赵宝山, 邢志君, 孙光蕊, 张志民, 张旭, 梁宗英. 甲基转移酶DNMT3b和SPG20甲基化在肺腺癌中的相关性研究[J]. 中国肿瘤临床, 2021, 48(19): 973-978. DOI: 10.12354/j.issn.1000-8179.2021.20211076
引用本文: 赵宝山, 邢志君, 孙光蕊, 张志民, 张旭, 梁宗英. 甲基转移酶DNMT3b和SPG20甲基化在肺腺癌中的相关性研究[J]. 中国肿瘤临床, 2021, 48(19): 973-978. DOI: 10.12354/j.issn.1000-8179.2021.20211076
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Baoshan Zhao, Zhijun Xing, Guangrui Sun, Zhimin Zhang, Xu Zhang, Zongying Liang. Correlation of methyltransferase DNMT3b and SPG20 methylation in lung adenocarcinoma[J]. CHINESE JOURNAL OF CLINICAL ONCOLOGY, 2021, 48(19): 973-978. DOI: 10.12354/j.issn.1000-8179.2021.20211076
Citation: Baoshan Zhao, Zhijun Xing, Guangrui Sun, Zhimin Zhang, Xu Zhang, Zongying Liang. Correlation of methyltransferase DNMT3b and SPG20 methylation in lung adenocarcinoma[J]. CHINESE JOURNAL OF CLINICAL ONCOLOGY, 2021, 48(19): 973-978. DOI: 10.12354/j.issn.1000-8179.2021.20211076
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甲基转移酶DNMT3b和SPG20甲基化在肺腺癌中的相关性研究

Correlation of methyltransferase DNMT3b and SPG20 methylation in lung adenocarcinoma

    摘要
  • 摘要:
      目的  探讨肺腺癌组织中DNA甲基转移酶3b（DNMT3b）表达与SPG20甲基化水平，分析二者相关性及在肺腺癌发病机制中的临床意义。
      方法  收集2020年4月至2021年4月于承德医学院附属医院收集的70例肺腺癌和癌旁组织，采用焦磷酸测序检测SPG20基因甲基化水平，采用实时荧光定量PCR（RT-qPCR）检测SPG20和DNMT3b mRNA的表达，采用免疫组织化学法及Western blot检测SPG20和DNMT3b的蛋白表达。统计分析SPG20甲基化水平与DNMT3b相关性及两者与临床病例特征的相关性。构建DNMT3b下调si-RNA，采用脂质体介导法，将DNMT3b si-RNA、阴性对照si-RNA和空白对照组转染A549细胞。使用RT-qPCR和Wesrern blot检测DNMT3b mRNA和蛋白表达水平，采用焦磷酸测序检测各组SPG20基因甲基化水平。
      结果  在癌组织和癌旁组织中，SPG20基因甲基化率分别为76.25%±5.74%和13.45%±2.41%，SPG20 mRNA相对表达量为0.18±0.03和1.21±0.17，差异具有统计学意义（均P<0.05）。DNMT3b mRNA相对表达量为1.62±0.27，高于癌旁组织的0.32±0.05；免疫组织化学检测结果显示SPG20蛋白表达阳性率分别为27.14%和72.86%，差异具有统计学意义（P<0.05）；DNMT3b蛋白表达阳性率分别为75.71%和22.86%，差异具有统计学意义（P<0.05）。Western blot检测结果显示DNMT3b在癌组织中表达为68.57%±3.18%，高于癌旁组织25.29%±1.35%；SPG20在癌组织中表达为16.87%±1.56%，低于癌旁组织的58.75%±2.68%，差异均具有统计学差异（均P<0.05）。肺腺癌组织中SPG20甲基化和DNMT3b具有相关性（r=0.437，P<0.05）；SPG20甲基化和DNMT3b与肿瘤TNM分期、组织分化和淋巴结转移密切相关（P<0.05）。体外实验显示，RNA干扰后DNMT3b si-RNA组中DNMT3b mRNA表达量最低，SPG20基因甲基化率明显下降。
      结论  在肺腺癌病变过程中，DNMT3b与SPG20基因甲基化存在明显的相关性，DNMT3b高表达可能是SPG20基因甲基化前重要的分子事件，在肺腺癌早期病变诊治中具有潜在应用价值。

     

    Abstract:
      Objective  To investigate the expression of DNA methyltransferase 3b (DNMT3b) and SPG20 methylation in lung adenocarcinoma tissue, and to analyze their correlation and clinical significance in the pathogenesis of lung adenocarcinoma.
      Methods  Seventy cases of lung adenocarcinoma and paracancerous tissue were collected in The Affiliated Hospital of Chengde Medical College from April 2020 to April 2021. The methylation level of SPG20 was detected by pyrophosphate sequencing; the expressions of SPG20 and DNMT3b mRNA were detected by a real-time fluorescence quantitative polymerase chain reaction, and the expressions of the SPG20 and the DNMT3b proteins were detected by immunohistochemical tests and Western blot. The correlation of the SPG20 methylation level and the DNMT3b, and the clinical case characteristics were statistically analyzed. The Si-RNA of downregulated DNMT3b was constructed and then transfected with A549 cells, using liposome-mediated methods in DNMT3b si-RNA negative control si-RNA, and blank controls. RT-qPCR and Western blot were used to detect DNMT3b mRNA and protein expression levels. SPG20 gene methylation was detected by pyrophosphate sequencing.
      Results  In the cancerous and paracancerous tissue, the methylation rates of the SPG20 were 76.25%±5.74% and 13.45%±2.41%. The relative expression of the SPG20 mRNA was 0.18±0.03 and 1.21±0.17, and the differences were statistically significant (P<0.05). The relative expression of DNMT3b mRNA was 1.62±0.27, higher than in the paracancerous tissue 0.32±0.05. Immunohistochemical results showed that the positive expression rate of the protein was 27.14% and 72.86% in the cancerous and paracancerous tissue respectively. This difference was statistically significant (P<0.05). Western blot results showed that the expression of DNMT3b was 68.57%±3.18% in cancerous tissue and 25.29%±1.35% in paracancerous tissue. The expression of SPG20 was 16.87%±1.56% in cancerous tissue and 58.75%±2.68% in paracancerous tissue. The differences were statistically significant (P<0.05). Hence, SPG20 methylation and DNMT3b were associated in lung adenocarcinoma tissue (r=0.437, P<0.05). SPG20 methylation and DNMT3b were closely related to tumor TNM staging, tissue differentiation, and lymph node metastasis (P<0.05). The results of in vitro experiments had further revealed that both the DNMT3b mRNA expression in the DNMT3b si-RNA group after RNA interference, and the SPG20 methylation rate, decreased significantly.
      Conclusions  DNMT3b is significantly related to SPG20 gene methylation during lung adenocarcinoma lesions. A higher DNMT3b expression may be an important molecular event before the methylation of the SPG20 gene and has important potential value in the diagnosis and treatment of early lesions of lung adenocarcinoma.

     

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