LINC01134 regulates cell cycle and senescence to inhibit the proliferation of lung adenocarcinoma by competitively binding to CTCF and inhibiting CTCF-mediated p21 transcription

Long non-coding RNAs (lncRNAs) have garnered significant attention as potential therapeutic targets for treating lung adenocarcinoma (LUAD). This study focused on LINC01134, delving into the mechanism by which its overexpression promoted the proliferation of A549 cells. Gene set enrichment analysis (GSEA) revealed significant enrichment of LINC01134 in p21 pathway. Bioinformatics screening highlighted the role of the transcription factor CTCFas a potential candidate for mediating the connection between LINC01134 and p21. Cell proliferationand cycle were assessed using CCK-8, colony formation and Flow cytometry. SA-β-galactosidase staining kit was used to assess cellular senescence. Optical microscopy was done to examine cell morphology. Cell cycle-related proteins were analyzed using Western blotting to observe the effects of LINC01134 overexpression on the cell cycle, senescence, and proliferation. The aforementioned methods were employed to detect LINC01134-mediated regulation of p21 on cell cycle, senescence, and proliferation, and the LINC01134-mediated regulation of CTCF on p21, cell cycle, senescence, and proliferation. RNA pulldown and RIP were conducted to detect interaction between LINC01134 and CTCF. ChIP was used to measure CTCF-p21 interactions and the effect of LINC01134 overexpression on this direct interaction. FISH was used to assess the effect of LINC01134 overexpression on the colocalization of CTCF protein and p21 DNA. The results confirmed that LINC01134 competitively interacted with CTCF to inhibit CTCF-mediated transcription of p21to accelerate G1/S cycle transition, delay cellular senescence, and promote the proliferation of A549 cells. LINC01134 is anticipated to serve as a potential biomarker for recurrence and prognosis in patients with LUAD.长链非编码RNA（long non-coding RNAs, lncRNAs）作为治疗肺腺癌（lung adenocarcinoma, LUAD）的潜在治疗靶点，已受到广泛关注。本研究聚焦于LINC01134，深入探讨其过表达促进A549细胞增殖的分子机制。基因集富集分析（gene set enrichment analysis, GSEA）结果显示，LINC01134在p21通路中显著富集。生物信息学筛选表明，转录因子CTCF是介导LINC01134与p21之间相互关联的潜在候选因子。采用CCK-8法、克隆形成实验及流式细胞术检测细胞增殖与细胞周期；使用衰老相关β-半乳糖苷酶染色试剂盒评估细胞衰老状态；通过光学显微镜观察细胞形态；采用蛋白质印迹法分析细胞周期相关蛋白的表达水平，以探究LINC01134过表达对细胞周期、细胞衰老及细胞增殖的影响。采用上述实验方法，检测LINC01134介导的p21对细胞周期、细胞衰老及细胞增殖的调控作用，以及LINC01134介导的CTCF对p21、细胞周期、细胞衰老及细胞增殖的调控效应。采用RNA下拉实验与RNA结合免疫沉淀（RNA immunoprecipitation, RIP）实验检测LINC01134与CTCF之间的相互作用；采用染色质免疫共沉淀（chromatin immunoprecipitation, ChIP）实验检测CTCF与p21的相互作用，以及LINC01134过表达对该直接相互作用的影响；采用荧光原位杂交（fluorescence in situ hybridization, FISH）实验评估LINC01134过表达对CTCF蛋白与p21 DNA共定位的影响。研究结果证实，LINC01134可通过竞争性结合CTCF，抑制CTCF介导的p21转录，从而加速G1/S期转换、延缓细胞衰老并促进A549细胞增殖。LINC01134有望成为肺腺癌患者复发与预后评估的潜在生物标志物。