假基因HMGB1P10调控同源基因HMGB1参与非小细胞肺癌吉非替尼耐药的机制

Drug resistance to TKI in non-small cell lung cancer has greatly troubled clinical therapy, pseudogene and tumor drug reistance has not been reported. Via analyzing the differentially expressed pseudogenes in the gefitinib-resistant cells and gefitinib-sensitive cells in the GEO database, We found that the expression of HMGB1P10 in the drug resistant cells and its homologous gene HMGB1 were upregulated and it had been verified in the resistant tissues. Inhibition of HMGB1P10 could significantly enhance resistant cells sensibility to gefitinib, the level of autophagy protein Beclin 1 and LC3-II were decreased. Bioinformatics analysis and luciferase reporting experiments proved that the miR-142-3p/218/129 can bind to both HMGB1P10 and HMGB1. RIP assays confirmed HMGB1P10 can bind to WDR5 protein, the level of HMGB1 decreased after knock down of WDR5. Accordingly, the hypothesis is proposed : HMGB1P10 regulate the expression of HMGB1 by mediating the histone methylation through recruiting WDR5 and sponging miRNA at the transcriptional and posttranscritional level, thus inducing autophagy that leads to the drug resistance to TKI. We will verify the aboved hypothesis by use of clinical samples, RNA immunoprecipitation（RIP）, chromatin immunoprecipitation(ChIP), luciferase reporter and other techniques. Our work will provide a new basis for targeted therapy to reserve TKI-resistance.

TKI耐药极大地困扰非小细胞肺癌临床治疗，假基因与肿瘤耐药的研究未见报道。我们通过分析GEO数据库中吉非替尼耐药细胞和敏感细胞中差异表达的假基因，发现耐药细胞中HMGB1P10及其同源基因HMGB1表达上调，并在耐药组织中得到了验证。干扰HMGB1P10后耐药细胞对吉非替尼敏感性增加，自噬相关蛋白Beclin 1和LC3-II表达下调。生物信息学分析及荧光素酶实验证实miR-142-3p/218/129能同时与HMGB1P10和HMGB1结合。RIP实验证实HMGB1P10与WDR5蛋白绑定，干扰WDR5后HMGB1表达下调。据此提出假设： HMGB1P10通过募集WDR5介导组蛋白甲基化及吸附miRNA在转录及转录后水平共同调控HMGB1，诱发自噬导致细胞对TKI的耐药。本课题组将通过临床样本验证，运用RIP、ChIP及荧光素酶等技术证实上述假设，为逆转TKI耐药的靶向治疗提供新依据。

EGFR-TKI耐药极大地困扰非小细胞肺癌(NSCLC)临床治疗，是NSCLC患者治疗失败的主要原因。我们通过分析GEO数据库中吉非替尼耐药细胞和敏感细胞中差异表达的假基因，发现耐药细胞中HMGB1P10及其同源基因HMGB1表达上调，并在耐药组织中得到了验证。干扰HMGB1P10后耐药细胞对吉非替尼敏感性增加，自噬相关蛋白Beclin 1和LC3-II表达下调。生物信息学分析及荧光素酶实验证实miR-142-3p/218/129能同时与HMGB1P10和HMGB1结合，竞争性吸附miR-142-3p/218/129 拮抗其对HMGB1 蛋白的翻译抑制作用，促进HMGB1 的表达。RIP、ChIP实验证实HMGB1P10与WDR5蛋白结合，HMGB1P10与WDR5结合促进HMGB1的转录，诱发自噬导致细胞对EGFR-TKI的耐药。本研究探索了NSCLC吉非替尼耐药的分子机制，为逆转TKI耐药提供新依据。