SIK2在食管癌放疗抵抗中的作用及相关机制研究

Salt-induced kinase 2 (SIK2) is a key regulator of glucose and lipid metabolism in human body. It is closely related to cell rhythm, cell cycle and cell proliferation. Studies have shown that SIK2 is highly expressed in various tumors and is involved in tumor development and progression. However, little is known about the relationship between SIK2 and the tumor radiosensitivity, and the related mechanism also remains unclear. The interaction between PI3K-Akt-mTOR pathway and Hippo-YAP pathway is the key of tumor cell radioresistance. Our previous study suggested that esophageal cancer patients with high SIK2 expression showed poor radiosensitivity and disappointing prognosis. The expression of SIK2 in esophageal cancer ECA cell lines was higher than that of the other four cell lines, and the expressions of p-Akt and YAP were also increased at the same time with the elevated SIK2 expression after radiation. However, we observed increased apoptosis and declined p-Akt and YAP expression after treating ECA cancer cell line with SIK2 inhibitor ARN-3236. Therefore, we speculate that SIK2 may regulate the radioresistance of esophageal cancer by interacting with the two signal transduction pathways of PI3K-Akt-mTOR and Hippo-YAP. To verify this hypothesis, we will take ECA109, KYSE150 and other esophageal cancer cell lines and tumor-bearing nude mice as models. We will use quantitative PCR, Western blot, lentivirus transfection, RNA interference and other techniques to expolre the relationship bewteen SIK2 and tumor cell radiosensitivity from the molecular, cellular and animal levels. We will try to find the mechanism of esophageal cancer radioresistance from the new view of the interaction between SIK2 and PI3K-Akt-mTOR pathway and Hippo-YAP pathway, providing new ideas to improve the radiosensitivity of tumors.

盐诱导激酶2（SIK2）是糖脂代谢的调控因子，在多种肿瘤中高表达并参与肿瘤发生发展，但其与肿瘤放疗敏感性的关系仍不明确。我们的前期研究发现：高表达SIK2的食管癌患者放疗疗效较差；食管癌ECA109细胞株受照射后SIK2表达增加的同时 p-Akt与YAP上调；经SIK2抑制剂处理后，ECA109细胞受照射后的凋亡增加，克隆形成能力减弱，且p-Akt、YAP表达降低。故推测SIK2可能通过与PI3K-Akt-mTOR和Hippo-YAP为核心的两条信号通路相互作用介导食管癌放疗抵抗。为验证此假说，我们将以食管癌细胞株及荷瘤裸鼠为模型，采用定量PCR、免疫印迹、慢病毒转染、RNA干扰等技术，从分子、细胞及动物整体水平探讨SIK2与肿瘤细胞放射敏感性的关系，并力图从SIK2与PI3K-Akt-mTOR和Hippo-YAP相互作用这一新视点探讨食管癌放疗抵抗的机制，为提高肿瘤放射敏感性提供新思路。

背景：我们在对SIK2蛋白研究中发现其上游miR-450a-5p对食管癌的放疗敏感性影响更为明显，故我们着重研究了miR-450a-5p介导食管癌放疗敏感性的分子机制。.方法：通过梯度照射构建放疗抵抗细胞株，并进行转录组测序，比较ECA-109R与其亲本细胞的miRNA表达谱差异；慢病毒转染用于构建miR-450a-5p稳定敲低和过表达ESCC细胞株；克隆形成实验、CCK8以及EdU实验用于探究其对ESCC放疗后细胞增殖水平的影响；免疫荧光染色用于检测放疗后ESCC细胞中γ-H2AX foci形成情况；流式细胞术用于检测放疗后ESCC的细胞凋亡水平；生物信息学分析用于揭示miR-450a-5p前体表达水平与ESCC自噬相关通路之间的潜在联系； Western blot实验用于检测相关通路蛋白的表达水平；构建小鼠皮下成瘤模型比较其在体内环境对ESCC放疗敏感性的影响。.结果：我们成功构建放疗抵抗细胞株ECA-109R，放射生物学参数显示ECA-109R的D0、Dq及SF2显著高于亲本ECA-109，并通过体内实验验证；通过转录组测序，我们发现miR-450a-5p在ESCC放疗抵抗细胞株ECA-109R中表达显著降低，并通过qRT-PCR加以验证；构建miR-450a-5p稳定敲低及过表达细胞系，克隆形成实验证明其在ESCC细胞株中可以产生放疗增敏的作用； CCK8及EdU实验结果显示其抑制ESCC细胞辐照后的细胞增殖；γ-H2AX foci点检测表明miR-450a-5p可以加剧放疗导致的ESCC细胞DNA双链断裂；流式细胞术显示其能导致放疗后ESCC的调亡比例增加；GSEA分析显示miR-450a-5p与自噬相关通路的潜在联系，并通过Western blot加以验证；公共数据库分析结合测序数据进一步筛选，并用qRT-PCR与双荧光素酶报告实验加以验证，最终筛选出DUSP10是miR-450a-5p的直接下游靶基因，并通过功能回复实验加以证明，随后的western blot实验显示miR-450a-5p/DUSP10可通过调控MAPK信号通路影响ESCC细胞的放疗敏感性；小鼠皮下成瘤模型进一步验证miR-450a-5p对ESCC细胞放疗敏感性的调控。.结论：miR-450a-5p/DUSP10轴通过调控MAPK信号通路的转导进而影响ESCC细胞自噬水平及放疗敏感性。