ABSTRACT

Dysregulated microRNAs (miRNAs) are involved in carcinoma progression, metastasis, and poor prognosis. We demonstrated that in nasopharyngeal carcinoma (NPC), transactivated MIR106A-5p promotes a malignant phenotype by functioning as a macroautophagy/autophagy suppressor by targeting BTG3 (BTG anti-proliferation factor 3) and activating autophagy-regulating MAPK signaling. MIR106A-5p expression was markedly increased in NPC cases based on quantitative real-time PCR, miRNA microarray, and TCGA database analysis findings. Moreover, MIR106A-5p was correlated with advanced stage, recurrence, and poor clinical outcomes in NPC patients. In addition to three-dimensional cell culture assays, zebrafish and BALB/c mouse tumor models revealed that overexpressed MIR106A-5p targeted BTG3 and accelerated the NPC malignant phenotype by inhibiting autophagy. BTG3 promoted autophagy, and its expression was correlated with poor prognosis in NPC. Attenuation of autophagy, mediated by the MIR106A-5p-BTG3 axis, occurred because of MAPK pathway activation. MIR106A-5p overexpression in NPC was due to increased transactivation by EGR1 and SOX9. Our findings may lead to novel insights into the pathogenesis of NPC.

Abbreviations: ACTB: actin beta; ATG: autophagy-related; ATG5: autophagy related 5; BLI: bioluminescence; BTG3: BTG anti-proliferation factor 3; CASP3: caspase 3; ChIP: chromatin immunoprecipitation; CQ: chloroquine; Ct: threshold cycle; DAPI: 4ʹ,6-diamidino-2-phenylindole; DiL: 1,1ʹ-dioctadecyl-3,3,3ʹ,3ʹ-tetramethylindocarbocyanine perchlorate; EBSS: Earle’s balanced salt solution; EGR1: early growth response 1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GEO: Gene Expression Omnibus; GFP: green fluorescent protein; IF: immunofluorescence; IHC: immunohistochemistry; ISH: in situ hybridization; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MIR106A-5p: microRNA 106a-5p; miRNAs: microRNAs; MKI67: marker of proliferation ki-67; mRNA: messenger RNA; MTOR: mechanistic target of rapamycin kinase; NPC: nasopharyngeal carcinoma; qRT-PCR: quantitative real-time PCR; siRNA: small interfering RNA; SOX9: SRY-box transcription factor 9; SQSTM1: sequestosome 1; TCGA: The Cancer Genome Atlas; WB: western blot.

摘要

失调的微小 RNA (miRNA) 与癌症进展、转移和预后不良有关。我们证明，在鼻咽癌 (NPC) 中，反式激活的 MIR106A-5p通过靶向BTG3（BTG 抗增殖因子 3）和激活自噬调节 MAPK 信号传导作为巨自噬/自噬抑制因子来促进恶性表型。基于定量实时 PCR、miRNA 微阵列和 TCGA 数据库分析结果，NPC 病例中MIR106A-5p 的表达显着增加。此外，MIR106A-5p与 NPC 患者的晚期、复发和不良临床结果相关。除了三维细胞培养试验，斑马鱼和 BALB/c 小鼠肿瘤模型显示过表达的 MIR106A-5p靶向BTG3并通过抑制自噬加速 NPC 恶性表型。BTG3促进自噬，其表达与NPC预后不良相关。由MIR106A-5p -BTG3 轴介导的自噬减弱是由于 MAPK 通路激活而发生的。NPC 中的 MIR106A-5p过表达是由于 EGR1 和 SOX9 的反式激活增加。我们的研究结果可能会导致对 NPC 发病机制的新见解。

缩写： ACTB：肌动蛋白β；ATG：自噬相关；ATG5：自噬相关5；BLI：生物发光；BTG3：BTG抗增殖因子3；CASP3：半胱天冬酶 3；ChIP：染色质免疫沉淀；CQ：氯喹；Ct：阈值循环；DAPI：4ʹ,6-二脒基-2-苯基吲哚；DiL：1,1′-二十八烷基-3,3,3′,3′-四甲基吲哚羰花青高氯酸盐；EBSS：厄尔平衡盐溶液；EGR1：早期生长反应 1；GAPDH：3-磷酸甘油醛脱氢酶；GEO：基因表达综合；GFP：绿色荧光蛋白；IF：免疫荧光；IHC：免疫组化；ISH：原位杂交；MAP1LC3B：微管相关蛋白 1 轻链 3 β；MIR106A-5p：microRNA 106a-5p；miRNA：微小RNA；MKI67：增殖标志物ki-67；mRNA：信使RNA；MTOR：雷帕霉素激酶的机制靶点；NPC：鼻咽癌；qRT-PCR：定量实时 PCR；siRNA：小干扰RNA；SOX9：SRY-box 转录因子 9；SQSTM1：隔离体 1；TCGA：癌症基因组图谱；WB：蛋白质印迹。