Dicer and Drosha have been reported to be dysregulated in TNBC [21, 23]. However, these studies used very few normal tissues in their analysis and the correlation with LN metastases was not examined. This study aimed to evaluate whether the relative expression of Dicer and Drosha was altered in TNBC and whether it was associated with clinical features and progression to LN metastases in this subtype.
Our results have shown that Drosha was expressed at significantly higher levels than Dicer in TNBC and that the expression of these two genes was highly correlated with one another, in agreement with the results of Passon et al. and Dedes et al. [21, 23]. Decreased expression of Dicer has been noted in several cancers, while increased expression has been observed in ovarian, prostate and colorectal cancer [12–14]. We observed a significant decrease in Dicer expression in breast cancer when compared to matched NAT. The proportion of cases with decreased Dicer expression in this study (72%) is similar to that previously reported by Passon et al. (61.3%) and Dedes et al. (77.7%) [21, 23]. In contrast, we observed a significant increase in Drosha expression in 78% of the tumour tissues analysed when compared to matched NAT. This confirms previous findings that Drosha is increased in TNBC . The relevance of increased Drosha expression and decreased Dicer expression in TNBC is not known at present. It is possible that Dicer expression becomes decreased as a result of increased Drosha expression to limit miRNA biogenesis, thereby reducing miRNA function in the breast.
Reduced Dicer expression as well as over-expression has previously been reported to be associated with worse prognosis in breast and colorectal cancer respectively, indicating cancer specific differences in the prognostic value of this gene [14, 19]. Although we did not have outcome data on the patients used in this study, we were able to examine whether Dicer and Drosha were differentially expressed in LN positive patients compared to LN negative patients and whether this expression was related to clinical features in the highly aggressive TNBC subtype. While Dicer was reduced in primary breast cancers of both LN positive and LN negative patients; unexpectedly, we found its expression significantly increased in LN metastases when compared to matched primary tumours. However, we saw no association of either Dicer or Drosha with clinical features including age at diagnosis, tumour size or the number of positive lymph nodes. This may be due to the small sample size used in this analysis, but TNBC represents a very specific proportion of all breast cancers and these specimens are difficult to obtain. This is the only study to date that has analysed Dicer and Drosha in TNBC samples compared to matched normal adjacent tissue and matched lymph node metastases.
A limitation of our study is that it was performed only on cDNA derived from FFPE tissues. Although FFPE tissues contain fragmented RNA, our real-time RT-PCR assays were designed with small amplicon sizes (Dicer- 65 bp, Drosha- 66 bp and β2-Microglobulin- 75 bp) to circumvent the requirement for intact RNA. Additionally, random hexamers were incorporated in the reverse-transcription procedure, which do not require the RNA to be intact, in contrast to reverse-transcription with oligo dT. Given the inconsistent correlations between these enzymes at the mRNA and protein level in the literature, and that the interpretation/quantification of Dicer and Drosha immunohistochemical staining is problematic, we did not perform immunohistochemical analyses on this cohort of tumours [21, 22]. Although, the sample size used in this study is relatively small, the tumours are homogenous with regards to size, hormone receptor status and histological grade and this is the only study that has analysed matched TNBC cases and lymph node metastases.