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Renal cell carcinoma (RCC) has a high potential for bone metastasis; however, the molecular mechanisms underlying this metastasis have remained to be elucidated. The present study aimed to explore the expression levels of enhancer of zeste homolog 2 (EZH2), matrix metalloproteinase-2 (MMP2) and tissue inhibitor of metalloproteinase-2 (TIMP2) as determinants of RCC-associated bone metastasis. Their expression was evaluated in a newly generated RCC cell subline that has a high potential for bone metastasis, in tissue specimens from metastasized bone tissues from patients with RCC and in RCC tissues without metastasis. A total of 25 RCC tissue specimens without metastasis and 13 RCC tissue specimens with bone metastasis were acquired for immunohistochemical analysis of EZH2, MMP2 and TIMP2 protein expression. The expression levels of EZH2, MMP2 and TIMP2 mRNA and protein were analyzed in the ACHN and ACHN-BO5 cell lines using western blot and reverse transcription polymerase chain reaction (PCR) analyses. Methylation-specific PCR was also used to analyze TIMP2 promoter methylation. EZH2 and MMP2 proteins were found to be expressed at higher levels in tissues from patients where RCC had metastasized to the bone as compared with those in RCC patients without metastasis, whereas there was no significant difference in the expression of TIMP2 protein between the two tissues. Furthermore, the expression of EZH2 protein was correlated with MMP2 expression, but there was no significant correlation between the expression of EZH2 and TIMP2 proteins. The in vitro results using cell lines confirmed the ex vivo findings, indicating that the expression levels of EZH2 and MMP2 protein and mRNA were higher in ACHN-BO5 cells than those in ACHN cells. By contrast, TIMP2 protein and mRNA expression levels were lower in ACHN-BO5 cells than those in the parental ACHN cells. The TIMP2 promoter was highly methylated in ACHN-BO5 cells compared with that in ACHN cells. Upregulation of EZH2, MMP2 and TIMP2 expression was correlated with metastasis of RCC to bone tissues ex vivo and in vitro. Further studies are required in order to elucidate the mechanism underlying the altered expression of these genes.

molecular medicine reports

Alterations in enhancer of zeste homolog 2, matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-2 expression are associated with ex vivo and in vitro bone metastasis in renal cell carcinoma