LBX2-AS1 in acceleration of glycolysis and cancer angiogenesis via miR-491-5p/MDK signaling axis in colorectal cancer: an experimental study

Recent studies have identified various roles for long non-coding RNAs (lncRNAs) in cancer progression. However, an in-depth understanding of the regulatory mechanisms of lncRNAs in colorectal cancer (CRC) and their diagnostic significance is rather limited. The diagnostic value of LBX2 antisense RNA 1 (LBX2-AS1) was assessed using public databases and clinical samples. Cell-counting kit 8 (CCK-8), Colony Formation, transwell, and wound scratch assays were performed to assess CRC cell proliferation and metastasis <i>in vitro</i>. Glucose consumption, lactate production, adenosine triphosphate (ATP), extracellular acidification rate (ECAR), and oxygen composition rate (OCR) were evaluated in CRC cells. A dual luciferase assay was used to verify the target-binding relationship of LBX2-AS1/microRNA (miR)-491-5p/midkine (MDK). BALB/C nude mice were used to establish a subcutaneous xenograft mouse model. The role of the LBX2-AS1/MDK axis in CRC proliferation and angiogenesis was analyzed <i>in vivo</i>. As a marker of poor prognosis, LBX2-AS1 was highly expressed in CRC tumor tissues and cells (all <i>p</i> &lt; 0.01). Downregulation of LBX2-AS1 inhibited the proliferation, migration, and invasion of CRC cells (all <i>p</i> &lt; 0.01). Furthermore, the levels of glucose consumption, lactate production, ATP, ECAR, glycolytic-related indicators, and markers of angiogenesis were also inhibited by the downregulation of LBX2-AS2 expression (all <i>p</i> &lt; 0.01). Overexpression of MDK reversed the downregulation of LBX2-AS1 inhibition of these biological activities in CRC cells (all <i>p</i> &lt; 0.01). Downregulation of LBX-AS1 inhibited tumor growth and angiogenesis <i>in vivo</i> (all <i>p</i> &lt; 0.001). Overexpression of MDK reversed the downregulation of LBX2-AS1 inhibition of tumor growth and angiogenesis <i>in vivo</i> (all <i>p</i> &lt; 0.001). Mechanistically, LBX2-AS1 regulated MDK transcription by mediating miR-491-5p. This study demonstrates that LBX2-AS1 promotes CRC angiogenesis and activates the glycolysis <i>via</i> the miR-491-5p/MDK axis. LBX2-AS may serve as a novel diagnostic biomarker and potential therapeutic target for CRC.

既往研究已揭示长链非编码RNA（long non-coding RNAs，lncRNAs）在肿瘤发生发展中的多种调控作用。然而，目前对于长链非编码RNA在结直肠癌（colorectal cancer，CRC）中的调控机制及其诊断价值的深入认知仍较为匮乏。本研究通过公共数据库与临床样本，评估了LBX2反义RNA1（LBX2 antisense RNA 1，LBX2-AS1）的诊断价值。采用细胞计数试剂盒-8（Cell Counting Kit-8，CCK-8）、集落形成实验、Transwell小室实验以及划痕愈合实验，体外（in vitro）检测结直肠癌细胞的增殖与迁移侵袭能力。同时检测结直肠癌细胞的葡萄糖消耗量、乳酸生成量、三磷酸腺苷（adenosine triphosphate，ATP）、细胞外酸化率（extracellular acidification rate，ECAR）以及氧组成率（oxygen composition rate，OCR）水平。采用双荧光素酶报告基因实验，验证LBX2-AS1/微小RNA（microRNA，miR）-491-5p/中期因子（midkine，MDK）之间的靶向结合关系。选用BALB/C裸小鼠构建皮下移植瘤小鼠模型，体内（in vivo）探究LBX2-AS1/MDK轴在结直肠癌增殖与血管生成中的作用。作为不良预后标志物，LBX2-AS1在结直肠癌组织与细胞中呈高表达（所有组均p < 0.01）。下调LBX2-AS1的表达可抑制结直肠癌细胞的增殖、迁移与侵袭能力（所有组均p < 0.01）。此外，下调LBX2-AS2的表达还可抑制葡萄糖消耗、乳酸生成、ATP、ECAR、糖酵解相关指标以及血管生成标志物的水平（所有组均p < 0.01）。过表达MDK可逆转LBX2-AS1下调对结直肠癌细胞上述生物学活性的抑制作用（所有组均p < 0.01）。下调LBX-AS1的表达可在体内抑制肿瘤生长与血管生成（所有组均p < 0.001）。过表达MDK可逆转LBX2-AS1下调在体内对肿瘤生长与血管生成的抑制作用（所有组均p < 0.001）。机制层面，LBX2-AS1通过靶向调控miR-491-5p，进而影响MDK的转录。本研究证实，LBX2-AS1可通过miR-491-5p/MDK轴促进结直肠癌的血管生成并激活糖酵解过程。LBX2-AS有望成为结直肠癌新型诊断生物标志物与潜在治疗靶点。