LncRNA DLG1-AS1调控miR-203/ZEB2轴诱导甲状腺乳头状癌细胞恶性化生长和转移的机制研究-东南大学学报医学版

LncRNA DLG1-AS1调控miR-203/ZEB2轴诱导甲状腺乳头状癌细胞恶性化生长和转移的机制研究

单位：临沂市中医医院普外三科, 山东临沂 276000

关键词：LncRNA DLG1-AS1 miR-203 上皮间质转化甲状腺乳头状癌增殖转移

分类号：R736.1

出版年·卷·期（页码）：2021·40·第二期（133-141）

摘要：

目的：探讨LncRNA DLG1-AS1对甲状腺乳头状癌（PTC）细胞恶性化生长和转移的影响，并对其可能的分子机制进行研究。方法：体外培养PTC细胞（TPC1、KTC-1、B-CPAP、HTori-3）和正常甲状腺上皮细胞Nthy-ori3-1，采用实时荧光定量聚合酶链反应（qRT-PCR）检测细胞中DLG1-AS1和miR-203表达差异。将B-CPAP细胞分为空白对照组、阴性对照组（转染pcDNA3.1空载体和阴性对照序列）、shDLG1-AS1组（转染pcDNA3.1-shDLG1-AS1）、shDLG1-AS1+miR-203抑制物组（转染pcDNA3.1-shDLG1-AS1和miR-203抑制物）。通过CCK-8法和Transwell小室法检测细胞增殖、迁移和侵袭活性。蛋白质印迹法检测上皮间质转化（EMT）相关蛋白表达。双荧光素酶报告分析DLG1-AS1、miR-203、ZEB2之间的靶关系。结果：经qRT-PCR法检测，与Nthy-ori3-1细胞相比，TPC1、KTC-1、B-CPAP、HTori-3细胞中DLG1-AS1相对表达量均升高，同时miR-203相对表达量均降低（均P<0.05），选择DLG1-AS1相对表达量最高的B-CPAP细胞进行后续实验。与空白对照组和阴性对照组相比，shDLG1-AS1组细胞增殖、迁移、侵袭活性降低，ZEB2、N-cadherin、vimentin、β-catenin蛋白表达下调，同时E-cadherin表达上调（均P<0.05）。与shDLG1-AS1组相比，shDLG1-AS1+miR-203 inhibitor组细胞增殖、迁移、侵袭活性被逆转，ZEB2、N-cadherin、vimentin、β-catenin蛋白表达上调，同时E-cadherin表达下调（均P<0.05）。经双荧光素酶报告证实，DLG1-AS1可以直接调控miR-203，而ZEB2则是miR-203的下游靶基因。结论：LncRNA DLG1-AS1通过与miR-203竞争结合，消除miR-203对靶基因ZEB2表达的抑制，促进PTC细胞发生EMT，这可能是PTC恶性化生长和转移的重要机制。

Objective: To investigate the effect of LncRNA DLG1-AS1 on the proliferation and metastasis of papillary thyroid carcinoma(PTC) cells, and further to explore its mechanism. Methods: PTC cells TPC1,KTC-1,B-CPAP,HTori-3 and normal follicular cell Nthy-ori3-1 were used to detect DLG1-AS1 and miR-203 expressions by real time quantitative polymerase chain reaction(qRT-PCR) method. B-CPAP cells were divided into blank group, NC group(transfected pcDNA3.1-vector and negative control inhibitor), shDLG1-AS1 group(transfected pcdna 3.1-shDLG1-AS1), shDLG1-AS1+miR-203 inhibitor group(transfected pcdna3.1-shDLG1-AS1 and miR-203 inhibitor). The proliferation and invasion, migration activities of cells were detected by CCK-8 assay and transwell method. Western blotting was used to detect the relative expression of epithelial-mesenchymal transition(EMT) related proteins. The luciferase report was used to analyze the target relationship between DLG1-AS1, miR-203, and ZEB2. Results: qRT-PCR results showed compared with Nthy-ori3-1 cells, the relative expression of DLG1-AS1 in TPC1, KTC-1, B-CPAP and HTori-3 cells was increased, while the relative expression of miR-203 was decreased(P<0.05).B-CPAP cells with the highest relative expression of DLG1-AS1 were selected for follow-up study. Compared with blank group and NC group, the proliferation, migration and invasion of shDLG1-AS1 group cells were decreased, while the expression of ZEB2, N-cadherin, vimentin, β-catenin protein was down-regulated, and the expression of E-cadherin was up-regulated(P<0.05). Compared with shDLG1-AS1 group, shDLG1-AS1+miR-203 inhibitor group showed a reversal of cell proliferation, migration and invasion, while the expression of ZEB2, N-cadherin, vimentin, β-catenin protein was up-regulated and the expression of E-cadherin was down-regulated(P<0.05). DLG1-AS1 could directly regulate miR-203, and ZEB2 was the downstream target gene of miR-203. Conclusion: By combining with miR-203,LncRNA DLG1-AS1 eliminates the inhibition of miR-203 to ZEB2 expression, promotes the EMT of PTC cells, which may be an important mechanism of PTC malignant growth and metastasis.

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