miR-33a靶向HIF-1α抑制黑色素瘤有氧糖酵解的机制研究

The earlier experiments have found that the miR-33a significantly inhibited aerobic glycolysis and cell proliferation of the melanoma ,as HIF-1α is the direct target genes of miR-33a ,combining with document that show HIF-1α has closely relationship with erobic glycolysis of tumor cells,so research group consider that there may be a pathway of "miR-33a/HIF-1α/glycolysis" . .The subject : 1.Confirm that miR-33a has an effect on melanoma aerobic glycolysis and proliferation or apoptosis ; 2.Confirm that miR-33a regulate melanoma tumors glucose metabolism, proliferation and apoptosis through HIF-1α. 3. Analysis the regulation network of miR-33a/HIF-1α/glycolysis through the whole genome chip technology ; 4. Observe the expression level of miR-33a effect drug influence to tumor energy metabolism sensitivity, provides the basis for cancer treatment by the angle of metabolic. 5. Observe relationship of expressions of miR-33a and HIF-1α with prognosis in clinical cases, looking for new targets of melanoma diagnosis and treatment,providing scientific basis for the development of individual drug relating to glycolysis and gene therapy.

前期预实验发现miR-33a明显抑制了黑色素瘤的有氧糖酵解和细胞增殖，HIF-1α为miR-33a的直接靶基因，结合文献HIF-1α与肿瘤细胞有氧糖酵解有密切调控关系，课题组推测黑色素瘤细胞中可能存在"miR-33a/HIF-1/糖酵解"调控通路。本课题1.确定miR-33a对黑色素瘤有氧糖酵解及增殖凋亡的影响；2.明确miR-33a通过HIF-1调控黑色素瘤葡萄糖代谢并影响肿瘤细胞增殖及凋亡。3.通过全基因组芯片技术分析"miR-33a/HIF-1/糖酵解"之间的调控网络。4.观察miR-33a表达水平对药物作用肿瘤能量代谢敏感性的影响，为从代谢角度进行肿瘤治疗提供依据。5.观察miR-33a及HIF-1在临床病例中表达与预后关系，寻找黑色素瘤诊断和治疗的新靶点，为开展糖酵解相关个体化药物或基因治疗提供科学依据。

课题组前期用miR微阵列芯片发现let-7b、miR-33和miR-199a在恶性黑色素瘤中表达异常，let-7b和miR-199a抑制细胞增殖，miR-33a抑制黑色素瘤的侵袭和转移。将miR-33a转染人黑色素瘤细胞株A375后证实miR-33a抑制A375的葡萄糖消耗、乳酸生成和增殖，但其调控黑色素瘤Warburg效应的潜在的分子机制尚不清楚。课题组用生物信息学软件miRanda和Pictar对miR-33a行靶基因预测后发现HIF-1α是miR-33a的靶基因，并用双荧光素酶报告基因法证实了这个预测。前期的研究和文献报道都提示miR-33a可能靶向HIF-1α抑制黑色素瘤有氧糖酵解，本课题主要针对该假设进行验证。通过实时荧光定量PCR检测其3p片段和5p片段在不同恶性程度黑色素瘤细胞系中的表达发现miR-33a-3p表达量与黑色素瘤恶性程度无关，miR-33a-5p在WM451、A375、SK-MeL-1细胞中的表达量显著低于低度恶性、无转移潜能WM35细胞中的表达量，而作为深入研究microRNA。随后构建miR-33a-5p慢病毒过表达和干扰质粒，干预其表达水平并建立稳定表达细胞系，体内外水平通过MTT 法，化学比色法，Western-blot法等检测miR-33a-5p对黑色素瘤细胞增殖、糖酵解和HIF-1α表达的影响；构建HIF-1α过表达和干扰质粒，回复HIF-1α的表达后探讨其对miR-33a-5p介导的黑色素瘤细胞增殖和糖酵解的影响。研究发现：miR-33a-5p抑制黑色素瘤细胞增殖和糖酵解表型，并抑制相关蛋白HIF-1α、LDH-A、HK1和HK2的表达及LDH-A和HK的酶活性。HIF-1α能够逆转miR-33a-5p对黑色素瘤细胞的增殖和糖酵解抑制效应。miR-33a-5p能够显著抑制裸鼠种植瘤的生长，提高预后。miR-33a-5p能够在体内水平抑制乳酸生成，并抑制HIF-1α和LDH-A的表达，降低LDH-A和HK酶活性。回复HIF-1α的表达，能逆转体内miR-33a-5p对黑色素瘤乳酸生成的抑制作用。本课题初步探讨了microRNA影响肿瘤Warburg效应的潜在分子机制，分析了干预这一通路对肿瘤增殖的影响，丰富了miR-33a作为黑色素瘤治疗的靶点的理论体系，为黑色素瘤的代谢治疗提供了重要依据。