| Background:Esophageal cancer(EC)is the eighth most common cancer in the world,with esophageal squamous cell carcinoma(ESCC)predominating.Esophageal cancer is characterized by high malignant potential,poor prognosis and high lethality,which seriously threatens human life and health.Globally,more than 455,800 new cases occur each year,and nearly 400,200 death form EC.Although in recent years,great progress has been made in the surgery and preoperative radiotherapy and chemotherapy of esophageal cancer,and the survival rate of patients has been improved.Unfortunately,the majority of patients with EC are diagnosed at an advanced stage and unable to undergo surgical resection.In addition,tumor metastasis,recurrence,and resistance to chemotherapy and radiation therapy lead to poor prognosis in patients with EC.The median survival time for patients with EC is only 1.5 years,and the 5-year survival rate ranges from 15% to 20%.Consequently,it is urgent to identify predictive biomarkers and therapeutic targets for early diagnosis and treatment of EC.Increasing evidence demonstrates that a number of long non-coding RNAs(lnc RNAs)are involved in tumorigenesis and progression by transcriptional and post-transcriptional regulation.Among them,lnc RNA DLEU2(deleted in lymphocytic leukemia 2),also known as LINC00022,has been revealed to be dysregulated in various tumors and is closely related to tumor growth and metastasis.lnc RNA DLEU2 is down-regulated in laryngeal carcinoma and plays a role as a tumor suppressor by regulating mi R-16-1.lnc RNA DLEU2 expression in cervical cancer tissues and cells is lower than that in adjacent tissues and normal cervical cell lines,respectively,and lnc RNA DLEU2 overexpression can inhibits the proliferation,migration and invasion of cervical cancer cells and exerts the function as a tumor suppressor.However,lnc RNA DLEU2 has been shown to be up-regulated in hepatocellular carcinoma(HCC)tissues,and promotes the proliferation,migration and invasion of HCC cells by binding to EZH2,acting as a tumor promoter.lnc RNA DLEU2 is also up-regulated in pancreatic cancer tissues and cell lines,acting as an endogenous " mi R-455 sponge" to regulate the expression of SMAD2,promoting the proliferation and invasion of pancreatic cancer cells.Therefore,lnc RNA DLEU2 may play different roles in different tumors.However,little is known about the function of lnc RNA DLEU2 in the development of esophageal cancer.In addition,a number of studies have shown that lnc RNAs function in a competing endogenous RNAs(ce RNA)pattern to be involved in the development of tumors,which acts as a "mi RNA sponge" to adsorb mi RNAs and regulate the expression of target genes.In this study,we aimed to investigate the biological function of lnc RNA DLEU2 in the growth and metastasis of esophageal carcinoma,and to explore whether lnc RNA DLEU2 plays a role in the development of esophageal cancer in a ce RNA pattern to further analyze its mechanism.This study aimed to provide new therapeutic target for developing new therapeutic strategies of esophageal cancer.Method:(1)Expression and prognostic significance of lnc RNA DLEU2 in esophageal cancer1)The Gene Expression Profiling Interactive Analysis(GEPIA)was used to analyze the expression of lnc RNA DLEU2 in esophageal cancer tissues and normal tissues,the correlation of lnc RNA DLEU2 between the prognosis of patients with esophageal cancer was also analyzed by GEPIA.2)Expression of lnc RNA DLEU2 in ESCC cell lines Eca-109 and KYSE-150 and human normal esophageal epithelial cells HEEC was detected by RT-PCR.(2)The biological function of lnc RNA DLEU2 in the growth and metastasis of ESCC1)Eca-109 and KYSE-150 cells were transfected with si RNA-DLEU2 and the negative control,respectively.The expression of lnc RNA DLEU2 was detected by RT-PCR to verify knockdown efficiency.2)After transfection,CCK8,colony formation,wound-healing,transwell,and flow cytometry assays were performed to assess the effects of lnc RNA DLEU2 knockdown on the proliferation,migration,invasion,and apoptosis of Eca-109 and KYSE-150 cells.The effect of lnc RNA DLEU2 knockdown on apoptosis-related proteins(Bcl-2,Bax,cleaved Caspase 3 and active Caspase 9)and key components of the Akt signaling pathway(Akt,p-Akt and Cyclin D1)was examined by western blot analysis.(3)Study on the ce RNA pattern of lnc RNA DLEU21)Starbase was used to screen the possible mi RNAs(mi R-30e-5p)with binding sites of lnc RNA DLEU2.The dual-luciferase reporter gene vectors were constructed to verify whether lnc RNA DLEU2 binds to mi R-30e-5p by dual-luciferase reporter gene assay.2)The bioinformatics prediction website(Target Scan)was used to screen the target gene(E2F7)which can bind to mi R-30e-5p and the binding sites were similar to lnc RNA DLEU2/mi R-30e-5p.The dual-luciferase reporter gene vectors were constructed and the binding of mi R-30e-5p to E2F7 was verified by dual-luciferase reporter gene assay.Western blot analysis was used to detect the effects of mi R-30e-5p and mi R-30e-5p+pc DNA3.1-DLEU2 on the expression of E2F7 protein to verify the interaction of lnc RNA DLEU2/ mi R-30e-5p/E2F7.(4)The biological function of E2F7 in the progression of ESCC1)The expression of E2F7 in esophageal cancer tissues and normal tissues was analyzed by GEPIA database,the correlation of E2F7 between the prognosis of patients with esophageal cancer was also analyzed by GEPIA.Expression of E2F7 in esophageal cancer cell lines Eca-109 and KYSE-150 and human normal esophageal epithelial cells HEEC was detected by RT-PCR.2)Eca-109 and KYSE-150 cells were transfected with si RNA-E2F7 and the negative control,respectively.The expression of E2F7 was detected by RT-PCR and western blot analysis to verify knockdown efficiency.3)After transfection,CCK8,colony formation,wound-healing and transwell assays were performed to assess the effects of E2F7 knockdown on the proliferation,migration and invasion of Eca-109 and KYSE-150 cells.Flow cytometry was used to detect the effect of E2F7 knockdown on apoptosis of Eca-109 and KYSE-150 cells.The effect of E2F7 knockdown on apoptosisrelated proteins(Bcl-2,Bax,cleaved Caspase 3 and active Caspase 9)and key components of the Akt signaling pathway(Akt,p-Akt and Cyclin D1)was examined by western blot analysis.(5)Rescue experiments further verifies the correlation among lnc RNA DLEU2/mi R-30e-5p/E2F7Eca-109 and KYSE-150 cells were divided into four groups.They were transfected with si RNA-DELU2,si RNA-DELU2+mi R-30e-5p inhibitor,si RNA-DELU2+pc DNA3.1-E2F7,respectively;and one group transfected with empty vector was used as negative control.After transfection,E2F7 expression was examined by western blot analysis,cell proliferation was detected by CCK8 and colony formation assays,cell migration and invasion were assessed by wound-healing and Transwell assays.6.Statistical analysisAll data were expressed as mean ± SD from three independent experiments.Graph Pad Prism 7.0 software with Student’s t-test or one-way ANOVA was performed for Statistical analysis.P values less than 0.05 were considered statistically significant.Results:(1)Expression and prognostic significance of lnc RNA DLEU2 in esophageal cancerFrom the GEPIA,we observed that the expression of lnc RNA DLEU2 was significantly up-regulated in EC tissues,and its expression was correlated with prognosis of patients with EC,patients with high expression had worse prognosis.RT-PCR results also showed that the expression of lnc RNA DLEU2 in Eca-109 and KYSE-150 cells was higher than in HEEC cells.(2)The biological function of lnc RNA DLEU2 in the growth and metastasis of ESCC1)RT-PCR results showed that the expression of lnc RNA DLEU2 was significantly inhibited by si-DLEU2 in both Eca-109 and KYSE-150 cells.2)lnc RNA DLEU2 knockdown significantly inhibited the proliferation,migration and invasion of Eca-109 and KYSE-150 cells,as well as promoted apoptosis.The expression of anti-apoptotic protein Bcl-2 was significantly down-regulated by si-DLEU2,while the expression of pro-apoptotic proteins Bax,cleaved Caspase3 and active Caspase9 was markedly up-regulated.Additionally,activation of the Akt signaling pathway was also inhibited by lnc RNA DLEU2 knockdown by decreasing phosphorylation level of Akt(p-Akt),and the expression of its downstream protein Cyclin D1 was decreased correspondingly.(3)Study on the ce RNA pattern of lnc RNA DLEU21)With the help of the bioinformatics online database(Starbase),we found that mi R-30e-5p has a potential binding site targeting lnc RNA DLEU2.Dual-luciferase reporter assay showed that the luciferase activity of lnc RNA DLEU2(wt)was obviously diminished by mi R-30e-5p in both Eca-109 and KYSE-150 cells,but the luciferase activity of lnc RNA DLEU2(mut)was not changed.2)By the bioinformatics online database(Target Scan),E2F7 was considered to be a potential target of mi R-30e-5p,and the binding sites were similar to lnc RNA DLEU2/mi R-30e-5p.Dual-luciferase reporter assay showed that mi R-30e-5p significantly blocked the luciferase activity of the wild type E2F7,while did no effect on the mutant type of E2F7.As indicated by western blot analysis,mi R-30e-5p significantly inhibited the protein level of E2F7 in Eca-109 and KYSE-150 cells,while this tendency was significantly reversed when lnc RNA DLEU2 was up-regulated.These results suggest that lnc RNA DLEU2 can positively regulate the expression of E2F7 through competitive binding to mi R-30e-5p.(4)The biological function of E2F7 in the progression of ESCC1)From the GEPIA,we observed that the expression of E2F7 m RNA was significantly up-regulated in EC tissues,and its high expression was correlated with poor prognosis of patients with EC.RT-PCR results also showed that the expression of E2F7 m RNA in Eca-109 and KYSE-150 cells was higher than in HEEC cells.2)E2F7 knockdown by si RNA-E2F7 interference significantly inhibited the proliferation,migration and invasion of Eca-109 and KYSE-150 cells,as well as promoted apoptosis.The expression of anti-apoptotic protein Bcl-2 was significantly down-regulated by E2F7 knockdown,while the expression of pro-apoptotic proteins Bax,cleaved Caspase3 and active Caspase9 was markedly up-regulated.Additionally,E2F7 knockdown significantly inhibited activation of the Akt signaling pathway by decreasing phosphorylation level of Akt(p-Akt),and the expression of its downstream protein Cyclin D1 was decreased by si-E2F7.(5)Rescue experiments further verifies the correlation among lnc RNA DLEU2/mi R-30e-5p/E2F7As indicated by western blot analysis,the decrease in expression of E2F7 protein caused by si-DLEU2 was reversed by mi R-30e-5p inhibitor compared to cells transfected with si-DLEU2.Cell function experiments showed that the inhibitory effects of si-DLEU2 on cell proliferation,migration and invasion were significantly reversed by mi R-30e-5p inhibitor or up-regulation of E2F7 in both Eca-109 and KYSE-150 cells.Conclusion:Our study shows that lnc RNA DLEU2 is up-regulated in ESCC and its high expression is associated with poor prognosis of patients with ESCC.As a ce RNA,lnc RNA DLEU2 regulates the expression of E2F7 by competitive binding to mi R-30e-5p,affecting the proliferation,migration and invasion of ESCC cells.E2F7 also plays a role as a tumor promoter in the growth and metastasis of ESCC.lnc RNA DLEU2 can be used as a potential biomarker for predicting the progression and prognosis of ESCC patients,and function as an effective target for anti-cancer therapy. |
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