Li Jia, Shen Jie, Qin Lan, Lu Dongyan, Ding Enci
Department of Nuclear Medicine, Tianjin First Central Hospital, School of Medicine Nankai University, Tianjin, China.
Front Genet. 2021 Nov 11;12:765033. doi: 10.3389/fgene.2021.765033. eCollection 2021.
Thyroid cancer is a frequent endocrine tumor in women. It is of great significance to investigate the molecular mechanism of progression of thyroid cancer. Gene expression data set and clinical data were downloaded from The Cancer Genome Atlas database for differential expression analysis. The triplet of downstream transcription factors (TFs) and modulatory genes of target lncRNA in thyroid cancer was predicted by the lncMAP database. mRNA and protein expression of lncRNA LBX2-AS1, RARα, and FSTL3 were detected by qRT-PCR and western blot. The localization of lncRNA LBX2-AS1 in cells was tested by Fluorescence in situ hybridization assay. The RNA immunoprecipitation assay was applied to verify the binding relationship between lncRNA LBX2-AS1 and FSTL3. ChIP and dual-luciferase assays were used to prove the binding relationship between RARα and FSTL3. Cell function experiments were used to test cell proliferation, migration and invasion in each treatment group. The role of lncRNA LBX2-AS1 in thyroid cancer progression was also confirmed in nude mice. Bioinformatics analysis indicated that lncRNA LBX2-AS1, RARα, FSTL3 were remarkably fostered in thyroid cancer tissue, and LBX2-AS1 was evidently correlated with clinical features. The LncMAP triplet prediction showed that LBX2-AS1 recruited TF RARα to modulate FSTL3. RIP assay confirmed that LBX2-AS1 was prominently enriched on RARα. ChIP and dual-luciferase report assays unveiled that RARα bound to the promoter region of FSTL3 and functioned as a TF. Cell function experiments uncovered that LBX2-AS1 boosted the progression of thyroid cancer. The rescue experiments showed that LBX2-AS1 recruited the TF RARα to hasten the transcription activity of FSTL3 and thus promoted the development of thyroid cancer. The integrative results demonstrated that LBX2-AS1 activated FSTL3 by binding to TF RARα to hasten proliferation, migration and invasion of thyroid cancer.
甲状腺癌是女性常见的内分泌肿瘤。研究甲状腺癌进展的分子机制具有重要意义。从癌症基因组图谱数据库下载基因表达数据集和临床数据进行差异表达分析。通过lncMAP数据库预测甲状腺癌中靶lncRNA的下游转录因子（TFs）三联体和调控基因。采用qRT-PCR和蛋白质免疫印迹法检测lncRNA LBX2-AS1、RARα和FSTL3的mRNA和蛋白表达。通过荧光原位杂交试验检测lncRNA LBX2-AS1在细胞中的定位。应用RNA免疫沉淀试验验证lncRNA LBX2-AS1与FSTL3之间的结合关系。采用染色质免疫沉淀和双荧光素酶试验证明RARα与FSTL3之间的结合关系。通过细胞功能实验检测各治疗组细胞的增殖、迁移和侵袭能力。lncRNA LBX2-AS1在甲状腺癌进展中的作用也在裸鼠中得到证实。生物信息学分析表明，lncRNA LBX2-AS1、RARα、FSTL3在甲状腺癌组织中显著上调，且LBX2-AS1与临床特征明显相关。LncMAP三联体预测显示，LBX2-AS1招募TF RARα来调节FSTL3。RNA免疫沉淀试验证实LBX2-AS1在RARα上显著富集。染色质免疫沉淀和双荧光素酶报告试验表明，RARα与FSTL3的启动子区域结合并作为TF发挥作用。细胞功能实验发现，LBX2-AS1促进了甲状腺癌的进展。挽救实验表明，LBX2-AS1招募TF RARα以加速FSTL3的转录活性，从而促进甲状腺癌的发展。综合结果表明，LBX2-AS1通过与TF RARα结合激活FSTL3，从而加速甲状腺癌的增殖、迁移和侵袭。
