HNRNPL上调circ_0001964通过RNA甲基化调控非小细胞肺癌顺铂耐药的机制研究

Clinical treatment of non-small cell lung cancer (NSCLC) has been plagued by chemotherapy resistance. Researches concerning circRNA and cisplatin resistance have not been reported. We found that the expression of circ_0001964 in NSCLC cisplatin resistant cells was significantly higher than that in parental cells by high-throughput sequencing technology. Inhibition of circ_0001964 significantly increased the sensitivity of resistant cells to cisplatin and downregulated STMN1 which regulated autophagy. Bioinformatics analyses found the binding sites between the parental gene of circ_0001964 and the alternative splicing factor HNRNPL. The expression of circ_0001964 was downregulated after inhibition of HNRNPL. RIP and RNA pulldown assays confirmed that circ_0001964 could bind to the RNA methyltransferase METTL3. Therefore, we propose a scientific hypothesis: HNRNPL upregulated the expression of circ_0001964, which promoted the translation of STMN1 by mediating RNA methylation through binding with METTL3, further inducing autophagy to enhance the drug resistance of NSCLC cisplatin resistant cells. Our research group will verify the above assumptions through clinical samples validations, RIP, MeRIP, RNA pulldown and other experiments to provide new theoretical basis and molecular target for clinical reversal of drug resistance.

化疗耐药困扰非小细胞肺癌（NSCLC）临床治疗，circRNA与顺铂耐药的研究未见报道。我们通过测序技术发现NSCLC顺铂耐药细胞较亲本细胞circ_0001964表达显著上调，干扰circ_0001964显著增加耐药细胞的化疗敏感性，并下调STMN1，STMN1调控细胞自噬。生物信息学发现circ_0001964的亲本基因和可变剪接因子HNRNPL有结合位点，干扰HNRNPL后circ_0001964的表达下调，RIP和RNA pulldown证实circ_0001964可以与RNA甲基转移酶METTL3结合。据此提出假设：HNRNPL上调circ_0001964，结合METTL3介导RNA甲基化促进STMN1的翻译，进而诱发细胞自噬增强NSCLC的耐药性。本课题组将通过临床样本验证，运用RIP、MeRIP、RNA pulldown等实验证实上述假设，为逆转化疗耐药提供新依据及靶标。

我国肺癌的发病率和死亡率均排在恶性肿瘤的首位，以顺铂为基础的联合化疗是一线治疗的基石，然而肿瘤细胞的耐药性常导致治疗失败，给临床带来极大的困扰。课题组通过高通量测序技术对NSCLC顺铂耐药细胞及亲本细胞进行circRNA测序，在化疗耐药组织和敏感组织中进一步分析差异表达的circRNA，发现circ_0001964在化疗耐药组织、顺铂耐药细胞中显著高表达。进一步通过体内外实验证实敲低circ_0001964可逆转NSCLC对顺铂的耐药性，增加敏感性。生物信息学分析结合可变剪切报告基因实验证实可变剪接因子HNRNPL可以通过前mRNA反向剪接促进circ_0001964的形成。通过RNA pulldown及质谱分析发现，circ_0001964可与甲基转移酶METTL3结合。在顺铂耐药细胞中进行全转录组RNA甲基化测序(MeRIP-seq)，发现STMN1的3’UTR区存在明显的m6A峰，STMN1在耐药细胞中的m6A修饰水平明显高于敏感细胞。MeRIP-qPCR和Western blot实验证实METTL3通过m6A依赖的方式促进耐药细胞中STMN1的蛋白翻译。STMN1在耐药组织中显著高表达并参与调控细胞自噬。研究阐明了circ_0001964可促进METTL3与STMN1的结合能力，使STMN1的m6A水平上调，促进STMN1的蛋白翻译，诱发细胞自噬增强NSCLC的耐药性。通过本项研究为临床NSCLC精准治疗及逆转化疗耐药提供新依据及靶标。