背景

       N6-甲基腺苷（N6-methyladenosine，m6A）修饰是真核生物mRNA中丰度最高的表观遗传学修饰方式，是一种动态可逆的过程。研究发现，在不同肿瘤中m6A修饰可能发挥致癌或抑癌的作用。先前对m6A修饰研究仅限于肺癌、肝癌、乳腺癌等，针对食管癌和卵巢癌的研究尚不完善，鉴于本课题组的研究方向以及预实验的结果，探究m6A调控基因在食管癌和卵巢癌中的生物学功能，并鉴定其修饰的靶基因有助于进一步探究肿瘤的致病机理从而开发新的治疗方法。

       食管癌（Esophageal carcinoma，ESCA）是一类全球范围高发且难治的恶性肿瘤之一，中国作为食管癌高发地区，也是全世界新发病例比例最高的国家。因此，有必要详细探究食管癌的相关致病机制从而为食管癌的发病以及治疗提供理论依据。

       卵巢癌（Ovarian cancer，OC）以侵袭性强、早期诊断难和易复发为特征，本课题组先前基于数据库等生物信息学分析及部分实验初步探究了卵巢癌中调控m6A修饰的关键酶为ALKBH5。本研究第二部分则进一步从临床样本、体外细胞水平分析ALKBH5在卵巢癌中的调控m6A修饰的作用，并通过高通量测序鉴定m6A修饰的靶基因，完善卵巢癌中ALKBH5介导m6A修饰的作用机制。

方法

       本研究分为两个部分。第一部分我们系统地分析了TCGA数据库中25个m6A调控因子在食管癌中的表达情况及相关性。利用一致性聚类方法和LASSO算法构建食管癌患者的分子分型以及预后评分模型，并通过分析测序数据筛选关键调控因子HNRNPA2B1在食管癌中的潜在调控通路和靶基因。利用免疫组化实验和荧光定量PCR检测食管癌组织以及外周血中HNRNPA2B1和潜在靶miRNAs的表达，并通过RNA免疫沉淀实验、蛋白质印迹实验和细胞增殖实验探究HNRNPA2B1对靶miRNAs的互作和调控机制。

       第二部分我们通过免疫组化实验检测ALKBH5蛋白表达和m6A总体水平，并分析其与卵巢癌患者临床特征和预后的相关性。利用细胞增殖、细胞周期、平板克隆和细胞迁移实验检测敲降和敲除ALKBH5的卵巢癌细胞表型的变化。对敲低ALKBH5的卵巢癌细胞进行m6A RNA甲基化测序和转录组测序，分析ALKBH5介导的m6A修饰的关键靶基因和通路，并通过荧光定量PCR和RNA免疫沉淀验证ALKBH5与m6A修饰靶基因的相互作用。

结果       

   在本研究的第一部分中，我们发现ESCA中25个m6A调节因子中大部分呈显著高表达且调控因子间呈正相关。根据 HNRNPA2B1、ALKBH5 和 HNRNPG 表达水平建立预后评分模型，其中风险分数与ESCA患者的浸润深度和临床结局有很强的相关性。该风险分数可以作为 ESCA 的新型预后预测指标。HNRNPA2B1高表达提示ESCC患者预后较差，并与远处转移和淋巴结分期呈正相关。食管癌细胞中敲低HNRNPA2B1可降低 miR-17、miR-18a、miR-20a、miR-93和miR-106b的表达水平，抑制 ESCC 细胞增殖。

   第二部分我们发现在卵巢癌患者中ALKBH5水平升高，而m6A修饰的总体水平降低。ALKBH5敲降和敲除抑制卵巢癌细胞增殖、细胞周期和迁移。此外，ALKBH5调控的m6A RNA修饰主要影响卵巢癌细胞的RNA剪接功能。SRSF10是通过ALKBH5-m6A参与可变剪接调节的关键靶基因。敲低ALKBH5后SRSF10外显子5的保留增多，转录本SRSF10-211表达量显著降低。

结论

       ALKBH5、HNRNPG和HNRNPA2B1构建的预后评分模型中风险分数可作为食管癌新型预后指标。食管鳞癌中高表达HNRNPA2B1提示患者总生存期较差。HNRNPA2B1靶基因miR-17在食管鳞癌患者外周血中显著升高，且与肿瘤浸润深度和淋巴结转移相关。HNRNPA2B1通过调节miR-17-92簇表达影响食管癌的预后。

       ALKBH5在卵巢癌组织中高表达且与卵巢癌患者临床特征和预后相关。卵巢癌细胞 A2780 中 m6A 修饰的基因主要富集在选择性剪接通路上。ALKBH5-m6A 可调节卵巢癌细胞中SRSF10外显子5的跳跃事件。该发现提示了ALKBH5可通过调控RNA剪接参与肿瘤发生发展，有利于针对卵巢癌开发新的治疗方法。

Background

       N6-methyladenosine (m6A) modification is the most abundant epigenetic modification in eukaryotic mRNA, and it is a dynamic and reversible process. It has been found that m6A modification may play an oncogenic or tumor-suppressive role in different tumors. In view of that research direction of this subject and the result of the preliminary experiment, it is helpful to explore the biological function of m6A regulatory gene in esophageal and ovarian cancer and identify the target gene modified by m6A, which is helpful to further explore the pathogenesis of tumor and develop new therapeutic methods.

        Esophageal cancer (ESCA) is one of the most common and refractory malignant tumors in the world. As a high incidence area of esophageal cancer, China has the highest proportion of new cases in the world. Therefore, it is necessary to explore the pathogenesis of esophageal cancer in detail so as to provide theoretical basis for the pathogenesis and treatment of esophageal cancer.

        Ovarian cancer (OC) is characterized by strong invasiveness, difficult early diagnosis and easy recurrence. Based on bioinformatics analysis such as database and some experiments, our research group previously explored that the key enzyme regulating m6A modification in ovarian cancer is ALKBH5. In the second part of this study, the role of ALKBH5 in regulating m6A modification in ovarian cancer was further analyzed from clinical samples and in vitro cell level, and the target genes of m6A modification were identified by high-throughput sequencing, so as to improve the mechanism of ALKBH5-mediated m6A modification in ovarian cancer.

Methods

       This study is divided into two parts. In the first part, we systematically analyzed the expression and correlation of 25 m6A regulatory factors in esophageal carcinoma from TCGA database. The molecular typing and prognostic scoring models of esophageal cancer patients were constructed by consensus clustering method and LASSO algorithm, and the potential regulatory pathways and target genes of key regulatory factor HNRNPA2B1 in esophageal cancer were screened by analyzing sequencing data. The expression of HNRNPA2B1 and potential target miRNAs in esophageal cancer tissues and peripheral blood were detected by immunohistochemistry and fluorescence quantitative PCR. The interaction and regulation mechanism of HNRNPA2B1 on target miRNAs were explored by RNA immunoprecipitation, western blot and cell proliferation assay.

        In the second part, we detected ALKBH5 protein expression and m6A levels by immunohistochemistry, and analyzed their correlation with clinical features and prognosis of patients with ovarian cancer. Cell proliferation, cell cycle, plate cloning and cell migration assays were used to detect the phenotypic changes of ovarian cancer cells with and without ALKBH5 knockdown. The ALKBH5 knockdown ovarian cancer cells were subjected to m6A RNA methylation sequencing and transcriptome sequencing to analyze the key target genes and pathways of m6A modification mediated by ALKBH5, and the interaction between ALKBH5 and m6A modification target genes was verified by fluorescence quantitative PCR and RNA immunoprecipitation. 

Results    

       In the first part of this study, we found that most of the 25 m6A regulators were significantly overexpressed and positively correlated in ESCA. Prognostic scoring models were developed based on HNRNPA2B1, ALKBH5, and HNRNPG expression levels, where risk scores were strongly correlated with tumor size and clinical outcome in ESCA patients. The risk score may be an independent prognostic factor for ESCA. The high expression of HNRNPA2B1 in ESCA patients is associated with poor prognosis, distant metastasis and lymph node staging. Knockdown of HNRNPA2B1 in esophageal cancer cells decreased the expression levels of miR-17, miR-18a, miR-20a, miR-93 and miR-106b, and inhibited ESCA cell proliferation.

   In the second part, we found that ALKBH5 levels were elevated in ovarian cancer patients, while overall levels of m6A modifications were reduced. ALKBH5 knockdown or knockout inhibits ovarian cancer cell proliferation, cell cycle progression and migration. In addition, ALKBH5-regulated m6A RNA modification mainly affects RNA splicing function in ovarian cancer cells. SRSF10 is a key target gene involved in alternative splicing regulation through ALKBH5-m6A. ALKBH5 knockdown resulted in increased retention of SRSF10 exon 5 and decreased expression of transcript SRSF10-211.

Conclusions

       The prognostic score model constructed by ALKBH5, HNRNPG and HNRNPA2B1 can be used to predict the 3-year survival rate of esophageal cancer patients. Overexpression of HNRNPA2B1 in esophageal squamous cell carcinoma suggests poor overall survival. HNRNPA2B1 affects the prognosis of esophageal cancer by regulating the expression of miR-17-92 cluster. HNRNPA2B1 target gene miR-17 was significantly increased in peripheral blood of patients with esophageal squamous cell carcinoma, and was associated with tumor invasion depth and lymph node metastasis. 

       ALKBH5 was highly expressed in ovarian cancer tissues, and m6A was generally expressed at a low level, which was negatively correlated with the clinical characteristics and prognosis of ovarian cancer patients. In ovarian cancer cell line A2780, m6A-modified genes were mainly enriched in alternative splicing pathways. ALKBH5-m6A regulates exon skipping events in SRSF10 exon 5 in ovarian cancer cells.