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[摘要]
目的:通过检测食管鳞状细胞癌(esophageal squamous cell carcinoma,ESCC)中长链非编码RNA XLOC_002319(long non-colding RNA XLOC_002319,lncRNA XLOC_002319)基因的表达及其甲基化状态,探讨XLOC_002319基因在ESCC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)的方法检测DNA甲基化转移酶抑制剂5-氮杂-2′-脱氧胞苷(5-aza-2′-deoxycitydine,5-aza-dC)处理前后的食管癌细胞株(TE1、TE13、Yes-2、Eca109和T.TN)、ESCC组织以及癌旁正常组织、食管上皮内瘤变(esophageal intraepithelial neoplasia,EIN)组织中XLOC_002319基因的表达和甲基化状态。结果:未经5-aza-dC处理的5种食管癌细胞中XLOC_002319基因的表达均呈阴性或弱阳性,经5-aza-dC的5种食管癌细胞中 XLOC_002319基因的表达均增高。5株食管癌细胞在5-aza-dC处理前表现为XLOC_002319高甲基化状态,处理后,Eca109和T.TN细胞系中XLOC_002319基因甲基化程度降低,其余3株细胞系中XLOC_002319基因均表现为非甲基化状态。XLOC_002319基因在ESCC组织中的表达显著低于食管上皮内瘤变组织和癌旁正常组织(P<0.01),并与组织学分化程度和TNM分期密切相关(P<0.05)。ESCC组织中XLOC_002319基因启动子区甲基化率为63.75%(51/80),显著高于食管上皮内瘤变组织和癌旁正常组织(P<0.01),并与淋巴结转移、组织学分化程度和TNM分期密切相关(P<0.05)。发生XLOC_002319基因甲基化的ESCC组织中XLOC_002319基因表达显著低于未发生甲基化的ESCC组织(P<001)。结论:XLOC_002319基因在ESCC中的低表达可能与ESCC的发生密切相关,且其启动子区甲基化可能是导致其表达沉默的机制之一。
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[Abstract]
Objective:To investigate the expression and methylation status of long non-coding RNA XLOC_002319 (lncRNA XLOC_002319) in esophageal squamous cell carcinoma (ESCC), and to elucidate its role in the progression of ESCC. Methods: Reverse transcription polymerase chain reaction (RT-PCR) and methylation specific PCR (MSP) were used respectively to detect the expression and methylation status of XLOC_002319 in esophageal cancer cell lines (TE1, TE13, Yes-2, Eca109, T.TN) treated or untreated with DNA methyltransferase inhibitor 5-aza-2′-detoxycytidine (5-aza-dC), and in cancer-adjacent normal tissues, esophageal intraepithelial neoplasia (EIN) tissues and ESCC tissues. Results:Negative or weak positive expression of XLOC_002319 was detected in the five esophageal cancer cell lines untreated with 5-aza-dC; however, after the treatment with 5-aza-dC, the expression of XLOC_002319 was enhanced. The methylation status of XLOC_002319 was highly expressed in esophageal cancer cell lines before the treatment of 5-aza-dc, however, after the treatment, the methylation level was decreased in Eca109 and T.TN cell lines, while the status in other three cell lines was negative. The expression of XLOC_002319 gene in ESCC tissues was significantly reduced compared to cancer-adjacent tissues and EIN tissues (P<0.01), and was closely associated with pathological differentiation and TNM stage (P<0.05). The methylation frequency of XLOC_002319promoter in ESCC tissues(63.75% \[51/80\]) was significantly higher than that in EIN tissues and adjacent normal tissues (P<0.01), and it was also associated with lymph node metastasis, pathological differentiation and TNM stage (P<0.05). The expressions of XLOC_002319 in ESCC tumor tissues with methylation of the gene was significantly lower than that in tumor tissues with unmethylation of the gene (P<0.01). Conclusion: Aberrant low expression of lncRNA XLOC_002319 was closely related to the development and occurrence of ESCC, and promoter methylation might be one of the mechanisms for inactivation of XLOC_002319 in ESCC.
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[基金项目]
国家自然科学基金资助项目(No.81572441);河北省自然科学基金资助项目(No. H2015206196)