| Background and ObjectiveEsophageal squamous cell carninoma(ESCC)is one of the high-incidence tumors in our country,and the current five-year survival rate of ESCC patients is still less than20%.Although we have made some progress in the diagnosis and treatment of ESCC,our focus is mainly on a small part of the DNA sequence responsible for encoding proteins.With the widespread application of next-generation sequencing technology,a large number of long non-codingRNA(lncRNA)molecules have been identified and found to be closely related to the occurrence and development of tumors.Therefore,elucidating the mechanism of tumor genesis and development from the perspective of lncRNA is expected to be a new breakthrough point in overcoming tumors.Our research group previously collected 93 pairs of ESCC tissues and their paracancerous tissues and performedRNA sequencing(RNA-Seq)on them,and DSCAM-AS1 was the lncRNA whose expression level is significantly increased in ESCC tissues.Studies have shown that DSCAM-AS1 can regulate tumor progression through binding withRNA-binding proteins or as a competitive endogenousRNA(ceRNA).However,the expression,function and mechanism of DSCAM-AS1 in ESCC are still unclear.Recent studies have shown that lncRNA can regulate the expression of target mRNA through sponging microRNA(miRNA).Through the online database Starbase v3.0,we predicted that DSCAM-AS1 could sponge miR-376c-3p to promote the expression of SOX4.It has been reported that miR-376c-3p is a cancer-related miRNA,and the expression of miR-376c-3p is down-regulated in various tumors.However,no research has focused on the expression and function of miR-376c-3p in ESCC and its relationship with DSCAM-AS1.Sex-determining region Y-related high-mobility group box 4(SOX4)is a transcription factor closely related to tumor development.More and more evidences showed that SOX4 regulated the occurrence and development of a variety of tumors,including ESCC.Interestingly,recent studies have shown that SOX4 can act as a binding target for a variety of miRNAs in human cancers.However,no research has focused on the relationship between SOX4 and miR-376c-3p.This study will explore the biological function and molecular mechanism of DSCAM-AS1 in ESCC from the following three aspects.(1)The expression and clinical significance of DSCAM-AS1 in ESCC;(2)The effect of DSCAM-AS1 on the proliferation,invasion and migration of ESCC cells;(3)The molecular mechanism that DSCAM-AS1 promotes SOX4 expression through sponging miR-376c-3p.Methods1 The expression and clinical significance of DSCAM-AS1 in ESCCThe expression level of DSCAM-AS1 in ESCC tissues and cells was detected byRNA-Seq and quantitative reverse transcription PCR(RT-qPCR).Correlations between DSCAM-AS1 expression and clinicopathological features and prognosis were analyzed.2 The effect of DSCAM-AS1 on ESCC cell proliferation,invasion and migrationCCK8,clone formation and Transwell experiments were used to detect the DSCAM-AS1 effect on the proliferation,invasion and migration of ESCC cells after knocking down or overexpressing DSCAM-AS1.Western blot experiment was used to detect DSCAM-AS1 effect on the expression of epithelial-mesenchymal transition(EMT)related proteins after knocking down or overexpressing DSCAM-AS1.3 The molecular mechanism of DSCAM-AS1 in ESCC3.1 Localization of DSCAM-AS1 in ESCC cellsFluorescence in situ hybridization(FISH)and nucleo-cytoplasmic separation experiments were used to detect the specific location of DSCAM-AS1 in ESCC cells.3.2 The combination between DSCAM-AS1 and miR-376c-3pThe online database Starbase v3.0 was used to predict the combination between DSCAM-AS1 and miR-376c-3p,and the dual luciferase reporter experiment was used to confirm their combination in ESCC cells.Finally,RT-qPCR was used to further confirm the correlation between DSCAM-AS1 and miR-376c-3p by detecting the effect of knockdown or overexpression of DSCAM-AS1 on the expression of miR-376c-3p in ESCC cells.3.3 The expression of miR-376c-3p in ESCC cellsRT-qPCR detected the expression level of miR-376c-3p in ESCC cells.Then,the expression correlation between miR-376c-3p and DSCAM-AS1 in ESCC cells was analyzed.3.4 The miR-376c-3p effect on the proliferation,invasion and migration of ESCC cells and rescue effect on the malignant phenotype of ESCC cells mediated by DSCAM-AS1CCK8,clone formation and Transwell experiments were used to detect the miR-376c-3p effect on the proliferation,invasion and migration of ESCC cells after knocking down or overexpressing miR-376c-3p.The functional rescue experiment was used to detect the effect of miR-376c-3p inhibitor or mimic on the malignant phenotype of ESCC cells mediated by DSCAM-AS1 knockdown or overexpression.3.5 The combination between miR-376c-3p and SOX4 mRNAThe online database Starbase v3.0 was used to predict the combination between miR-376c-3p and SOX4 mRNA,and the dual luciferase reporter experiment was used to confirm their combination in ESCC cells.Finally,RT-qPCR and Western blot were used to further confirm the correlation between miR-376c-3p and SOX4 by detecting the effect of miR-376c-3p inhibitor or mimic on the expression of SOX4 in ESCC cells.3.6 The expression of SOX4 in ESCCRNA-Seq,RT-qPCR and Western blot experiments were used to detect the expression level of SOX4 in ESCC tissues and cells.Then,the expression correlations between SOX4 and miR-376c-3p and DSCAM-AS1 in ESCC cells and tissues were analyzed.3.7 The expression correlations between DSCAM-AS1 and miR-376c-3p and SOX4To verify the expression correlations between DSCAM-AS1 and miR-376c-3p and SOX4,RT-qPCR and Western blot experiments were used to detect the effect of DSCAM-AS1 down-regulation or up-regulation on the SOX4 expression and rescue effects of miR-376c-3p inhibitor or mimic on the SOX4 expression mediated by DSCAM-AS1 knockdown or overexpression in ESCC cells.Results1 The expression and clinical significance of DSCAM-AS1 in ESCCCompared with adjacent tissues and normal esophageal epithelial cell Het-1A,the expression of DSCAM-AS1 in ESCC tissues and cells is significantly increased(P<0.05).Increased expression of DSCAM-AS1 is associated with advanced TNM stage,lymph node metastasis and poor overall survival of ESCC patients(P<0.05).2 The effect of DSCAM-AS1 on ESCC cell proliferation,invasion and migrationThe results of biological behavioral experiments showed that knockdown of DSCAM-AS1 inhibited the proliferation,invasion and migration of ESCC cells(P<0.05).Furthermore,overexpression of DSCAM-AS1 promoted the proliferation,invasion and migration of ESCC cells(P<0.05).Western blot results revealed that DSCAM-AS1 down-regulation enhanced E-cadherin level and reduced the levels of Ncadherin and Vimentin proteins(P<0.05).Furthermore,DSCAM-AS1 up-regulation suppressed E-cadherin level and promoted the levels of N-cadherin and Vimentin proteins in ESCC cells(P<0.05).3 The molecular mechanism of DSCAM-AS1 in ESCC3.1 The localization of DSCAM-AS1 in ESCC cellsThe results of FISH and nucleo-cytoplasmic separation assays showed that DSCAM-AS1 was mainly distributed in the cytoplasm of ESCC cells.3.2 The combination between DSCAM-AS1 and miR-376c-3pThe online database Starbase v3.0 predicted that DSCAM-AS1 can sponge miR-376c-3p.The dual luciferase reporter experiment confirmed the interaction between DSCAM-AS1 and miR-376c-3p.The results of RT-qPCR assay showed that DSCAMAS1 down-regulation enhanced miR-376c-3p level(P<0.05)and DSCAM-AS1 upregulation suppressed miR-376c-3p level in ESCC cells(P<0.05).3.3 The expression of miR-376c-3p in ESCC cellsRT-qPCR results showed that the expression of miR-376c-3p in ESCC cells was significantly lower than that in normal esophageal epithelial cell,which was negatively correlated with the expression of DSCAM-AS1 in ESCC cell lines(P<0.05).3.4 The miR-376c-3p effect on the proliferation,invasion and migration of ESCC cells and rescue effect on the malignant phenotype of ESCC cells mediated by DSCAM-AS1The results of biological behavioral experiments showed that knockdown of miR-376c-3p promoted the proliferation,invasion and migration of ESCC cells(P<0.05).Furthermore,overexpression of miR-376c-3p inhibited the proliferation,invasion and migration of ESCC cells(P<0.05).Interestingly,miR-376c-3p reversed the effect on the malignant phenotype of ESCC cells mediated by DSCAM-AS1(P<0.05).3.5 The combination between miR-376c-3p and SOX4 mRNAThe online database Starbase v3.0 predicted that miR-376c-3p can sponge SOX4 mRNA.The dual luciferase reporter experiment confirmed the interaction between miR-376c-3p and SOX4 mRNA.The results of RT-qPCR and Western blot assays showed miR-376c-3p down-regulation enhanced SOX4 level(P<0.05)and miR-376c-3p up-regulation reduced SOX4 level in ESCC cells(P<0.05).3.6 The expression of SOX4 in ESCCRNA-Seq result showed that the expression of SOX4 mRNA in ESCC tissues was significantly higher than that in adjacent tissues,which was positively correlated with the expression of DSCAM-AS1 in ESCC tissues(P<0.05).RT-qPCR result showed that the expression of SOX4 mRNA in ESCC cells was significantly higher than that in normal esophageal epithelial cell,which was negatively correlated with the expression of miR-376c-3p in ESCC cells(P<0.05).Western-blot result showed that the expression of SOX4 protein in ESCC cells was significantly higher than that in normal esophageal epithelial cell(P<0.05).3.7 The expression correlation between DSCAM-AS1 and miR-376c-3p and SOX4The results of RT-qPCR and Western blot assays showed that DSCAM-AS1 knockdown reduced SOX4 level of ESCC cells(P<0.05).Furthermore,DSCAM-AS1 overexpression enhanced the SOX4 level of ESCC cells(P<0.05).In addation,miR-376c-3p reversed the effect on high SOX4 level of ESCC cells mediated by DSCAMAS1(P<0.05).conclusions1 DSCAM-AS1 is up-regulated in ESCC tissues and cells,and its high expression is tightly associated with TNM stage,lymph node metastasis and poor prognosis of ESCC patients.2 DSCAM-AS1 promotes SOX4 expression by sponging miR-376c-3p,which results in the promotion of cell proliferation,invasion and migration in ESCC cells. |
PDF Full Text Request