微小RNA-1275对食管癌EMT获得与放射抵抗形成的调控机制

Acquired radioresistance during radiotherapy has significantly affected the treatment efficacy in esophageal cancer. Many of radioresistant cancer cells demonstrated epithelial-mesenchymal transition (EMT), which has been believed to link to the suppressed apoptosis signal pathway, enhanced DNA repair ability and growth factors. However, it remains unclear for the role of EMT in radio－resistant acquirement in esophageal cancer. By fractional radiation on KYSE-150, we established radio-resistant esophageal cancer cell line KYSE-150R which demonstrated an EMT-like phenotype, together with the abnormal activation of Wnt/β-catenin signal pathway. We further revealed that there was the suppressed miR-1275 expression in above radio-resistant cell line by microRNA chip screening. Bioinformatics analysis indicated that wnt1 in Wnt signal pathway could be the possible target gene. The purpose of this study is to study if miR-1275 could suppress protein expression of WNT1 in vivo and in vitro and whether the decreased expression of miR-1275 subsequently activate Wnt/β-catenin signal pathway, induce EMT, and acquire radioresistence in esophageal cancer cells. The results derived from this study will reveal mechanism of miR-1275 in regulating radioresistance of esophageal cancer for the first time, and improve our understanding on target therapy with microRNAs.

获得性放射抵抗的形成是影响食管癌治疗效果的重要因素。前期研究认为多种肿瘤细胞可通过 抑制细胞凋亡、激活DNA损伤修复通路和生长因子，引起上皮间质转换(EMT )而使放射抵抗性 得到增强。然而，EMT参与食管癌放射抵抗过程的机制尚不明确。在前期研究中，基于分次照 射处理我们构建了放射抵抗食管癌细胞株KYSE-150R，发现该细胞株发生了EMT，并伴随Wnt/β catenin信号通路异常激活。此外，通过芯片并筛选到miR-1275可能起放疗增敏作用。通过生物信息学分析，推测Wnt信号通路重要分子wnt1可能是其潜在靶基因。本项目拟通过体内外实验，确立miR-1275是否通过调控wnt1的蛋白表达而激活Wnt信号通路，从而导致食管癌发生EMT和放射抵抗的形成。研究结果将首次揭示miR-1275在食管癌放射抵抗形成过程中的机制，为该microRNA在食管癌临床靶向性治疗中的应用用奠定基础。

获得性放疗抵抗的发生，是食管癌局部复发和远处转移的主要原因之一。最近的研究表明，microRNAs（miRNAs）在恶性肿瘤中的放射抵抗形成起重要作用。本课题研究miR-1275在获得性放射抵抗和上皮-间质转化（EMT）中的作用。首先，采用梯度剂量法照射建立一个放射抵抗细胞系KYSE-150R。与亲本细胞相比，KYSE-150R细胞中的miR-1275表达下调。随后分别向KYSE-150R和KYSE-150转染miR-1275 mimic和inhibitor，通过克隆形成实验检测miR-1275对细胞放射敏感性的影响；MTT法检测细胞增殖能力的影响；Transwell小室和划痕实验检测细胞迁移能力的影响;Western blot检测EMT表型的调控。结果显示过表达miR-1275抑制食管癌细胞的增殖、迁移能力，提高放射敏感性，逆转EMT表型。干扰miR-1275后，食管癌细胞的增殖、迁移能力提高，放射敏感性下调，细胞转向EMT表型。进一步通过荧光定量PCR及双荧光素酶报告验证WNT1是miR-1275的靶基因。TOP／FOP实验、Western blot实验验证miR-1275与下游调控基因WNT1和WNT/β-catenin通路之间的关系。采用体外细胞功能回复实验验证干扰WNT1表达能否逆转miR-1275对食管癌细胞放射敏感性及EMT表型的影响，选取KYSE-150R细胞分别转染NC-mimic、miR-1275 mimic或miR-1275 mimic+pcDNA3.1/WNT1，选取KYSE-150细胞分别转染NC-inhibitor、miR-1275 inhibitor或miR-1275inhibitor+si-WNT1。结果显示敲除WNT1通过抑制EMT减弱了miR-1275抑制对KYSE-150细胞放射抵抗能力的促进作用，而WNT1过表达则挽救了miR-1275上调EMT，从而降低KYSE-150R细胞对辐射的敏感性。最后动物实验证实miR-1275对食管癌细胞的生长具有抑制作用，并通过抑制体内EMT增强了肿瘤对放射治疗的敏感性。.本课题表明miR-1275通过靶向WNT1激活的WNT/β-catenin信号抑制EMT，促进食管癌细胞的放射敏感性，为食管癌放射抵抗的治疗提供了新的途径。