lnc-SNHG1竞争性结合miR-216b-5p上调NF-κB1促进卵巢癌化疗耐药的作用研究

Ovarian cancer is the most lethal of the gynecological cancers, with approximately 200,000 new cases and more than 100,000 deaths reported every year. Patients routinely undergo a debulking surgery before chemotherapy with Taxol and platinum-based drugs. However, chemoresistance is recurrent, and the 5-year survival rate is approximately 30%. Therefore, the elucidation of the underlying mechanisms in ovarian cancer is an indispensable prerequisite.The regulation of lncRNA and miRNA and its downstream target genes has become in focus of the field , including gynecological tumors. Our study is based on the lncRNA-miRNA-mRNA gene regulation network in searching the cause of ovarian cancer chemo-resistance. In this study,we first aim to evaluate the differential expression of lnc-SNHG1 and miR-216b-5p between epithelial ovarian cancer chemo-resistant tissues and chemo-sensitive tissues. By implying in situ hybridization method we aim to detect the expression of lnc-SNHG1, miR-216b-5p. The qRT-PCR and TaqMan real-time PCR method were applied to confirm the result. The relationship between the expression of lnc-SNHG1 and miR-216b-5p, lnc-SNHG1 and NF-κB1 mRNA were analyzed. By applying luciferase reportor assay along with cell co-transfection assay we aim to confirm that NF-κB1 could be a direct target of miR216b-5p. And by applying CCK-8 assay, cell apoptosis assay, cell cycle assay,we are to clarify the role of miR-216b-5p regulating NF-κB1 in chemo-resistance of ovarian cancer. Further, by knocking down the expression of lnc-SNHG1 in ovarian cancer chemo-resistant cell line SKOV3/Taxol or A2780/Tax,then testing cell proliferation, cell apoptosis and cell cycle, we aim to clarify the effect of lnc-SNHG1 on chemo-sensitivity in ovarian cancer. Finally, by applying the luciferase reportor assay along with RIP method, we aim to detect the combination of lnc-SNHG1 with miR-216b-5p in ovarian cancer resistant cells. Taken together, we aim to provides a new hypothesis that lncRNA-SNHG1 contributes to ovarian cancer chemo-resistance through inhibition of miR-215b-5p and there after NF-κB1 up-regulation.

对卵巢癌化疗耐药机制的研究是国内外妇科恶性肿瘤研究者一直关注和亟待攻克的难题。最近研究表明lncRNA 可作为ceRNA与miRNA结合对肿瘤化疗耐药产生重要影响。项目申请人前期通过lncRNA及miRNA芯片发现在卵巢癌化疗耐药细胞中lnc-SNHG1上调，miR-216b-5p呈下调趋势；软件预测两者有结合位点。同时NF-κB1在卵巢癌化疗耐药组织及细胞系中高表达。本项目拟在前期研究工作基础上通过RIP, 荧光素酶报告基因，基因转染，耐药相关生物学行为检测等方法明确低表达的miR-216b-5p是否通过转录后调控NF-κB1引起卵巢癌化疗耐药；进一步研究lnc-SNHG1竞争性结合miR-216-5p从而上调NF-κB1表达在卵巢癌化疗耐药细胞系中的作用。本项目立足于lncRNA-miRNA-mRNA这一新的基因调控模式，为卵巢癌化疗耐药研究提供新的研究思路和新的基因治疗靶点。

对卵巢癌化疗耐药机制的研究是国内外妇科恶性肿瘤研究者一直关注和亟待攻克的难题。最近研究表明lncRNA 可作为ceRNA与miRNA结合对肿瘤化疗耐药产生重要影响。课题组在前期工作基础上，首先利用组织芯片通过FISH方法证实lnc-SNHG1在卵巢癌化疗耐药组织中过表达，同时miR-216b-5p低表达，免疫组化证实Pak2过表达。lnc-SNHG1高表达与卵巢癌患者总生存期（OS）及无病生存期（DFS）缩短显着相关。miR-216b-5p表达水平低的患者OS和DFS亦显著缩短。miR-216b-5p低表达和FIGO分期是卵巢癌患者总生存期缩短的独立危险因素。免疫组化方法表明在卵巢癌化疗耐药组织中Pak2表达阳性率较化疗敏感组显著升高且其主要表达于胞浆。通过细胞转染，在卵巢癌耐药细胞系中沉默lnc-SNHG1表达、过表达miR-216b-5p、双抑制表达lnc-SNHG1及miR-216b-5p、敲减Pak2表达，对细胞增殖、凋亡、划痕、克隆形成等化疗耐药生物学行为检测。通过荧光素酶报告基因及RIP等方法明确卵巢癌化疗耐药细胞中lnc-SNHG1与Pak2 mRNA竞争性结合miR-216b-5p的作用，从而进一步明确lnc-SNHG1作为ceRNA竞争性结合miR-216b-5p而上调Pak2表达在卵巢癌化疗耐药中的作用。本项目以lncRNA通过ceRNA方式竞争性结合miRNA为研究立足点，为卵巢癌化疗耐药的机制研究提供新的研究思路和新的基因治疗靶点。