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. 2013;10(6):676-82.
doi: 10.7150/ijms.5528. Epub 2013 Apr 3.

MicroRNA-34a inhibits human osteosarcoma proliferation by downregulating ether à go-go 1 expression

Xinyu Wu  1 , Daixing Zhong, Quan Gao, Wenliang Zhai, Zhenqi Ding, Jin Wu
Affiliations

MicroRNA-34a inhibits human osteosarcoma proliferation by downregulating ether à go-go 1 expression

Xinyu Wu et al. Int J Med Sci. 2013.

Abstract

Aberrant expression of MicroRNAs (miRNAs) has been implicated in several types of cancer. As a direct target gene of p53, miR-34a has been suggested to mediate the tumor suppressor function of p53. Ether à go-go 1 (Eag1) channel is overexpressed in a variety of cancers and plays important roles in cancer progression. However, the link between miR-34a and Eag1 in cancer is unclear. In this study, we used human osteosarcoma as the model to demonstrate that miR-34a was significantly downregulated in osteosarcoma tissues and cell lines compared with normal brain tissues and osteoblastic cell line. Next we evaluated the role of miR-34a in the regulation of osteosarcoma cell proliferation by CCK-8 and colony formation assays. The results showed that overexpression of miR-34a inhibited the proliferation of MG-63 and Saos-2 cells. Furthermore, xenograft nude mice model showed that miR-34a inhibited osteosarcoma growth in vivo. Mechanistically, we found that overexpression of miR-34a led to decreased Eag1 expression in osteosarcoma cells while inhibition of miR-34a increased Eag1 expression. Taken together, our results suggest that miR-34a could inhibit osteosarcoma growth via the down regulation of Eag1 expression.

Keywords: Ether à go-go1 (Eag1); MicroRNA-34a; osteosarcoma; proliferation; target gene..

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Conflict of interest statement

Competing Interests: The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
miR-34a is downregualted in osteosarcoma tissues and cells. A: The miR-34a expression level was measured in ten pairs of osteosarcoma tissues and normal bone tissues by stem-loop RT-PCR. B: Considerably reduced miR-34a expression was observed in MG-63 and Saos-2 cells, compared to hFOB 1.19 cells. The miR-34a level in normal bone tissues and hFOB 1.19 cells was normalized to 100%. *** P<0.001.
Figure 2
Figure 2
Construction of pcDNA/miR-34a eukaryotic expression vector. Cartoon of miR-34a expression vector.
Figure 3
Figure 3
miR-34a inhibits the proliferation of osteosarcoma cells. A: The proliferation of osteosarcoma cells was determined by CCK-8 assay at 24, 48, 72, and 96 h after transfection of miR-34a into MG-63 and Saos-2. The proliferation of osteosarcoma cells was significantly reduced after transfection wtih miR-34a. Data were presented as mean ± SD (n = 6). B: The tumorigenicity of osteosarcoma cells was determined by colony formation assay. The tumorigenicity of osteosarcoma cells was significantly reduced after transfection wtih miR-34a. Data were presented as mean ± SD (n = 3). ** P<0.01, *** P<0.001.
Figure 4
Figure 4
miR-34a inhibits osteosarcoma growth in vivo. The length and width of tumor were measured weekly after inoculation and the volume of tumor was calculated. After 5 weeks, the tumor volume growth curve was drafted. *** P<0.001 vs. control.
Figure 5
Figure 5
miR-34a regulates Eag1 expression. A: Western blot analysis showed that the expression level of Eag1 in the miR-34a group was significantly lower than that in the blank group or control group, while the expression level of Eag1 was increased in the miR-34a-2'-O-Me group. B: Grey-value analysis of the bolts with actin as the internal reference. The results were expressed as mean ± SD (n = 3). *** P<0.001.
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