非编码长链RNA LOC100130476在食管鳞癌中的表达及其甲基化状态

Expression and m ethylation status of long non-coding RNA LOC100130476 in esophageal squamous cell carcinoma

目的 :检测人食管癌细胞系及食管鳞状细胞癌(esophageal squamous cell carcinoma,ESCC)组织中长链非编码RNA(long non-coding RNA,lnc RNA)LOC100130476的表达情况及其甲基化状态,并探讨lnc RNA LOC100130476在食管鳞癌发生及发展中的作用。方法 :分别应用RT-PCR以及甲基化特异性PCR(methylation specific PCR,MSP)法检测DNA甲基化转移酶抑制剂5-氮杂-2’-脱氧胞苷(5-aza-2’-deoxycytidine,5-Aza-d C)处理前后的食管癌细胞系TE1、TE13、T.TN和Ec109细胞中以及食管鳞癌组织及其相应癌旁正常组织中lnc RNA LOC100130476的表达情况和甲基化状态。结果:未经5-Aza-d C处理的食管癌TE1、TE13和T.TN细胞中lnc RNA LOC100130476的表达均为阴性,仅在Ec109细胞中为弱阳性表达;用5-Aza-d C处理后,TE1、TE13和T.TN细胞中lnc RNA LOC100130476均转变为阳性表达,而Ec109细胞中lnc RNA LOC100130476的表达水平也有所上调。TE1、TE13、T.TN和Ec109细胞中,在5-Aza-d C未处理前lnc RNA LOC100130476均表现为高甲基化的状态,应用5-Aza-d C处理后,Ec109细胞中lnc RNA LOC100130476甲基化程度明显降低,TE1、TE13和T.TN细胞中的lncR NA LOC100130476则均表现为非甲基化状态。LncR NA LOC100130476在食管鳞癌组织中的表达水平为0.49±0.09,明显低于癌旁正常组织的0.56±0.08(P<0.05),并与组织分化程度和TNM分期密切相关(P值均<0.05)。食管鳞癌组织中lnc RNA LOC100130476第一外显子区的甲基化率为69.44%(50/72),明显高于癌旁正常组织的26.39%(19/72)(P<0.05),并与组织分化程度和TNM分期密切相关(P值均<0.05)。发生lnc RNA LOC100130476甲基化的食管鳞癌组织中lnc RNA LOC100130476的表达量为0.47±0.08,明显低于未发生甲基化的食管鳞癌组织的0.55±0.08(P<0.05)。结论 :Lnc RNA LOC100130476在食管鳞癌中的异常低表达与食管鳞癌的发生和发展密切相关,其第一外显子区的甲基化可能是导致其表达沉默的机制之一。

Objective： To detect the expression and methylation status of long non-coding RNA （IncRNA） LOC100130476 in esophageal cancer cell lines and esophageal squamous cell carcinoma （ESCC） tissues, and to explore its role in occurrence and development of ESCC.Methods： The expression level of IncRNA LOC100130476 and its methylation status in esophageal cancer cell lines （TEl, TE13, T.TN and Ec109） treated or not treated with DNA methyltransferase inhibitor 5-aza-2＇-deoxycytidine （5-Aza-dC）, ESCC tissues and the corresponding para-cancerous normal tissues were detected by RT-PCR and methylation- specific PCR （MSP）.Results： The expressions of IncRNA LOC100130476 were negative in TEl, TE13 and T.TN cells without 5-Aza-dC treatment, but the IncRNA LOC1 00130476 demonstrated weak expression in Ec109 cells. After 5-Aza-dC treatment, positive expression of IncRNA LOC1 00130476 was detected in TEl, TEl3 and T.TN cells, and the expression level of IncRNA LOC100130476 in Ec109 cells was increased. Hypermethylation of IncRNA LOC100130476 before 5-Aza- dC treatment was detectable in TE1, TE13, T.TN and Ec109 cells. However, after 5-Aza-dC intervention, the methylation level of IncRNA LOC100130476 was reduced in Ec109 cells, but unmethylated status in TE1, TE13 and T.TN cells. The IncRNA LOC100130476 expression level in ESCC tissues was significantly lower than that in para-cancerous normal tissues （0.49± 0.09 vs 0.56±0.08, P 〈 0.05）, and it was closely correlated with pathological differentiation and TNM stages （both P 〈 0.05）. The methylation frequency in first exon region of IncRNA LOC100130476 in ESCC tissues （69.44%, 50/72） was significantly higher than that in the corresponding para-cancerous normal tissues （26.39%, 19/72） （P 〈 0.05）, and it was closely correlated with pathological differentiation and TNM stages （both P 〈 0.05）. The expression of IncRNA LOC100130476 in ESCC tissues with IncRNA LOC100130476 methylation （0.47±0.08） was significantly lower than that in ESCC tissues without methylation of the gene （0.55±0.08） （P 〈 0.05）.