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Influence of osteopontin short hairpin RNA on the proliferation and invasion of human renal cancer cells

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Summary

The influence of short hairpin RNA (shRNA)-mediated osteopontin (OPN) gene silencing on the proliferation and invasion of human renal cancer ACHN cells was investigated. Four types of OPN shRNA recombinant plasmids were constructed and RT-PCR assays were used to screen the most highly functional shRNA recombinant plasmids, which were transferred into the cultured ACHN cells by Lipofectamine™ 2000. The cells transfected by shRNA expression vectors (ACHN/OPN) were visualized under an inverted microscope and screened by G418. Untreated cells (ACHN) and cells transfected by mock vectors (ACHN/Vect) were used as control groups. The expression levels of OPN mRNA and protein were detected by real-time PCR and Western blot respectively. The cell cycle and ratios of apoptotic cells were assessed by flow cytometry. MTT method was used for drawing the growth curve and observing cell proliferation in vitro. The abilities of migration and invasion in three groups were measured by Transwell chamber test. The expression levels of matrix metalloproteinase (MMP)-2 and MMP-9 in three groups were examined by Western blot. Our results showed that the recombinant plasmid could be successfully transferred into ACHN cells by LipofectamineTM 2000. Compared with untreated cells, the expression levels of OPN mRNA and protein in ACHN/OPN cells were decreased by 59.68% and 76.42%, respectively (P<0.05), ACHN/OPN cells were blocked in S phase and apoptotic ratio increased significantly (P<0.05), however, no significant differences were found between ACHN/Vect and ACHN. Recombinant plasmid significantly attenuated expression levels of MMP-2 and MMP-9 proteins and suppressed the proliferation, migration, and invasion of ACHN cells. This study suggested that OPN may play an important role in the growth and invasion of human renal cancer ACHN cells, and these processes are correlated with the activations of MMP-2 and MMP-9. Our data provided preliminary experimental evidence for the feasibility of RNA interference technology in gene therapy of human renal cancer.

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Authors and Affiliations

  1. Department of Orthopedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China

    Hao Liu  (柳 昊), Anmin Chen  (陈安民), Fengjing Guo  (郭风劲) & Lin Yuan  (袁 林)

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  1. Hao Liu  (柳 昊)
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  2. Anmin Chen  (陈安民)
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  3. Fengjing Guo  (郭风劲)
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  4. Lin Yuan  (袁 林)
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Corresponding author

Correspondence to Anmin Chen  (陈安民).

Additional information

This project was supported by a grant from the Major State Basic Research Development Program of China (973 Program) (No. 2002CB513100).

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Liu, H., Chen, A., Guo, F. et al. Influence of osteopontin short hairpin RNA on the proliferation and invasion of human renal cancer cells. J. Huazhong Univ. Sci. Technol. [Med. Sci.] 30, 61–68 (2010). https://doi.org/10.1007/s11596-011-0111-7

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  • DOI: https://doi.org/10.1007/s11596-011-0111-7

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