前列腺素E2激活YAP正反馈回路促进小鼠结肠炎后结肠再生及癌变的分子机制

发布时间：2018-05-07 18:10

本文选题：前列腺素E + 信号通路　；参考：《中国老年学杂志》2017年15期

【摘要】：目的研究前列腺素(PGE2)激活Yes信号通路相关蛋白(YAP)正反馈回路促进小鼠结肠炎后结肠再生及癌变的分子机制。方法将DLD1结肠癌细胞株或基因稳定敲除细胞株,在有/无信号通路特异性抑制剂的条件下,采用重组PGE2或PBS孵育,采用免疫印迹法、免疫荧光法、定量PCR、转录和增殖法进行比较分析。采用DSS法诱导C57/BL6小鼠结肠炎模型(对照鼠),同时构建羟基前列腺素脱氢酶基因敲除鼠(15-PGFH-KO)、YAP基因敲除鼠(YAP-KO)、和双基因敲除(DKO)小鼠,采用吲哚美辛抑制PGE2合成信号通路,检测下游基因表达及结肠再生和癌变功能。结果 PGE2处理结肠癌细胞株导致YAP mRNA转录及蛋白表达水平升高,15-PGDH-KO组鼠结肠组织中PGE2的表达水平较对照敲除细胞(WT)组增长了2.5倍,吲哚美辛处理后15-PGDH-KO组鼠YAP的表达水平及下游靶基因水平均较WT鼠显著升高。吲哚美辛处理显著降低PG过氧化物合成酶(COX)-2及PGE受体4基因(EP4)的转录水平,转染YAP持续激活突变体(5SA)可显著增加COX-2和EP4的蛋白表达水平和转录水平,敲除YAP后上述现象消失。对照鼠口服葡聚糖硫酸钠(DSS)导致结肠炎,注射PGE2促进小鼠发生结肠再生;YAP敲除鼠并未发生结肠再生,同时口服DSS后迅速死亡;15-PGFH敲除鼠结肠组织再生比对照鼠更快,且体重恢复更快、结肠长度和结肠炎组织学得分更高。上述现象可以被注射吲哚美辛逆转。YAP敲除鼠或15-PGFH敲除鼠诱导结肠炎后未发现自发性肿瘤,YAP/15-PGDH双基因敲除鼠出现息肉并最终发展成原位癌。口服吲哚美辛可有效防止双敲除鼠自发性肿瘤的形成。结论 PGE2可增加YAP的表达水平和转录活性,从而促进COX-2及EP4的表达水平增加,这一信号回路可促进扩散的结肠癌细胞系增殖和结肠炎小鼠结肠组织再生,持续性激活进一步可导致息肉和小鼠结肠肿瘤的形成。
[Abstract]:Aim to study the molecular mechanism of prostaglandin PGE2 (PGE2) activation of Yes signal pathway associated protein (YAP) positive feedback loop in promoting colon regeneration and carcinogenesis after colitis in mice. Methods DLD1 colon cancer cell line or gene stable knockout cell line was incubated with recombinant PGE2 or PBS in the presence or absence of signal pathway specific inhibitors. Quantitative PCR, transcription and proliferation were compared and analyzed. The colitis model of C57/BL6 mice was induced by DSS method (control mice). The hydroxyl prostaglandin dehydrogenase gene knockout mice, 15-PGFH-KOAP gene knockout mice, and double-gene knockout mice were constructed. Indomethacin was used to inhibit the PGE2 synthesis signal pathway. The downstream gene expression and colon regeneration and carcinogenesis were detected. Results the expression of PGE2 in colon tissue of 15-PGDH-KO group was increased by 2.5-fold compared with that of control group. After indomethacin treatment, the expression level of YAP and the downstream target gene level in 15-PGDH-KO group were significantly higher than those in WT group. Indomethacin treatment significantly reduced the transcription level of COX-2 and PGE receptor 4 gene (EP4). Transfection of YAP continuously activated mutants (p5SA4) significantly increased the protein expression and transcription level of COX-2 and EP4, which disappeared after YAP knockout. The colitis was induced by oral dextran sodium sulfate in control mice. The colonic regeneration was not observed in mice induced by PGE2 injection, and the colonic tissue regeneration of 15-PGFH knockout mice was faster than that of control mice after oral DSS. Weight recovered faster, colon length and colitis histological scores were higher. These phenomena can be reversed by injection of indomethacin. YAP knockout mice or 15-PGFH knockout mice induced colitis. Oral administration of indomethacin can effectively prevent the formation of spontaneous tumor in double knockout mice. Conclusion PGE2 can increase the expression level and transcription activity of YAP, thus promote the expression of COX-2 and EP4. This signal loop can promote the proliferation of diffuse colon cancer cell line and the regeneration of colonic tissue in colitis mice. Continuous activation may further lead to polyp and colon tumor formation in mice.
【作者单位】：牡丹江医学院第二附属医院消化内科;
【分类号】：R574.62

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