| Background and ObjectiveEsophageal cancer is one of the most common cancers in the world.In 2018,it is estimated that there were 572034 new cases and 508585 patients died of esophageal cancer worldwide.China is one of the countries with the highest incidence rate and mortality rate of esophageal cancer.There are approximately 246 thousand new cases and 188 thousand deaths annually.It is ranked the sixth and fourth in terms of incidence rate and mortality rate respectively.Esophageal squamous cell carcinoma(ESCC)is the most common type of esophageal cancer in China,accounting for 90%of esophageal cancer.The poor prognosis of esophageal squamous cell carcinoma is due to its difficult early diagnosis and rapid progress.To further reveal the critical molecular mechanisms underlying the initiation and development of ESCC would promote the findings of novel therapeutic targets and prognostic biomarkers for ESCC.Long non-coding RNAs(lncRNAs)and microRNAs(miRNAs)are two class of important regulatory RNAs,which have been revealed to play critical roles in various pathophysiological statuses.Accumulating evidences have demonstrated that many lncRNAs are dysregulated in various diseases,including cancers.Furthermore,these dysregulated lncRNAs may have important roles in these diseases.As to cancers,lncRNAs have been revealed to regulate cancer cells’ growth,cell cycle,apoptosis,migration,invasion,senescence,drug resistance,and so on.The mechanisms of action of lncRNAs are diverse.lncRNAs can directly bind proteins,mRNAs,miRNAs,and/or DNAs,and further modulate the location,expression,and/or functions of interacted partners.Consistent with lncRNAs,many miRNAs are also revealed to be dysregulated and have important roles in various diseases.Some studies have shown that miRNA plays a critical role in lncRNA biological function,lncRNA can regulate miRNA in several ways.miRNAs negatively regulate the expression of their target genes via binding to the 3’-untranslated regions(3’UTRs)of target mRNAs,and further causing translation inhibition and/or target mRNAs degradation.LncRNA LincIN(long intergenic non-coding RNA between ITGB1 and NRP1)was recently identified to be overexpressed in human breast tumors and correlated with poor prognosis of breast cancer patients.Furthermore,LincIN was revealed to promote breast tumor cell migration and invasion via binding nuclear factor 90(NF90).However,the expression,clinical significances,biological roles,and mechanisms of action of LincIN in ESCC are still unknown.Thus,the purposes of the study were to determine the expression,functions,and mechanisms of action of LincIN in ESCC.Methods1.Exploring the expression pattern of LincIN and the correlation of LincIN expression and prognosis in Esophageal squamous carcinomawe measured the expression of LincIN in 56 pairs of ESCC tissues and adjacent noncancerous tissues using qRT-PCR.In addition,the expression of LincIN in immortalized normal esophageal epithelial cell line Het-1 A and ESCC cell lines TE-1,KYSE150,and Eca-109 was measured using qRT-PCR.Wilcoxon signed-rank test,one-way ANOVA followed by Dunnett’s multiple comparison test were performed to illustrated the difference of LincIN expression in tumor tissues and corresponding non-tumor tissues,and in different cell lines.Further more,the correlations between LincIN expression and clinicopathological characteristics in these 56 ESCC cases were analysed by Pearson chi-square test.Besides,Kaplan-Meier survival analysis followed by Log-rank test was used to analysed the correlation between LincIN expression and overall survival of these 56 ESCC patients.2.Exploring the biological roles of LincINLincIN stably overexpressed and control TE-1 cells were constructed via stable transfection of LincIN overexpression and control plasmids,and LincIN stably depleted and control Eca-109 cells were constructed via stable transfections of two independent LincIN specific shRNAs.CCK-8 assays,EdU incorporation assays,Transwell migration assays and Transwell invasion assays were applied to demonstrated the ability of cell growth,cell migration,cell invasion in these cell lines.The data was analysed by Student’s t-test,one-way ANOVA followed by Dunnett’s multiple comparison test.3.Exploring the effect of LincIN overexpression on downstream molecule miR-7LincIN stably overexpressed and control TE-1 cells,LincIN stably depleted and control Eca-109 cells were constructed.RNA Immunoprecipitation assay followed by qRT-PCR were used to explore the binding between LincIN and NF90,and the binding between NF90 and pri-miR-7.qRT-PCR were applied to explore the expression of pri-miR-7 and miR-7.The data was analysed by Student’s t-test,one-way ANOVA followed by Dunnett’s multiple comparison test.Besides,the expression of miR-7 in 56 pairs of ESCC tissues and adjacent noncancerous tissues was detected by qRT-PCR,and the results were analysed by Wilcoxon signed-rank test.What’s more,the correlation between miR-7 and LincIN expression level in 56 ESCC tissues was analysed by Pearson correlation analysis.4.Exploring the effect of miR-7 under-expression on downstream molecule HOXB13LincIN stably overexpressed TE-1 cells,LincIN overexpression plasmid and miR-7 mimics co-transfected cells and control TE-1 cells,LincIN stably depleted and control Eca-109 cells were constructed.In those cell lines,dual-luciferase reporter assay was applied to detect the activity of HOXB13 3’UTR,qRT-PCR was used to detect the expression of HOXB13 mRNA,and Western blot was employed to detect the expression of HOXB13 protein.Results are analysed by one-way ANOVA followed by Dunnett’s multiple comparison test.5.Exploring the effect of miR-7 Overexpression or HOXB13 depletion on the roles of LincINWestern blot was applied to detect the expression of HOXB13 protein in the following cell lines,miR-7 and LincIN simultaneously stably overexpressed TE-1 cells,HOXB13 stably depleted and simultaneously LincIN stably overexpressed TE-1 cells,LincIN stably overexpressed TE-1 cells and controlled TE-1 cells.Then,CCK-8 assays,EdU incorporation assays,Transwell migration assays and Transwell invasion assays were applied to demonstrated the ability of cell growth,cell migration,cell invasion in these cell lines.The data was analysed by one-way ANOVA followed by Dunnett’s multiple comparison test.Results1.LincIN was increased in ESCC and correlated with poor prognosis of ESCC patientsWe measured the expression of LincIN in 56 pairs of ESCC tissues and adjacent noncancerous tissues using qRT-PCR.The results displayed that the expression of LincIN was significantly increased in ESCC tissues compared with that in adjacent noncancerous tissues.Analyses of the correlations between LincIN expression and clinicopathological characteristics in these 56 ESCC cases revealed that increased expression of LincIN was positively correlated with advanced tumor invasion depth,lymph node metastasis,and TNM stages.Further analysis of the correlation between LincIN expression and overall survival of these 56 ESCC patients revealed that ESCC patients with higher expression of LincIN have shorter survival time than that with lower expression of LincIN.In addition,the expression of LincIN in immortalized normal esophageal epithelial cell line Het-1A and ESCC cell lines TE-1,KYSE150,and Eca-109 was measured using qRT-PCR.The results displayed that the expression of LincIN was significantly increased in ESCC cell lines compared with that in normal esophageal epithelial cell line.Collectively,these results suggested that LincIN was increased in ESCC tissues and cell lines,and correlated with advanced clinical stages and poor prognosis of ESCC patients.2.Overexpression of LincIN promoted ESCC cell growth,migration,and invasionLincIN stably overexpressed and control TE-1 cells were constructed via stable transfection of LincIN overexpression and control plasmids.CCK-8 assays demonstrated that overexpression of LincIN promoted ESCC cell growth.EdU incorporation assays further verified the roles of LincIN overexpression in promoting ESCC cell growth.Transwell migration assays demonstrated that overexpression of LincIN promoted ESCC cell migration.Transwell invasion assays demonstrated that overexpression of LincIN promoted ESCC cell invasion.Collectively,these results suggested that overexpression of LincIN promoted ESCC cell growth,migration,and invasion.3.Knockdown of LincIN inhibited ESCC cell growth,migration,and invasionLincIN stably depleted and control Eca-109 cells were constructed via stable transfections of two independent LincIN specific shRNAs.CCK-8 assays demonstrated that knockdown of LincIN inhibited ESCC cell growth.EdU incorporation assays further verified the repressive roles of LincIN knockdown in ESCC cell growth.Transwell migration assays demonstrated that knockdown of LincIN inhibited ESCC cell migration.Transwell invasion assays demonstrated that knockdown of LincIN inhibited ESCC cell invasion.Collectively,these results suggested that knockdown of LincIN inhibited ESCC cell growth,migration,and invasion.4.LincIN enhanced the suppressive roles of NF90 on miR-7 biogenesisRIP assays displayed that LincIN was specifically enriched in NF90 antibody group,suggesting the binding between LincIN and NF90.RIP assays revealed that overexpression of LincIN promoted the binding between NF90 and pri-miR-7.RIP assays also revealed that knockdown of LincIN repressed the binding between NF90 and pri-miR-7.These results suggested that LincIN enhanced the binding between NF90 and pri-miR-7.Next,the expression levels of pri-miR-7 and mature miR-7 in LincIN stably overexpressed and control TE-1 cells were measured using qRT-PCR.The results displayed that overexpression of LincIN induced the accumulation of pri-miR-7 and downregulated mature miR-7 in ESCC cells.The expression levels of pri-miR-7 and mature miR-7 in LincIN stably depleted and control Eca-109 cells were also measured using qRT-PCR.The results displayed that knockdown of LincIN downregulated the accumulation of pri-miR-7 and upregulated mature miR-7 in ESCC cells.Collectively,these results suggested that LincIN enhanced the suppressive roles of NF90 on miR-7 biogenesis and therefore downregulated mature miR-7 level.5.miR-7 was decreased and inversely correlated with LincIN in ESCC tissuesWe measured the expression of miR-7 in the same 56 pairs of ESCC tissues and adjacent noncancerous tissues.Conversely to the expression pattern of LincIN,the expression of miR-7 was significantly decreased in ESCC tissues compared with that in adjacent noncancerous tissues.Analysis of the correlation between miR-7 and LincIN expression levels in these 56 ESCC tissues revealed a statistically significant inverse correlation between miR-7 and LincIN expression levels in ESCC(r=-0.4024,P=0.0021).6.LincIN upregulated HOXB13 via suppressing miR-7Dual-luciferase reporter assays displayed that overexpression of LincIN significantly upregulated the 3’UTR activity of HOXB13,which was reversed by concurrent overexpression of miR-7.Reciprocally,knockdown of LincIN significantly reduced the 3’UTR activity of HOXB13.qRT-PCR assays displayed that overexpression of LincIN significantly upregulated the mRNA level of HOXB13,which was reversed by concurrent overexpression of miR-7.Reciprocally,knockdown of LincIN significantly reduced the mRNA level of HOXB13.Western blot assays displayed that overexpression of LincIN significantly upregulated the protein level of HOXB13,which was reversed by concurrent overexpression of miR-7.Reciprocally,knockdown of LincIN significantly reduced the protein level of HOXB13.Collectively,these results suggested that LincIN upregulated HOXB13 via suppressing miR-7.7.Overexpression of miR-7 or depletion of HOXB13 attenuated the roles of LincIN in promoting ESCC cell growth,migration,and invasionwe stably overexpressed miR-7 or depleted HOXB13 in LincIN stably overexpressed TE-1 cells.CCK-8 assays demonstrated that both miR-7 overexpression and HOXB13 depletion attenuated the roles of LincIN in promoting ESCC cell growth.EdU incorporation assays further verified that both miR-7 overexpression and HOXB13 depletion attenuated the pro-proliferative roles of LincIN.Transwell migration assays demonstrated that both miR-7 overexpression and HOXB13 depletion attenuated the roles of LincIN in promoting ESCC cell migration.Transwell invasion assays demonstrated that both miR-7 overexpression and HOXB13 depletion attenuated the roles of LincIN in promoting ESCC cell invasion.Collectively,these results suggested that overexpression of miR-7 or depletion of HOXB 13 both attenuated the roles of LincIN in promoting ESCC cell growth,migration,and invasion.ConclusionTaken together,in the present study,we found that lncRNA LincIN is highly expressed in ESCC,and correlates with advanced clinical stages and poor prognosis of ESCC patients.LincIN promotes ESCC cell growth,migration,and invasion via binding NF90,suppressing the biogenesis of miR-7,and upregulating HOXB 13.Our data implied that LincIN may be a promising prognostic biomarker and therapeutic target for ESCC. |
