exosome/miR-221/Bmf细胞间通讯轴在解毒颗粒抑制肝癌生长中的作用及机制研究

Human hepatocellular carcinoma(HCC) is one of the most common malignant cancers worldwide and there is still no satisfactory treatment.Jiedu granule is the preparation developed under the guidance of TCM "qingrejiedu" anticancer therapeutic principle. Our long-term clinical application confirms that it has good anti-cancer effect on HCC, but the related molecular mechanism remains unclear. Recent researches have shown that different from traditional intracellular signal transduction, exosome mediated intercellular transfer of microRNAs and intercellular communication play an important role in HCC. MiR-221 is one of the most important “oncomiRs” for HCC which can target and degrade the tumor suppressor gene Bmf and thus promote the growth of HCC cells. The expression of miR-221 was significantly increased in HCC tissue compared to normal tissue. Recently it was reported the serum levels of exosomal miR-221 was significantly higher in patients with HCC than those with chronic hepatitis B (CHB) or liver cirrhosis (LC),which showed exosome/miR-221 may closely related to the process of HCC and suggested that serum exosomal miR-221 may be used as a novel serological biomarker for HCC. Our preliminary experiments showed the exosomal miR-221 was enriched in the urine of patients with HCC, Jiedu granule could reduce the exosome secretion in urine of HCC patients and the expression of exosomal miR-221 was also decreased. Moreover, the drug serum of Jiedu granule could inhibit the proliferation of HCC cells and increase the Bmf expression.So on the basis of the above research,we plan to analyze the regulatory mechanism on exosome/miR-221/Bmf intercellular communication pathway of Jiedu granule and further discuss its anti-HCC effect. In the present study,we will explore the detail molecular mechanism at different levels such as animals,cells, molecules by realtime fluorescence quantitative PCR,small animal in vivo imaging technology and other methods both in vitro and in vivo. This research could be contribute to clarify the anti-HCC mechanism of Jiedu granule, offer valuable reference for clinical treatment of HCC and further provide experimental evidence for related anticancer therapeutic principle of TCM.

肝癌目前仍缺乏令人满意的治疗方法，解毒颗粒是本科室在中医“清热解毒”抗癌方法指导下开发的院内制剂，长期临床应用证实其具有良好的抗肝癌作用，但机制尚不清楚。近年研究表明，不同于传统的细胞内信号传导，exosome介导的miRNA转运这一新型细胞间通讯方式在肝癌生长中具有重要意义。miR-221是一种与肝癌密切相关的促癌miRNA，能够靶向降解抑癌基因Bmf从而促进肝癌增殖。我们的前期预实验发现解毒颗粒能够降低肝癌患者尿液中exosome的含量及其中miR-221的表达，其含药血清能够抑制肝癌细胞增殖并上调其中Bmf分子的表达。本研究拟以exosome/miR-221/Bmf细胞间通讯方式为切入点，利用荧光定量PCR、小动物活体成像等方法在动物、细胞、分子等不同水平探讨解毒颗粒对该通讯轴的调控作用及分子机制。本课题有助于明确解毒颗粒的抗肝癌机理，并为中医相关抗癌理论和方法提供实验依据。

解毒颗粒是本课题组在“清热解毒”中医抗癌法则指导下研发的院内制剂，临床实践已证实其对于肝癌具有良好的疗效，但相关的分子机制尚不清楚。本项目着眼于细胞间通讯调控模式，从动物、细胞、分子等不同水平对解毒颗粒的抗肝癌效应及对exosome/miR-221/Bmf细胞间通讯轴的调节机制开展研究。结果显示，解毒颗粒能够抑制多种肝癌细胞的生长并诱导肝癌细胞凋亡，抑制exosome/miR-221的表达，并促进Bmf的表达。与对照相比，上调miR-221的水平能够部分逆转解毒颗粒的抗肝癌生长效应及其诱导的Bmf的升高。相应的，下调miR-221的水平能够进一步促进解毒颗粒的抗肝癌生长效应，并能够提高解毒颗粒诱导的Bmf的表达。下调Bmf的表达能够部分逆转解毒颗粒的抗肝癌效应，上调Bmf的水平能够进一步促进解毒颗粒的抗肝癌效应。进一步机制研究发现，解毒颗粒能够活化Vps4A分子，下调Vps4A分子水平能够部分逆转解毒颗粒的抗肝癌效应及其诱导的Bmf的升高，提示Vps4A是解毒颗粒可能的上游作用靶点之一。因此，我们推测解毒颗粒可能通过调控Vps4A分子参与调节exosome/miR-221/Bmf细胞间通讯轴并发挥抗肝癌作用。此外，我们还发现，解毒颗粒能够调节MAPK、NFκB、PI3K/Akt等肝癌生长相关信号通路的活化及c-Myc、VEGF等分子的表达，并能够抑制肝癌细胞的迁移和侵袭。上述结果有助于明确解毒颗粒的抗肝癌机理，为其进一步临床推广应用提供实验依据，并能够为肝癌的临床治疗提供潜在的治疗靶点和干预途径，具有一定的科学意义和应用价值。