HNF4α-miR-541-自噬相关基因调控通路在肝癌中的作用

Hepatocyte nuclear factor 4α (HNF4α) could induce the differentiation of hepatocellular carcinoma (HCC). Our previous study shown that HNF4α transcriptionally up-regulated the expression of miR-379-656 cluster in the DLK1-DIO3 imprinted region. Functional assays demonstrated that, of these miRNAs within the cluster, miR-541 displayed the strongest inhibitory effect on the proliferation of hepatocellular carcinoma (HCC) cells. Moreover, the expression of miR-541 was significantly reduced in HCC tissues and mammospheres. Furthermore, several algorithm predictions indicated autophagy related genes ATG2A and RAB1B as potential targets of miR-541.The following reporter assays demonstrated that miR-541 could bind to the 3’ untranslation region of RAB1B and ATG2A. To date, little has been known about the function of miR-541. Moreover, the effect of miR-541 on HCC also remains unknown. Based on these findings, the present study aims to 1) detect the level of miR-541 in the serum of chronic liver diseases patients and HCC patients and investigate whether it can be used as a diagnostic marker; detect the expression of miR-541 in the liver tissues and serum from HCC patients and analyze its relationship with the clinical malignant phenotype and prognosis of patients; 2) investigate the effects of miR-541 on the malignant biological behavior of HCC cells both in vitro and in vivo; 3) verify the effect of miR-541 on autophagy through regulating ATG2A and RAB1B; 4) study whether HNF4α could affect autophagy by regulating miR-541. Taken together, the research will reveal the consequence of HNF4α-miR-541-ATG2A/RAB1B pathway dysregulation in the development of HCC, which may help to unveil the underlying molecular mechanism and provide novel targets for the diagnosis and treatment of HCC.

肝细胞核因子4α（HNF4α）可诱导肝癌分化。我们前期研究发现它可转录调控DLK1-DIO3印记基因区上的miR-379-656簇，对该簇miRNA进行功能研究发现miR-541抑制肝癌增殖的作用最强，且其在肝癌组织和成球细胞中下调。多种软件均预测自噬相关基因ATG2和RAB1B为其靶基因，报告基因实验证实它可结合二者的3’非翻译区。既往对miR-541的研究较少，其对肝癌的作用亦无报道。本研究将检测肝病患者及肝癌患者血清miR-541表达，分析其作为诊断标志物的可能性，检测肝癌患者肝脏和血清miR-541表达，分析其与临床恶性表型和预后的关系；研究其在体内外对肝癌细胞恶性生物学行为的作用；探讨miR-541是否通过调控RAB1B和ATG2影响自噬以及HNF4α是否通过调控miR-541影响自噬，从而首次探明HNF4α、miR-541、自噬相关调控通路在肝癌中的作用，为其诊治提供新靶点。

肝细胞癌（以下简称肝癌）是常见的恶性实体肿瘤。由于其发病隐匿，进展期有效治疗手段少，目前预后仍较差。索拉菲尼是目前唯一被FDA批准用于进展期肝癌治疗的分子靶向药物，但耐药和副作用等问题影响了其临床收益。近年来，自噬成为肿瘤研究领域的热点。自噬可促进肿瘤细胞转移、复发，导致肿瘤细胞对化疗、放疗和靶向治疗产生耐药性。我们前期研究发现位于DLK1-DIO3印记基因区的一簇microRNA可能具有抑制肝癌的作用，其中miR-541对肝癌细胞增殖的抑制作用最强，但其在肝癌中的作用未见报道。本研究主要探讨miR-541对肝癌的抑制作用及机制以及其在临床标本中的表达和临床意义。结果显示miR-541在肝癌患者癌组织中表达显著低于癌旁组织，且其表达下调预示患者预后不良。miR-541可在体内外抑制肝癌细胞的生长和转移，且可下调肝癌细胞的自噬水平。为明确miR-541作用机制，我们利用靶基因预测软件预测与自噬相关的潜在靶基因。六个靶基因预测软件均预测出自噬相关基因2同源体A（autophagy related 2 homolog A, ATG2A）和Ras相关蛋白Rab-1B（Ras-related protein Rab-1B, RAB1B）为miR-541潜在的靶基因。进一步研究发现miR-541可下调ATG2A和RAB1B的mRNA和蛋白表达，报告基因实验证实二者是miR-541的直接靶基因，且在临床标本中表达与miR-541呈负相关。Kmplot数据库预测发现ATG2A和RAB1B在肝癌组织中表达上调提示预后不佳。且ATG2A和RAB1B的小干扰RNA均可抑制肝癌细胞的增殖、克隆形成、迁移和侵袭，并可逆转miR-541 inhibitor对肝癌细胞的促进作用，提示ATG2A和RAB1B部分介导了miR-541在肝癌中的作用。进一步研究发现miR-541可增加肝癌细胞的敏感性，并在小鼠体内增强索拉菲尼对肝癌的抑制作用。以上研究结果表明，miR-541与肝癌患者预后相关，且可能成为肝癌治疗的新靶点，并可能作为增敏剂增强索拉菲尼的作用，具有良好的临床应用前景。