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Plasminogen activator inhibitor 2 (PAI2) inhibits invasive potential of hepatocellular carcinoma cells in vitro via uPA- and RB/E2F1-related mechanisms

  • Ye Jin
  • Zhi-Yong Liang
  • Wei-Xun Zhou
  • Li ZhouEmail author
Original Article

Abstract

Background

Plasminogen activator inhibitor 2 (PAI2) has been shown to be associated with invasive phenotypes and prognosis in hepatocellular carcinoma (HCC). However, its biological roles and underlying mechanisms in invasion of HCC have not been explored. The present study aimed to address the issues.

Methods

First, sub-lines in that PAI2 was stably overexpressed and silenced were established based on MHCC97H and BEL7402 cell lines, respectively. Wound-healing and transwell assays were applied to evaluate cell migration and invasion. Urokinase-type plasminogen activator (uPA) activity was measured using an ELISA kit. Real-time RT-PCR and western blotting were used to show gene expression at mRNA and protein levels. E2F1 expression in human specimens was determined by tissue microarray-based immunohistochemical staining.

Results

The sub-lines, MHCC97H-PAI2 and BEL7402-siPAI2, were successfully established. The two sub-lines carried much lower and higher migration and invasion powers, respectively, in contrast to the controls. In MHCC97H-PAI2 sub-line, intra-medium uPA activity was significantly decreased, while RB expression was obviously elevated, compared with the controls. The BEL7402-siPAI2 sub-line presented the opposite trend. To identify the role of RB/E2F1 pathway, we transiently overexpressed E2F1 in MHCC97H-PAI2 sub-line, and largely reversed the inhibitory effects of PAI2 on cell migration and invasion, through regulating multiple matrix metalloproteinases and epithelial–mesenchymal transition. In HCC specimens, E2F1 expression was much higher in tumor than in non-tumor tissues, and was significantly related to Edmondson–Steiner grade, overall as well as tumor-free survival.

Conclusions

Our data suggest that PAI2 inhibits invasive potential of HCC cells via uPA- and RB/E2F1-related mechanisms.

Keywords

Hepatocellular carcinoma Plasminogen activator inhibitor 2 Urokinase-type plasminogen activator RB/E2F1 Prognosis 

Notes

Acknowledgements

This study was supported by the National Natural Science Foundation of China (81301845).

Compliance with ethical standards

Conflict of interest

Ye Jin, Zhi-Yong Liang, Wei-Xun Zhou and Li Zhou declare that they have no conflict of interest.

Ethical standards

All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (Institutional and National) and with the Helsinki Declaration of 1975, as revised in 2008. This study was approved by the institutional ethics committee and informed consent was obtained from all patients.

Supplementary material

12072_2018_9920_MOESM1_ESM.tif (1.2 mb)
Fig. S1 The baseline cell migration and PAI2 expression in MHCC97H and BEL7402 cell lines. (A) Significantly enhanced migration in MHCC97H as contrast to BEL7402 cells; (B) Significantly lower PAI2 protein expression in MHCC97H as contrast to BEL7402 cells. ***: P<0.001. (TIFF 1266 kb)
12072_2018_9920_MOESM2_ESM.tif (335 kb)
Fig. S2 The impacts of PAI2 stably overexpression and silence on cell proliferaion. (A) Slightly decreased proliferation in MHCC97H-PAI2 sub-line (PAI2 overexpression, not statistically significant); (B) Slightly enhanced proliferation in BEL7402-siPAI2 sub-line (PAI2 silence, not statistically significant). (TIFF 335 kb)
12072_2018_9920_MOESM3_ESM.tif (380 kb)
Fig. S3 Significantly decreased expression of E-cadherin and enhanced expression of N-cadherin and Vimentin after E2F1 transient overexpression in MHCC97H-PAI2 sub-line. (TIFF 380 kb)
12072_2018_9920_MOESM4_ESM.tif (166 kb)
Fig. S4 The difference of E2F1 H-scores in different Edmondson-Steiner grades (Grade I-II vs Grade III-IV, Mann-Whitney U-test; P=0.006). (TIFF 166 kb)

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Copyright information

© Asian Pacific Association for the Study of the Liver 2019

Authors and Affiliations

  1. 1.Clinical Research Laboratory, Peking Union Medical College HospitalChinese Academy of Medical Sciences/Peking Union Medical CollegeBeijingChina
  2. 2.Department of Pathology, Peking Union Medical College HospitalChinese Academy of Medical Sciences/Peking Union Medical CollegeBeijingChina
  3. 3.Department of General Surgery, Peking Union Medical College HospitalChinese Academy of Medical Sciences/Peking Union Medical CollegeBeijingChina

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