LncRNA-SNHG6活化NF-κB通路促进骨肉瘤发病转移及益气化瘀清热解毒法的疗效机制研究

Osteosarcoma is a most common cancer in children and adolescents, the 5-year survival rate is less than 30% with metastasis. With pulmonary metastasis or not at diagnosis is the key prognosis indicator of the 5-year survival rate. .Long non-coding RNAs (lncRNAs) are newly discovered cancer regulatory factors, and can preferably mark the tumor inherent characteristics as the effector molecules, treatment targets lncRNAs will have fewer side effects, but their regulatory roles in osteosarcoma are not clear. NF-κB signaling is involved in many aspects of cancer incidence, and anticancer therapy targeted NF-κB pathway is an effective strategy for improving the activity of anti-osteosarcoma drugs. Expression of SNHG6 (lncRNA), Ikkβ and XIAP (NF-κB pathway critical factors) was significantly upregulated in human osteosarcoma tissues detected by lncRNAs microarray..In this project, SNHG6 gene knockout osteosarcoma cells will be generated by the CRISPR/Cas9 technique. RT-PCR, Northern Blot, RIP, RNA EMSA, in situ hybridization, immunohistochemistry, and other techniques will be used to study and reveal the importance of NF-κB activated by SNHG6 in osteosarcoma incidence and metastasis, and the effective mechanisms of "Qichong sanchi pulvis " for anti-osteosarcoma incidence and lung metastasis in vivo, with the models of wild-type and SNHG6 knockout osteosarcoma cells, in situ and lung metastasis osteosarcoma nude mice, SNHG6 knock-in mice.

骨肉瘤是儿童青少年最常见恶性肿瘤，转移后5年存活率不足30％，诊断时是否伴肺转移是预测5年存活率关键指标。.长链非编码RNA是新发现的癌症调控因子，作为效应分子能更好标记肿瘤特征，针对它的治疗副作用较少，但其对骨肉瘤调控作用不清楚。NF-κB参与肿瘤发病的各个环节，针对该通路的靶向治疗是提高抗癌药物对骨肉瘤活性的有效方法。芯片检测发现人骨肉瘤组织中长链非编码RNA-SNHG6、NF-κB通路关键因子Ikkβ和XIAP显著上调。.本项目用CRISPR/Cas9技术敲除骨肉瘤细胞中SNHG6基因，在野生型及SNHG6敲除骨肉瘤细胞、原位和肺转移裸鼠骨肉瘤、SNHG6敲入小鼠模型中运用RT-PCR、Northern Blot、RIP、RNA EMSA、原位杂交、免疫组化等方法研究，揭示SNHG6激活NF-κB通路对骨肉瘤发病和转移的重要性，以及“芪重三七散”体内外抗骨肉瘤发病和肺转移的效应机制。

骨肉瘤肺转移患者5年生存率不足30％，因无早期特异筛查策略，确诊时往往已肺转移。.本项目用芯片及qPCR检测证实骨肉瘤病人组织、血液和细胞SNHG6显著上调；测序及qPCR证实143B细胞中以转录本1为主；Kaplan-Meier生存分析发现SNHG6高表达骨肉瘤患者总生存期显著短。 .用shRNA或Crisp/Cas9包装慢病毒敲降或敲除骨肉瘤细胞SNHG6，并将标记GFP和Luciferase的细胞注射到裸鼠胫骨内制备原位骨肉瘤模型，通过检测细胞增殖、克隆形成、凋亡、周期、迁移和浸润能力及相应分子标记，体内肿瘤体积和重量、X-RAY和μCT检测骨组织破坏、荧光检测肺转移克隆，证实了SNHG6缺失显著抑制骨肉瘤生长和肺转移。.原位杂交激光共聚焦和RNA核质分离检测发现SNHG6主要在细胞浆内。测序后GSEA分析表明敲降143B中SNHG6下调了HIPPO靶基因，并用qPCR证实。.构建了HIPPO信号报告基因元件用报告基因检测HIPPO信号活性、qPCR检测对HIPPO靶基因表达、Western Blot检测对HIPPO上游激酶MST1和LATS1磷酸化及转录共激活子TAZ和YAP入核影响，证明了SNHG6促进MST1和LATS1磷酸化和TAZ入核。用shRNA分别敲降发现MST1、LATS1、YAP和TAZ均参与SNHG6诱导的HIPPO报告基因活性及SNAI表达。.过表达SNHG6不同长度截短序列，用报告基因检测HIPPO活性、Western Blot检测MST1和LATS1磷酸化和SNAI表达，证实SNHG6的1-150bp是其催化HIPPO信号主要活性位点。.通过RNA pull-down和RIP检测发现SNHG6主要与LATS1和TAZ结合；通过分析骨肉瘤组织mRNA芯片m6A相关酶、SNHG6敲降对m6A相关酶影响、m6A相关酶过表达对SNHG6表达影响、突变SNHG6甲基化位点对SNHG6及Tead表达影响证实SNHG6的m6A修饰参与Hippo信号调控骨肉瘤发生和肺转移。.本研究还证实“芪重三七散”主要有效成分单独或协同抑制SNHG6降低骨肉瘤细胞存活率。.因此，本研究揭示了骨肉瘤发生及肺转移分子机制，可为骨肉瘤临床诊治提供新思路、为研发早期诊断、防治和预后等策略提供分子靶标，促进临床转化应用，从根本上提高骨肉瘤患者生存率、改善其临床预后。