Derivate Isocorydine (d-ICD) Suppresses Migration and Invasion of Hepatocellular Carcinoma Cell by Downregulating ITGA1 Expression

Abstract

In our previous studies, we found that isocorydine (ICD) could be a potential antitumor agent in hepatocellular carcinoma (HCC). Derivate isocorydine (d-ICD), a more effective antitumor agent, has been demonstrated to inhibit proliferation and drug resistance in HCC. In order to investigate the potential role of d-ICD on HCC cell migration and its possible mechanism, wound healing assay, trans-well invasion assay, western blot analysis, and qRT-PCR were performed to study the migration and invasion ability of HCC cells as well as relevant molecular alteration following d-ICD treatment. Results indicated that the migration and invasion ability of HCC cells were suppressed when cultured with d-ICD. Meanwhile, the expression level of ITGA1 was markedly reduced. Furthermore, we found that ITGA1 promotes HCC cell migration and invasion in vitro, and that ITGA1 can partly reverse the effect of d-ICD-induced migration and invasion suppression in HCC cells. In addition, dual luciferase reporter assay and chromatin immunoprecipitation assay were used to study the expression regulation of ITGA1, and found that E2F1 directly upregulates ITGA1 expression and d-ICD inhibits E2F1 expression. Taken together, these results reveal that d-ICD inhibits HCC cell migration and invasion may partly by downregulating E2F1/ITGA1 expression.

Keywords: ITGA1; d-ICD; hepatocellular carcinoma; invasion; migration.

Figure 1

Derivate isocorydine (d-ICD) inhibits hepatocellular carcinoma (HCC) cell migration and invasion and ITGA1 expression. (A) Various HCC cell lines were incubated with d-ICD, and the wound-healing assay was performed to analyze the migration ability (scale bar stand for 200 μm); (B) The Trans-well assay was performed to analyze the invasion ability of HCC cells after d-ICD treatment (scale bar stand for 100 μm); (C) RT-PCR and western blotting analyses were performed to examine ITGA1 expression in HCC cells with d-ICD treatment for 0 h, 12 h, and 24 h; (D) Western blot analyzed endogenous ITGA1 expression in HCC cells. (* p < 0.05, ** p < 0.01).

Figure 1

Derivate isocorydine (d-ICD) inhibits hepatocellular carcinoma (HCC) cell migration and invasion and ITGA1 expression. (A) Various HCC cell lines were incubated with d-ICD, and the wound-healing assay was performed to analyze the migration ability (scale bar stand for 200 μm); (B) The Trans-well assay was performed to analyze the invasion ability of HCC cells after d-ICD treatment (scale bar stand for 100 μm); (C) RT-PCR and western blotting analyses were performed to examine ITGA1 expression in HCC cells with d-ICD treatment for 0 h, 12 h, and 24 h; (D) Western blot analyzed endogenous ITGA1 expression in HCC cells. (* p < 0.05, ** p < 0.01).

Figure 2

Overexpression of ITGA1 promotes HCC cell migration and invasion in vitro. (A) The efficiency of ITGA1 overexpression were analyzed via RT-PCR and western blot; (B) The in vitro migration ability of MHCC-97L, MHCC-LM3 and HCC-LY10 cells stably transfected with ITGA1 or vector were assessed using the wound-healing assay (scale bar stand for 200 μm); (C) The in vitro invasion ability of those ITGA1-overexpressed HCC cells were assessed using the trans-well assay (scale bar stand for 100 μm). (*p < 0.05, ** p < 0.01).

Figure 3

Silencing ITGA1 expression inhibits HCC cell migration and invasion in vitro. (A) qRT-PCR and western blot analyzed the silencing efficiency of ITGA1 expression; (B) The in vitro migration ability of SMMC-7721, Huh7 and HCC-LY5 cells stably transfected with ITGA1 ShRNA or ShNC or MOCK cells were assessed using wound-healing assay (scale bar stand for 200 μm); (C) The in vitro invasion ability of those ITGA1-silencing HCC cells were assessed by trans-well assay (scale bar stand for 100 μm). (“ns” indicates no statistical significance, * p < 0.05, ** p < 0.01).

Figure 4

ITGA1 partly rescues d-ICD-induced migration and invasion suppression in HCC cells; (A) The wound-healing assay was performed to detect the migration ability of MHCC-97L, MHCC-LM3 and HCC-LY10 empty cells after treating with or without d-ICD, and MHCC-97L, MHCC-LM3, and HCC-LY10 stably transfected with ITGA1 or vector after treating with d-ICD (scale bar stand for 200 μm); (B) The Trans-well assay was performed to detect the invasion ability of those HCC cells (scale bar stand for 100 μm). (“ns” indicates no statistical significance, * p < 0.05, ** p < 0.01).

Figure 5

E2F1 transcriptional upregulates ITGA1 expression and d-ICD inhibits E2F1 expression in HCC cells. (A) Identification of potential E2F1 binding sites (double arrow marked) in the ITGA1 promoter (arrow direction indicates sequence from 5′ to 3′) according to four TF-binding prediction websites (QIAGEN, ALGGEN PROMO, TFBIND, GENE REGULATION), and the mutant sites are marked (arrow direction indicates transcriptional direction of the downstreem gene); (B) Dual luciferase reporter gene studies were performed to detect the ITGA1 promoter and its truncated and mutant construct activity in 293T cells; (C) Binding of E2F1 to the ITGA1 promoter in HCC cells was analyzed by chromatin immunoprecipitation. qRT-PCR was used to analyze the ITGA1 promoter, and agarose gel electrophoresis was used to analyze the crosslinking status; (D) qRT-PCR and western blotting analyzed E2F1 and ITGA1 expression in HCC cells with transiently transfected E2F1 plasmid; (E) Western blotting analyses of E2F1 expression in HCC cells treated with d-ICD at different time points. (* p < 0.05, ** p < 0.01).