| 【Background】Esophageal cancer(EC)is seventh in terms of incidence and sixth in cancer deaths worldwide.In 2020,more than 600000 patients were dignosed with EC.Due to the lack of effective early diagnostic approaches and therapy methods,the five-year survival rate of EC patients is extremely low.Therefore,it is of great significance to develop specific diagnostic marker molecules for EC patients and establish individualized targeted therapy strategies for improving the level of diagnosis and treatment for EC.In recent years,lnc RNAs,as the important regulators in tumorgenesis and development,has gradually received attention from researchers.However,the function of lnc RNAs and their potential mechanisms in tumor progression have been only partially elucidated,and most of them remain unknown.In terms of ESCC,there are still a large number of related lnc RNAs that have not been discovered.Previously,we identified a total of 597 differential expressed lnc RNAs in EC by analyzing the date from TCGA database.Among them,LINC01234 is highly expressed in ESCC and is associated with the outcome of patients,but its role and mechanism in ESCC are still unclear.Studies on this molecule may contribute to further understanding of the pathogenesis of ESCC and lay a foundation for targeted therapy.【Aims】1.To detect the expression level of LINC01234 in ESCC cells and tissues,as well as to analyze the relationship between the expression level of LINC01234 and prognosis of patients.2.To explore the functions of LINC01234 in regulating the malignant phenotypes of ESCC.3.To investigate the molecular mechanism of LINC01234 in promoting the proliferation and metastasis of ESCC.【Methods】1.Bioinformatics analysis and q RT-PCR detection were used to analyze the expression pattern of LINC01234 in ESCC cells and tissues.The relationship between LINC01234 expression and prognosis of patients were also explored.2.ESCC cell models with increased or decreased LINC01234 expression were established by transfection of overexpression plasmid or sh RNA lentivirus,respectively.The effects of LINC01234 on the proliferation and colony formation of ESCC were investigated by CCK-8,Ed U and colony formation assays.Flow cytometry was used to detect the effects of LINC01234 on the cell cycle and apoptosis of ESCC cells.Transwell assay was used to observe the effects of LINC01234 on invasion and migration of ESCC cells.The effects of LINC01234 on tumorigenesis and metastasis of ESCC cells in vivo were observed by nude mice xenograft assay and tail vein metastatic assay.3.Bioinformatics analysis,q RT-PCR,luciferase reporter assay,and Ch IP experiments were used to confirm that whether transcription factor SOX2 activates transcription of LINC01234 by binding to its promoter.4.The downstream genes of LINC01234 were screened and verified by bioinformatics analysis,RNA-pulldown,mass spectrometry,RNA-sequencing,q RT-PCR,Western Blot and dual-luciferase reporter gene assays.5.In addition,we also screened some m RNAs and mi RNAs regulated by LINC01234.The underlying mechanisms of LINC01234 in regulating the proliferation and metastasis of ESCC were explored.【Results】1.Bioinformatics analysis showed that the expression of LINC01234 in ESCC tissues was higher than that in adjacent tissues.Compared with Het-1A,LINC01234 was highly expressed in ESCC cell lines.And high expression of LINC01234 predicts poor prognosis of ESCC patients.2.LINC01234 expression was successfully modulated either by stable transfection using sh RNA lentivirus infection or by transient transfection using overexpression plasmid.CCK-8,Ed U and colony formation assays showed that overexpression of LINC01234 promoted proliferation and colony formation of ESCC cells in vitro,downregulation of LINC01234 inhibited proliferation and colony formation of ESCC cells in vitro.Transwell assay showed that overexpression of LINC01234 could promote the invasion and migration of ESCC cells in vitro,whereas downregulation of LINC01234 could inhibit the invasion and migration of ESCC cells in vitro.Overexpression of LINC01234 promoted cell cycle progression as detected by the flow cytometry,whereas downregulation of LINC01234 inhibited cell cycle progression.Nude mice xenograft assay revealed that downregulation of LINC01234 could inhibit the growth of ESCC cells in vivo.Tail vein metastatic assay showed that downregulation of LINC01234 inhibited ESCC cells from forming metastatic sites in the lung.3.The transcription factor SOX2 can directly bind to the promoter region of LINC01234 to promote transcriptional activation of LINC01234.4.Through bioinformatics analysis,RNA-pulldown,mass spectrometry,q RT-PCR and Western Blot,PCBP2 was identified as an important RNA-binding protein that could bind to LINC01234.5.In addition,we also screened some m RNA and mi RNA molecules regulated by LINC01234.Functional and molecular biological experiments have confirmed that LINC01234/PCBP2 complex can interact with Dicer and regulate the maturation of mi R-512-3p,thus promoting the proliferation of ESCC.Moreover,LINC01234 can also interact with mi R-140-3p to regulate metastasis of ESCC.【Conclusion】This study confirmed that LINC01234 is highly expressed in ESCC cells and tissues,and its high expression predicts poor prognosis of patients,suggesting that the increased expression of LINC01234 may be related to the occurrence and development of ESCC.SOX2 directly binds to the promoter region of LINC01234 to promote its expression.Through a series of functional experiments,LINC01234 has been confirmed to play an oncogentic role in ESCC.Finally,this study proposed and clarified that LINC01234 can regulate the maturation of mi R-512-3p by binding PCBP2,thus regulating the proliferation of ESCC cells.In addition,mi R-140-3p may be involved in the LINC01234-mediated ce RNA mechanism to regulate metastasis of ESCC cells.This study provides a theoretical basis for further understanding the role and regulatory mechanism of LINC01234 in the malignant phenotypes of ESCC,and LINC01234 is promising to become a new therapeutic target and prognostic marker of ESCC. |
