| BackgroundOesophageal squamous cell carcinoma(OSCC)is a prevalent malignancy with high morbidity and mortality due to early metastasis and poor prognosis.Although early screening and detection programmes have brought a decline in age-standardised mortality in recent years,oesophageal cancer continues to rank sixth among the most common cancer-related deaths worldwide as access to effective treatment diminishes over time.lncRNAs participate in a variety of important regulatory processes including chromosome silencing,chromatin modification,transcriptional activation and silencing.In terms of tumours,several studies reported that aberrant lncRNA expression and functions affect biological properties such as proliferation,apoptosis and drug resistance,which in turn regulate the development of many tumours including OSCC,and provide reference for tumour diagnosis,prognosis and targeted therapy.LINC01116,as an intergenic lncRNA,has been reported to be involved in the development of various tumours such as lung cancer,osteosarcoma,glioma,prostate cancer,breast cancer and ovarian cancer.However,the specific role of LINC01116 in the progression of oesophageal squamous carcinoma has not been clarified.AGO1,as a member of the Argonaute protein family,is a critical component of the RNA-induced silencing complex.Several studies have reported connections between AGO family proteins and tumours such as colorectal cancer,pancreatic cancer and hepatocellular cancer.Nonetheless,AGO1 is an understudied area of investigation in the field of oesophageal squamous carcinoma.In this study,we identified the expression of LINC01116 in OSCC based on the sequencing data and online database,investigated the role of LINC01116 in the biological behaviour of OSCC in vitro and in vivo,and analysed the correlation between LINC01116 expression and clinical parameters.We also applied molecular biological methods to identify its targeting and regulating relationship with AGO1 and to explore the specific role of AGO1 in the malignancy of OSCC.In addition,we sought to identify the role of AGO1 to gain a deeper understanding of its function and regulation,providing new insights for molecular targeted therapy.Methods1.The lncRNA expression data of OSCC and corresponding clinical information were obtained through RNA-sequencing and GEO database and were analysed by R language.Data from the Cancer Genome Atlas(TCGA)database were utilised for subsequent validation.The expression of LINC01116 in OSCC tissues and intraepithelial neoplasia tissues was examined by qRT-PCR.2.siRNA was used to interfere with the expression of LINC01116 and p EX-3 plasmid was used for ectopic expression of LINC01116.CCK-8 assay,clone formation assay,flow cytometry,Transwell assay and western blot were applied to observe the proliferation,apoptosis,migration,invasion and EMT process of OSCC cells.Lentiviral stable transfection of cells were used for xenograft mouse model.ki67 expression was detected by immunohistochemistry.3.Subcellular localization of LINC01116 was predicted by online databases and validated by subcellular fractionation.Dual luciferase reporter gene assay was performed to verify the binding site of AGO1 and LINC01116.qRT-PCR and western blot were performed to detect alterations of AGO1 following changes in LINC01116 expression.4.qRT-PCR and GEPIA database were used to detect AGO1 expression in OSCC tissues.CCK-8 assay,clone formation assay,flow cytometry,Transwell assay and western blot were applied to observe the proliferation,apoptosis,migration,invasion and EMT process of OSCC cells after siRNA interference with AGO1.Immunohistochemistry was applyed to detect AGO1 expression in OSCC xenograft mouse model.Fluorescence in situ hybridization and immunofluorescence were used to validate colocalization of LINC01116 and AGO1.5.GSEA tool was applied to enrich potential LINC01116-related pathways.Results1.In this study,133 differential lncRNAs were detected by sequencing 4 pairs of OSCC and normal oesophageal tissues(|log2FC|>1,p<0.05 statistically significant),of which 63 were upregulated and 70 were down-regulated.A total of 1393 differential lncRNAs were detected in 143 OSCC and 133 normal oesophageal samples from integrated GEO datasets,of which 587 were upregulated and 806 were down-regulated(|log2FC|>1,p<0.05 was statistically significant).Venn diagram screening analysis showed that both LINC01116 and DLEU1 were up-regulated and 13 lncRNAs were down-regulated.qRT-PCR verified that the level of LINC01116 was significantly elevated in both OSCC surgical specimens(p=0.0191)and in intraepithelial neoplasia specimens obtained by endoscopic mucosal dissection(p=0.0266).The expression of LINC01116 was significantly correlated with tumour differentiation(p=0.034)and T-stage(p=0.036)in patients with oesophageal squamous carcinoma.2.CCK-8 assay suggested that siRNA interference with LINC01116 significantly the proliferation activity was significantly inhibited by downregulating LINC01116 starting from 48h(p<0.01),and was significantly promoted by overexpression of LINC01116 starting from 72h(p<0.05).Clone formation assays suggested that downregulating LINC01116 significantly reduced the number of clones formed by OSCC cells(p<0.01),while upregulating LINC01116 increased the number of clones(p<0.05).Flow cytometry showed a significant increase in apoptosis rate in OSCC cells interfering with LINC01116(p<0.05)and a significant decrease in apoptosis rate in the upregulating LINC01116 group(p<0.05).Transwell assays verified that knockdown of LINC01116 significantly decreased OSCC cell invasion(p<0.05)and western blot showed a significant increase in E-cadherin and a significant decrease in N-cadherin expression in the LINC01116 downregulating group,while the overexpression of LINC01116 significantly increased E-cadherin expression and decreased N-cadherin expression(p<0.05).In xenograft mouse model,the growth rate of OSCC subcutaneous tumors was significantly slowed in the downregulating LINC01116 group(p<0.001)with reduced ki67 expression level(p<0.01),while the stable overexpression LINC01116 significantly increased the growth rate of OSCC(p<0.01)with increased ki67 level(p<0.05).3.Both online databases and subcellular fractionation assay indicate that LINC01116 predominantly located in the cytoplasm of OSCC cells and that AGO1 is one of its interacting molecules.The double luciferase reporter gene assay confirmed that disruption or overexpression of LINC01116 decreased or increased the levels of the reporter gene containing the AGO1 5’-UTR binding sequence(p<0.05),and that disruption or overexpression of LINC01116 decreased or increased the levels of mRNA and protein expression of AGO1(p<0.05).4.Both GEPIA database and clinical samples exhibited that AGO1 was also significantly unregulated in OSCC(p<0.05).CCK-8 assay suggested that siRNA interference with AGO1 did not inhibit the proliferation of OSCC cells(p>0.05);flow-cytometry results showed that interference with AGO1 had no effect on the apoptosis rate in vitro(p>0.05);Transwell assay demonstrated that the number of migrating OSCC cells was significantly reduced in the AGO1 knockdown group(p<0.05)compared to the control group.Similarly,the invasing number of OSCC cells was also significantly reduced with AGO1 knockdown(p<0.05).Western blot results showed that E-cadherin expression was significantly increased in the AGO1 knockdown group,while N-cadherin expression was significantly reduced(p<0.05).5.Multiple tumor relating pathways are enriched in high LINC01116 expression group.ConclusionIn sum,we identified the upregulation of LINC01116 in OSCC and IEN tissues,along with its relevance with cliniopathological parameters,implicating an emerging early diagnostic value and prognostic value.We also demonstrated the functional relevance of LINC01116 is primarily exhibited in driving cell proliferation,migration and invasion and the EMT process by affecting the protein levels of E-cadherin and N-cadherin,as well as hindering apoptosis.At the mechanistic level,we robustly demonstrate that genetic inhibition of LINC01116 hampered AGO1-meadiated EMT process.Our data add another tractable player,LINC01116,for understanding the metastatic cascade and for searching novel therapeutic targets to prevent OSCC metastasis. |
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