环状MBOAT2通过miR-664b-3p介导对非小细胞肺癌中TLK1的调控的机制研究
Mechanistic insights into Circ-MBOAT2-mediated regulation of TLK1 through miR-664b-3p in non-small cell lung cancer.
作者信息
Zhao DanTing, Wang Cong, Zhang GuangCheng, Song ZhengChang, Luan ChunYu
机构信息
Department of Respiratory and Critical Care Medicine, The Second Affiliated Hospital of Shandong First Medical University, No. 366 Taishan Street, Taishan District, Tai'an City, Shandong Province, 271000, China.
出版信息
Hereditas. 2025 May 14;162(1):77. doi: 10.1186/s41065-025-00439-y.
BACKGROUND
Emerging evidence highlights the critical involvement of dysregulated circular RNAs (circRNAs) in non-small cell lung cancer (NSCLC) pathogenesis. Nevertheless, the precise functional role and mechanistic contributions of circ-MBOAT2 in NSCLC remain poorly characterized. The purpose of this study was to investigate the pathogenesis of NSCLC based on circ-MBOAT2.
METHODS
Our investigation focused on the interplay among circ-MBOAT2, miR-664b-3p, and Tousled-like kinase 1 (TLK1) mRNA in NSCLC tissues, along with their association with the clinical and pathological characteristics of NSCLC patients. Sequences or plasmids were transfected into A549 cells. Gene expressions were identified using RT-qPCR and Western blot analysis. NSCLC cells' cancerous characteristics were identified using CCK-8, EdU, AnnexinV-PI double staining, and Transwell, while their in vivo growth was assessed through a xenografted tumor assay. To monitor alterations in the CD8 T cell ratio and inflammatory factors in PBMCs, co-cultures were created with both normal human PBMCs and A549 cells. Evaluations using bioinformatics software, dual luciferase reporter tests, and RIP assays were performed to verify the connection between circ-MBOAT2 and miR-664b-3p, as well as the interaction between miR-664b-3p and TLK1.
RESULTS
Circ-MBOAT2 expression was up-regulated in NSCLC, and reducing circ-MBOAT2 hampered NSCLC cell proliferation, EMT, immune escape, and tumor growth in vivo. There was a negative correlation between miR-664b-3p expression and circ-MBOAT2, and miR-664b-3p could compete with circ-MBOAT2 for binding. miR-664b-3p downregulation impaired the anti-tumor effect of circ-MBOAT2 reduction on NSCLC cells. TLK1 expression was elevated in NSCLC specimens compared to adjacent normal tissues (p < 0.001), negatively correlated with miR-664b-3p (r=-0.351, p < 0.001), and positively correlated with circ-MBOAT2 (r = 0.341, p < 0.001). In vitro functional experiments showed that silencing TLK1 restrained NSCLC cell proliferation, EMT, and immune escape, whlie TLK1 overexpression rescued the inhibitory effects of miR-664b-3p on NSCLC cell malignant behaviors.
CONCLUSION
Circ-MBOAT2 promotes NSCLC cell proliferation, EMT and immune escape by competitively binding to miR-664b-3p to promote TLK1 expression.
背景
新出现的证据凸显了失调的环状RNA（circRNAs）在非小细胞肺癌（NSCLC）发病机制中的关键作用。然而，circ-MBOAT2在NSCLC中的确切功能作用和机制贡献仍不清楚。本研究旨在基于circ-MBOAT2探讨NSCLC的发病机制。
方法
我们的研究聚焦于NSCLC组织中circ-MBOAT2、miR-664b-3p和类Tousled激酶1（TLK1）mRNA之间的相互作用，以及它们与NSCLC患者临床病理特征的关系。将序列或质粒转染到A549细胞中。使用RT-qPCR和蛋白质免疫印迹分析鉴定基因表达。使用CCK-8、EdU、AnnexinV-PI双染和Transwell实验鉴定NSCLC细胞的癌性特征，同时通过异种移植瘤实验评估其体内生长情况。为了监测外周血单核细胞（PBMCs）中CD8 T细胞比例和炎性因子的变化，将正常人PBMCs与A549细胞共同培养。使用生物信息学软件、双荧光素酶报告基因检测和RNA免疫沉淀实验（RIP）进行评估，以验证circ-MBOAT2与miR-664b-3p之间的联系，以及miR-664b-3p与TLK1之间的相互作用。
结果
Circ-MBOAT2在NSCLC中表达上调，降低circ-MBOAT2可抑制NSCLC细胞增殖、上皮-间质转化（EMT）、免疫逃逸及体内肿瘤生长。miR-664b-3p表达与circ-MBOAT2呈负相关，且miR-664b-3p可与circ-MBOAT2竞争结合。下调miR-664b-3p可削弱circ-MBOAT2降低对NSCLC细胞的抗肿瘤作用。与癌旁正常组织相比，NSCLC标本中TLK1表达升高（p < 0.001），与miR-664b-3p呈负相关（r = -0.351，p < 0.001），与circ-MBOAT2呈正相关（r = 0.341，p < 0.001）。体外功能实验表明，沉默TLK1可抑制NSCLC细胞增殖、EMT和免疫逃逸，而TLK1过表达可挽救miR-664b-3p对NSCLC细胞恶性行为的抑制作用。
结论
Circ-MBOAT2通过竞争性结合miR-664b-3p促进TLK1表达，从而促进NSCLC细胞增殖、EMT和免疫逃逸。
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