Dysregulation of miRNA expression has been linked to the formation and progression of many types of cancers 
. A number of studies have shown that profiles of miRNA expression can serve as phenotypic signatures of different cancer types. For example, analysis of a panel of more than 200 miRNAs from 334 chronic lymphocytic leukemia samples indicated that miRNA profiles reflected the developmental lineage and differentiation status of this neoplasm 
. A similar study that involved analysis of 540 solid tumors identified miRNA signatures composed largely of over-expressed miRNAs 
. In addition, miRNA expression profiling of 60 cancer-derived cell lines (NCI-60 cell lines) allowed the clustering of the cell lines according to their tissue of origin and identified specific miRNAs in different tumor types as candidate oncogenes and tumor-suppressor genes 
. Another recent study identified miRNA expression patterns that appeared to be associated with steps during in vivo
neoplastic development in a mouse pancreatic tumor model 
; this study found that miRNA profiling can be employed to detect miRNA dysregulation at different stages of neoplastic progression in vivo
(hyper-proliferation, angiogenesis, primary tumor, and metastasis).
Reported here are the results of the first miRNA expression-profiling study of non-human primate cells that transformed spontaneously during serial passage in cell culture. We evaluated patterns of miRNA expression in pAGMK cells and in derivatives of the 10-87 VERO cell line (10-87 LP cells, 10-87 HP cells, and 10-87 T cells) in an attempt to identify the miRNAs whose altered expression might correlate with, and perhaps be involved in, the evolution of the neoplastic phenotypes that occurred during passage of these AGMK cells in tissue culture. Hierarchical clustering of miRNA expression profiles revealed that many miRNAs were differentially expressed in the tumorigenic 10-87 HP cells and tumor-derived 10-87 T cells when compared with pAGMK cells. In contrast, fewer miRNAs were differentially expressed when the non-tumorigenic 10-87 LP cells were compared with pAGMK cells. These results suggested that the dysregulation of the majority of miRNAs occurred in the 10-87 cell lineage during the transition from a non-tumorigenic to a tumorigenic phenotype. Furthermore, when the expression levels of the up-regulated miRNAs were organized according to their cell line-determined expression patterns, a correlation emerged between the relative expression (compared with levels in pAGMK cells) of representative miRNAs and the evolution of the 10-87 VERO cells into cells that could form tumors in nude mice (). From this correlation, we suggest that a sequence of changes in miRNA expression occurred during the tissue-culture passage-induced neoplastic transformation of 10-87 VERO cells. The initial changes in miRNA expression, which appear to be associated with the initiation of neoplastic development and cell immortalization, occurred prior to p148 (a passage level at which 10-87 LP cells were shown to be non-tumorigenic). These initial changes consisted of the down-regulation of multiple miRNAs, highlighted by the down-regulation of miR-31, miR-200c, and miR-218. The up-regulation of specific miRNAs occurred between p148 and p256 (); p256 was a passage level at which 10-87 HP cells expressed the ability to form tumors in newborn nude mice. The miRNA-expression patterns obtained from immortalized non-tumorigenic CV-1 cells and BSC-1 cells and the tumorigenic A4497 VERO cells at p165 supported the evolving increase in miRNA expression found between 10-87 LP cells at p148 and 10-87 HP cells at p256 (). In contrast, the pattern of expression of the down-regulated miRNAs did not appear to correlate with the change in VERO cells to a tumorigenic phenotype. In view of the evidence that many tumors occurring in nature arise from the selection of cells with genetic and/or epigenetic changes 
, one interpretation of our data is that the in vitro
passage of VERO cells selected for the expansion of cells bearing alterations in the expression of specific miRNAs. These findings lead us to propose that the miRNA signatures identified in early-passage VERO cells (10-87 LP) and late-passage VERO cells (10-87 HP and 10-87 T cells) were indicative of the progressive changes in VERO cells that were undergoing neoplastic transformation during serial passage in cell culture.
Correlation between expression of miRNAs and the neoplastic phenotype.
To determine whether the overexpression of the identified signature miRNAs could increase the levels of cell migration or invasion – phenotypes that correlated with the capacity to form tumors in animals – stable cell lines expressing the pre-miRNA sequences of some miRNAs were generated from the non-tumorigenic 10-87 LP cells. These experiments demonstrated that miR-376a and miR-376abc were sufficient to confer the cell migration and invasion phenotype. No change in migration and invasion phenotypes was observed in cells expressing miR-382 or miR-299-5p. Recently, a target and function of miR-376a has been identified. A human cytomegalovirus (HCMV) miRNA acts synergistically with a cellular miR-376a to suppress one of the major histocompatibility complex class I polypeptide-related sequences (MICB) during HCMV infection 
. The MICB is a known ligand for natural killer cell activating receptor NKG2D, which is over-expressed on cell surfaces after stress such as that induced by viral infection and cell transformation 
. It has been documented that overexpression of microRNAs binding to the MICB 3′ UTRs results in enhanced tumorigenicity through the downregulation of MICB expression, which enables tumors to avoid immune recognition 
. Furthermore, it was found that NKG2D-mediated tumor rejection can be effective at early stages of tumor growth 
. While the specific effect of miR-376a on tumorigenicity has not yet been demonstrated, it is plausible to suggest that the 10-87 VERO cells might use a similar mechanism as does HCMV to escape the immune system during tumor growth in athymic nude mice.
When we compared experimental results from several published studies on the differential expression of miRNA in different cancers with our results, 30% (18 miRNAs) of the dysregulated miRNAs identified in our study () have an association with a variety of other cancer types, including renal, prostate, lung, bladder, breast, ovarian and retinoblastoma 
. When compared with expression profiles of miRNAs associated with renal cancer, there was a 15% overlap with the miRNAs described in a recent report 
; however, only miR-376a 
out of the highly upregulated miRNAs, and miR-638, miR-200b and miR-200c from the highly downregulated miRNAs, were found to be in common between our studies and other studies 
. Although, there are differences in our in vitro
model of neoplastic development in VERO cells, which were derived from the kidney of African green monkey, and tumors of the kidney that originated in vivo
in humans, the finding of similar patterns of dysregulation of miRNAs in both systems suggests that similar molecular processes may be underway during neoplastic development in cells from both species irrespective of their microenvironments.
The other observation was the chromosomal clustering of the genes for some of the miRNAs up-regulated in 10-87 HP cells. Importantly, all of the genes for the 10 up-regulated miRNAs in 10-87 HP cells are clustered in a homologous chromosome region of rhesus monkeys and humans. The genes for the 10 miRNAs highly expressed in 10-87 HP cells and 10-87 T cells are spread over 47.6 kb on rhesus chromosome 7. This highly conserved chromosomal region is homologous to human 14q32.31. Interestingly, specific up-regulation of some miRNAs whose genes are located in the human 14q32 imprinted domain has been reported in acute myeloid leukemia 
. Clustering of these miRNA genes suggests that their expression may be co-regulated and that they might play a role in a common molecular process. However, genes for some miRNAs included in the same region did not show an increased expression in 10-87 HP cells, suggesting a complex mechanism of gene regulation in this region. It has been suggested that some miRNA genes in this region act as tumor-suppressor genes and that changes in the methylation status of their promoters can trigger cancer development 
. Furthermore, epigenetically regulated expression of miRNA genes associated with oncogenic activity has been described for some miRNAs 
. Thus, it is possible that the increased expression of some miRNAs but not others in the cluster in 10-87 HP VERO cells could be due to epigenetic changes within this chromosomal region. Another possibility is that the altered level of mature miRNA could be due to differential miRNA processing in 10-87 LP cells and 10-87 HP cells, as reported in different stages of colorectal neoplasia 
. Studies are being initiated to evaluate the mechanism of the regulation of these miRNAs.
The phenomena of neoplastic development and neoplastic transformation, whether they occur in vivo
or in vitro
, are thought to represent the accumulation of a series of genetic and epigenetic alterations that disrupt the normal processes of cell division and tissue integrity 
. These processes convey upon the cells in which they occur the propensity to outgrow unaffected cells and either expand locally to produce tumor masses or spread and gradually replace unaffected cells in tissue cultures. As these genetic and epigenetic alterations accumulate, the affected cells assume further growth advantages that allow them to invade local tissues in their host or overgrow unaffected cells in vitro
, at some point producing cells that form invasive tumors or cells that possess the capacity to form tumors when injected into animals. In the VERO cell model, we have found that miRNA down-regulation occurs during the initial stages of neoplastic transformation that are associated with immortalization and some yet-to-be defined forms of neoplastic progression that are not reflected in a currently detectable phenotype by VERO cells at p140. miRNA up-regulation occurs as the immortalized cells are undergoing further neoplastic progression through the conversion, during 116 passages (from p140 to p256) in cell culture, to the expression of a tumorigenic phenotype. We have identified miR-376a and the polycistronic miR-376abc as having a functional role in establishing the enhanced cell migration and cell invasion phenotypes, which are correlated with the expression of the VERO cell tumorigenic phenotype. Studies are underway to determine whether one or more of these over-expressed miRNAs can confer on non-tumorigenic VERO cells the capacity to form tumors in animals and consequently can be used as biomarkers for the expression of the VERO-cell tumorigenic phenotype.
The associations between neoplastic processes and miRNA dysregulation in the VERO-cell model presented in this report provide additional support for the concept that neoplastic development in vitro and in vivo have similarities. If further evidence for these similarities is forthcoming, the VERO-cell model may provide a format for the systematic comparison of neoplastic development in vivo and neoplastic transformation in vitro across tissues, organs, and species.