沉默Versican减轻放疗引发的肿瘤微环境炎性改变的作用及机理研究

At present,radiotherapy has become a common anti-cancer therapy. However, accumulating evidence suggests that radiotherapy is not satisfactory for radioresistance, and even has a risk of tumor recurrence and metastasis. This phenomenon is associated with a series of reactive changes that have occurred in the tumor microenvironment, especially resulting in the inammatory state of tumor microenvironment. Versican, a ‘bridge’ connecting inflammation with tumor progression as well as playing a central role in the generation of an inammatory tumor microenvironment is a promising candidate for the intervention of inammatory changes in the tumor microenvironment elicited by radiotherapy. Our recent study have found that, radiotherapy elicited an increase in versican in tumor tissue. We speculate that, versican, overexpressed in tumor stroma after radiotherapy, plays a key role in the generation of an inammatory tumor microenvironment. To test this hypothesis, we plan to study the effects of versican on the inammatory changes in the tumor microenvironment after radiotherapy. An foundation stone might be established by this study in the understanding of profound mechanism of radioresistance, the inevitable link between the changes of inflammatory state of tumor microenvironment and the tumor recurrence and metastasis. And this study might provide the new perspective expanding on cancer research and the potential clinical application of radiotherapy.

放疗抵抗及随之出现的肿瘤复发转移成为目前放射治疗在临床中面临的主要困境，这涉及放疗后肿瘤微环境的炎症性改变。Versican—介导炎症与肿瘤转移的桥梁分子，是个具有干预肿瘤微环境炎症性改变作用的候选者。我们前期研究发现，Versican在营造抗肿瘤血管生成后的肿瘤炎性微环境中发挥关键作用；放疗后肿瘤组织中Versican的表达较未放疗组明显增高。我们推测，放射治疗后，作为炎症关键因子的Versican的上调，可能是放疗引发肿瘤微环境发生一系列炎性改变的主要促发因素之一。为验证该假说，本项目在原有研究的基础上，拟开展沉默Versican减轻放疗引发的肿瘤微环境炎性改变的作用及机理研究。这对进一步阐明放疗抵抗相关机理，Versican与放疗后肿瘤微环境变化，炎症与肿瘤复发转移的关系，拓展肿瘤放射治疗新思维具有创新科学意义，亦具潜在临床应用价值。

放疗抵抗及随之出现的肿瘤复发转移成为目前放射治疗在临床中面临的主要困境，这涉及放疗后肿瘤微环境的炎症性改变。本项目拟开展沉默Versican减轻放疗引发的肿瘤微环境炎性改变的作用及机理研究。按研究计划构建结直肠癌细胞株（CT26）和肺癌细胞株（LLC）小鼠皮下瘤模型，发现在CT26模型中，辐照3天后RT组中的MDSCs、TAM的比例均显著上调，WB发现肿瘤组织中RT组Versican蛋白的表达增高，完成了验证放射治疗后结直肠癌肿瘤微环境发生以炎性反应为主的改变，其中伴随Versican表达上调，Versican可能在细胞增殖及炎性反应中发挥重要作用；进一步构建shRNA-Versican载体，基于前期CT26荷瘤小鼠模型结果继续构建肠癌Versican稳转株，由于Versican-shRNA在肠癌细胞株中敲低效果差，部分已敲除细胞增殖存活影响较大，故进行结直肠癌其他类似生物标志物筛选：通过结直肠差异表达基因的生信分析，构建PPI网络鉴定了10个枢纽基因，其中HCAR3和INSL5此前未见报道，并在TCGA结直肠癌(COADREAD)数据库中探索结直肠癌患者的HCAR3和INSL5的 mRNA表达和总生存期数据分布，发现INSL5为结直肠癌中的下调基因，且其高表达与结直肠癌的预后正相关，进一步在临床结直肠癌患者肿瘤样本及结直肠癌过表达INSL5稳转细胞株中进行了相应的表达量检测及生物学功能验证，检测选择INSL5较低且其受体RXFP4表达量较高的两株结直肠癌细胞株构建INSL5过表达稳转株，QPCR、WB和免疫组化验证了INLS5在临床肿瘤样本中的差异表达，分别采用CCK8、细胞周期实验、细胞凋亡实验及凋亡通路验证，检测过表达INSL5可显著抑制CRC细胞的增殖，促进PARP的剪切，初步阐明INSL5在结直肠癌中的生物学功能及可能机制，为发现结直肠癌治疗潜在新靶点奠定基础；同时，结合本课题“抗炎”结合“抗肿瘤”研究思路策略，发现新型抗生素——可利霉素（CAM）通过免疫调节相关抗肿瘤机制，CAM可上调M1巨噬细胞TNF-α和iNOS的水平，下调M2中Arg-1的水平，CAM抑瘤率为41.6%,促进M1巨噬细胞数量增加，提示CAM可能通过诱导巨噬细胞向M1巨噬细胞极化发挥抗肿瘤作用。为进一步探索“抗炎”联合“抗肿瘤”治疗新策略奠定基础。