AZGP1在老年结直肠癌患者中的预后价值及其下调后对结直肠癌细胞的影响

Prognostic value of AZGP1 in elderly colorectal cancer and the effect on colorectal cancer cells by knockdown of AZGP1

目的探讨锌-α2-糖蛋白1(AZGP1)在老年结直肠癌组织中的表达和临床意义,及其对结直肠癌细胞恶性增殖能力的影响和可能的机制。方法选取行手术切除的结直肠癌存档蜡块和癌旁正常组织(距病变部位>5 cm处,且证实为无癌浸润)石蜡标本各70例,应用免疫组织化学法检测结直肠癌组织及癌旁组织中AZGP1蛋白的表达,分析蛋白的表达与结直肠癌临床病理特征间的关系,并进行生存分析。然后通过慢病毒感染的方式成功敲低HCT116、SW480细胞系中AZGP1的表达水平,采用MTT实验检测AZGP1敲低后细胞的增殖情况;采用克隆形成实验检测AZGP1敲低后细胞的克隆形成能力;采用流式细胞技术检测敲低AZGP1后细胞周期的改变;Western blotting检测细胞G_(1)/S期调控分子cyclin D1、促凋亡分子active-caspase-3及PI3K/AKT信号通路蛋白PI3K、AKT和磷酸化AKT(p-AKT)表达情况。结果AZGP1蛋白在结直肠癌组织中的表达较癌旁正常组织明显上调,蛋白表达与结直肠癌患者临床分期、肿瘤浸润程度、肿瘤大小、肝脏转移显著相关,而与患者性别、淋巴结转移、肿瘤分化程度无关。生存分析结果提示AZGP1高表达组的患者生存率明显低于低表达组。RT-PCR实验显示:感染慢病毒后HCT116、SW480细胞中AZGP1表达水平显著下降。MTT实验显示:敲低AZGP1可降低结直肠癌细胞的增殖能力,差异有统计学意义。细胞克隆实验显示:敲低AZGP1可降低结直肠癌细胞的克隆形成能力。流式细胞术检测结果显示:敲低AZGP1后G_(1)期细胞数增加,S期细胞数减少,差异有统计学意义。Western blotting实验显示:敲低AZGP1可降低cyclin D1表达,增加active-caspase-3表达;同时发现,敲低AZGP1后,PI3K和p-AKT蛋白表达下降。结论AZGP1蛋白在结直肠癌组织中高表达,并可能与结直肠癌的转移及不良预后相关。敲低AZGP1可阻断结直肠癌细胞G_(1)/S期,抑制结直肠癌细胞的恶性增殖,促进凋亡;并且PI3K/AKT信号通路可能参与其中。

Objective To explore the expression of knockdown Zinc-α2-glycoprotein 1(AZGP1)in elderly colorectal cancer tissues and its clinical significance,as well as its effect on the malignant proliferation of colorectal cancer cells and possible mechanisms.Methods Selected surgically resected colorectal cancer archive wax block and adjacent normal tissues(>5 cm from the lesion site,and confirmed no cancerous infiltration)paraffin specimens each of 70 cases,using immunohistochemistry to detect human colorectal cancer tissues and adjacent normal tissues to analyze the relationship between the expression of the protein and the clinicopathological characteristics of colorectal cancer,and performed survival analysis.Then the expression level of AZGP1 in the HCT116 and SW480 cell lines was successfully knocked down by lentivirus infection,and the MTT experiment was used to detect the proliferation of cells after knocking down AZGP1;the clone formation experiment was used to detect the clonogenic ability of cells after knocking down AZGP1;Flow cytometry was used to detect expression situation of cell cycle changed after knocking down AZGP1;Western blotting was used to detect expression situation of cell G1/S phase regulatory molecules cyclin D1,pro-apoptotic molecules active-caspase-3 and PI3K/AKT signaling pathway proteins PI3K,AKT and phosphorylated AKT(p-AKT).Results The expression of AZGP1 protein in colorectal cancer tissues was significantly up-regulated compared with adjacent tissues.The expression of protein was significantly correlated with clinical stage,tumor invasion,tumor size and liver metastasis,but not with the patient’s gender,lymph node metastasis and the degree of tumor differentiation.The results of survival analysis suggested that the survival rate of patients in the AZGP1 high expression group was significantly lower than that in the low expression group.RT-PCR experiments showed that the expression level of AZGP1 in HCT116 and SW480 cells decreased significantly after infection with lentivirus.MTT experiments showed that knocking down AZGP1 can reduce the proliferation ability of colorectal cancer cells,with statistical differences.Cell cloning experiments showed that knocking down AZGP1 could reduce the cloning ability of colorectal cancer cells.The results of flow cytometry showed that the number of cells in G1 phase increased after knocking down AZGP1,while the number of cells in S phase decreased,and there was a statistical difference.Western blotting experiments showed that knocking down AZGP1 could reduce the expression of cyclin D1 and increase the expression of active-caspase-3.At the same time,the expression of PI3K and p-AKT protein decreased after knocking down AZGP1.Conclusion AZGP1 protein is highly expressed in colorectal cancer tissues,and may be related to colorectal cancer metastasis and poor prognosis.Knockdown of AZGP1 can block the G1/S phase of colorectal cancer cells,inhibit the malignant proliferation of colorectal cancer cells,and promote apoptosis;and the PI3K/AKT signaling pathway may be involved.