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. 2012 Nov;13(11):884-93.
doi: 10.1631/jzus.B1200037.

ST13, a Proliferation Regulator, Inhibits Growth and Migration of Colorectal Cancer Cell Lines

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Free PMC article

ST13, a Proliferation Regulator, Inhibits Growth and Migration of Colorectal Cancer Cell Lines

Rui Bai et al. J Zhejiang Univ Sci B. .
Free PMC article

Abstract

Background and objective: ST13, is the gene encoding the HSP70 interacting protein (HIP). Previous research has shown that ST13 mRNA and protein levels are down-regulated in colorectal cancer (CRC) tissues compared with adjacent normal tissues. This study aims at the role of ST13 in the proliferation and migration of CRC cells.

Methods: The transcript level of ST13 in different CRC cell lines was evaluated by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). ST13-overexpressed and ST13-knockdown CRC cells were constructed respectively by lentiviral transduction, followed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay, plate colony formation, cell-cycle analysis, and migration assays to evaluate the influence of ST13 on proliferation and migration in vitro. Moreover, a mouse xenograft study was performed to test in vivo tumorigenicity of ST13-knockdown CRC cells.

Results: Lentivirus-mediated overexpression of ST13 in CRC cells inhibited cell proliferation, colony formation, and cell migration in vitro. In contrast, down-regulation of ST13 by lentiviral-based short hairpin RNA (shRNA) interference in CRC cells significantly increased cell proliferation and cloning efficiency in vitro. In addition, down-regulation of ST13 expression significantly increased the tumorigenicity of CRC cells in vivo.

Conclusions: ST13 gene is a proliferation regulator that inhibits tumor growth in CRC and may affect cell migration.

Figures

Fig. 1
Fig. 1
ST13 expression in colorectal cancer cell lines (a) qRT-PCR shows relative transcript levels of ST13 in six colorectal cancer cell lines, including RKO, HT29, SW480, SW620, LOVO, and LS174T, and HEK293. The relative quantity was normalized to HEK293 cells, and β-actin was used as an internal control. (b) qRT-PCR shows relative transcript levels of ST13 in ST13-lentivirus-infected Lenti-ST13, control lentivirus-infected Lenti-Mock, and SW620. The relative quantity was normalized to Lenti-Mock cells, and β-actin was used as an internal control. (c) qRT-PCR shows relative transcript levels of ST13 in SW620, control-shRNA-lentivirus-infected shRNA-Mock, and ST13-shRNA-lentivirus-infected shRNA-ST13. The relative quantity was normalized to shRNA-Mock cells, and β-actin was used as an internal control. (d) Western blot results show that ST13 was expressed in SW620 cells with different groups, including Lenti-ST13, Lenti-Mock, SW620, shRNA-Mock, and shRNA-ST13. β-actin was served as loading control. Error bars indicate SEM (n=3 experiments)
Fig. 2
Fig. 2
Effect of ST13 expression on SW620 cell growth in vitro (a) Effect of ST13 overexpression on SW620 cell proliferation determined by MTT assay. Error bars indicate SEM (n=3 experiments). ** P<0.01, Lenti-ST13 vs. Lenti-Mock and SW620 (one way ANOVA and Dunnett’s test). (b) Colony formations of Lenti-ST13, Lenti-Mock, and SW620 cells in vitro. (c) Quantitative analyses of colony formations of Lenti-ST13, Lenti-Mock, and SW620 groups. Error bars indicate SEM (n=3 experiments). * P<0.05, Lenti-ST13 vs. Lenti-Mock and SW620 (one way ANOVA and Dunnett’s test). (d) Effect of ST13 knockdown on SW620 cell proliferation determined by MTT assay. Error bars indicate SEM (n=3 experiments). ** P<0.01, shRNA-ST13 vs. shRNA-Mock (Student’s t-test). (e) Colony formations of shRNA-ST13 and shRNA-Mock cells. (f) Quantitative analyses of colony formations of shRNA-ST13 and shRNA-Mock groups. Error bars indicate SEM (n=3 experiments). * P<0.05 shRNA-ST13 vs. shRNA-Mock (Student’s t-test)
Fig. 3
Fig. 3
Effect of ST13 expression on SW620 cell cycling (a–e) Cell cycle progressions of Lenti-ST13, Lenti-Mock, SW620 cells, shRNA-ST13, and shRNA-Mock, respectively. (f) The relative frequency of different phases of Lenti-ST13, Lenti-Mock, and SW620 cells. Error bars indicate SEM (n=3 experiments). * P<0.05, Lenti-ST13 vs. Lenti-Mock and SW620; ** P<0.01, Lenti-ST13 vs. Lenti-Mock and SW620 (one way ANOVA and Dunnett’s test). (g) The relative frequency of different phases of shRNA-ST13 and shRNA-Mock cells. Error bars indicate SEM (n=3 experiments). * P<0.05, shRNA-ST13 vs. shRNA-Mock (Student’s t-test)
Fig. 4
Fig. 4
Effect of ST13 expression on SW620 cells migration in vitro Lenti-ST13, Lenti-Mock, SW620, shRNA-Mock, and shRNA-ST13 cells were cultured in the top well of a transwell system and migration of SW620 cells was measured by counting the number of SW620 cells migrating to the filter surface. (a) Quantitative measurement of invaded cells. Data are representatives of each group and expressed as mean±SEM of cells per six high power fields from three separated experiments. ** P<0.01, Lenti-ST13 vs. Lenti-Mock and SW620 (one way ANOVA and Dunnett’s test). (b) Images of Lenti-ST13, Lenti-Mock, SW620, shRNA-Mock, and shRNA-ST13 cells on the filter surface
Fig. 5
Fig. 5
Effect of ST13 expression on the development and growth of inoculated SW620 cells BALB/c nude mice (n=4 per group) were inoculated with 5×106 shRNA-ST13 or shRNA-Mock cells and the development of solid SW620 tumors was monitored every 3‒4 d. The mice were sacrificed 32 d post-inoculation and the tumors were taken at the same time. (a) The dynamics of shRNA-ST13 and shRNA-Mock tumor growth. Data are expressed as mean±SEM for each group. (b) The images of individual tumors

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