半枝莲调控lncRNA/miRNA-34a逆转大肠癌5-FU耐药的分子机制研究

Multi-drug resistance (MDR) of tumor is a major cause of failure of cancer chemotherapy. LncRNAs may act as competing endogenous RNAs to regulate target genes by sponging miRNAs, and thus play important roles in the process of MDR. Scutellaria barbata D. Don (SB) has long been used as a major component in numerous Chinese medical formulas to clinically treat colorectal cancer. Our previous studies showed that SB could enhance 5-FU sensitivity in colorectal carcinoma cells via miR-34a. Based on that, we suppose that SB could overcome 5-FU drug resistance via the regulation of lncRNAs/miRNAs signaling network. On account of this, by using the “lncRNAs/miR-34a axis” as breakthrough point, we will preliminarily screen some lncRNAs which might be possibly regulated by SB though bioinformatics analysis and second-generation sequencing technology. Thus, we will investigate the interaction mechanisms between target lncRNA and miR-34a as well as its relationship with MDR. Eventually, we will illuminate the molecular mechanism of SB reversing 5-FU drug resistance via modulation of lncRNAs/miR-34a in vivo and vitro. The aim of this study can extend the relationship between lncRNAs/miRNAs signaling network and the drug resistance. Meanwhile, it will also illustrate the mechanism of anti-drug resistance of SB to provide a scientific foundation for the development of its clinical application.

肿瘤耐药是大肠癌化疗失败的重要原因。长链非编码RNA(lncRNAs)能充当分子“海绵”，通过竞争性结合miRNAs调控基因的表达，参与肿瘤耐药。我们前期研究发现：在临床上治疗大肠癌疗效确切的半枝莲能逆转大肠癌5-FU耐药且该过程与miR-34a密切相关。这些提示半枝莲可能通过调控lncRNAs/miRNAs信号网络逆转大肠癌5-FU耐药。因此，本项目拟以lncRNA/miR-34a轴为切入点，初步筛选出半枝莲调控的与耐药相关的lncRNAs/miR-34a的信号网络；并验证靶lncRNA与miR-34a的互作机制及其与大肠癌5-FU耐药的关系；最后通过体内外实验探讨半枝莲调控lncRNA/miRNA-34a信号通路逆转大肠癌5-FU耐药的分子机制。本项目的研究拓展了lncRNAs/miRNAs与肿瘤耐药的关系；同时也阐明了半枝莲逆转大肠癌5-FU耐药的作用机制，为其临床应用提供实验依据。

大肠癌是一种常见的恶性肿瘤，其发病率和死亡率在中国均位居第五。5-FU是大肠癌最常用的化疗药物。然而，患者对5-FU产生的耐药限制了其治疗的有效性。许多文献报道lncRNAs通过竞争性结合miRNAs，从而调控其基因的表达，参与肿瘤耐药。半枝莲具有清热解毒、消肿散结、活血化瘀、扶正祛邪的功效，符合中医对大肠癌的疗法。我们前期研究发现半枝莲通过“多环节、多途径、多靶点”方式起到抗大肠癌的作用。但半枝莲是否能通过调控lncRNAs/miRNAs信号轴，从而逆转大肠癌细胞对5-FU的耐药有待进一步研究。本项目主要通过以下几个方面展开研究：一、从体内外水平验证HCT-8/5-FU细胞对5-FU的耐药性，确定耐药细胞模型的可行性。二、半枝莲逆转HCT-8/5-FU细胞对5-FU的耐药性。三、半枝莲对HCT-8/5-FU细胞内miR-34a表达的影响。四、半枝莲对HCT-8/5-FU细胞内linc01843/miR-34a信号轴及其下游靶蛋白表达的影响。研究结果显示：HCT-8/5-FU细胞对5-FU存在显著耐药，而对半枝莲不存在耐药。半枝莲能促进HCT-8/5-FU细胞的凋亡，抑制其增殖。半枝莲还能显著升高HCT-8/5-FU细胞内5-FU的含量，显著抑制HCT-8/5-FU细胞内耐药相关基因（LPR、TOP2α、BCRP）的表达。证明半枝莲能有效逆转HCT-8/5-FU细胞对5-FU的耐药。本研究还发现miR-34a参与了大肠癌对5-FU的耐药性，且半枝莲逆转大肠癌5-FU耐药的作用机制之一是通过调控miR-34a的表达。进一步利用Targetscan和RNAhybird软件并结合lncRNA测序筛选出半枝莲可能调控与耐药相关的lncRNAs/miR-34a的信号网络。通过细胞功能实验筛选出linc01843可能是通过竞争性结合miR-34a，而调控了其下游的靶蛋白，介导了肿瘤细胞增殖凋亡相关途径，进而参与了大肠癌对5-FU的耐药。半枝莲通过调控linc01843/miR-34a信号轴及其下游靶蛋白（Notch1，Notch2，Jagged，Sirt-1，CyclinD1，CDK4，CDK6和Bcl-2），逆转了大肠癌对5-FU的耐药性。本研究拓展了lncRNAs/miRNAs与肿瘤耐药的关系；阐明了半枝莲逆转大肠癌5-FU耐药的作用机制，为其临床应用提供实验依据。