南方医科大学学报

南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (3): 329-335.doi: 10.12122/j.issn.1673-4254.2021.03.03

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m6A修饰调控的LDB2抑制肺腺癌细胞增殖

翟东凤，王 鸽，李 蕾，贾小婷，郑国沛，尹 江   

  • 出版日期:2021-03-20 发布日期:2021-04-04

LIM-domain binding protein 2 regulated by m6A modification inhibits lung adenocarcinoma cell proliferation in vitro

  • Online:2021-03-20 Published:2021-04-04

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摘要：

目的 研究LIM域结合蛋白2（LDB2）在肺腺癌中的表达模式及作用。方法 从mRNA和蛋白表达数据库中分析在肺腺癌中表达下调基因的交集。通过qRT-PCR、Western blot和免疫组化的方法验证LDB2在肺腺癌中的表达模式。在GEO和TCGA数据库中分析LDB2与肺腺癌患者预后的相关性。在H1299细胞中过表达LDB2，通过细胞计数实验、软琼脂克隆实验及流式细胞术证明LDB2在肺腺癌中的作用。生信预测结合后期MeRIP-qPCR实验证实LDB2转录本上存在m6A结合位点。通过qRT-PCR和Western blot实验检测YTHDC2对LDB2的转录调控。RIP实验验证YTHDC2能否与LDB2转录本结合。结果 通过分析TCGA、GEO及CPTAC 3个数据集中在肺腺癌中低表达的差异基因，最终聚焦于LDB2基因。免疫组化结果证实该基因在80例肺腺癌组织中的表达量也显著低于17例正常癌旁组织的表达量。与正常肺上皮细胞16HBE相比，LDB2在肺腺癌细胞中的表达量亦明显降低。生信分析提示高表达的LDB2与肺腺癌患者良好的预后正相关。在H1299细胞中过表达LDB2后，发现LDB2可诱导H1299细胞发生S期阻滞，从而抑制该细胞的增殖能力和软琼脂克隆形成能力。生信预测结合后期MeRIP-qPCR实验证实LDB2 转录本上存在m6A位点。GEPIA数据库提示YTHDC2在肺腺癌组织中低表达，且与LDB2在肺腺癌中表达正相关（r=0.22，P<0.0001）。在H1299细胞中过表达YTHDC2，可明显增加LDB2的表达。最后，在过表达YTHDC2的H1299细胞中开展RIP实验，定量结果显示LDB2在YTHDC2组的含量是其在IgG组含量的19.35倍，YTHDC2可结合在LDB2转录本上调控其表达。双荧光素酶报告基因实验证实YTHDC2可上调野生型而不是缺失型报告基因载体活性。结论 m6A编码器 YTHDC2在肺腺癌中上调LDB2的表达。过表达LDB2可明显诱导肺腺癌细胞发生S期阻滞，抑制肺腺癌细胞增殖能力。

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Abstract:

Objective To investigate the role and expression pattern of LIM-domain binding protein 2 (LDB2) in lung adenocarcinoma. Methods We studied the expression pattern of LDB2 in lung adenocarcinoma based on data from the online databases TCGA, GEO and CPTAC, and the results were verified in lung adenocarcinoma tissues and cells using
immunohistochemistry, qRT-PCR and Western blotting. The relationship between LDB2 and the prognosis of patients with lung adenocarcinoma was analyzed using GEPIA and GEO databases. We further analyzed the role of LDB2 in regulating cell behaviors in a H1299 cell model over-expressing LDB2 using cell counting, soft agar colony forming assay and flow cytometry. The m6 A binding sites on LDB2 were confirmed by bioinformatics analysis and MeRIP-qPCR assays. The effect of YTHDC2 on LDB2 was examined using qRT-PCR and Western blotting, and the binding of YTHDC2 to the transcript of LDB2 was verified with RIP-qPCR assays. Dual luciferase reporter assay was performed to verify YTHDC2 functioning via m6 A sites. Results LDB2 expression was significantly decreased in lung adenocarcinoma in comparison with normal tissues based on data from TCGA, GEPIA and CPTAC, and the same results were obtained from 80 lung adenocarcinoma tissues and 17 adjacent normal tissues. Similarly, LDB2 expression was decreased in lung adenocarcinoma cells as compared with 16HBE cells. The data from Prognoscan and GEPIA suggested that a high LDB2 expression was positively correlated with a more favorable outcome of lung adenocarcinoma patients. LDB2-overexpressing H1299 cells showed a significant inhibition of proliferative activity with cell cycle arrest in S phage. Bioinformatics analysis and MeRIP-qPCR assay confirmed the presence of m6 A sites on LDB2. The m6 A reader YTHDC2 was positively related with LDB2 in lung adenocarcinoma based on data from GEPIA (r=0.22). Overexpression YTHDC2 significantly enhanced LDB2 expression in H1299 cells by about 19.35 folds. Dual luciferase reporter assay showed that YTHDC2 enhanced the promoter activity in the wild-type group but not in deletion group. Conclusion LDB2 expression can be up-regulated by m6 A reader YTHDC2 in lung adenocarcinoma to inhibit the proliferation of the tumor cells in vitro.

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