EBV-miR-BART7-3p靶向SMAD7促进鼻咽癌细胞“干性”的作用及分子机制

We previously found that EBV-miR-BART7-3p was highly expressed in nasopharyngeal carcinoma (NPC) and promoted the proliferation, metastasis and EMT of NPC cells, indicating that Epstein–Barr virus (EBV) can influence the occurrence and development of host tumor by encoding its own miRNA. It is known that cancer stem cell is one of the major causes for recurrence and treatment resistance of tumor. Recently, we have preliminary discovered that EBV-miR-BART7-3p can probably affect the stemness of NPC cells, leading to NPC recurrence and resistance to therapy. To confirm this, the present project is firstly going to carry out a series of experiments including culturing of tumor spheres, sorting of side population cells and ALDH1+/PKH26+cells, analyzing of chemotherapeutic drug sensitivity, in vivo tumorigenicity tests, and chemo-therapeutic experiments, etc., validating that this viral miRNA indeed promote NPC stemness. Subsequently, luciferase reporter analyses, IHC, IFC and clinical sample analyses will be used to further define SMAD7 as a novel target gene of EBV-miR-BART7-3p, and then explore the regulation of its downstream signals. This project is expected to newly reveal the possible action and mechanism of EBV-encoding miRNA in cancer stem cells, providing potential new targets for NPC therapy.

我们前期发现EBV-miR-BART7-3p在鼻咽癌（NPC）中高表达并促进NPC细胞的增殖、转移及EMT，表明EB病毒能通过编码自身miRNA影响宿主肿瘤的发生发展过程。众所周知，肿瘤干细胞是肿瘤复发和治疗抵抗的重要原因，我们近期实验又初步新发现EBV-miR-BART7-3p有可能通过影响NPC细胞“干性”进而导致NPC复发及治疗抵抗。为证实该发现，本项目将首先通过肿瘤球培养、侧群细胞分选、ALDH1+/PKH26+细胞分选、化疗药物敏感性、体内成瘤和化疗药物治疗实验，证实EBV-miR-BART7-3p确实具有促进NPC细胞“干性”的作用；然后，应用荧光素酶实验、IHC、IFC等，结合临床标本分析，进一步明确SMAD7是EBV-miR-BART7-3p新的靶基因，并探讨其下游调控的分子机制。本项目有望揭示EB病毒编码miRNA在肿瘤干细胞中的可能作用及机制，为治疗提供新的潜在靶标。

我们前期研究发现并报道EBV-miR-BART7-3p在鼻咽癌（NPC）中高表达并促进NPC细胞的增殖、转移及EMT，表明EB病毒能通过编码自身miRNA影响宿主肿瘤的发生发展过程。肿瘤干细胞是肿瘤复发和治疗抵抗的重要原因，我们提出本课题研究假说：EBV-miR-BART7-3p有可能通过影响NPC细胞“干性”进而导致NPC复发及治疗抵抗。为证实该假说，本研究小组首先利用病毒感染的方法构建稳定过表达EBV-miR-BART7-3p的鼻咽癌细胞株及对照细胞株，接着通过体外实验（肿瘤球培养、SP 细胞分选、western blot、qPCR 及MTT 检测临床化疗药物敏感性等） 和体内实验（裸鼠皮下成瘤率实验、化疗药物治疗实验）明确EBV-miR-BART7-3p能够促进鼻咽癌细胞“干性”。接下来通过芯片分析、生物信息学、荧光素酶报告基因、western blot、qPCR、IHC等实验，结合临床标本验证，明确 EBV-miR-BART7-3p 直接调控宿主靶基因SMAD7。最后查阅文献及利用qPCR、western blot、IHC、IFC 等实验明确EBV-miR-BART7-3p 通过靶向SMAD7 的表达而激活TGFbeta pathway，诱导p-smad2和p-smad3的核定位进而促进鼻咽癌细胞“干性”。相关研究成果于2019 年10 月发表于Front Genet. 2019 Oct 17;10:939. 本项目丰富EB病毒编码miRNA在肿瘤中的作用及机制，为临床提供新的治疗靶点和预后指标。.同时研究小组还在本课题的资助下，我们通过功能实验及机制研究发现并验证EBV-miR-BART8-3p 通过激活ATM/ATR 促进鼻咽癌的放疗抵抗，相关研究成果于2019.09发表于Biosci Rep. 2019 Sep 30; 39(9): BSR20190415..