苯转化的恶性细胞外泌体miR-221在细胞增殖和凋亡抑制信号传导中的作用及机制

Benzene is a confirmed chemical carcinogen, but the mechanism is not clear. We have recently found that population environmentally exposed to benzene had increased expression of lymphocyte miR-221. As an oncogene, miR-221 has enhanced expression level in many malignant tumors and mediates cell proliferation and apoptosis inhibition by targeting PTEN to activate the PI3K/Akt pathway. It has been newly discovered that miRNA in exosomes of tumor cells mediated signal transduction among cells and thus influenced biological functions of receptor cells. In this study, malignant TK6-t cells will be obtained by transforming normal TK6 cells with benzene. The expression levels of miR-221 and the target protein of PTEN, PI3K and p-Akt in these two kinds of cells and TK6-t exosomes will be analyzed by using qRT-PCR and Western blot. Transfect TK6-t exosomes to TK6 and endocytosis will be confirmed by using laser confocal microscopy. Then the changes of expressions of miR-221 and target protein will be analyzed. Cell proliferation and apoptosis of TK6 will be tested by using CCK-8 and flow cytometry analysis, respectively. Animal test of tumor formation in nude mice will be conducted and the expression levels of miR-221 , PTEN, PI3K and p-Akt of tumor will be detected. Finally,LNA-antimiR will be used to block the signal of exosome miRNA-221 and then to confirm its function in reverse. This study aims to explore the effects and mechanisms of exosome miR-221 in the process of signal transduction of proliferation and apoptosis inhibition in benzene induced malignant cells.

苯是确认的化学致癌物，但机制未明。我们近期发现，环境苯暴露人群淋巴细胞miR-221高表达。miR-221 作为癌基因在多种恶性瘤中增强表达，并靶向PTEN激活PI3K/Akt通路介导细胞增殖和凋亡抑制。研究发现，肿瘤细胞外泌体miRNA介导细胞间信号传导而影响受体细胞生物学功能。本课题以苯转化人TK6细胞获恶性TK6-t；以qRT-PCR、Western blot分析两种细胞和TK6-t外泌体miR-221及靶蛋白PTEN、PI3K、p-Akt表达; 以TK6-t外泌体处理TK6并确认被胞吞；分析TK6 miR-221及靶蛋白表达变化；以CCK-8、流式细胞分析TK6增殖及凋亡；做移植成瘤动物模型，分析瘤体miR-221、靶蛋白、增殖及凋亡；最后以锁定的反义寡核苷酸阻断外泌体miR-221信号反证其功能。课题旨在探索苯转化的恶性细胞外泌体miR-221传导增殖和凋亡抑制信号的作用及机制。

苯是一种重要的化工原料，也是一种确认的致癌性环境污染物。到目前为止，其致癌作用机制仍未清楚。我们前期的研究发现，环境苯暴露人群淋巴细胞 miR-221 高表达。miR-221作为癌基因在多种恶性瘤中增强表达，并能靶向 PTEN， 激活 PI3K/Akt 信号通路，介导细胞增殖和凋亡抑制。最近的研究发现，肿瘤细胞外泌体（exosomes）能介导细胞间信号传导，从而影响受体细胞的生物学功能。本课题以苯转化人支气管上皮细胞（16HBE），获取恶性细胞16HBE-t；以 qRT-PCR、Western blot分析正常16HBE，16HBE-t，16HBE-t exosomes miR-221及靶蛋白 PTEN、PI3K、p-Akt 等的表达；以 16HBE-t exosomes处理16HBE，使16HBE摄取16HBE-t exosomes；分析16HBE摄取16HBE-t exosomes之后，其miR-221及上述靶蛋白的表达变化；以CCK-8、流式细胞分析 16HBE摄取16HBE-t exosomes之后，其增殖及凋亡的变化情况等等。最后以锁定的反义寡核苷酸阻断16HBE-t exosomesmiR-221信号，重复上述研究过程，以反证16HBE-t exosomes miR-221的功能。本课题旨在探索苯转化的恶性细胞的exosomes miR-221对细胞增殖和凋亡抑制信号的传递作用及机制。通过本课题的研究，我们证实了苯转化的恶性细胞的细胞增殖和凋亡抑制性状可以通过 exosomes 传递给正常细胞；证实了苯转化的恶性细胞 exosomes miRNA-221在细胞增殖和凋亡抑制性状传递中的促进作用；证实了苯转化的恶性细胞 exosomes miRNA-221 可以通过抑制受体细胞 PTEN 表达，激活 PI3K/Akt 信号通路，诱导正常受体细胞的细胞增殖和凋亡抑制。本研究从表观遗传学角度探索了苯的毒性作用调控机制，为苯中毒的预防和控制工作提供了重要的基础性资料。