ANG Promotes Proliferation and Invasion of the Cell of Lung Squamous Carcinoma by Directly Up-Regulating HMGA2

Abstract

Objective: To determine the mechanism of Angiogenin(ANG) function involved in the carcinogenesis of lung squamous cell carcinoma.

Methods: 12 patients' normal tissue and cancerous tissue were collected. ANG expression in the squamous cell carcinoma of the lung was evaluated by qRT-PCR and western-blot. The regulation of ANG on proliferation, migration, invasion, and apoptosis of SK-MES-1 cells were analyzed by Cell Counting Kit-8, Transwell migration chamber, Transwell invasion chamber, and Annexin V-FITC assay, respectively. PCR array was utilized for screening potential target genes of ANG. Chromatin immunoprecipitation(ChIP) assays and luciferase assay were adopted for investigation of ANG's direct regulation on HMGA2.

Results: ANG expression is increased in the squamous cell carcinoma of the lung tissue. In vitro experiments results indicated that overexpression of ANG promotes proliferation and invasion capability of SK-MES-1 cells. The candidate proliferation, migration, and invasion related ANG target gene found was HMGA2, expression levels of which were also enhanced in lung squamous cell carcinoma tissue. The direct regulation of ANG on HMGA2 was verified by ChIP and luciferase assay results. Furthermore, down-regulating HMGA2 significantly alleviated the suppression effects of ANG on proliferation, migration, and invasion of SK-MES-1 cells.

Conclusions: Our data illustrated the mechanisms that ANG promoted the cell of SQCLC proliferation, migration, and invasion capacity via directly up-regulating HMGA2.

Keywords: Angiogenin; HMGA2.; lung squamous cell carcinoma.

Figure 1

ANG expression levels in the SQCLC cancerous tissues versus adjacent normal tissues, and verification of ANG overexpression in Ad-ANG SK-MES-1s. A, RT-PCR showed that ANG mRNA levels were significantly higher in cancerous tissues than in adjacent normal tissues in SQCLC patients (n = 3 experiments for all tests). * P < 0.05 versus adjacent normal. B, Local expressions of ANG and HMGA2 protein were upregulated in cancerous tissues. C, The transfection efficiency of adenovirus vectors in SK-MES-1s, which was assessed under fluorescence microscope, reached ≤ 70%. D, mRNA level of ANG was significantly enhanced in Ad-ANG SK-MES-1s (n = 3 for each cell group). * P < 0.05 versus control. Ad-GFP served as control. E, Protein levels of ANG and HMGA2 were significantly upregulated in Ad-ANG SK-MES-1s. All experiments were carried out in triplicate.

Figure 2

ANG is an important regulator of proliferation, migration, invasion, and apoptosis of SK-MES-1s. A, A higher optical density at 450 nm (OD 450) in the CCK8 assay was observed in Ad-ANG SK-MES-1s at 24, 48, and 72 hours (n = 3 for each cell group). * P < 0.05 versus control. B and E, Results of the migration chamber assay showed that migration capability was significantly higher in the Ad-ANG SK-MES-1s. Quantified data were presented as the number of migrated cells per high-power field (HPF) (n = 3 for each cell group). * P < 0.05 versus control. C and F, Transwell invasion results showed that invasion capability was significantly higher in Ad-ANG SK-MES-1s. Quantified data were presented as the number of invasion cells per HPF (n = 3 for each cell group). * P < 0.05 versus control. D and G, Cells were stained with Annexin V-FITC and propidium iodide (PI). Flow cytometric contour plots showed the percentage of the stained cells. Histogram showed the percentage of Annexin V⁺/PI^- and Annexin V⁺/PI⁺ cells. Ad-ANG cells showed a lower apoptotic rate (n = 3 each cell group). * P < 0.05 versus control. All experiments were carried out in triplicate.

Figure 3

Knocking down of ANG suppresses proliferation and migration abilities of SK-MES-1s. A and B, Successful knockdown of ANG in SK-MES-1s via shANG. HMGA2 protein expression was decreased in shANG SK-MES-1s, as compared with shScramble SK-MES-1s (n = 3 for each cell group). * P < 0.05 versus control. C, shANG SK-MES-1s showed a lower proliferation capacity at 24, 48, and 72 hours, as compared with shScramble SK-MES-1s (n = 3 for each cell group). * P < 0.05 versus control. D and E, migration capability was significantly lower in the shANG SK-MES-1s. Quantified data were presented as the number of migrated cells per HPF (n = 3 for each cell group). * P < 0.05 versus control. All experiments were carried out in triplicate.

Figure 4

PCR array analysis of candidate ANG target genes, ANG regulates HMGA2 expression in SK-MES-1s. A, Names of the 84 proliferation, migration, and invasion related genes. B, Heat map of the variations in the expression of the 84 genes between Ad-ANG and Ad-GFP SK-MES-1s. C, HMGA2 mRNA levels were significantly higher in cancerous tissues than in adjacent normal tissues in SQCLC patients (n = 3 experiments for all tests). * P < 0.05 versus adjacent normal. D, HMGA2 mRNA expression was activated in Ad-ANG SK-MES-1s (n = 3 for each cell group). * P < 0.05 versus control. E, HMGA2 mRNA expression was decreased in shANG SK-MES-1s, as compared with shScramble SK-MES-1s (n = 3 for each cell group). * P < 0.05 versus control. F, ICC results showed that HMGA2 expression levels were decreased in the nucleus of shANG SK-MES-1s. All experiments were carried out in triplicate.

Figure 5

HMGA2 is a direct target of ANG. A, ChIP results showed that ANG was recruited to the promoter of HMGA2 during transcription activity (n = 3 experiments for all tests). B, Construction of plasmid psiCHECK-2-HMGA2-WT and plasmid psiCHECK-2-HMGA2-MU. C, Overexpression of ANG significantly enhanced the relative luciferase activity in psiCHECK-2-HMGA2-WT group. However, the relative luciferase activity difference in psiCHECK-2-HMGA2-MU group did not show statistical significance by overexpression of ANG (n = 3 experiments for all tests). * P < 0.05 versus control. ^▲ P > 0.05 versus control. All experiments were carried out in triplicate.

Figure 6

HMGA2 is a mediator of ANG in regulating proliferation, migration, and invasion of SK-MES-1s. A and B, Successful knockdown of HMGA2 in Ad-ANG SK-MES-1s. Ad-ANG SK-MES-1s transfected with shScramble served as negative control (n = 3 for each cell group). * P < 0.05 versus control. C, CCK8 assay after HMGA2 knockdown in Ad-ANG SK-MES-1s. A lower OD 450 value was observed in Ad-ANG/shHMGA2 SK-MES-1s at 24, 48, and 72 hours (n = 3 for each cell group). * P < 0.05 versus control. D and F, Ad-ANG/shHMGA2 cells showed a lower migration ability, as compared to Ad-ANG/shScramble SK-MES-1s. Quantified data were presented as the number of migrated cells per HPF (n = 3 for each cell group). * P < 0.05 versus control. E and G, Transwell invasion results showed that invasion capability was significantly lower in Ad-ANG/shHMGA2 SK-MES-1s. Quantified data were presented as the number of invasion cells per HPF (n = 3 for each cell group). * P < 0.05 versus control. H, The apoptotic rates difference in Ad-ANG/shHMGA2 and Ad-ANG/shScramble SK-MES-1s showed no statistical significance (n = 3 for each cell group). ^▲ P > 0.05 versus control. All experiments were carried out in triplicate.