MSP58在肝细胞癌中表达和作用的机制研究

MSP58 is upregulated in many tumors including glioma, colorectal cancer, gastric cancer and hepatocellular carcinoma, Downregulation of MSP58 inhibits the survival and proliferation of tumor cells. But the mechanisms underlying the roles and regulation of MSP58 during the initiation and progression of HCC remain to be decided. Our primary data demonstrated MSP58 expression is increased in HCC samples compared to adjacent non-cancer tissues; the expression of MSP58 is regulated by transcriptional factor AP-1 activating agent TPA and by kinase CKII; and MSP58 interacts with mitosis regulators Wee1 and BUB3. Based on the findings and previous studies, we hypothesize that during the initiation and progression of HCC, MSP58, upregulated by AP-1 and stabilized by CKII, regulates the cell cycle progress to favor the survival and proliferation of HCC cells through interacting with Wee1 and BUB3, and promote the initiation and progression of HCC. In this proposal, we are to investigate the expression and roles of MSP58 on the four HCC mouse models using immunohistochemistry, and explore the mechanisms underlying regulation of HCC cell cycle by MSP58 using HCC cell line HepG2 and PLC. We are also to investigate how MSP58 is regulated by AP-1 and CKII in HCC cells using luciferase reporter assays, EMSA, CHIP-PCR and liquid chromatography/tandem mass spectrometry. Moreover, we are to explore the relationship between the expression of MSP58 and Wee1, BUB3,AP-1 subunits or CKII on the HCC tissues or adjacent noncancerous tissues; using small molecular compounds related to the mechanisms we proposed, we treat the HCC mouse to examine effects of the mechanism obtained from this study. Our study will open a new avenue for developing not only targeted therapies, but also preventive strategies of the HCC with increased expression of MSP58.

MSP58在多种肿瘤中表达升高，促进细胞生存和增殖，但在肝细胞癌（HCC）中表达和作用的机制不清楚。前期已经发现MSP58在HCC中表达升高，受激酶CKII和转录因子AP-1激活剂TPA调节，与细胞周期调节激酶Wee1和纺锤体检测点蛋白BUB3互作。基于此，我们假设：在癌变中，AP-1和CKII上调MSP58；MSP58通过与Wee1和BUB3互作调控细胞周期, 促进癌变细胞的生存和增殖。为此，以HCC小鼠模型、肝癌组织及HepG2和PLC细胞为材料，从分子、细胞和动物水平上，明确MSP58在HCC发生发展中的动态表达和作用；MSP58通过与Wee1和BUB3互作对HCC细胞周期调控的具体分子机制；CKII在翻译后水平和AP-1在转录水平对MSP58调节机制；MSP58在HCC中表达与Wee1和BUB3等的相关性。本课题的实施将揭示HCC发生发展的新机制，为HCC的治疗和预防提供新的思路。

肝细胞肝癌（HCC）是一种很难治疗的恶性肿瘤，目前的靶向治疗效果非常不理想，因此深入了解HCC发生发展的分子机制对其治疗预防意义深远。在本研究中，我们发现MSP58在64%（58/90）HCC组织中较癌旁组织明显高；过表达MSP58的肝癌细胞HepG2和HUH7生长明显快于，使用shRNA敲低MSP58，生长明显慢于原始细胞；在小鼠肝脏中仅过表达MSP58可导致HCC发生。这些数据显示MSP58是促进HCC发生发展的重要因子。为进一步探索MSP58在HCC发生发展中的作用机理，我们通过免疫共沉淀结合质朴分析的方法发现MSP58能够和细胞周期蛋白Wee1和BUB3相互作用，免疫共沉淀实验证实了MSP58能够和Wee1及BUB3相互作用； 通过与Wee1的相互作用，MSP58促进Wee1降解，从而减少CyclinB/CDC2抑制性磷酸化，激活该复合物，促进细胞进入分裂期；通过与BUB3的相互作用，MSP58激活纺锤体检查点，维持CyclinB和Securin的高表达，有利于染色体的正常分离，维持基因组的稳定。我们的研究发现MSP58通过Wee1和BUB3调节细胞周期进程，促进HCC发生发展，提示可以通过干预细胞周期干预HCC的发生发展。