Angio-associated migratory cell protein is highly expressed in hepatocellular carcinoma and promotes tumor cell invasion and metastasis

doi:10.12122/j.issn.1673-4254.2025.12.10

Abstract

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

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.

Figures/Tables 9

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.

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

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.

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.

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.

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

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).

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.

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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