成纤维细胞来源外泌体LINC00152介导的食管鳞癌细胞放疗抵抗研究

How to overcome the radiation resistance of esophageal squamous cell carcinoma(ESCC) has always been a hot yet intricate topic, since radiation therapy acts as one major modality in treating ESCC and radiation resistance is a most serious problem for its treatment outcome. Cancer associated fibroblast(CAF) is an important part of tumor microenvironment, and it can influence the malignant phenotype of tumor cells by multiple pathways. However, the change of CAF after radiation therapy and how it would influence the radiation sensitivity of ESCC have yet to be researched and demonstrated. We have found that the radiation resistance of ESCC can be induced by exosomes released from CAF after radiation therapy, which is associated with the increased level of LINC00152 in our previous studies. Calculations conducted by software have provided the hint that LINC00152 can inhibit miR-138 and miR-138 can activate AKT pathway, which is also supported by our preliminary experiments. Besides, we have proved activation of AKT pathway can lead to radiation resistance of ESCC by previous studies. Thus, we presume that LINC00152 in exosomes released from the CAF after radiation therapy can induce radiation resistance by modulating miR-138/AKT pathway. Based on that presumption, we designed a series of experiments to verify the presumption on molecular, cellular, clinical samples levels, hoping to look further into the mechanism existing in the radiation resistance of ESCC, and to provide solid evidence for novel therapeutic targets against radiation resistance and better prognostic markers for radiation therapy.

放疗是食管鳞癌的重要治疗手段，而如何克服食管鳞癌放疗抵抗一直是研究的难点与热点。肿瘤相关成纤维细胞(cancer associated fibroblast, CAF)是肿瘤微环境的重要组成部分，可以多途径调控肿瘤细胞的恶性表型，但放疗后CAF的变化及其对食管鳞癌放射敏感性的影响尚无研究报道。我们在前期研究中发现，放疗后CAF来源的外泌体促进食管鳞癌细胞放疗抵抗，且与LINC00152增加相关，我们通过软件预测和初步实验证实其可抑制细胞内miR-138表达继而激活AKT通路，此外我们已有研究表明AKT通路激活可导致食管鳞癌放疗抵抗。由此我们提出假设：放疗后CAF来源外泌体内LINC00152通过调控食管鳞癌细胞miR-138/AKT通路诱导放疗抵抗。本研究中我们将从分子、细胞、临床样本等多水平对该假设验证，以探寻食管鳞癌放疗抵抗新机制，为临床寻找放疗抵抗新的干预靶点及放疗疗效标志物。

食管癌是全世界高发的恶性肿瘤之一。在我国90%的食管癌病理类型为食管鳞癌（Esophageal squamous cell carcinoma, ESCC）。放疗是ESCC的重要治疗手段，而复发转移严重影响了放疗后ESCC的预后。寻找提高ESCC放疗疗效的干预靶点具有重要临床意义。肿瘤相关成纤维细胞（cancer associated fibroblast, CAF）是肿瘤微环境的重要组成部分。CAF能通过外泌体传递信号从而介导肿瘤细胞表型变化。外泌体内含的非编码RNA可以作为信使实现细胞间的信息传递与功能调控。我们的前期研究显示：照射后CAF可通过外泌体向ESCC细胞传递促侵袭转移信号，外泌体中长链非编码RNA（long non-coding RNA，lncRNA）表达量较少。相较于lncRNA，外泌体中包含的microRNA（miRNA）发挥更重要的作用。我们对未照射与照射后CAF分泌的外泌体进行了测序，发现13个差异表达的miRNA。其中，miR-193a-3p与miR-197-3p经验证在照射后CAF分泌的外泌体中显著上调，且miR-193a-3p上调更为明显。我们通过miRNA敲除、Transwell、Western blot 、双荧光素酶报告基因等实验在分子和细胞水平探讨了外泌体miR-193a-3p对ESCC侵袭转移的影响与可能机制。结果如下: 在CAF中敲低miR-193a-3p可逆转照射后CAF来源外泌体对ESCC的促侵袭转移作用，在ESCC细胞中过表达miR-193a-3p能促进细胞侵袭转移。miR-193a-3p可通过靶向PTEN激活AKT/Snail通路，从而诱导上皮-间质转化促进ESCC侵袭转移。裸鼠移植瘤模型进一步证实了照射后CAF来源外泌体miRNA-193a-3p及其介导的下游通路在ESCC侵袭转移中的作用。综上所述，我们认为外泌体miR-193a-3p有潜力成为提高ESCC放疗疗效的干预靶点，是构成ESCC生物学复杂度的有前景的新组分。