TFFl甲基化和甲基转移酶SETDB1表达在肺腺癌中的相关性研究

Study on the correlation between TFF1 methylation and methyltransferase SETDB1 expression in lung adenocarcinoma

    摘要
  • 摘要:
    目的 探究SET结构域分支型1（SET domain bifurcated 1，SETDB1）表达与三叶因子1（trefoil factor 1，TFF1）基因甲基化的相关性及临床意义。
    方法 选取于河北省承德医学院附属医院就诊的55例肺腺癌患者的肺腺癌及远癌组织，焦磷酸测序法检测TFF1基因甲基化水平，实时荧光定量聚合酶链反应（qRT-PCR）检测TFF1 mRNA相对表达，免疫组织化学方法检测TFF1和SETDB1蛋白表达。统计分析TFF1甲基化水平与SETDB1的相关性及临床意义。采用脂质体介导法将SETDB1 siRNA和阴性对照siRNA转染至肺腺癌A549细胞，应用qRT-PCR和Wesrern blot检测SETDB1 mRNA和蛋白表达水平；采用焦磷酸测序检测各组TFF1基因甲基化水平。
    结果 肺腺癌组织和远癌组织中TFF1基因甲基化率分别为（70.16±6.32）% 和（12.48±2.22）%；TFF1 mRNA 相对表达量分别为0.56±0.17和1.56±0.22，差异均具有统计学意义（均P<0.05）。免疫组织化学结果显示，TFF1蛋白阳性表达率分别为29.09%（16/55）和65.45%（36/55）；SETDB1蛋白阳性表达率分别为 74.55%（41/55） 和23.64%（13/55），差异均有统计学意义（均P<0.05）。Western blot 结果显示 SETDB1在癌组织中表达为（72.89±5.27）%，高于远癌组织（24.27±2.37）%；TFF1 在癌组织中表达为（15.38±2.33）%，低于远癌组织（72.72±4.48）%，差异均具有统计学意义（均 P<0.05）。肺腺癌组织中TFF1甲基化和SETDB1表达具有相关性（r=0.486，P<0.05）；TFF1甲基化和SETDB1与肿瘤TNM分期、组织分化和淋巴结转移密切相关（P<0.05）。下调SETDB1后，TFF1基因甲基化率上升（P<0.05）。
    结论 在肺腺癌病变进程中，SETDB1的表达与TFF1基因甲基化水平有相关性，SETDB1高表达可能是TFF1基因甲基化的催化酶。

     

    Abstract:
    Objective To investigate the relationship between SET domain bifurcated 1 (SETDB1) expression and trefoil factor 1 (TFF1) gene methylation, along with its clinical significance.
    Methods Fifty-five lung adenocarcinoma samples and normal tissues of distant cancer were collected from the Affiliated Hospital of Chengde Medical College Hebei Province. TFF1 gene methylation levels were measured by pyrosequencing, relative TFF1 mRNA expression was assessed using quantitative real-time polymerase chain reaction (qRT-PCR), and TFF1 and SETDB1 protein expression were quantified via immunohistochemistry. The clinical significance and correlation between TFF1 methylation levels and SETDB1 protein expression were analyzed statistically. In vitro, SETDB1 siRNA or negative control siRNA was transfected into A549 cells. Following transfection, SETDB1 mRNA expression was analyzed using qRT-PCR, and SETDB1 protein expression was evaluated via Western blot. TFF1 methylation was reassessed via pyrosequencing.
    Results In lung adenocarcinoma and normal tissues of distant cancer, TFF1 gene methylation rates were (70.16±6.32)% and (12.46±2.22)%, respectively. TFF1 mRNA relative expression levels were 0.56±0.17 for cancer tissues and 1.56±0.22 for the normal tissues of distant cancer. All detected differences were statistically significant (all P<0.05). TFF1 protein expression rates were 29.09% (16/55) for cancer tissues and 65.45% (36/55) for the normal tissues of distant cancer. Additionally, relative positivity rates for SETDB1 protein expression were 74.55% (41/55) and 23.64% (13/55), respectively, and all differences were statistically significant (all P<0.05). Western blot analysis showed that SETDB1 protein expression was significantly higher in cancer tissues (72.89±5.27)%, compared to normal tissues of distant cancer (24.27±2.37)%. In contrast, TFF1 protein expressed was markedly lower in cancer tissues (15.38±2.33)% than in normal tissues of distant cancer (72.72±4.48)%. All differences were statistically significant (all P<0.05). TFF1 methylation and SETDB1 expression were associated with lung adenocarcinoma (r=0.486, P<0.05). Both TFF1 methylation and SETDB1 expression were closely associated with tumor TNM stage, tissue differentiation, and lymph node metastasis (P<0.05). Furthermore, TFF1 methylation increased following SETDB1 downregulation (P<0.05).
    Conclusions During lung adenocarcinoma progression, SETDB1 expression correlates with TFF1 methylation, and the highly expressed SETDB1 may play a role in catalyzing TFF1 methylation.

     

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