甘露糖结合凝集素相关丝氨酸蛋白酶1对肝癌细胞增殖和迁移的作用及机制研究

Effect and mechanism of mannan-binding lectin-associated serine protease 1 on the proliferation and migration of hepatocellular carcinoma cells

目的探究甘露糖结合凝集素相关丝氨酸蛋白酶1(MASP1)对肝癌细胞生物学行为的影响及可能分子机制。方法选取2022年1月10日至10月25日在南通大学附属医院行肝癌切除手术患者的20对新鲜肝癌组织和癌旁(距癌组织3 cm)正常组织标本并分析MASP1的表达情况。收集2012年3月1日至2017年5月20日南通大学附属医院病理科组织标本库中67例肝癌患者的癌组织及对应的癌旁(距癌组织3 cm)正常组织,分析MASP1的表达水平与肝癌患者临床资料的相关性。培养人肝癌细胞系SK-Hep1、Hep3B并构建MASP1过表达和MASP1敲减细胞株,设立SK-Hep1阴性对照、SK-Hep1 MASP1过表达组与Hep3B阴性对照和Hep3B MASP1敲减组。通过裸鼠皮下移植瘤实验分析MASP1对肝癌细胞增殖的影响。通过蛋白质印迹法检测MASP1对上皮-间质转化相关蛋白质[神经钙黏素、基质金属蛋白酶9(MMP9)、上皮钙黏素]表达的影响,以及MASP1对磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路相关蛋白质的表达及其磷酸化水平的影响。统计学方法采用独立样本t检验、配对t检验和卡方检验。结果20对新鲜组织标本的免疫组织化学染色结果显示,MASP1在肝癌组织中的表达低于癌旁正常组织(3.73±1.03比6.76±1.46),差异有统计学意义(t=16.97,P<0.001)。67例肝癌患者的临床资料与MASP1的表达水平相关性分析显示,MASP1的表达水平与肿瘤有无侵犯血管有关,差异有统计学意义(χ^(2)=5.20,P=0.023)。裸鼠皮下移植瘤实验显示,SK-Hep1 MASP1过表达组小鼠的皮下肿瘤比SK-Hep1阴性对照组体积更小、重量更轻[(165.42±149.08)mm^(3)比(260.42±179.78)mm^(3)、(0.13±0.12)g比(0.18±0.12)g],差异均有统计学意义(t=5.15、3.89,均P<0.05)。蛋白质印迹法显示,SK-Hep1 MASP1过表达组神经钙黏素和MMP9表达低于SK-Hep1阴性对照组,上皮钙黏素表达高于SK-Hep1阴性对照组(0.73±0.01比1.02±0.02、0.40±0.01比0.69±0.01、0.91±0.02比0.78±0.02),差异均有统计学意义(t=24.23、36.87、9.27,均P<0.001)。Hep3B MASP1敲减组神经钙黏素和MMP9表达高于Hep3B阴性对照组,上皮钙黏素表达低于Hep3B阴性对照组(1.04±0.01比0.31±0.01、0.54±0.02比0.04±0.01、0.56±0.01比0.93±0.01),差异均有统计学意义(t=163.20、53.16、60.74,均P<0.001)。SK-Hep1 MASP1过表达组的磷酸化PI3K、磷酸化Akt、磷酸化mTOR的表达低于SK-Hep1阴性对照组(0.59±0.01比1.02±0.01、0.64±0.01比1.12±0.02、0.10±0.01比1.05±0.02),Hep3B MASP1敲减组的磷酸化PI3K、磷酸化Akt、磷酸化mTOR的表达高于Hep3B阴性对照组(0.96±0.01比0.55±0.01、0.99±0.01比0.38±0.01、0.93±0.02比0.06±0.01),差异均有统计学意义(t=40.87、40.91、87.30、44.17、107.50、67.28,均P<0.001)。结论MASP1在肝癌组织中低表达,并且与肝癌患者的不良预后及肿瘤血管侵袭的发生显著相关,其可能通过调控PI3K/Akt/mTOR信号通路影响肝癌细胞的增殖和迁移能力,提示MASP1有望成为治疗肝癌的关键基因。

ObjectiveTo investigate the effect and possible molecular mechanism of mannan-binding lectin-associated serine protease(MASP)1 on the biological behavior of hepatocellular carcinoma(HCC)cells.MethodsFrom January 10 to October 25,2022,20 pairs of fresh liver cancer tissue and adjacent(3 cm from cancer tissue)normal tissue samples of patients who underwent hepatic cancer resection at the Affiliated Hospital of Nantong University were collected,and the expression of MASP1 was analyzed.From March 1,2012 to May 20,2017,the cancer tissue and adjacent(3 cm from cancer tissue)normal tissue samples of 67 patients with HCC were collected from the tissue sample bank of the Department of Pathology,the Affiliated Hospital of Nantong University and the correlation between the expression level of MASP1 and clinical data of patients with HCC were analyzed.Human hepatoma cells lines SK-Hep1 and Hep3B were cultured,and MASP1 overexpression and MASP1 knockdown cell lines were constructed.SK-Hep1 negative control group,SK-Hep1 MASP1 overexpression group,Hep3B negative control group and Hep3B MASP1 knockdown group were set up.The effect of MASP1 on the proliferation of HCC cells was detected by subcutaneous tumor transplantation experiment in nude mice.The effect of MASP1 on the expression of epithelial-mesenchymal transition related proteins(N-cadherin,matrix metalloproteinase 9(MMP9),and E-cadherin),and the effects of MASP1 on the expression of phosphoinositide 3-kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR)pathway related proteins and their phosphorylation levels were detected by Western blotting.Independent sample-t test,paired-t test and chi-square test were used for statistical analysis.ResultsThe results of immunohistochemical staining of 20 pairs of fresh tissue samples showed that the expression level of MASP1 in liver cancer tissue was lower than that in adjacent normal tissue(3.73±1.03 vs.6.76±1.46),and the difference was statistically significant(t=16.97,P<0.001).The correlation analysis of MASP1 expression level and clinical data of 67 patients with HCC revealed that the expression level of MASP1 was related to vascular invasion of the tumor,and the difference was statistically significant(χ^(2)=5.20,P=0.023).The subcutaneous tumor transplantation experiment in nude mice showed that the volume and weight of subcutaneous tumor of mice in SK-Hep1 MASP1 overexpression group were lower than those of the SK-Hep1 negative control group((165.42±149.08)mm^(3)vs.(260.42±179.78)mm^(3),(0.13±0.12)g vs.(0.18±0.12)g),and the differences were statistically significant(t=5.15 and 3.89,both P<0.05).The results of Western blotting showed that the expression levels of N-cadherin and MMP9 in SK-Hep1 MASP1 overexpression group were lower than those of SK-Hep1 negative control group,while the expression level of E-cadherin was higher than that of SK-Hep1 negative control group(0.73±0.01 vs.1.02±0.02,0.40±0.01 vs.0.69±0.01,0.91±0.02 vs.0.78±0.02),and the differences were statistically significant(t=24.23,36.87 and 9.27,all P<0.001).The expression levels of N-cadherin and MMP9 in Hep3B MASP1 knockdown group were higher than those in Hep3B negative control group,and the expression levels of E-cadherin was lower than that in Hep3B negative control group(1.04±0.01 vs.0.31±0.01,0.54±0.02 vs.0.04±0.01,0.56±0.01 vs.0.93±0.01),and the differences were statistically significant(t=163.20,53.16,60.74,all P<0.001).The expression levels of phosphoinositide PI3K,phosphoinositide Akt,and phosphoinositide mTOR of SK-Hep1 MASP1 overexpression group were lower than those of SK-Hep1 negative control group(0.59±0.01 vs.1.02±0.01,0.64±0.01 vs.1.12±0.02,0.10±0.01 vs.1.05±0.02);the expression levels of phosphoinositide PI3K,phosphoinositide Akt,and phosphoinositide mTOR of Hep3B MASP1 knockdown group were higher than those of Hep3B negative control group(0.96±0.01 vs.0.55±0.01,0.99±0.01 vs.0.38±0.01,0.93±0.02 vs.0.06±0.01),and the differences were statistically significant(t=40.87,40.91,87.30,44.17,107.50,67.28,all P<0.001).ConclusionsThe expression level of MASP1 is low in HCC tissue,and is significantly correlated with the poor prognosis of HCC patients and the occurrence of tumor vascular invasion.MASP1 may affect the proliferation and migration of liver cancer cells by regulating the PI3K/Akt/mTOR signaling pathway,suggesting that MASP1 may become a key gene in the treatment of HCC.