| Objective:Due to its high incidence and low survival rate,esophageal cancer ranks among the top 10 causes of malignance-related death worldwide.Compared with the esophageal adenocarcinoma in developed countries,esophageal squamous cell carcinoma(ESCC)account for the majority of the esophageal cancer patients in China.There is a similar trend in other developing countries.ESCC also has a has regional distribution in China.Henan,Hebei and Chongqing are the areas with high incidence of ESCC in China,which may be related to the dietary habits of local residents.Due to the lack of specific and sensitive biological therapeutic targets,there has been no breakthrough progress in the early diagnosis and treatment of esophageal cancer,and patients are prone to local invasion and distant metastasis in the late stage of the disease,resulting in poor prognosis.Therefore,in order to diagnose and treat ESCC early and improve the survival rate of patients,we urgently need to further explore and study the occurrence and development of ESCC.Long non-coding RNA(lncRNA)are a class of non-coding RNAs with a length of more than 200 nucleotides.LncRNAs play a variety of positive or negative effects in the occurrence and development of multiple diseases,especially in the malignant tumors.They are not only present in tissues but also widely present in serum,plasma,body fluids and cerebrospinal fluid.Therefore,we selected 4 pairs of ESCC and corresponding paracancerous tissues for RNA-sequencing.Based on the results of differential expression,we analyzed,screened and verified that lncRNA VPS9D1-AS1 was differentially expressed in ESCC tissues.Previous studies have shown that lncRNA VPS9D1-AS 1,also known as lncRNA MYU,can act as an oncogene or a tumor suppressor gene in different cancer types.However,the function of VPS9D1-AS1 in ESCC was unclear.In order to further research its relationship with clinical characteristics and prognosis in ESCC patients,as well as its biological function and mechanism of action in the occurrence and development of ESCC,we conduct an indepth study.Methods:RNA-sequencing was performed on 4 pairs of ESCC and matched adjacent normal tissues(NATs)to screen lncRNAs with different expressions.The expression of VPS9D1-AS1 in 92 pairs of ESCC tissues and adjacent normal tissues was detected by qRTPCR.The expression of VPS9D1-AS1 in ESCC cell lines were detected by the same method.The expression of VPS9D1-AS1 in ESCC tissues was analyzed,and the correlation between VPS9D1-AS1 and the clinicopathological information was analyzed,so as to explore the clinical significance of VPS9D1-AS1,further to determine the prognosis and guide the treatment.Compared with the need for invasive surgery to obtain tissue specimens,peripheral blood specimens are more convenient and non-invasive.By detecting the VPS9D1-AS1 expression in peripheral blood,the sensitivity and specificity of VPS9D1AS1 were determined in the diagnosis of ESCC,which provide the basis for early diagnosis.Furthermore,to investigate its mechanism of action,we performed proliferation,cell cycle,migration and invasion experiments of ESCC cell lines after the knockdown of VPS9D1AS1,as well as tumorigenesis experiments in nude mice,and immunohistochemical analysis was performed to determine the biological function changes in the development of ESCC in vivo and in vitro.Western blot was used to detection the key proteins of its major pathways,and rescued experiments were used to identify the relevant influencing pathways.Results:1.Based on the sequencing results(p<0.05 and log2 fold change>2 or<0.5),3,514 lncRNAs were differentially expressed in ESCC tumors and NATs),among which 2,079 were downregulated and 1,435 were upregulated.All different lncRNAs were ranked based on their log2 fold change values.First,we removed lncRNAs with longer than 4000 nucleotides and then eliminated the molecules that have already been studied in ESCC.Then,the candidates were verified using TCGA and ENCORI databases.Finally,VPS9D1-AS1 was selected among these lncRNAs for further studies.It can be detected in human tissues,cells and peripheral blood,according to the RNAlocate database.VPS9D1-AS1 was markedly upregulated in tumor tissues compared with NATs.2.Cox analysis demonstrated that the level of VPS9D1-AS1 was significantly correlated with histopathological grade and clinical stage of ESCC patients,and its high expression was positively correlated with high histopathological grade,deep invasion,local lymph node metastasis,and advanced clinical stages.The patients in the high-VPS9D1-AS 1 expression group had a shorter PFS and OS than those in the lower-expression group.High expression of VPS9D1-AS1 lead to poor prognosis especially in patients with advanced clinical stages.3.The expression of VPS9D1-AS1 in plasma of ESCC patients was higher than patients with benign esophageal diseases.It suggested that VPS9D1-AS1 could be a potential diagnostic biomarker of ESCC.4.Knockdown of VPS9D1-AS1 negatively affects the proliferation,migration,and invasion of ESCC cells and the cell cycle was also changed.The G1-S phase transformation was inhibited,and the expression of CDK4,CDK6 and Cyclin D1 were decreased.Combined with biological analysis,it was concluded that VPS9D1-AS1 may regulate the cell cycle via the Wnt/β-catenin signaling pathway.Expression of the key proteins in the pathway β-catenin,c-Myc,and cyclin D1 decreased after knockdown of VPS9D1-AS1.However,the rescued assay shown that the expressions of these proteins lncreased after the addition of pathway agonist CT99021.It was also found in the colony formation assay that the proliferation and cloning ability of tumor cells which had been significantly inhibited by knockdown of VPS9D1-AS1 was significantly improved after the addition of CT99021.Expression of VPS9D1-AS1 was positively correlated with β-catenin and c-Myc in ESCC tissues,which was consistent with in ESCC cells.Conclusion:VPS9D1-AS1 can act as an oncogene or an antioncogene in different types of malignancies.The present study demonstrated that it was significantly upregulated in ESCC,and its biological function and clinical prognosis were assessed.Whole transcriptome sequencing was conducted in four pairs of ESCC tissues and normal adjacent tissues.In this study,compared with normal controls,VPS9D1-AS1 was highly expressed in ESCC and cell lines.The upregulation of VPS9D1-AS1 was significantly correlated with the histopathological grade and clinical stage of ESCC.The analyses showed poor prognosis in ESCC patients with high VPS9D1-AS1 expression.Knockdown of VPS9D1-AS1 not only inhibited tumor proliferation,migration and invasion,but also downregulated the Wnt/β-catenin signaling pathway by key proteins such as β-catenin and c-myc.Thus,VPS9D1-AS1 was highly expressed in ESCC,and the high level of expression could lead to poor prognosis.It could be a potential biomarker for diagnosis of ESCC and played a major role in the progression and metastasis of ESCC through the Wnt/β-catenin signaling pathway. |
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