| Background:Non-small cell lung cancer(NSCLC)has a high morbidity and high mortality rate,which poses a serious threat to the health and life of patients.With the development of molecular biology technology,targeted therapy has become a hot spot for NSCLC treatment.Thus,t he elucidation of the pathogenesis of NSCLC can provide potentially effective molecular targets for the treatment of NSCLC.Purpose:The function of LINC01619 in human malignant tumors has not been clearly elucidated,and there is no study of its mechanism in NSCLC.This study aimed to explore the impact of LINC01619 on NSCLC development.Furthermore,the intrinsic molecular mechanism of LINC01619 regulating the development of NSCLC has been studied in detail.Methods:This study included 63 patients with NSCLC,and their pathological characteristics were recorded in detail.Tumor tissues and adjacent normal tissues were collected during surgery.After surgery,patients were followed up for 2000 days.LINC01619 expression in tumor tissues and adjacent normal tissues of NSCLC patients were detected using quantitative real-time polymerase chain reaction(q RT-PCR)and in situ hybridization(ISH).The relationship between LINC01619 expression and the prognosis of NSCLC patients was analyzed,including tumor size,,lymph node metastasis,TNM stage and overall survival rate of 2000 days after surgery.Immunohistochemistry and Western blot were used to detect the expression of PAX6 protein in tumor tissues and adjacent normal tissues.Human lung bronchial epithelial cell line(BEAS-2B)and 7NSCLC cell lines(A549,SPCA1,H1299,H1975,H1703,SK-7MES-1,H520)were cultured.LINC01619 expression in these cell lines was researched by q RT-PCR.A549 cells were transfected by LINC01619sh RNA and its negative control.SPCA1 cells were transfected by pcDNA3.1-LIN-C01619 overexpression vector and pcDNA3.1 empty vector.Cell counting kit-8(CCK-8)assay and cell colony formation experiment were used to detect the viability and proliferation of A549 and SPCA1.Nude mouse xenograft tumor model was established by subcutaneous injecting the transfected SPCA1 and A549 cells in to the back of nude mice.The volume and weight of xenograft tumors were measured in order to detect the effect of LINC01619 on SPCA1 and A549cells growth in vivo.ALDH~+cells from SPCA1 and A549 cells were screened by flow cytometry.LINC01619 expression in ALDH~+cells were regulated by transfection.Flow cytometry,sphere formation assay,q RT-PCR and Western blot were used respectively to detect the percentage of ALDH~+cells,sphere formation,and the expression of cancer stem cell markers m RNA and protein(including SOX2,NANOG,OCT4,CD133 and CD44).Dual luciferase reporter gene assay and RNA-binding protein immunoprecipitation were used to verify the relationship between LINC01619 and miR-129-5p.The co-localization of LINC01619 and miR-129-5p in cells was confirmed by RNA fluorescence in situ hybridization.miR-129-5p expression in tumor tissues of NSCLC patients and the transfected SPCA1 and A549 cells was explored by q RT-PCR.Pearson correlation analysis was used to detect the correlation between LINC01619 and miR-129-5p expression in NSCLC tumor tissues.The relationship between miR-129-5p and PAX6 was detected dual luciferase reporter gene assay.SPCA1 cells and ALDH~+cells derived from SPCA1 cells were co-transfected by miR-129-5p mimics and pcDNA3.1-LINC01619 overexpression vector,or by pcDNA3.1-LINC01619 overexpression vector and PAX6 sh RNA.In addition,A549cells and ALDH~+cells derived from A549 cells were co-transfected by INC01619 si RNA and miR-129-5p inhibitors,or by LINC01619 si RNA and pcDNA3.1-PAX6 overexpression vector.The expression of PAX6m RNA and protein in SPCA1 and A549 cells after transfection was detected by q RT-PCR and Western blot.Pearson correlation analysis was used to study the correlation between PAX6 and LINC01619 expression or between PAX6 and miR-129-5p expression in tumor tissues.CCK-8assay and cell colony formation experiment were used respectively to detect the viability and proliferation of the co-transfected SPCA1 and A549 cells.After being co-transfected,ALDH~+cells sphere formation and cancer stem cell markers m RNA and protein(including SOX2,NANOG,OCT4,CD133 and CD44)expression was researched by sphere formation assay,q RT-PCR and Western blot.Results:The expression of LINC01619 in tumor tissues of NSCLC patients was significantly higher than that in adjacent normal tissues(P<0.0001).High LINC01619 expression indicated severely poor prognosis for NSCLC patients,including larger tumor size,lymph node metastasis,later clinical TNM stage and lower overall survival rate of 2000 days after surgery.After LINC01619 being overexpressed,the viability,malignant proliferation and growth in vivo of SPCA1 cells were significantly enhanced(P<0.01).Simultaneously,the percentage of ALDH~+cells(derived from SPCA1 cells),sphere formation and cancer stem cell markers expression(including ALDH,SOX2,NANOG,OCT4,CD133 and CD44)were all enhanced after LINC01619 expression in ALDH~+cells being overexpressed(P<0.01).In addition,after LINC01619 being silenced,the viability,malignant proliferation and growth in vivo of A549 cells were significantly inhibied(P<0.01).Meanwhile,the percentage of ALDH~+cells(derived from A549 cells),sphere formation and cancer stem cell markers expression(including ALDH,SOX2,NANOG,OCT4,CD133 and CD44)were all weakened after LINC01619 expression in ALDH~+cells being silenced(P<0.01).Dual luciferase reporter gene assay and RNA binding protein immunoprecipitation showed that miR-129-5p was a target gene of LINC01619,and its expression was directly inhibited by LINC01619.RNA fluorescence in situ hybridization experiment showed that LINC01619 and miR-129-5p were co-expressed in the cytoplasm.In the tumor tissues of NSCLC patients,the expression of miR-129-5p was obviously decreased,and miR-129-5p and LINC01619 expression showed a significant negative correlation(P<0.0001).Moreover,PAX6was a target gene of miR-129-5p,and its expression was directly inhibited by miR-129-5p.In the tumor tissues of NSCLC patients,PAX6expression was significantly up-regulated(P<0.0001).PAX6 and LINC01619 expression exhibited a significant positive correlation,while PAX6 and miR-129-5p expression presented a significant negative correlation(P<0.0001).High PAX6 expression was obviously related to the later TNM stage of NSCLC patients(P<0.05).The rescue experiment results showed that LINC01619 promoted the in vitro malignant phenotype of NSCLC cells by sponging the miR-129-5p/PAX6axis,including the enhanced cell viability,malignant proliferation,sphere formation and cancer stem cell markers expression(including ALDH,SOX2,NANOG,OCT4,CD133 and CD44)(P<0.05 or P<0.01).Conclusion:LINC01619 promoted the malignant development of NSCLC by sponging the miR-129-5p/PAX6 axis. |
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