miRNA介导LncRNA AC003092.1通过TFPI-2调控胶质瘤替莫唑胺耐药及其分子机制

The temozolomide-resistance of glioma is the main factor affecting the efficacy in postoperative treatments of patients, and the mechanism is still not clearly demonstrated. In our previous study, a novel LncRNA AC003092.1 that lowly expressed in U87 Temozolomide-Resistant glioma cells was identified through LncRNA microarray and initially comfirmed that AC003092.1 may regulate glioma temozolomide resistance through TFPI-2. Combined the competing endogenous RNA (ceRNA) regulation theory and bioinformatics analysis, we assumed that miRNA may involve in this regulation, and revealed that five miRNA were highly expressed in U87TR cells, which had co-existence ofmiRNA binding sites with LncRNA AC and TFPI-2. In this study, we aim from in vitro and in vivo levels: 1) further comfirm the regulatory role of LncRNA AC in TMZ resistance of glioma through TFPI-2; 2) clear the miRNA mediation in LncRNA AC-TFPI-2 regulatory via co-transfection and the luciferase reporter technology; 3) finally, investigate the possible mechanisms of miRNA-mediated LncRNA AC in regulating glioma TMZ resistance through TFPI-2 using RIP、MSP、BSP、ChIP-qPCR methods. Our research would provide a new theoretical basis for mechanism elucidating and the reversal of glioma TMZ resistance.

替莫唑胺(TMZ)耐药是胶质瘤患者术后疗效主要影响因素，但机制尚不明确。课题组前期通过LncRNA芯片发现LncRNA AC003092.1在耐TMZ胶质瘤细胞U87TR中差异低表达,并初步证实其可能通过TFPI-2调控胶质瘤TMZ耐药；结合竞争性内源RNA机制，我们推测此调控过程可能有miRNA参与，生物信息学分析发现5个与LncRNA AC和TFPI-2存在共同结合位点的miRNA在U87TR高表达。本项目拟从细胞、动物及临床组织标本：1）进一步证实LncRNA AC通过TFPI-2调控胶质瘤TMZ耐药；2)共转染、荧光素酶报告等明确miRNA介导LncRNA AC对TFPI-2的调控；3)应用RIP、n-MSP、BSP、ChIP-qPCR等方法探讨miRNA介导LncRNA AC通过TFPI-2调控胶质瘤TMZ耐药的可能机制。以期为胶质瘤TMZ耐药机制阐明及临床逆转提供新的理论依据。

替莫唑胺(TMZ)耐药是制约胶质瘤患者术后疗效的主要影响因素，但机制尚不明确。本项目采用基因芯片分析体外构建的耐TMZ胶质瘤细胞系，筛选出差异表达于耐TMZ细胞中的LncRNAs与mRNAs。通过体内外研究验证耐TMZ差异分子靶标表达并构建共表达网络，进一步深入探讨差异表达基因对胶质瘤化疗抵抗的影响及其分子调控机制。通过研究得到以下研究结果：一、成功筛选胶质瘤耐TMZ细胞中差异表达的LncRNAs与mRNAs，并对耐药相关通路富集分析；二、体内外证实LncRNA AC003092.1通过TFPI-2介导细胞凋亡增强TMZ敏感性，阐明lncRNA AC003092.1通过ceRNA机制负性调节miR-195促进TFPI-2表达；三、明确差异高表达LncRNA SOX2OT在胶质瘤化疗敏感性中的作用，阐明去甲基化转移酶ALKBH5介导SOX2OT表观调控SOX2表达促进TMZ耐药的分子机制；四、明确TMZ诱导CTGF表达增加，干扰CTGF增强胶质瘤对TMZ敏感性，阐明CTGF通过维持胶质瘤细胞干性发挥TMZ化疗抵抗作用，且证实Smad和ERK通路参与TMZ诱导的TGF-β1/CTGF信号轴激活。项目研究初步提示了胶质瘤TMZ耐药是多因素共同参与的复杂分子调控网络；不同层面分子靶标均可导致胶质瘤化疗抵抗；本研究为胶质瘤TMZ化疗抵抗的治疗提供了新靶点，进一步丰富与完善了胶质瘤TMZ耐药的分子调控机制。