miR-211调控的lncRNA(s)鉴定及其在皮肤黑色素瘤发生发展中的作用研究

Cutaneous melanoma is a highly invasive cancer. Advanced melanoma patients have poor prognosis due to the lack of effective therapies. Our earlier studies have already shown that the expression of miR-211 is greatly decreased in both melanoma cells and melanoma tissue samples compared to melanocytes and benign nevi. Moreover, our other studies also find that ectopic overexpression of miR-211 in melanoma cells repressed both colony formation in methylcellulose and migratory ability in matrigel, an in vitro surrogate assay for invasiveness. These findings indicate that miR-211 acts as tumor suppressors in melanomagenesis. Recent studies have shown lncRNAs being targets of microRNAs mediate oncogenic or tumor-suppressing effects and may be a new class of cancer biomarkers and therapeutic targets. Thus, this project tries 1) to screen miR-211 regulated lncRNA(s) that influence the melanomagenesis through lncRNA microarray, 2) to verify the selected lncRNA(s) in melanoma through qPCR and ISH, 3) to characterize the functions of the selected lncRNA(s) in melanoma progression by cell proliferation, apoptosis and migration efficacy tests. This project aims to look for miR-211 regulated lncRNA(s) and investigate their roles in melanoma development and progression, which potentially offers a novel therapeutic strategy for melanoma patients.

皮肤黑色素瘤是一种侵袭性很高的恶性肿瘤，发生转移的黑色素瘤因缺乏有效的治疗手段而预后极差。我们先前的研究显示miR-211在黑色素瘤细胞和组织中非常一致的低表达；通过提高其在黑色素瘤的表达发现肿瘤细胞的侵袭力显著下降，提示miR-211在黑色素瘤中肿瘤抑制因子的作用。近期研究发现lncRNA可作为微小RNA的靶分子起到致瘤或抑瘤的作用，并成为新的肿瘤标志物和治疗靶点。因此，本课题将通过1）lncRNA基因芯片技术筛选受miR-211调控的与黑色素瘤发生发展相关的lncRNA(s)；2）qPCR和原位杂交技术鉴定目标lncRNA(s)在黑色素瘤的表达；3）一系列功能实验对目标lncRNA(s)表达调控后的黑色素瘤细胞进行肿瘤增生、凋亡及侵袭转移等的作用研究。该课题旨在寻找与miR-211相关的靶lncRNA(s)并深入研究目标lncRNA(s)的作用，从而为黑色素瘤的治疗提供新的研究方向。

黑色素瘤是一种侵袭性很高的最多见于皮肤的恶性肿瘤。发生转移的进展期黑色素瘤预后极差归因于没有有效的治疗手段。我们的研究表明miR-211在黑色素瘤细胞和组织中非常一致的低表达，并通过提高其在黑色素瘤细胞的表达发现肿瘤细胞的侵袭能力下降，提示miR-211在黑色素瘤中肿瘤抑制因子的作用。本研究同过 1）lncRNA基因芯片技术在野生型和miR-211过表达A375细胞上进行特异表达的lncRNA筛选。在得到的特异表达的lncRNA后，结合文献阅读，我首先选择lncRNA——Neat1进行后续的研究。2）qPCR技术验证了miR-211在黑色素瘤A375细胞中抑制Neat1的表达，而Neat1又负调节miR-211在黑色素瘤A375细胞中的表达，两者负反馈抑制对方的表达。并通过双荧光素酶实验进行miR-211与Neat1相关关系的研究发现两者表达变化是通过其他途径发生的，Neat1并非miR-211的直接靶分子。3）一系列功能实验包括：凋亡实验结果显示NC组显示两组的凋亡率均低于5%，提示两组均未发生明显的细胞凋亡；MTT细胞增殖实验结果显示siRNA-Neat1组从Day1-Day5 的细胞增殖均高于NC组，从Day3天开始两组增殖情况比较有差异(P<0.05)；克隆形成能力siRNA Neat1组较NC组显著降低 （P<0.05）；然而在侵袭试验方面两者结果无明显差异。从上述实验结果显示miR-211抑制Neat1的细胞增殖和克隆形成，未影响细胞的侵袭转移。本课题将第一次深入研究lncRNA与miR-211相互调控及其在黑色素瘤发生发展的作用，从而为黑色素瘤提供新型诊断分子和新的研究靶点。