作者：邹岩1，李辉2，姜桂春2，谢艳敏1

单位：1. 辽宁省肿瘤医院内镜中心，辽宁 沈阳 110042；2. 辽宁省肿瘤医院护理部，辽宁 沈阳 110042

Authors: Zou Yan1, Li Hui2, Jiang Guichun2, Xie Yanmin1

Unit:  1.Endoscopy Center, Liaoning Province Cancer Hospital & Institute, Shenyang 110042, Liaoning, China；2.Nursing Department, Liaoning Province Cancer Hospital & Institute, Shenyang 110042, Liaoning, China

摘要：

目的  探讨AlkB同源蛋白5（AlkBhomolog5, ALKBH5）在肝细胞癌（hepatocellularcarcinoma，HCC）的细胞增殖中的作用。方法通过癌症基因组图谱数据库分析ALKBH5 和前mRNA 加工因子6（pre-mRNA processing factor 6，PRPF6）在HCC组织中的表达水平。采用实时荧光定量聚合酶链反应和蛋白质印迹法检测ALKBH5 和PRPF6在HCC细胞系（Huh7和Hep3B）中的mRNA和蛋白表达水平。荧光原位杂交确定ALKBH5和PRPF6在HCC细胞系（Huh7和Hep3B）中的亚细胞定位。在免疫沉淀复合物中检测PRPF6的N6-甲基腺苷甲基化水平。通过过表达和敲低基因表达的方法，将Huh7细胞和Hep3B细胞分别进行转染，分组为sh-NC组（阴性对照转染细胞）、sh-ALKBH5实验组（ALKBH5干扰序列转染细胞）、sh-ALKBH5+ex-PRPF6实验组（ALKBH5干扰序列转染+PRPF6 pcDNA 3.1 过表达质粒转染细胞），并采用CCK-8和染色增殖试验检测各组细胞的增殖情况。裸鼠异种移植瘤实验验证ALKBH5和PRPF6在体内的生物学功能。蛋白质印迹法检测各组细胞中AKT/mTOR 通路相关蛋白的表达水平。结果  HCC 细胞中ALKBH5的表达上调，可以去除PRPF6的甲基化修饰。体外和体内实验结果显示，ALKBH5可通过调节PRPF6的表达参与HCC细胞的恶性增殖。此外，敲低ALKBH5会抑制丝氨酸/苏氨酸蛋白激酶（serine -threonine kinase, AKT）和哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin, mTOR）的磷酸化，而PRPF6的过表达则有助于AKT和mTOR的磷酸化。结论  ALKBH5能促进HCC细胞的增殖，相关机制是通过ALKBH5/PRPF6/AKT/mTOR轴实现的。

关键词： Alk B同源蛋白5；前mRNA 加工因子6；N6-甲基腺苷；肝细胞癌；增殖

Abstract：

Objective  This study intends to explore the role of AlkB homolog5 (ALKBH5) in proliferation of hepatocellular carcinoma (HCC) cells. Method  The expression levels of ALKBH5 and pre-mRNA processing factor 6 (PRPF6) in HCC tissues were analyzed via the cancer genome atlas database. Real-time fluorescent quantitative polymerase chain reaction and western-blot were used to determine the mRNA and protein expression levels of ALKBH5 and PRPF6 in HCC cell lines (Huh7 and Hep3B). Fluorescence in situ hybridization determined the subcellular localization of ALKBH5 and PRPF6 in HCC cell lines (Huh 7 and Hep3B). The N6 -methyladenosine methylation level of PRPF6 was detected in the immunoprecipitation complex. By up-regulation and knockdown of the gene expression, Huh7 cells and Hep3B cells were transfected respectively and were divided into sh-NC group (negative control transfected cells), sh-ALKBH5 group (short hairpin-ALKBH5 transfected cells) and sh-ALKBH5+ex-PRPF6 group (short hairpin-ALKBH5 transfected + overexpressed pcDNA 3.1-PRPF6 transfected cells). Then, CCK -8 assay and dyeing proliferation test were used to determine the proliferation of the cells in different groups. Tumor xenograft in nude mice checked the biological function of ALKBH5 and PRPF6 in vivo. Besides, the expression levels of AKT/mTOR pathway related proteins in the cells of different groups were detected by western-blot. Result  ALKBH5 was up-regulated in HCC cells, which could remove the methylation modification of PRPF6. In vitro and in vivo experiments have showed that ALKBH5 could participate in the malignant proliferation of HCC cells by regulating the expression of PRPF6. Moreover, the knockdown of ALKBH5 could inhibit the phosphorylation of serine -threonine kinase (AKT) and mammalian target of rapamycin (mTOR), while the overexpression of PRPF6 contributed to the phosphorylation of AKT and mTOR. Conclusion  ALKBH5 can promote the proliferation of HCC cells, and the related mechanism is achieved through the ALKBH5/PRPF6/AKT/mTOR axis.

Key Words:  Alk B homolog 5; Pre -mRNA processing factor 6; N6 -methyladenosine; Hepatocellular carcinoma; Proliferation

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