We have developed a workflow for analyzing deep sequencing data focusing on miRNA. This workflow has revealed several trends in miRNA expression profile for hES-MSCs. The majority of sequenced transcripts were identified by mapping to known small RNA libraries and a considerable proportion of these were miRNAs. Several studies have showed that certain miRNAs are associated with specific stem cell types and those that are regulated during stem cell differentiation. Characterizing miRNA expression profile for the purpose of extracting biological function from sequencing data can be a challenge.
Raw sequencing data of our samples showed distinct distribution patterns across a large range of magnitude for transcripts counts. The necessity of a minimum threshold of transcript count for biological significance was addressed by adaptive thresholding exploiting KS statistics amongst biological replicates giving a lower bound value for transcript counts. Currently KS statistics are used primarily for comparing distributions and have been used in areas such as image processing for examining pixel distribution. In our study, we employed KS statistics to estimate the lower bound where distributions of transcripts peaks begin to differ. Using a transcript count of 32 as a lower bound, we were able to define a threshold for miRNA expression based on transcript abundance. Further comparisons (as shown in Figure 4) of top ranked genes between our sequenced data with microarray data showed that there is a considerable degree of overlap between the two especially in the intracellular samples. miRNAs from the extra-cellular samples form a subset of those expressed in the intracellular samples. Among the most abundantly expressed transcripts across both intra and extra-cellular environment (Tables 1 & 2) the let-7 family of miRNAs was the only overexpressed family of known miRNAs.
Amongst other over expressed miRNAs, miR199b has transcription factors like SOX4 and has been shown to be involved in liver cancer and muscular dystrophy [10, 11]. miR143 has recently been shown to cooperate with miR 145 to regulate plasticity and cell fate determination of smooth muscle cells via regulation of expression of transcription factors like Kruppel like factor 4 (Klf-4), myocardin and Elk-1 .
let-7 mutant phenotype studied in C. elegans has been associated with a lack of terminal differentiation and ongoing cell proliferation, both of which are characteristics of stem cells and cancer. let-7 targets include cell cycle regulators such as CDC25A and CDK6 ; promoters of growth including RAS and c-myc [14, 15] and a number of early embryonic genes including HMGA2, Mlin-41 and IMP-1 [16, 17]. Further, let-7 also targets Dicer [18, 19] which is the protein responsible for miRNA maturation. Therefore it is possible that the let-7 family of miRNA acts as a master regulator of miRNA function. Since the let-7 family of miRNAs were abundantly expressed in MSC and given their central role in controlling cellular differentiation and miRNA regulation, we decided to focus on this family of miRNAs for further investigations.
miRNA mediated translational repression might play an important role in stem cell self-renewal. In our study, a high level of let-7 family of miRNA transcripts was predominant in both intra and extra cellular samples for our hES-MSC. This was true for other abundantly expressed miRNAs in the extra cellular space as well. We were unable to find miRNAs that were expressed only in the extra cellular space or were expressed more abundantly in the extra cellular space compared to the intra cellular space. This suggests that the process of secretion of miRNAs is most likely a passive one unlike that of proteins, where signal peptide containing proteins are transported out of the cell as soon as they are synthesized.
Moreover, the elevated presence of miRNAs in the extra cellular environment suggests a possible intercellular signaling mechanism. Recent literature has also supported that genetic exchange of mRNA and miRNA between cells can be accomplished through microvesicles transfer [7, 8]. miRNAs like miR 21 , miR 133 and miR 30  have been shown to play a role in cardiac remodelling in infracted hearts. Since miRNA can affect cardiac remodelling and these miRNAs are transported in micro vesicles by transplanted MSC (Lim SK personal communication), it is possible that transplanted MSC regulate remodelling of the surrounding myocardium through secreted miRNAs in addition to secreted cytokines and soluble factors.
HNF4A is a transcription factor that has also been shown to be essential for morphological and functional differentiation of hepatocyte and for liver morphogenesis . HNF4A is also one of the physiological factors in the liver to activate apoB gene expression at the AF-1 site . Elevated levels of apoB have been correlated with an increased risk of atherosclerosis and coronary heart disease . HNF4A is also involved in the regulation of serum lipid levels and is linked to elevated serum cholesterol and triglyceride levels in Finnish combined familial hyperlipidemia patients . However to date there is no evidence of miRNA regulation of HNF4A. None of the target prediction algorithms predict the regulation of HNF4A by let-7 family of miRNAs. There are no predicted miRNA binding sites in the untranslated or intronic regions of the HNF4A gene. However combining the networks generated by sequence alignment of expressed miRNAs and Targetscan, we predict that HNF4A is indirectly regulated by the let-7 family of miRNAs. We were able to confirm this indirectly by measuring the levels of let-7 family of miRNAs and HNF4A in undifferentiated MSC and HEPG2 cells. In undifferentiated MSC when let-7 miRNAs are highly expressed, expression of HNF4A is very low. Conversely in HEPG2 cells where a high level of HNF4A is expressed, we find very low expression of let-7 family miRNAs. Since HNF family of transcription factors have been reported to be upregulated in hepatocytes derived from adipose tissue MSC , it is possible that let-7 regulates HNF4A levels during this process. Since genes in the HNF4A alignment network also show a similar expression profile to HNF4A in HEPG2 and hES-MSC cells, it is possible that the let-7 family miRNA regulation of HNF4A is mediated through genes in this network. It is also interesting to note that there is no predicted miRNA target site in the UTR of HNF4A. Thus miRNA based regulation of HNF4A may be indirectly achieved through regulation of genes in the network that interact with HNF4A. It will be interesting to study the effect of Lin28 overexpression on differentiation of hepatocytes from hES-MSC since Lin 28 is a transcription factor that inhibits function of let-7 family miRNAs.
let-7f miRNA is known to exert pro-angiogenic effects . Human MSC conditioned medium has been shown to reduce infarct size in patients with acute myocardial infraction . Since MSC conditioned medium contains exosomes with let-7 family miRNAs and these let-7 family miRNAs may regulate HNF4A (based on our network and expression analysis), it is highly likely that MSC conditioned medium mediated reduction of infarct size is achieved by indirect regulation of HNF4A mediated by the let-7 family of miRNAs.
Thus, our study suggests the possibility of let-7 family of miRNAs indirectly regulating this particular transcription factor to achieve physiological changes. This is in line with recent paradigm where miRNAs regulate self-renewal and differentiation pathways of embryonic stem cells by forming an integral biological network with transcription factors .