miRNA are non-coding RNA that play important roles in post-transcriptional regulation. During oncogenesis, dysregulated or dysfunctional miRNA can result in increased translation of oncoprotein(s) and/or decreased translation of tumor suppressor protein(s). In the current study, we used a previously validated microarray-based methodology 
to evaluate the expression levels of 328 human miRNA in a series of extensively characterized ovarian surface epithelial tumors and normal fallopian tube samples. Several biologically important patterns and correlations emerged from our analysis of the data. Ovarian surface epithelial tumors as a group possessed a miRNA expression profile that was highly dissimilar to the miRNA expression profiles of soft tissue tumors. Given the documented roles of miRNA in cellular differentiation 
, some of these observed differences likely relate to underlying differences in differentiation into different cellular lineages, i.e. epithelial versus mesenchymal.
While a number of recent studies have characterized the expression profiles of miRNA in ovarian carcinomas including high grade serous carcinomas, none has examined the miRNA levels of high grade serous carcinomas with differing BRCA1/2 abnormalities. BRCA1 or BRCA2 abnormalities are present in most high grade serous carcinomas and lead to chromosomal instability through loss of the ability to repair double strand breaks by homologous recombination 
. In the current study, miR-29a and miR-29b were found to be more highly upregulated in high grade serous carcinomas with any demonstrable BRCA1/2 loss compared to the group lacking demonstrable BRCA1/2 abnormalities by both microarray and qRT-PCR analyses. Notably, high grade serous carcinomas carrying BRCA1 mutation were previously reported by us to possess lower PTEN mRNA levels 
, and PTEN is a among the top 20 predicted targets of miR-29a. It is plausible that the high level of miR-29a may cause increased degradation of PTEN mRNA, resulting in the decreased level of PTEN mRNA observed in this group of tumors. It is important to note that only a small number of BRCA2 mutation-carrying tumors was included in this series and our analysis will likely not reveal subtle differences in this group compared to other groups. Overall, very few differences were observed in the comparisons between high grade serous carcinomas possessing mutation in BRCA1, mutation in BRCA2, epigenetic silencing of BRCA1, and no demonstrable loss in BRCA1. It is possible that all of the tumors reach the same endpoint during oncogenesis (i.e. chromosomal instability), despite the presence of different underlying molecular abnormalities. The near identical miRNA expression profiles between the genotypic subgroups may then reflect this end state.
All of the ovarian carcinoma miRNA profiling analysis reported to date have used non-neoplastic ovarian tissue samples and/or cultured ovarian surface epithelial cells for comparison as normal tissue counterpart 
. There is increasing evidence to suggest most high-grade serous carcinomas, including those with bulky unilateral or bilateral ovarian disease, do in fact arise from the tubal fimbriae or secondary mullerian epithelial inclusions in the ovaries 
. In patients with familial BRCA1/2 mutation, intraepithelial carcinoma at the tubal fimbriae represents the most common earliest evidence of neoplasm in prophylactic bilateral salpingooophrectomy specimens 
, whereas analogous precursor lesion on ovarian surface has not been consistently demonstrated despite careful examination. In sporadic cases of ovarian serous carcinomas, there is frequently evidence of similar appearing intraepithelial carcinoma involving the tubal fimbriae, though in these cases (and the cases included in this study), one cannot be certain whether the tubal neoplasm represents the primary site of the disease or site of secondary involvement 
. Furthermore, the immunophenotypic profile of high grade ovarian serous carcinomas appears to more closely resemble that of normal tubal epithelium than ovarian surface epithelium 
. Given that mullerian type epithelium from the tubal fimbriae or ovarian mullerian inclusions is now regarded as the more likely epithelium of origin for high grade ovarian serous carcinomas, we believe that the use of tubal fimbriae as normal tissue counterpart for comparison is most appropriate. Moreover, high grade serous carcinomas generally are composed predominantly of neoplastic epithelium with relatively little stroma. Comparison to normal ovarian tissue, which contains an overwhelming predominance of stromal tissue is likely not an ideal comparison. The use of cultured ovarian surface epithelial cells for comparison may also be problematic as it would require an assumption that the miRNA expression profiles of normal ovarian surface epithelial cells are unaltered in culture. Although only three samples of normal fallopian tube tissue were analyzed, they showed highly uniform expression profiles, leading us to conclude that analysis of additional samples would be non-contributory.
In the current study a total of 28 differentially expressed miRNA were identified with 19 miRNA being upregulated and 9 miRNA being downregulated in high grade serous carcinoma versus normal fallopian tubes by microarray analysis. Selected dysregulated miRNA identified by microarray analysis were also confirmed to be dysregulated by qRT-PCR analysis. Nine of the 19 upregulated miRNA and all 9 downregulated miRNA were reported to be dysregulated in the same manner in earlier analyses 
. miR-145 and/or miR-143, the most significantly downregulated miRNA found in the current analysis were also frequently observed to be downregulated in carcinomas of prostate, colon, and breast, relative to the respective normal tissue 
and in B-cell malignancies 
. In addition, low grade serous carcinomas and serous borderline tumor also demonstrated similarly low levels of miR-145 and miR-143 in the current series. It is important to note that when the precursor or mature form of miR-145 were transfected into colon cancer and Burkitt lymphoma cell lines, dose-dependent growth inhibition was observed 
. miR-145 and miR-143 in these instances appear to function as a tumor suppressor and similar tumor suppressor roles for these miRNA may be speculated in the case of high grade serous carcinomas and serous borderline tumors/low grade serous carcinomas. The absence of upregulated miRNA in high grade serous versus serous borderline tumor/low grade serous carcinoma parallel the findings made when mRNA expression profiles were compared between these groups 
. A possible explanation includes the rapid divergence of chromosomally unstable high grade serous carcinomas after the early events during oncogenesis (typically loss of BRCA1 or BRCA2 and p53 loss) that results in heterogeneity within this group of tumors, such that no consistent pattern of miRNA upregulation emerged across the group as a whole, compared to the low grade tumors.
Prognostically, the level of miR-422b and the level of miR-34c were both found to be positive predictors of patient survival by microarray analysis. miR-422b was among the 19 miRNA that were found to be significantly upregulated in high grade serous carcinomas compared to normal fallopian tubes. Little is known about the functional importance of miR-422b at the present. miR-34c was among the 9 miRNA that were found to be downregulated in high grade serous carcinomas compared to fallopian tubes and high grade serous carcinomas with greater downregulation of miR-34c were associated with more aggressive clinical behavior. These findings point to a tumor suppressor role for miR-34c in high grade serous carcinomas. Functionally, miR-34c expression is known to be regulated by p53 and low level of miR-34c was observed in p53 deficient ovarian carcinoma cell lines 
. Mutation of p53 is common in high grade ovarian serous carcinomas, occurring in about 80% of cases based on the literature 
and in the current series (85%), while low grade ovarian serous carcinomas and serous borderline tumors typically do not possess p53 mutation 
. However, the lack of significant association between miR-34c downregulation and p53 mutation in our current series indicate that additional influences are likely important in regulating miR-34c expression in high grade serous carcinomas.
With regard to the prognostic significance of miR-34c, analysis based on the microarray data demonstrated significant association between low-level miR-34c expression and decreased disease-specific survival, while analysis based on qRT-PCR data showed a similar trend but did not reach statistical significance with a p-value of 0.06. Because the input miRNA samples for both microarray and qRT-PCR analysis were derived from the same extraction batch, the observed discrepancies likely reflect differences in methodology. A number of studies/reviews have addressed the issues of reproducibility between microarray and qRT-PCR quantification 
; while there is in general a good correlation between microarray and qRT-PCR data, there is some variability and the correlation is not perfect. In this study, the downregulation of miR-34c in high grade serous carcinomas shown by microarray analysis was confirmed by qRT-PCR analysis but inter-method variability appears to have affected the reproducibility of demonstration of a statistically significant miR-34c prognostic association, indicating borderline prognostic significance, based on this cohort. Further study will be needed to verify the prognostic significance of miR-34c downregulation in high grade serous carcinoma.
In summary, miRNA expression analysis of the current series of ovarian serous tumors and normal fallopian tubes has provided us with several important insights. High grade serous carcinomas of different BRCA1/2 status exhibit highly similar miRNA expression patterns, with very few differences present. Comparison between high grade serous carcinomas and normal tubal epithelium revealed several dysregulated miRNA, including miR-34c and miR-422b, the levels of which are both associated with prognostic importance in our current series. This improved understanding of the pattern and the significance of miRNA dysregulation in high grade serous carcinoma will allow us to better understand the oncobiology of this aggressive disease.