Regulation of microRNA biosynthesis and expression in 2102Ep embryonal carcinoma stem cells is mirrored in ovarian serous adenocarcinoma patients

Abstract

Background: Tumours with high proportions of differentiated cells are considered to be of a lower grade to those containing high proportions of undifferentiated cells. This property may be linked to the differentiation properties of stem cell-like populations within malignancies. We aim to identify molecular mechanism associated with the generation of tumours with differing grades from malignant stem cell populations with different differentiation potentials. In this study we assessed microRNA (miRNA) regulation in two populations of malignant Embryonal Carcinoma (EC) stem cell, which differentiate (NTera2) or remain undifferentiated (2102Ep) during tumourigenesis, and compared this to miRNA regulation in ovarian serous carcinoma (OSC) patient samples.

Methods: miRNA expression was assessed in NTera2 and 2102Ep cells in the undifferentiated and differentiated states and compared to that of OSC samples using miRNA qPCR.

Results: Our analysis reveals a substantial overlap between miRNA regulation in 2102Ep cells and OSC samples in terms of miRNA biosynthesis and expression of mature miRNAs, particularly those of the miR-17/92 family and clustering to chromosomes 14 and 19. In the undifferentiated state 2102Ep cells expressed mature miRNAs at up to 15,000 fold increased levels despite decreased expression of miRNA biosynthesis genes Drosha and Dicer. 2102Ep cells avoid differentiation, which we show is associated with consistent levels of expression of miRNA biosynthesis genes and mature miRNAs while expression of miRNAs clustering to chromosomes 14 and 19 is deemphasised. OSC patient samples displayed decreased expression of miRNA biosynthesis genes, decreased expression of mature miRNAs and prominent clustering to chromosome 14 but not 19. This indicates that miRNA biosynthesis and levels of miRNA expression, particularly from chromosome 14, are tightly regulated both in progenitor cells and in tumour samples.

Conclusion: miRNA biosynthesis and expression of mature miRNAs, particularly the miR-17/92 family and those clustering to chromosomes 14 and 19, are highly regulated in both progenitor cells and tumour samples. Strikingly, 2102Ep cells are not simply malfunctioning but respond to differentiation specifically, a mechanism that is highly relevant to OSC samples. Our identification and future manipulation of these miRNAs may facilitate generation of lower grade malignancies from these high-grade cells.

Figure 1

Expression of miRNA biosynthesis genes and mature miRNAs in undifferentiated NTera2 and 2102Ep EC cells. The relative expression levels of miRNA biosynthesis genes Drosha, Dicer and eIF6 in undifferentiated 2102Ep and NTera2 EC cells is shown. In each case, data represents expression in 2102Ep cells compared to NTera2. While the expression of eIF6 is almost identical, that of Dicer was downregulated slightly (4.3 fold) and of Drosha was substantially lower (74.5 fold) in undifferentiated 2102Ep EC cells compared to undifferentiated NTera2 EC cells.

Figure 2

The comparative expression levels of miRNAs in undifferentiated 2102Ep cells compared to undifferentiated NTera2 cells. The relative expression of mature miRNAs in undifferentiated 2102Ep cells compared to Ntera2 cells is shown. Despite the qualitative similarities of the miRNA profiles expressed, our data indicated that the majority of miRNAs are expressed at higher levels in 2102Ep cells compared to NTera2.

Figure 3

The comparative expression levels of 17 of the highest upregulated and 18 of the highest downregulated miRNAs in undifferentiated 2102Ep cells compared to undifferentiated NTera2 cells. The relative expression of the top mature miRNAs in undifferentiated 2102Ep cells compared to Ntera2 cells is shown These results demonstrate a substantial bias towards increased expression of mature miRNAs in 2102Ep cells. The miRNAs and levels of expression are listed in Additional file 1.

Figure 4

Differentiation status and miRNA biosynthesis gene expression in differentiated NTera2 and 2102Ep cells. The expression of markers of pluripotency (Oct4 and Nanog) and of endoderm (Afp), ectoderm (Ncam1) and mesoderm (Eno3) differentiation is shown. In each case, data represents changes in expression in the differentiate state compared to undifferentiated state. In NTera2 cells, differentiation status is confirmed by decreases in expression of Oct4 and Nanog and increases in expression of Afp, Ncam1 and Eno3. 2102Ep cells alter expression of these genes less than two-fold, confirming nullipotency.

Figure 5

The expression levels of the miRNA biosynthesis genes Drosha, Dicer and eIF6 in differentiated cells compared to undifferentiated cells. Expression of Drosha decreases 65 fold while that of Dicer decreases 2.6 fold and of eIF6 decreased less than 2 fold upon differentiation of NTera2 cells. In contrast, the level of differential expression of each gene is within 1.0 fold in both undifferentiated and differentiated 2102Ep cells. Maintained expression of miRNA biosynthesis genes, therefore, is associated with the 2102Ep nullipotent phenotype.

Figure 6

Comparison of the expression levels of miRNAs altered in differentiated NTera2 and 2102Ep cells: Group 1 miRNAs. miRNAs were grouped according to their expression patterns upon differentiation of NTera2 and 2102Ep cells. 15 Group 1 miRNAs are similarly altered in both cell types. We propose that Group 1 miRNAs likely act upstream of any lesion in the 2102Ep differentiation mechanism. miRNAs in each group are listed in Additional file 3.

Figure 7

Comparison of the expression levels of miRNAs altered in differentiated NTera2 and 2102Ep cells: Group 2 miRNAs. miRNAs were grouped according to their expression patterns upon differentiation of NTera2 and 2102Ep cells. 85 Group 2 miRNAs are altered in NTera2 cells but unaltered in 2102Ep cells. We propose that Group 2 miRNAs likely act downstream of any lesion in the 2102Ep differentiation mechanism. miRNAs in each group are listed in Additional file 3.

Figure 8

Comparison of the expression levels of miRNAs altered in differentiated NTera2 and 2102Ep cells: Group 3 miRNAs. miRNAs were grouped according to their expression patterns upon differentiation of NTera2 and 2102Ep cells. 13 Groups 3 miRNAs are altered in an opposite fashion in each cell line. Group 3 miRNAs represent a 2102Ep-specific response to differentiation that is independent of NTera2 mechanisms. miRNAs in each group are listed in Additional file 3.

Figure 9

Comparison of the expression levels of miRNAs altered in differentiated NTera2 and 2102Ep cells: Group 4 miRNAs. 29 Group 4 miRNAs are altered in RA-treated 2102Ep cells but unaltered in differentiated Ntera2 cells. Group 4 miRNAs represent a 2102Ep-specific response to differentiation that is independent of Ntera2 mechanisms. miRNAs in each group are listed in Additional file 3.

Figure 10

Comparison of miRNA expression in OSC patient samples. The venn diagram shows the number of differentially expressed miRNAs identified in the current study and the number of miRNAs identified in six previous studies [26,36-40]. These overlap substantially.

Figure 11

Validation of OSC patient sample miRNA data. Validation of training set: Log10 RQ values for miR-429, mir-141 and let-7f in both the pilot study (RQp) and the larger independent FFPE cohort (RQv). There was modest correlation between both sets of results (r = 0.50).

Figure 12

Top 10 miRNAs up- and downregulated in OSC patient samples. Barchart of the mean logarithmic fold change (RQ) of the top ten up and downregulated miRNAs in OSC samples relative to normal ovary.

Figure 13

Comparison of miRNA expression in EC cells and OSC patient samples. Hierarchal clustering of the differentially expressed miRNAs in EC cells and ovarian tumour samples is shown. Hierarchal clustering was performed on miRNAs altered upon differentiation of nullipotent (2102Ep) and pluripotent (NTera2) EC cells and in ovarian tumour samples (TS) compared to normal tissue. Clustering indicates that alterations in miRNA expression during differentiation of the nullipotent EC cell type are more similar to tumour samples than alterations in miRNA expression during differentiation of the pluripotent cells.