长链非编码RNA NEAT1调控miR-124-3p/CTNNB1轴对胰腺癌细胞生物学功能的影响

Long noncoding RNA NEAT1 regulates the miR-124-3p/CTNNB1 axis to affect the biological functions of pancreatic cancer cells

摘要:

背景与目的 长链非编码RNA（lncRNA）核富集转录体1（NEAT1）是一种致癌lncRNA，可通过竞争性内源性RNA（ceRNA）机制促进多种癌症进展。笔者前期通过TargetScan网站发现，NEAT1与微小RNA-124-3p（miR-124-3p），以及miR-124-3p与钙黏蛋白相关蛋白（CTNNB1）之间存在结合位点。因此，本研究探讨NEAT1、miR-124-3p、CTNNB1在胰腺癌中的表达，以及它们之间相互作用对胰腺癌细胞功能的影响。方法 双荧光素酶报告基因试验验证NEAT1、miR-124-3p、CTNNB1的关系。检测胰腺癌组织及癌旁组织、胰腺癌PANC-1细胞及正常胰腺上皮H6C7细胞中NEAT1和miR-124-3p的表达水平，以及CTNNB1蛋白的表达情况。将NEAT1 siRNA转染或与miR-124-3p抑制物同时转染PANC-1细胞，评估转染后PANC-1细胞生物学功能、上皮-间充质转化相关蛋白表达以及在小鼠体内生长能力的变化。结果 双荧光素酶报告基因试验证实NEAT1与miR-124-3p、miR-124-3p与CTNNB1之间存在靶向关系。胰腺癌组织（vs.癌旁组织）以及PANC-1细胞（vs. H6C7细胞）中，NEAT1与CTNNB1表达升高，miR-124-3p表达降低（均P<0.05）。转染NEAT1 siRNA后，PANC-1细胞NEAT1、CTNNB1表达降低，miR-124-3p表达增高，而同时转染miR-124-3p抑制物后，PANC-1细胞中miR-124-3p与CTNNB1上述表达变化被抑制（均P<0.05）。转染NEAT1 siRNA后，PANC-1细胞的增殖、迁移与侵袭能力明显减弱，凋亡率明显升高，E-cadherin蛋白表达上调，而N-cadherin、vimentin蛋白表达下调，在小鼠体内的生长能力明显降低（均P<0.05）；同时转染miR-124-3p抑制物PANC-1细胞上述指标的变化明显减弱（均P<0.05）。结论 NEAT1可能通过ceRNA机制与miR-124-3p竞争性结合，削弱miR-124-3p对CTNNB1的抑制作用，从而上调CTNNB1表达，促进胰腺癌细胞的恶性生物学行为。

Abstract:

Background and Aims Long noncoding RNA (lncRNA) nuclear-enriched abundant transcript 1 (NEAT1) is an oncogenic lncRNA that promotes the progression of various cancers through the competing endogenous RNA (ceRNA) mechanism. Using the TargetScan database, we previously identified binding sites between NEAT1 and microRNA-124-3p (miR-124-3p), as well as between miR-124-3p and catenin beta-1 (CTNNB1). Therefore, this study was conducted to investigate the expression of NEAT1, miR-124-3p, and CTNNB1 in pancreatic cancer and their interactions affecting pancreatic cancer cell functions.Methods A dual-luciferase reporter assay was used to validate the relationships among NEAT1, miR-124-3p, and CTNNB1. The expression levels of NEAT1 and miR-124-3p, as well as CTNNB1 protein expression, were detected in pancreatic cancer tissues and adjacent normal tissues, as well as in pancreatic cancer PANC-1 cells and normal pancreatic epithelial H6C7 cells. PANC-1 cells were transfected with NEAT1 siRNA alone or co-transfected with a miR-124-3p inhibitor. After transfection, changes in PANC-1 cell biological functions, epithelial-mesenchymal transition related protein expression, and tumor growth ability in mice were assessed.Results The dual-luciferase reporter assay confirmed the targeting relationships between NEAT1 and miR-124-3p, as well as between miR-124-3p and CTNNB1. NEAT1 and CTNNB1 expression levels were significantly upregulated, while miR-124-3p expression was downregulated in pancreatic cancer tissues (vs. adjacent tissues) and in PANC-1 cells (vs. H6C7 cells) (all P<0.05). NEAT1 siRNA transfection led to decreased NEAT1 and CTNNB1 expression and increased miR-124-3p expression in PANC-1 cells. However, co-transfection with a miR-124-3p inhibitor suppressed the expression changes in miR-124-3p and CTNNB1 (all P<0.05). NEAT1 siRNA transfection significantly reduced PANC-1 cell proliferation, migration, and invasion, while promoting apoptosis. Additionally, E-cadherin protein expression was upregulated, whereas N-cadherin and vimentin protein expression were downregulated. Tumor growth in mice was also significantly inhibited (all P<0.05). These changes were attenuated upon co-transfection with the miR-124-3p inhibitor (all P<0.05).Conclusion NEAT1 may act as a ceRNA by competitively binding to miR-124-3p, thereby attenuating miR-124-3p-mediated inhibition of CTNNB1. This leads to CTNNB1 upregulation, ultimately promoting the malignant biological behavior of pancreatic cancer cells.