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Effects of legumain as a potential prognostic factor on gastric cancers

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Abstract

Although legumain has been found to be a prognostic factor in both breast cancer and colorectal cancer, its effects on gastric cancer are unknown. In this study, we investigated effects of legumain on gastric cancer and the correlation between legumain expression and prognosis of gastric cancer patients. SGC7901 cells were transduced with legumain cDNA (SGC7901-hLeg) for overexpression of legumain or with legumain shRNA to knock down legumain. In vitro tumor migration was examined by wound healing assay. Furthermore, a tumorigenicity and metastasis mouse model was used to examine legumain function in vivo; asparaginyl endopeptidase inhibitor (AEPI, an inhibitor of legumain) was injected to the mice (i.p.) to evaluate its therapeutic effect. Tissue microarray analysis from 112 gastric cancer patients was performed to evaluate the association between legumain expression and the cumulative survival time. Legumain was highly expressed in gastric cancer patients and some gastric cancer cell lines. Legumain promoted gastric cell migration in vitro and promoted gastric tumor growth and metastasis in vivo, and these effects were reversed by knockdown of legumain with shRNA or treated with AEPI. In gastric cancer clinical samples, legumain expression in tumor was significantly higher than in non-tumor and was negatively associated with the cumulative survival rate. In conclusion, legumain was highly expressed in gastric adenocarcinoma; legumain promoted gastric cancer tumorigenesis and metastasis in vitro and in vivo. Legumain expression in tumor was a poor prognostic factor for gastric cancer patients, and legumain could be a potential target molecule for gastric cancer therapy in clinic.

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Acknowledgments

This work was supported by the National Natural Science Foundation of China (#30971137 and # 31171308) and Shanghai Commission for Science and Technology (11DZ1910200).

Conflict of interests

All authors indicated no potential conflicts of interests, and all authors could fully access the primary data and agree to allow the journal to review the data if requested.

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Authors and Affiliations

  1. Institute of Health Sciences, Shanghai Jiao Tong University School of Medicine and Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

    Na Li & Bin Lan

  2. Nanomedicine Center, Shanghai Advanced Research Institute, Chinese Academy of Sciences, Shanghai, 201210, China

    Qiaoling Liu, Chongyang Wei, Jianyong Wang, Ying Yu, Baowei Peng, Xiangming Yan & Fang Guo

  3. Department of General Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Guoqing Bao

  4. Department of Thoracic Surgery, Songjiang Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Qi Su

  5. Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, USA

    Fei Yan

  6. Department of Pathology, Feinberg School of Medicine, Northwestern University, Chicago, USA

    Ju Qiu

  7. Department of Pathology, First Affiliated Hospital of Fujian Medical University, Fuzhou, 350000, Fujian Province, China

    Sheng Zhang

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  1. Na Li
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  2. Qiaoling Liu
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  3. Qi Su
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  4. Chongyang Wei
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  5. Bin Lan
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  6. Jianyong Wang
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  7. Guoqing Bao
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  9. Ying Yu
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  10. Baowei Peng
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  11. Ju Qiu
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  12. Xiangming Yan
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  13. Sheng Zhang
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  14. Fang Guo
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Corresponding authors

Correspondence to Sheng Zhang or Fang Guo.

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12032_2013_621_MOESM1_ESM.pdf

Supplemental figure 1. (A) Supernatants were collected and immunoblotted to detect the efficiency that stable transfection of legumain to SGC7901 cells, 56KD was prolegumain, while 36KD was active legumain. β-actin was used as an internal control for equal loading. (B) Wound healing assay was used to explore cell migration at 0 and 24 hour. The image demonstrated that, at 24 hour, cell migration was promoted in the legumain overexpression group compared to control group. (C) Number of migrated SGC7901 cells was much higher in legumain overexpressed cells than control cells. (D) MTS was used to detect SGC7901 cell proliferation at 24h, 48h, 72h. P value has no significance between control cells and legumain overexpressed SGC7901 cells. (E) Western blot assay was used to detect the efficiency that transient transfection of legumain to MKN45 cells, 56 KDa was prolegumain, while 36 KDa was active legumain. β-actin was used as an internal control for equal loading. (F) Wound healing assay show that overexpression of legumain promotes the migration of MKN45 cells. (G) Number of migrated cells in legumain overexpressed MKN45 cells were higher than control MKN45 cells, p = 0.0065. (H) AEPI-1 structure.

12032_2013_621_MOESM2_ESM.pdf

Supplemental figure 2. (A1, A2) MMP-2 negative in non-tumor tissues; magnifications were ×100, ×400, respectively. (B1, B2-D1, D2)) MMP-2 was highly expressed in tumor tissues; magnifications were ×100, ×400, respectively. (E) Number distribution of MMP-2 expression (low, medium, high) in non-tumor and tumor tissues. (F) MMP-2 expression between non-tumor and tumor tissues ( p< 0.0001). (PDF 368592 kb)

12032_2013_621_MOESM3_ESM.pdf

Supplemental figure 3. (A) Number distribution of legumain and MMP-2 coordinate expression. (B) Correlation between legumain expression and MMP-2 expression in tumor, r2 = 0.8640, p < 0.0001. (C) Kaplan-Meier curves used to analyze the association between MMP-2 expression in tumor and cumulative survival rate, p < 0.0001. (PDF 1892 kb)

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Li, N., Liu, Q., Su, Q. et al. Effects of legumain as a potential prognostic factor on gastric cancers. Med Oncol 30, 621 (2013). https://doi.org/10.1007/s12032-013-0621-9

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  • DOI: https://doi.org/10.1007/s12032-013-0621-9

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