miR-92b、miR-181和miR-375调控网络靶向NLK在非小细胞肺癌增殖中的作用研究

Xuanwei has the highest morbidity and mortality of lung cancer in China. However, the pathogenesis is still poorly understood. Recent investigations have demonstrated that miRNAs played a vital role in the process of tumorigenesis. Therefore, through a miRNA array with Xuanwei NSCLC tissues, we uncover miR-92b, miR-181 and miR-375 expression decreased. Bioinformatics analysis depicts the convergent targeting of the 3`UTRs of NLK by miR-92b, miR-181 and miR-375. Moreover we have identified that depression of these 3 miRNAs increases the expression of NLK. Meanwhile cell proliferation is significantly reduced , cell-cycle G1/S arrest and Cyclin D3 expression decreases in XWLC-05 cell line, which was derived from surgical resection tissue of a patient with lung cancer from Xuanwei. Notably, NLK expression was highly related with tumor volume in Xuanwei NSCLC tissues. Based on these results, we hypothesize that NLK mediated by a miRNA network leads to NSCLC cell abnormal proliferation. In this study, we further use Luciferase Assay and etc. to obtain reliable evidences on the hypothesis so as to identify the key target genes, and to provide a novel scientific proof for targeted therapy on NSCLC.

云南宣威肺癌发病居全国首位，其发病机制远未阐明。新近研究表明，miRNA在肿瘤发生过程中起至关重要的作用。通过miRNA芯片我们在宣威NSCLC中发现miR-92b、miR-181和miR-375表达降低，软件预测：NLK是其共同作用靶点；进一步研究发现，在宣威肺癌细胞系XWLC-05导入上述miRNAs，NLK表达降低，细胞增殖能力下降, G1/S期阻滞，Cyclin D3表达降低；在宣威肺癌组织中，NLK与肿瘤大小密切相关，提示上述miRNAs靶向NLK调控CylinD3促进肺癌细胞增殖。由此，我们提出肺癌细胞增殖可能存在的信号转导机制：上述miRNAs调控网络靶向NLK调控Cyclin D3，促进NSCLC恶性增殖。本项目拟进一步用荧光素酶报告基因系统等技术，旨在获得上述miRNAs调控网络靶向NLK调控Cyclin D3促进NSCLC增殖的证据，阐明肺癌增殖信号转导通路作用机制。

云南宣威独特的二叠纪煤成矿环境和传统的燃煤习惯，使宣威成为特有的肺癌高发现场。新近研究表明，miRNA在肿瘤发生过程中起至关重要的作用。为了深入研究云南宣威肺癌的发病机制，我们研究小组利用前期利用miRNA Assay 技术，在云南宣威NSCLC 组织中筛选出miR-92b、miR-181和miR-375等一系列差异表达miRNAs，且miR-92b、miR-181和miR-375表达具有高度相关性、均与nemo 样激酶（nemo like kinase, NLK）的3’UTR 端存在结合位点，引起我们的极大关注。.我们研究小组后期通过细胞功能实验、分子实验研究表明：过表达miR-92b、miR-181和mi R-375可降低NLK的表达，而过表达miR-92b、miR-181和mi R-375，或敲减NLK均可抑制NSCLC恶性增殖，下调细胞周期相关蛋白Cyclin D3导致细胞周期出现G0/G1期阻滞，初步阐明miR-92b、miR-181和mi R-375靶向NLK导致细胞周期紊乱促进NSCLC恶性增殖；动物实验证实，过表达miR-92b、miR-181和miR-375，或敲减NLK可减慢NSCLC细胞在裸鼠体内生长，在体内环境表明miR-92b、miR-181和miR-375可影响NLK促进NSCLC恶性增殖。.更为重要的是，人体组织标本实验表明miR-92b、miR-181和mi R-375在NSCLC中表达降低，NLK表达升高，miR-92b、miR-181和mi R-375表达与NLK呈负相关，且NLK与肿瘤大小密切相关，进一步证明上述miRNAs调控网络靶向NLK促进NSCLC恶性增殖。.通过本研究，从细胞水平、动物水平和临床标本角度证明miR-92b、miR-181 和miR-375 调控网络靶向NLK 导致细胞周期紊乱，进而导致NSCLC 异常增殖的作用机理，阐明宣威非小细胞肺癌恶性增殖的信号转导通路作用机制，发现与宣威非小细胞肺癌恶性增殖相关的关键靶分子，为非小细胞肺癌新靶点治疗提供科学依据。