| With the in-depth study and understanding of human genome,more and more long non-coding RNAs(lnc RNAs)have been discovered,and their role in tumorigenesis has also been continuously studied.Based on the results of the laryngeal squamous cell carcinoma tissue chip performed in the previous work,our group found that LINC00886 is a lnc RNA that has been screened for differential expression in tumor tissues and normal tissues.Due to the overall pattern of mutation spectrum of esophageal squamous cell carcinoma(ESCC)is similar with head and neck squamous cell carcinoma,so we speculated that LINC00886,which was differentially expressed in laryngeal squamous cell carcinoma,may also play an important role in the initiation and progression of ESCC.This study examined the expression level of LINC00886 in esophageal cancer cell lines and ESCC tissues,and its effect of methylation status on transcriptional level,an overexpression plasmid was constructed to investigate its effect on the proliferation,migration,and invasion of esophageal cancer cells in vitro,analyzed the mechanism of its effect on the biological behavior of esophageal cancer cells initially.The main research contents and results are as follows: Part one Expression level of LINC00886 in esophageal cancer cells and esophageal squamous cell carcinoma tissuesObjective: To study the expression of LINC00886 in esophageal cancer cell lines and esophageal squamous cell carcinoma tissues.And to analyze the relationship between its expression level and clinical pathological parameters.Methods:1.Quantitative real-time reverse transcription polymerase chain reaction(q RT-PCR)method was used to detect the expression of LINC00886 in 4 esophageal cancer cell lines(Eca109,TE1,Kyse150 and YES2).2.q RT-PCR method was used to detect the expression of LINC00886 in 62 cases of esophageal squamous cell carcinoma and its corresponding adjacent normal tissues.And the analysis between its expression and clinical pathological parameters was conducted.3.The nucleus and cytoplasm of esophageal cancer cell lines were isolated by Paris kit,and the nucleus and cytoplasm localization of LINC00886 in esophageal cancer cell lines was detected by q RT-PCR.Results:1.The expression of LINC00886 in esophageal cancer cell lines: q RT-PCR results showed that LINC00886 was all down-regulated in 4 esophageal cancer cell lines(P < 0.01).2.The expression of LINC00886 in ESCC tissues: q RT-PCR results showed that the expression of LINC00886 in ESCC tissues was low.It was associated with lymph node metastasis and TNM stage(P < 0.05).3.Subcellular localization of LINC00886 in esophageal cancer cell lines: q RT-PCR results showed that in Eca109,Kyse150 and YES2 cells,LINC00886 is mainly distributed in the nucleus,and in TE1 cells,LINC00886 is mainly distributed in the cytoplasm.Part Two Methylation status of LINC00886 in esophageal squamous cell carcinoma and the effect on transcriptional activityObjective: To analyze the methylation status of Cp G island near the promoter region of LINC00886 in esophageal cancer cell lines and esophageal squamous cell carcinoma tissues,and to investigate the methylation effect on gene transcriptional activity.Methods:1.Esophageal cancer cell lines(Eca109,TE1,Kyse150,and YES2)were treated with TSA and 5-Aza-d C separately and sequentially,and the changes of LINC00886 expression level were detected by q RT-PCR.2.The methylation status of Cp G islands in different regions of LINC00886 genomic DNA in Eca109 and Kyse150 cells was detected by bisulfite genomic sequencing(BGS).3.Methylation specific polymerase chain reaction(MSP)was used to detect the methylation status of 4 esophageal cancer cell lines(Eca109,TE1,Kyse150 and YES2)before and after 5-Aza-d C treatment,29 cases of esophageal squamous cell carcinoma and their corresponding adjacent tissues in each region of the Cp G island of LINC00886.The Cp G island of LINC00886 is divided into three regions,namely the distal promoter region,the proximal promoter region and the intron region.4.The effect of methylation status of each region of the Cp G island of LINC00886 on transcriptional activity was examined by dual-luciferase reporter gene method.Three reporter gene vectors in total was constructed of LINC00886.Region one is the distal promoter region and the proximal promoter region(p GL3-Region 1),region two is the proximal promoter region(p GL3-Region 2),and region three is the intron region(p GL3-Region 3).Results:1.The expression level of LINC00886 before and after TSA and 5-Aza-d C alone and sequential treatment: q RT-PCR results showed that in addition to TSA treatment group of YES2 cells,the expression levels of LINC00886 in Eca109,TE1 and Kyse150 cells were increased after alone and sequential treatment with TSA,5-Aza-d C(P < 0.05 or P < 0.01).2.The methylation status of LINC00886 in the Cp G island near the promoter region in Eca109 and Kyse150 cells: BGS results showed that the CG sites in the proximal promoter region and the intron region of LINC00886 were highly methylated in Eca109 and Kyse150 cells.The distal promoter region was unmethylated in Eca109 cells and hypomethylated in Kyse150 cells.3.Methylation status of LINC00886 in esophageal cancer cell lines: 3 regions were hypermethylated in each cell line before 5-Aza-d C treatment,and the methylation degree of proximal promoter region and intron region was reduced after 5-Aza-d C treatment.While in distal promoter region,the degree of methylation of TE1,Kyse150 and YES2 cell lines was reduced,but the methylation status of Eca109 before and after 5-Aza-d C treatment was no significant changes.4.The methylation status of LINC00886 in ESCC and its adjacent normal tissues: the methylation rate of the proximal promoter region and intron region of LINC00886 in ESCC tissues is higher than that in adjacent normal tissues(P < 0.01),and associated with lymph node metastasis(P < 0.05).While in the distal promoter region,the methylation rate was no significant difference(P > 0.05).5.The relationship between the methylation status near the promoter region of LINC00886 and its expression in ESCC tissues: the degree of methylation in the proximal promoter region was negatively correlated with expression(P < 0.05),while there was no significant correlation between the methylation status of the distal promoter and the intron region and expression(P > 0.05).6.The effect of methylation status of each region near the promoter region of LINC00886 on gene transcriptional activity: methylation status of region 1 and region 2 can inhibit gene transcription level,and the inhibitory effect of region 2 was significant(P < 0.01),and the methylation status of region 3 had no significant effect on the transcription level(P > 0.05).Part Three Effect of the expression level of LINC00886 on biological behaviors of esophageal cancer cellsObjective: To study the effect of overexpression of LINC00886 on the biological behaviors of esophageal cancer cells.Methods:1.The overexpression vector pc DNA3.1-LINC00886 was constructed and transfected into esophageal cancer cells Eca109 and Kyse150,and the transfection efficiency was verified by q RT-PCR.2.MTS assay and clone formation assay were used to detect the effect of overexpression of LINC00886 on proliferation ability of esophageal carcinoma cells in vitro.3.The wound healing assay was used to detect the effect of overexpression of LINC00886 on migration ability of esophageal carcinoma cells in vitro.4.The Transwell chamber invasion assay was used to detect the effect of overexpression of LINC00886 on invasion ability of esophageal carcinoma cells in vitro.5.The effect of overexpression of LINC00886 on the m RNA expression of EMT-related markers(E-cadherin,N-cadherin,Vimentin,?-catenin?Twist?Snail?ZEB1)was detected by q RT-PCR.6.Western blot assay was used to detect the protein expression of EMT-related markers(E-cadherin,N-cadherin,Vimentin,?-catenin)after overexpression of LINC00886.Results:1.Construction of overexpression vector pc DNA3.1-LINC00886 and validation of transfection efficiency: the overexpression vector pc DNA3.1-LINC00886 was successfully constructed and transfected into esophageal cancer cells Eca109 and Kyse150,and the transfection efficiency was verified by q RT-PCR.The relative expression of the LINC00886 was significantly higher than that of the control group(P < 0.01).2.The effect of overexpression of LINC00886 in vitro on proliferation ability of esophageal cancer cells: MTS assay and clone formation assay showed that overexpression of LINC00886 significantly inhibited the proliferation ability of esophageal cancer cells Eca109 and Kyse150 in vitro(P < 0.05 or P < 0.01).3.The effect of overexpression of LINC00886 on the migration ability of esophageal carcinoma cells in vitro: the results of wound healing assay showed that overexpression of LINC00886 significantly inhibited the migration ability of esophageal cancer cells Eca109 and Kyse150 in vitro(P < 0.05 or P < 0.01).4.The effect of overexpression of LINC00886 in vitro on invasion ability of esophageal cancer cells: the Transwell chamber invasion assay showed that overexpression of LINC00886 significantly inhibited the invasion ability of esophageal cancer cells Eca109 and Kyse150 in vitro(P < 0.05 or P < 0.01).5.The effect of overexpression of LINC00886 on the m RNA expression of EMT-related markers: q RT-PCR results showed that the m RNA expression level of E-cadherin was increased after overexpression of LINC00886(P < 0.01)and N-cadherin,Vimentin,?-catenin and ZEB1 were decreased in Eca109 and Kyse150 cells.And the m RNA expression levels of Twist and Snail were decreased in Kyse150 cells(P < 0.05 or P < 0.01).6.The effect of overexpression of LINC00886 on protein expression level of EMT-related markers: Western blot assay showed that the protein expression level of E-cadherin was elevated after overexpression of LINC00886 in Eca109 and Kyse150 cells,N-cadherin,Vimentin and ?-catenin had a reduced level of protein expression(P < 0.01).Conclusions:1.Long non-coding RNA LINC00886 is lowly expressed in esophageal cancer cells and ESCC tissues,and is associated with lymph node metastasis and TNM stage,it may be involved in the development process of ESCC.2.The abnormal hypermethylation state was presented at near the promoter region of LINC00886,and hypermethylation of the proximal promoter region may be one of the main mechanism leading to its silence.3.Overexpression of LINC00886 can significantly attenuate the malignant biological behaviors of esophageal cancer cells in vitro,and may affect the invasion and metastasis of esophageal cancer cells by regulating the expression of EMT pathway related markers. |
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