miR-224-RASSF8预测T1期食管鳞癌转移和预后及其分子机制研究

Endoscopic resection is safe, easy, and esophagus-preserving, less invasive than esophagectomy, and can get a cure as well as esophagectomy for stage T1 esophageal squamous cell carcinoma (ESCC), but patients with risk of metastasis and poor prognosis should not be treated with endoscopic resection or need additional treatment after endoscopic resection. Screening metastasis and prognosis related biomarkers can help to identify stage T1 ESCC patients at a high risk of metastasis and poor prognosis and can direct reasonable individual therapy. MicroRNAs (miRNAs) are stable in paraffin embedded specimens and plasma, and are important in the study of biomarkers in early-stage cancer. Both miRNAs and their target genes can be biomarkers. Our previous study analyzed 30 T1 ESCC paraffin embedded specimens using miRNA array, and found 13 differentially expressed miRNAs between metastatic group and non-metastatic group. We validated one of these miRNAs, miR-224, with paraffin embedded specimens using qualitative real-time PCR (qRT-PCR), and found its expression was correlated with metastasis and prognosis. It was also validated in cell lines, whose abilities of proliferation, migration and invasion were increased after mimic transfection and decreased after inbibitor transfection. RASSF8 was predicted and confirmed as one of the target genes of miR-224 with predicting softwares, qRT-PCR, Western blot, Luciferase reporter and siRNA assays. RASSF8 expression was correlated with metastasis and prognosis using tissue microarray based immunohistochemistry. We will analyze miR-224 expression in the plasma and RASSF8 expression in the paraffin embedded tumor tissue of 200 stage T1 ESCC patients, and evaluate the effectiveness of these two biomarkers in clinicopathological testing using multivariate analysis to identify stage T1 ESCC patients at a high risk of metastasis or poor prognosis. Then we will use ESCC cell lines to study the upstream and downstream mechanisms of miR-224 and RASSF8. Finally, we will use nude mice to study effect of miR-224 on tumor growth and metastasis in vivo. This study can provide a theoretical basis for reasonable individual therapy of stage T1 ESCC patients and is valuable theoretically and clinically.

T1期食管鳞癌内镜下切除安全微创，但成功治疗的前提是需预测转移和预后进行个体化治疗。miRNA在石蜡组织和血中稳定且易检测，是难获得新鲜组织标本的早期癌的理想研究手段，miRNA及其靶基因均可成为转移和预后的标志物。我们前期用miRNA芯片分析30例T1期食管鳞癌石蜡组织发现了13个转移组和非转移组差异miRNA，经验证石蜡组织中miR-224表达与转移和预后相关。miR-224能促进食管癌细胞系增殖、迁移和侵袭。预测并确认的靶基因RASSF8的表达与转移和预后负相关。我们将检测血浆miR-224的表达，联合临床病理指标多因素分析评价血浆miR-224和组织RASSF8检测的临床应用价值；然后用细胞系研究miR-224和RASSF8上、下游调控机制；最后用裸鼠模型验证miR-224对食管鳞癌生长和转移的影响。本课题能为T1期食管鳞癌个体化治疗方式选择提供理论基础，有较好的科学性和临床价值。

准确预测T1期食管鳞癌转移和预后是进行个体化治疗的前提。miRNA在石蜡组织和血中稳定且易检测，是难获得新鲜组织标本的早期癌的理想研究手段，miRNA及其靶基因均可成为转移和预后的标志物。我们前期用miRNA芯片分析30例T1期食管鳞癌石蜡组织发现13个转移组和非转移组差异miRNA，经验证石蜡组织中miR-224表达与转移和预后相关。我们研究发现miR-224能促进食管癌细胞的增殖、迁移和侵袭以及淋巴管的生成。预测并确认miR-224的靶基因RASSF8，且免疫组化结果显示RASSF8的表达与T1b期食管癌淋巴结转移和预后负相关。我们还发现miR-224在食管癌患者血清中的表达水平可预测T1b期食管癌淋巴结的转移情况。miR-224通过下调其靶基因RASSF8促进转录因子NF-κB入核，而NF-κB可激活miR-224基因的转录，从而促进miR-224的表达，NF-κB-miR-224-RASSF8- NF-κB可形成正反馈通路；miR-224可刺激肿瘤细胞分泌VEGFC增加，可刺激淋巴内皮细胞中VEGFR3表达上调，从而诱导淋巴管的生成，促进食管癌淋巴结转移。动物实验结果显示，miR-224可促进食管癌的生长、肺转移和淋巴结转移。本课题能为T1期食管鳞癌个体化治疗方式选择提供理论基础，有较好的科学性和临床价值。