<0.01,P<0.05).4)Beta-catenin knockdown reversed chemo-resistance induced by FOXC1 overexpression.Beta-catenin knockdown enhanced the inhibitory effects of cisplatin and docetaxel on the viability of FOXC1-overexpression cells.Conclusions: FOXC1 promotes beta-catenin expression by enhanceing beta-catenin promoter activity to enhance CSC-like properties in NSCLC. |
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