Identification of metastamirs as metastasis-associated microRNAs in clear cell renal cell carcinomas

Abstract

MicroRNAs (miRNAs) play a pivotal role in cancerogenesis and cancer progression, but their specific role in the metastasis of clear cell renal cell carcinomas (ccRCC) is still limited. Based on microRNA microarray analyses from normal and cancerous samples of ccRCC specimens and from bone metastases of ccRCC patients, we identified a set of 57 differentially expressed microRNAs between these three sample groups of ccRCC. A selected panel of 33 miRNAs was subsequently validated by RT-qPCR on total 57 samples. Then, 30 of the 33 examined miRNAs were confirmed to be deregulated. A stepwise down-regulation of miRNA expression from normal, over primary tumor to metastatic tissue samples, was found to be typical. A total of 23 miRNAs (miR-10b/-19a/-19b/-20a/-29a/-29b/-29c/-100/-101/-126/-127/-130/-141/-143/-145/-148a/-192/-194/-200c/-210/-215/-370/-514) were down-regulated in metastatic tissue samples compared with normal tissue. This down-regulated expression in metastatic tissue in comparison with primary tumor tissue was also present in 21 miRNAs. In cell culture experiments with 5-aza-2'-deoxycytidine and trichostatin A, epigenetic modifications were shown as one reason of this down-regulation. The altered miRNA profiles, comprising newly identified metastasis-associated miRNAs, termed metastamir and the predicted miRNA-target interactions together with the significant correlations of miRNAs that were either lost or newly appeared in the studied sample groups, afford a solid basis for further functional analyses of individual miRNAs in RCC metastatic progression.

Keywords: Metastasis.; Microarray; RT-qPCR; Renal cell carcinoma; microRNAs.

Figure 1

Expression of the 33 miRNAs validated by RT-qPCR in normal, primary tumor, and metastatic tissue samples of renal cell carcinoma. Columns (blank column: normal, non-malignant tissue, n=22; gray column: primary tumor tissue, n=22; black column: bone metastatic tissue, n=13) represent medians with 95% CIs. The reference miRNA combination of miR-28, miR-103, and miR-106a was used for normalization . For the sake of completeness, we also included in this figure of deregulated miRNAs the data of miR-19b and miR-141 previously reported . For reasons of clarity, miRNAs were listed according to their number and the left (L) and right (R) y-axis, as indicated in the upper part of the figure, was used to represent the expression levels of the various miRNAs. Statistical differences were calculated by the Mann-Whitney U test between the groups and indicated as follows: *, P<0.05; **, P<0.01; ***, at least P<0.001.

Figure 2

Expression of miR-127, miR-141, miR-145, and miR-514 in the renal cell carcinoma cell lines 786-O, A498, Caki-1, and ACHN treated with 5-aza-2'-deoxycytidine (Aza) and trichostatin A (TSA). Values are given as fold changes (mean ± SEM) in treated cells compared with the expression in untreated cells. The reference gene combination of RNU48 and RNU6B was used for normalization and the expression in the untreated cells was set one.