长链非编码RNA XLOC_005009在食管鳞状细胞癌中的表达及其甲基化状态

Expression and Methylation Status of Long Non-coding RNA XLOC_005009 in Esophageal Squamous Cell Carcinoma

[目的]通过检测长链非编码RNA XLOC_005009在人食管癌细胞系及食管鳞状细胞癌(ESCC)组织中的表达情况及其甲基化状态,探讨XLOC_005009基因在食管鳞癌发生、发展中的作用及表观遗传学失活机制。[方法]分别应用逆转录—聚合酶链反应以及甲基化特异性聚合酶链反应的方法检测XLOC_005009基因在DNA甲基化转移酶抑制剂5-Aza-d C处理前后的食管癌细胞系(TE1、TE13、T.Tn、Eca109)以及57例食管鳞癌组织及相应癌旁正常组织中的表达情况和各位点的甲基化状态。[结果]XLOC_005009基因的表达在5-Aza-d C处理后的4株细胞系中表达增高,在5-Aza-d C处理前的4株细胞系中XLOC_005009基因的3个位点均表现为高甲基化状态,在5-Aza-d C处理后,各位点甲基化程度均降低。XLOC_005009基因在ESCC中的表达明显低于对应癌旁正常组织(0.21±0.22 vs 0.32±0.29,P<0.05),其远端Cp G岛及第2外显子区甲基化率在ESCC与其癌旁正常组织中差异无统计学意义,且其在发生甲基化的ESCC与未发生甲基化的ESCC中的表达无统计学差异。第一外显子区甲基化率在ESCC中显著高于其癌旁正常组织[45.61%(26/57)vs 19.30%(11/57),P<0.05],并与淋巴结转移、组织分化程度及TNM分期密切相关,且此位点发生甲基化的ESCC中该基因的表达显著低于未发生甲基化的ESCC组织(0.06±0.06 vs 0.33±0.23,P<0.05)。[结论]XLOC_005009基因在食管癌细胞系和ESCC中的低表达与ESCC的发生、发展密切相关,且其第一外显子区甲基化异常增高可能是导致其表达沉默的机制之一。

[Purpose] To investigate the expression and methylation status of long non-coding RNA XLOC_005009 in human esophageal cancer cell lines and esophageal squamous cell carcinoma（ESCC） tissues. [Methods] Reverse transcription-polymerase chain reaction（RT-PCR） and methylation specific polymerase chain reaction（MSP） methods were applied to detect the expression and methylation status of XLOC_005009 in esophageal cancer cell lines（TE1,TE13,T.TN and Eca109） treated or untreated with DNA methylation transferase inhibitor 5-Aza-d C and ESCC tissues and the corresponding noncancerous tissues. [Results] The expression of XLOC_005009 in 4treated cell lines was increased. Each site of XLOC_005009 gene showed hypermethylation in the4 untreated cell lines,after 5-Aza-d C treatment,the methylation status of XLOC_005009 was decreased. The expression of XLOC_005009 in ESCC tissues was significantly lower than that in corresponding normal tissues（0.21 ±0.22 vs 0.32 ±0.29,P〈 0.05）. Methylation frequency of XLOC_0059009 in the distal Cp G islands and the exon 2 region was not significantly different between ESCC tissues and corresponding normal tissues. The expression of XLOC_005009 in ESCC tissues with XLOC_005009 methylation in the distal Cp G islands and the exon 2 region was not significantly different from that in ESCC tissues without methylation of both regions. Methylation frequency of XLOC_0059009 in the exon 1 region was significantly higher in ESCC tissues than that in corresponding normal tissues [45.61%（26/57） vs 19.30%（11/57）,P0.05],and was closely correlated with lymph node metastasis,pathological differentiation and TNM stages. The expression of XLOC_005009 in ESCC tissues with XLOC_005009 methylation in the exon 1 was significantlylower than that in ESCC tissues without methylation of this region（0.06 ±0.06 vs 0.33 ±0.23,P〈0.05）. [Conclusion] The decreased expression of XLOC_005009 is closely related to the occurrence and development of ESCC and its exon 1 region methylation may be one of the mechanisms to lead to the silence of XLOC_005009.