| Long non-coding RNAs may perform a vital role in human physiological and pathological processes,including cancer.According to the recent reports,ZNF667-AS1 was down-regulated in cervical cancer and showed abnormal hypermethylation status in breast cancer.However,the regulation mechanisms of ZNF667-AS1 in esophageal squamous cell carcinoma(ESCC)and its relevances with ZNF667 still remains unknown.In the present study,we explored the expression and DNA methylation status of ZNF667-AS1 in ESCC,analyzed its relations with the clinicopathological features,evaluated the impacts of the promoter region methylation status on gene transcription,and detected the cellular localization of ZNF667-AS1.Furthermore,we explored the effects of overexpression of ZNF667-AS1 on cells proliferation,migration and invasion in vitro and its biological functions.Meanwhile,we detected the expression and methylation levels of ZNF667 in ESCC,and analyzed its relations with the clinicopathological features,discussed the correlations between ZNF667-AS1 and ZNF667 in ESCC.The main research contents and results are as follows: Part one Expression of long non-coding RNA ZNF667-AS1 and ZNF667 in esophageal cancer cell lines and esophageal squamous cell carcinoma tissues and their correlationsObjectives: To detect the expression levels of ZNF667-AS1 and ZNF667 in esophageal cancer cell lines and ESCC tissues and their correlations and further analyze their relations with the clinicopathologic features of ESCC cases.Methods:1.qRT-PCR method was applied to detect the mRNA expression level of ZNF667-AS1 and ZNF667 in esophageal cancer cell lines(Kyse170,Eca109,TE1,TE13)and 54 esophageal squamous cell carcinoma tissues and corresponding normal tissues.2.The Paris kit was applied to separate the nuclear and cytoplasma of esophageal cancer cell lines(Kyse170,Eca109,TE1,TE13),and qRT-PCR method was applied to detect the cellular localization of ZNF667-AS1.Result:1.The mRNA expression level of ZNF667-AS1 in esophageal cancer cell lines.The expression level of ZNF667-AS1 was significantly lower in esophageal cancer cell lines(Kyse170,Eca109,TE1,TE13)than that in control(P<0.05).2.Cellular localization of ZNF667-AS1 in esophageal cancer cell lines(Kyse170,Eca109,TE1,TE13).ZNF667-AS1 was mainly located in the nucleus of esophageal cancer cells.3.The mRNA expression level of ZNF667 in esophageal cancer cell lines.The mRNA expression level of ZNF667 in esophageal cancer cell lines(Kyse170,Eca109,TE1,TE13)was significantly lower than that in control(P<0.05).4.The mRNA expression level of ZNF667-AS1 in ESCC tissues and corresponding normal tissues.The mRNA expression level of ZNF667-AS1 in ESCC tissues was notably lower than that in corresponding normal tissues(P<0.05),and was closely correlated with lymph node metastasis,pathological differentiation,and TNM stages(P<0.05).5.The mRNA expression level of ZNF667 in ESCC tissues and corresponding normal tissues.The mRNA expression level of ZNF667 in ESCC tissues was notably lower than that in corresponding normal tissues(P<0.05),and was closely correlated with lymph node metastasis,pathological differentiation,and TNM stages(P<0.05).6.The correlations of the mRNA expression level of ZNF667-AS1 and ZNF667 in ESCC tissues.There was a significant positive correlation between the mRNA expression levels of ZNF667-AS1 and ZNF667 in ESCC tissues.Part Two Methylation status of ZNF667-AS1 and ZNF667 in esophageal squamous cell carcinomaObjectives: To detect the methylation status of various regions of ZNF667-AS1 and ZNF667 CpG islands in esophageal cancer cell lines and tissues,and analyze the impacts of methylation status on gene transcription,and its relations with the clinicopathologic features.Methods:1.qRT-PCR method was applied to detect the expression levels of ZNF667-AS1 and ZNF667 in esophageal cancer cell lines(Kyse170,Eca109,TE1,TE13)before and after 5-Aza-dC treatment.2.MSP method was applied to detect the methylation status of various regions of ZNF667-AS1 and ZNF667 CpG islands in esophageal cancer cell lines(Kyse170,Eca109,TE1,TE13)treated or untreated with 5-Aza-dC,and 54 esophageal squamous cell carcinoma tissues and corresponding normal tissues.3.Dual-luciferase reporter assay systems method was applied to detect the impacts of methylation status of various regions of ZNF667-AS1 and ZNF667 CpG islands on gene transcription activity.Result:1.The mRNA expression level of ZNF667-AS1 and ZNF667 in esophageal cancer cell lines before and after 5-Aza-dC treament.ZNF667-AS1 and ZNF667 were up-regulated after 5-Aza-dC treatment in four esophageal cancer cell lines(P<0.05).2.The methylation status of ZNF667-AS1 and ZNF667 in esophageal carcinoma cell lines.The hypermethylation status of three regions of ZNF667-AS1 and ZNF667 was reversed after the four cell lines were treated with 5-Aza-dC.3.The methylation status of ZNF667-AS1 and ZNF667 in ESCC tissues and corresponding normal tissues.In ESCC tissues,the methylation status of every region of ZNF667-AS1 and ZNF667 were all higher than that in the corresponding normal tissues(P<0.05)and was closely correlated with pathological differentiation and TNM stages(P<0.05).4.The associations between the methylation status of different regions and ZNF667-AS1 and ZNF667 expression levels in ESCC tissues.The expression levels of ZNF667-AS1 and ZNF667 in the ESCC tissues with region 2 methylation were significantly lower than that with this region unmethylation(P<0.05).However,the methylation status of region 1 and region 3 in ESCC tissues was not associated with ZNF667-AS1 and ZNF667 expression(P>0.05).5.The impacts of different regions methylation status on ZNF667-AS1 and ZNF667 genes transcriptional activity.The distal CpG island and the proximal promoter region of ZNF667-AS1 could inhibit gene transcription(P<0.05),while the methylation frequency of exon1 was unrelated with gene transcription(P>0.05).Part Three Effects of ZNF667-AS1 on biological characteristics of esophageal cancer cell linesObjective: To detect the effects and mechanisims of over-expression of ZNF667-AS1 on cell proliferation,migration and invasion in ESCC.Methods:1.The pcDNA3.1-ZNF667-AS1 vector was transfected into Eca109 cell lines,and qRT-PCR method was employed to detect the transfection efficiency.2.MTS and clone formation assay were used to determine the effects of over-expressed ZNF667-AS1 on cell proliferation capacity of esophageal cancer cell lines in vitro.3.Transwell chamber migration assay was applied to detect the effect of over-expressed ZNF667-AS1 on cell migration capacity of esophageal cancer cell lines in vitro.4.Transwell chamber invasion assay was applied to detect the effects of over-expressed ZNF667-AS1 on cell invasion capacity of esophageal cancer cell lines in vitro.5.qRT-PCR method was applied to detect the impacts of over-expressed ZNF667-AS1 on mRNA expression of ZNF667.6.qRT-PCR method was used to detect the mRNA expression level of EMT-related genes(E-cadherin,Snail,Vimentin,Zeb1 and Twist1)in pcDNA3.1-ZNF667-AS1 transfected Eca109 cells.Result:1.Construction and validation of over-expressed ZNF667-AS1 gene cell lines.Successfully established Eca109 cell lines overexpressing ZNF667-AS1,qRT-PCR showed the relative expression of ZNF667-AS1 in Eca109 cell lines was significantly higher than that in the control group(P<0.05).2.Effects of overexpression of ZNF667-AS1 on the proliferative ability of esophageal cancer cells in vitro.Overexpression of ZNF667-AS1 could inhibit the proliferative activity of Eca109 cells in vitro detected by MTS and clonal formation assays(P<0.05).3.Effects of overexpression of ZNF667-AS1 on the migration ability of esophageal cancer cells in vitro.Over-expression of ZNF667-AS1 could inhibit the migration ability of Eca109 cells in vitro detected by transwell ventricular migration assay(P<0.05).4.Effects of overexpression of ZNF667-AS1 on the invasion ability of esophageal cancer cells in vitro.Over-expression of ZNF667-AS1 could inhibit the invasive ability of Eca109 cells in vitro detected by transwell ventricular invasion assay(P<0.05).5.Effects of over-expressed of ZNF667-AS1 on mRNA expression levels of ZNF667.qRT-PCR analysis showed that the relative mRNA expression level of ZNF667 in ZNF667-AS1 over-expressed Eca109 cells was significantly higher than that in the control group(P<0.05).6.Effects of ZNF667-AS1 on EMT-related genes after overexpression.The mRNA expression level of E-cadherin in ZNF667-AS1 over-expressed Eca109 cells was significantly increased compared with control cells(P<0.05),while the mRNA expression level of Snail,Vimentin,Zeb1,and Twist1 was significantly decreased in ZNF667-AS1 over-expressed Eca109 cells(P<0.05).Conclusions:1.ZNF667-AS1 and ZNF667 were downregulated in esophageal cancer cell lines and ESCC tissues,and may be closely related to the occurrence and development of ESCC.2.Promoter hypermethylation of ZNF667-AS1 and ZNF667 was detected in ESCC,and the abnormal hypermethylation of proximal promoter region may be one of the mechanisms in ZNF667-AS1 and ZNF667 silence.3.Overexpression of ZNF667-AS1 significantly inhibit esophageal cancer cells biological characteristics in vitro.ZNF667-AS1 may play vital roles in inhibiting esophageal cancer cells invasion and metastasis by regulating the expression of EMT-related genes. |
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