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N-myc downstream-regulated gene 1/Cap43 may function as tumor suppressor in endometrial cancer

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Abstract

Purpose

N-myc downstream-regulated gene 1 (NDRG1) reportedly regulates tumor progression in various cancers. Our previous studies showed that NDRG1 was aberrantly overexpressed in human endometrial cancer tissues. The purpose of the present study was to investigate the role of NDRG1 in endometrial carcinogenesis.

Methods

A short hairpin RNA (shRNA)-mediated gene silencing strategy was employed to stably suppress the expression of NDRG1 in endometrial cancer Ishikawa cells. The influence of NDRG1 silencing on cancer cell biological behaviors was examined through observing in vitro tumor cell proliferation, colony formation, cell migration and invasion. Moreover, the mammalian NDRG1 expression vector pcDNA3.1(+)/NDRG1 was constructed to determine the effects of NDRG1 overexpression on cell proliferation and migration. Additionally, gene expression microarray analysis was conducted to identify NDRG1 downstream target genes after NDRG1 knockdown.

Results

It was demonstrated that NDRG1 knockdown significantly enhanced Ishikawa cell proliferation and dramatically promoted cell migration and invasion. Furthermore, overexpression of NDRG1 in Ishikawa cells greatly inhibited cell proliferation and migration. Through microarray analysis and data mining, a large cohort of NDRG1-repressed target genes were identified. Additionally, through comparing the current microarray results with those obtained previously in studies of cervical and ovarian cancer cells conducted by us, 19 more specific common downstream target genes were identified.

Conclusions

It was demonstrated that NDRG1 might carry out a tumor suppressor function during endometrial carcinogenesis. The identification of downstream target genes should afford meaningful hints for prospective investigations. The tumor suppressor function of NDRG1 may open a new window for the target therapy of endometrial cancer.

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Acknowledgments

Special thanks to the faculty and staff of the Department of Pathology, School of Medicine, Shanghai Jiaotong University. Special thanks to Professor Guohui Fu (Professor and director of Department of Pathology, School of Medicine, Shanghai Jiaotong University) for her kind help during the whole experiment conduction. We also thank Dr. Yongchang Zhang (Ph.D candidate from Department of Pathology, School of Medicine, Shanghai Jiaotong University) for technical help. This work was under the support of medical improvement project of Shanghai Songjiang District (NO2011PD11).

Conflict of interest

We declare that we have no conflict of interest.

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Authors and Affiliations

  1. Department of Pathology, Shanghai Jiaotong University Affiliated Shanghai First People’s Hospital, NO.85, Wujin Road, Shanghai, 200080, People’s Republic of China

    Xiu-Hong Lv & Jia-Wei Chen

  2. Department of Pathology, Tianjin Cancer Hospital, Tianjin Medical University, Tianjin, 300060, People’s Republic of China

    Gang Zhao

  3. Department of Pathology, Bengbu Medical College, Bengbu, 233030, Anhui, People’s Republic of China

    Zhen-Zhong Feng

  4. Department of Pathology, Shanghai Jiaotong University Affiliated Songjiang Hospital, Shanghai, 201601, People’s Republic of China

    Dao-Hua Yang & Wei-Wei Sun

  5. Department of Pathology, Si Jing Hospital, Songjiang District, Shanghai, 201601, People’s Republic of China

    Jin-Shuang Fan

  6. Department of General Surgery, Shanghai Jiaotong University Affiliated Shanghai First People’s Hospital, Shanghai, 200080, People’s Republic of China

    Guang-Hui Zhu

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  1. Xiu-Hong Lv
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Correspondence to Jia-Wei Chen.

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432_2012_1249_MOESM1_ESM.jpg

Supplementary material 2-Fig. 2S. Effects of NDRG1 knockdown or over expression on p21 protein expression in Ishikawa cells. a. NDRG1 suppression resulted in inhibition of p21 protein level in Ishikawa cells. b. Over expression NDRG1 up regulated expression of p21 protein level in Ishikawa cells. The rabbit polyclonal p21 antibody was purchased from Proteintech group (1:400 dilution) (JPEG 856 kb)

432_2012_1249_MOESM2_ESM.jpg

Supplementary material 1-Fig. 1S Effects of suppression or over expression NDRG1 in endometrial cancer AN3CA cells on cell migration and invasion. a. Verification of NDRG1 suppression through shRNA-mediated gene silence strategy in AN3CA cells by Western Blot. AN3CA cells were transfected with shNDRG1 expression vector (pGPU/GFP/Neo-shRNA NDRG1) and negative control vector (pGPU/GFP/Neo-shRNA NC), respectively. After selection with 0.7 mg/ml G418 for 4 weeks, stable clones were selected, expanded and confirmed by Western Blot. b. Cell migration and invasion after NDRG1 suppression in AN3CA cells were determined by Transwell (Boyden Chamber) Cell Migration and invasion Assay. Briefly, 10 × 104 cells from each group were seeded into the upper chamber of the transwell inserts. 24 h later, Migrated and invaded cells were fixed and then stained with crystal violet. Photographs were taken using a light microscopic at 200 × magnification. Migrated or invaded cells from five random fields per visual field at 200 × magnification were counted. The bars represent the mean ± SD. * P < 0.05 and ** P < 0.01 as compared with negative control vector group. c. AN3CA cells were transiently transfected with pcDNA3.1(+) or pcDNA3.1(+)/NDRG1, respectively. 48 h after transfection, over expression of NDRG1 in AN3CA cells was confirmed by Western blot. d. 48 h after transfection, equal number of cells from each group [AN3CA-pcDNA3.1(+) and AN3CA-pcDNA3.1(+)/NDRG1] were collected and subjected to in vitro cell migration and invasion assay. Migrated or invaded cells were fixed and then stained with crystal violet. Photographs were taken using a light microscopic at 200 × magnification. Migrated or invaded cells from five random fields per visual field at 200 × magnification from each group were counted. The bars represent the mean ± SD. * *P < 0.01 as compared with negative control vector group (JPEG 259 kb)

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Lv, XH., Chen, JW., Zhao, G. et al. N-myc downstream-regulated gene 1/Cap43 may function as tumor suppressor in endometrial cancer. J Cancer Res Clin Oncol 138, 1703–1715 (2012). https://doi.org/10.1007/s00432-012-1249-4

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