血管相关迁移细胞蛋白在肝细胞癌中高表达并促进肝癌细胞侵袭转移

doi:10.12122/j.issn.1673-4254.2025.12.10

摘要/Abstract

摘要：

目的研究血管相关迁移细胞蛋白（AAMP）异常高表达在肝细胞癌（HCC）中的临床意义和对肝癌细胞侵袭转移的影响。方法基于网络数据库中HCC队列资料分析AAMP基因在肝癌和癌旁组织中的表达差异以及与临床分期和预后的关系。免疫组化检测60对HCC及癌旁组织标本中AAMP表达，分析AAMP表达与患者临床病理特征的相关性。通过慢病毒转染下调HCC细胞（Mahlavu和Huh-7）内AAMP表达水平。EdU掺入实验、流式细胞仪、划痕愈合实验、transwell迁移/侵袭小室和裸鼠肺转移瘤模型分别检测敲低AAMP表达对HCC细胞体外增殖、凋亡、迁移、侵袭和体内肺转移能力的影响。Western blotting和免疫荧光检测上皮间质转化标志物E-cadherin、N-cadherin、Vimentin、Snail表达情况。RT-qPCR和Western blotting检测敲低AAMP对RhoA mRNA和蛋白表达的影响。分别使用MG-132和CHX处理敲低AAMP的HCC细胞，Western blotting检测细胞内RhoA蛋白表达变化。免疫组化染色分析HCC组织中AAMP和RhoA蛋白的表达相关性。结果生物信息学分析显示，AAMP基因表达水平在HCC组织中升高（P<0.05），AAMP高表达与患者临床分期较晚和不良预后相关（P<0.05）。免疫组化结果显示，HCC组织的AAMP染色评分高于对应癌旁组织（P<0.05），AAMP高表达与Edmondson-Steiner分级（III+IV）、血管侵犯及TNM分期（III+IV期）相关（P<0.05）。敲低AAMP对HCC细胞增殖和凋亡无显著影响（P>0.05），但能抑制HCC细胞的迁移、侵袭和体内肺转移能力（P<0.05）。敲低AAMP后HCC细胞内E-cadherin蛋白表达升高而N-cadherin、Vimentin和Snail的表达水平降低（P<0.05），相应蛋白的免疫荧光染色强度变化趋势与Western blotting检测结果一致。敲低AAMP对RhoA基因mRNA表达水平无显著影响（P>0.05），但降低了RhoA蛋白的表达量（P<0.05），并且MG-132处理阻断了敲低AAMP导致的RhoA蛋白表达下调（P<0.05）。相关性分析显示，HCC组织内AAMP和RhoA蛋白的表达水平呈正相关关系（P<0.05）。结论 AAMP高表达与HCC恶性临床特征密切相关，AAMP可能通过维持RhoA蛋白稳定表达来促进HCC细胞发生上皮间质转化，进而增强HCC的侵袭转移能力。

Abstract:

Objective To investigate the clinical significance of abnormal expression of angio-associated migratory cell protein (AAMP) in hepatocellular carcinoma (HCC). Methods Bioinformatics analyses were used to analyze AAMP expression level in HCC and its prognosis value. In 60 pairs of HCC and adjacent tissues, AAMP expression was detected immunohistochemically and its correlation with clinicopathological characteristics of the patients was analyzed. In cultured Mahlavu and Huh-7 cells with lentivirus-mediated AAMP knockdown, the changes in cell proliferation, apoptosis, migration and invasion were observed, and their lung metastasis following tail vein injection in nude mice were assessed. In HCC cells with AAMP knockdown, Western blotting, immunofluorescence staining or RT-qPCR were used to examine the changes in expression levels of E-cadherin, N-cadherin, Vimentin and Snail and the effects of MG-132 and CHX on RhoA expression. The correlation between the expressions of AAMP and RhoA in HCC tissues was analyzed by immunohistochemistry. Results Bioinformatics analysis showed that AAMP expression was elevated in HCC tissues (P<0.05) in correlation with advanced clinical stage and poor prognosis (P<0.05). Immunohistochemistry results confirmed significant correlations of high AAMP expression with Edmondson-Steiner grade (III+IV), venous infiltration and TNM stage (III+IV) of HCC (P<0.05). In cultured HCC cells, AAMP knockdown did not significantly affect cell proliferation or apoptosis, but obviously suppressed cell migration and invasion in vitro and lung metastasis in nude mice. AAMP knockdown significantly increased E-cadherin expression, decreased N-cadherin, Vimentin and Snail expressions, and reduced RhoA protein levels without obviously affecting RhoA mRNA levels. MG-132 treatment blocked the inhibitory effect of AAMP knockdown on RhoA protein expression. The expressions of AAMP and RhoA showed a significant positive correlation in HCC tissues (P<0.05). Conclusion AAMP overexpression is associated with malignant clinical features of HCC and promotes epithelial-mesenchymal transition and metastasis of HCC cells partly by stabilizing RhoA expression.

Key words: angio-associated migratory cell protein, hepatocellular carcinoma, epithelial-mesenchymal tran-sition, invasion, metastasis, RhoA

李超, 殷国志, 程萧, 姜业臻. 血管相关迁移细胞蛋白在肝细胞癌中高表达并促进肝癌细胞侵袭转移[J]. 南方医科大学学报, 2025, 45(12): 2628-2638.

Chao LI, Guozhi YIN, Xiao CHENG, Yezhen JIANG. Angio-associated migratory cell protein is highly expressed in hepatocellular carcinoma and promotes tumor cell invasion and metastasis[J]. Journal of Southern Medical University, 2025, 45(12): 2628-2638.

图/表 9

图1 分析AAMP在HCC公共数据库中的表达及临床意义

Fig.1 Bioinformatics analysis of AAMP expression in HCC. A-D: Data from different HCC datasets from GEO (A, B), ICGC (C) and TCGA (D) databases all show up-regulated AAMP expression in HCC. ***P<0.001 vs tumor-adjacent tissues. E: AAMP expression is higher in advanced TNM stages. **P<0.01 vs I+II. F: High expression of AAMP predicted unfavorable prognosis.

图2 AAMP在HCC及癌旁组织中的表达

Fig.2 Immunohistochemical detection of AAMP expression in HCC and adjacent tissues (Original magnification: ×400). **P<0.01 vs Tumor-adjacent.

图3 慢病毒转染敲低HCC细胞中AAMP表达

Fig.3 Knockdown of AAMP in HCC cells by lentivirus transduction. A: Expression of AAMP in 4 HCC cell lines (Hep3B, Li-7, Huh-7, and Mahlavu). B, C: Lentivirus-mediated knockdown of AAMP in Mahlavu (B) and Huh-7 (C) cells, *P<0.05 vs sh-ctrl group.

图4 敲低AAMP对HCC细胞增殖凋亡无显著影响

Fig.4 Knockdown of AAMP does not affect proliferation or apoptosis of HCC cells. A, B: Knockdown of AAMP does not affect proliferation of Mahlavu (A) and Huh-7 (B) cells. C, D: Knockdown of AAMP does not affect apoptosis of Mahlavu (C) and Huh-7 (D) cells.

图5 敲低AAMP抑制HCC细胞迁移和侵袭

Fig. 5 Knockdown of AAMP inhibits migration and invasion of HCC cells. A, B: Knockdown of AAMP inhibits wound healing of Mahlavu (A) and Huh-7 (B) cells (*P<0.05 vs sh-ctrl group). C, D: Knockdown of AAMP attenuates Transwell migration ability of Mahlavu (C) and Huh-7 (D) cells (**P<0.01 vs sh-ctrl group). E, F: Knockdown of AAMP suppresses Transwell invasion ability of Mahlavu (E) and Huh-7 (F) cells (*P<0.05 vs sh-ctrl group, **P<0.01 vs sh-ctrl group). n=3 per group.

表1 AAMP高表达在肝细胞癌中的临床意义

Tab.1 Clinical significance of high AAMP expression level in HCC

Clinical features		AAMP expression		χ²	P
Clinical features		Low (n=30)	High (n=30)	χ²	P
Gender	Male	21	22	0.082	0.774
Gender	Female	9	8	0.082	0.774
Age (year)	<60	5	3	0.144	0.704
Age (year)	≥60	25	27	0.144	0.704
HBsAg	Negative	5	7	0.417	0.519
HBsAg	Positive	25	23	0.417	0.519
Liver cirrhosis	Absent	10	8	0.317	0.573
Liver cirrhosis	Present	20	22	0.317	0.573
Tumor size(cm)	≤5	15	10	1.714	0.190
Tumor size(cm)	>5	15	20	1.714	0.190
Venous infiltration	No	18	9	5.455	0.020
Venous infiltration	Yes	12	21	5.455	0.020
Edmondson-steiner grading	I+II	19	10	5.406	0.020
Edmondson-steiner grading	III+IV	11	20	5.406	0.020
Serum AFP level (ng/mL)	<400	16	9	3.360	0.067
Serum AFP level (ng/mL)	≥400	14	21	3.360	0.067
TNM stage	I+II	17	9	4.344	0.037
TNM stage	III+IV	13	21	4.344	0.037

图6 敲低AAMP抑制HCC细胞裸鼠肺转移瘤形成

Fig.6 Knockdown of AAMP suppresses HCC lung metastasis in nude mice. A, B: Knockdown of AAMP decreases lung metastatic nodule numbers formed by Mahlavu (A) and Huh-7 (B) cells in nude mice (×100). *P<0.05 vs sh-ctrl group (n=5).

图7 敲低AAMP抑制HCC细胞EMT相关蛋白表达

Fig.7 Knockdown of AAMP inhibits expressions of EMT-related proteins in HCC cells. A, C: AAMP knockdown increases E-cadherin expression and reduces N-cadherin, vimentin and Snail protein expressions in Mahlavu (A) and Huh-7 (C) cells (*P<0.05 vs sh-ctrl group, n=3). B, D: Immunofluorescence staining for detecting the proteins in Mahlavu (B) and Huh-7 (D) cells.

图8 敲低AAMP促进HCC细胞内RhoA蛋白降解

Fig.8 Knockdown of AAMP promotes degradation of RhoA protein in HCC cells. A: Knockdown of AAMP does not affect mRNA expression levels of RhoA in Mahlavu or Huh-7 cells. B: Knockdown of AAMP down-regulates protein expression levels of RhoA in Mahlavu and Huh-7 cells (*P<0.05 vs sh-ctrl group). C: MG-132 treatment restores protein expression levels of RhoA in Mahlavu and Huh-7 cells with AAMP knockdown. D: CHX treatment does not rescue RhoA protein expression in Mahlavu or Huh-7 cells with AAMP knockdown (**P<0.01 vs sh-ctrl group, n=3). E: RhoA and AAMP expressions are positively correlated in HCC tissues.

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