转谷氨酰胺酶3(TGM3)促进肝细胞肝癌(HCC)增殖侵袭的潜在机制及其临床意义

Transglutaminase 3 (TGM3) promotes hepatocellular carcinoma (HCC) development and its potential mechanism and clinical significance

目的研究转谷氨酰胺酶3(transglutaminase 3,TGM3)在肝细胞肝癌(hepatocellular carcinoma,HCC)发生、发展过程中的作用及其预测HCC预后的临床价值。方法收集复旦大学附属中山医院HCC患者标本和临床资料进行分析,用qRT-PCR和Western blot方法检测28例HCC患者癌和癌旁TGM3表达差异,利用组织芯片染色结果及临床随访资料对92例HCC患者行Kaplan-Meier及Cox回归分析,探究TGM3不同表达水平对患者预后的影响。shRNA下调HCC细胞系Huh7和97H的TGM3表达水平,利用CCK-8法、克隆形成实验、Transwell小室实验、裸鼠皮下成瘤实验分析干扰后细胞增殖、迁移、侵袭及皮下成瘤能力的变化,Westernblot检测PI3K/AKT、MAPK/EPK、NF-κB信号通路、上皮间质转化(epithelial-mesenchymal transition,EMT)及凋亡相关蛋白的变化。结果TGM3在HCC中高表达,Kaplan-Meier生存分析表明TGM3高表达组的中位复发时间和中位生存期均较低表达组显著缩短(15.00个月vs.49.25个月,P<0.05;38.63个月vs.未达到;P<0.05);多因素Cox回归分析发现TGM3表达水平是HCC患者术后复发和死亡的独立预后因素(HR=2.31,P=0.029;HR=2.78,P=0.009)。体内外实验表明TGM3表达下调可以抑制HCC细胞的增殖、侵袭及皮下成瘤能力。TGM3下调使AKT、ERK、p65蛋白磷酸化水平降低,cleavedcaspase3水平增高,EMT相关指标E-钙黏素表达增加,N-钙黏素、纤维连接蛋白及波形蛋白表达下降。结论TGM3可能通过影响多条信号通路和EMT过程促进HCC的增殖和侵袭,并可作为HCC患者潜在的预后指标及治疗靶点。

Objective To investigate the biological role of transglutaminase 3 (TGM3) in HCC development and its clinical value for predicting prognosis in hepatocellular carcinoma (HCC) patients. Methods Clinical information and specimens of patients with HCC from Zhongshan Hospital,Fudan University were obtained and analyzed. Quantitative RT-PCR and Western blot were conducted to detect the TGM3 expression difference between tumors and paired ajacent normal liver tissues of 28 HCC patients.The tissue microarray and clinical information of 92 HCC patients were used to investigate the relationship between patient prognosis and TGM3 expression level by Kaplan-Meier estimator and Cox proportional hazards model.By depleting TGM3 with two different shRNAs in Huh7 and 97H cell lines,we investigated the changes of cell proliferation,migration,invasion and tumorigenesis through CCK-8 assay,colony formation assay,Transwell chamber and subcutaneous xenotransplanted tumor model,respectively.The markers of PI3K/AKT,MAPK/ERK,NF-κB signaling pathways,epithelial-mesenchymal transition (EMT) and apoptosis were detected by Western blot. Results TGM3 was upregulated in HCC.Kaplan-Meier analysis showed that median time to recurrence and median survival time were significantly shorter in patients with high TGM3 expression versus those with low expression (15.00 months vs.49.25 months,P <0.05;38.63 months vs.not reached,P <0.05).Multivariate Cox regression identified TGM3 as an independent indicator for recurrence and death in patients who have received operations (HR=2.31,P =0.029;HR=2.78,P =0.009).Depletion of TGM3 decreased cell proliferation,migration,invasion and tumorigenesis,and inhibited AKT,ERK and p65 phosphorylation,while promoted cleaved caspase 3 level.Also,cells with TGM3 knockdown displayed increased E-cadherin that corresponded with decreased N-cadherin,fibronectin and vimentin protein levels. Conclusions TGM3 may affect multiple oncogenic pathways and EMT transition to promote HCC development.TGM3 may serve as a potential prognostic marker and therapeutic target for HCC.