We employed a novel microdissection strategy to study microRNA levels in ESCC patient-matched normal squamous epithelium and tumor cells (). By specifically dissecting two distinct epithelial populations, differentiated and basal, we aimed to more closely define microRNA expression levels in the normal tissue compartments as compared to bulk specimen analysis. Previously, it has been shown that LCM procured material displays a significantly different expression profile as compared to un-dissected whole tissue specimens from patients with either colorectal cancer [48
] or Barrett's esophagus [49
]. Thus, the present analysis provided the first precise measurement of microRNA levels in individual cell populations and allowed us to explore both normal-tumor and normal basal-normal differentiated cell differences.
At the outset of the study, identification of a high-throughput yet robust method to measure microRNA expression from a relatively small number of dissected cells was challenging. We first excluded methods that required large amounts of total RNA/microRNA input such as hybridization-based microarrays. We then reviewed the technical aspects of available assays and selected the ABI 7500 RT-qPCR platform (ABI 7500) approach with full control of the 96-well plate design, employing a pre-amplification RT-qPCR protocol to enhance the detection sensitivity as well as maximize the number of microRNAs investigated.
Also of importance in the study were the development of a consistent microdissection protocol, the use of uniform inputs of total RNA, and the selection of control microRNAs for normalization [16
]. To date, there are few recognized microRNAs that are expressed evenly across all tissue and cell types, thus several normalization strategies have been suggested, including the use of individual microRNAs such as miR-191 or miR-103 [50
], or the mean expression value of all expressed microRNAs in a given sample [51
]. However, individual microRNAs have not been widely applied due to the lack of extensive validation, and generating a mean expression value from a large number of microRNAs is not easily accomplished using laser microdissected samples. Therefore, we averaged the RT-qPCR data for three commonly used microRNA normalizers, U6, RNU44, and RNU48 with the assumption that they are biologically consistent across the samples studied.
The list of potential microRNA biomarkers that distinguish normal and diseased esophagus is beginning to accumulate [6
]. To further understand the role of these molecules in esophageal development and the formation of ESCC, we studied the expression levels of 18 microRNA known to be associated with ESCC within microdissected subpopulations of normal squamous epithelial cells and matched tumor. Five of the 18 microRNAs demonstrated a consistent change in expression (). Only miR-145 was differentially expressed (down-regulated) in the NB vs. ND comparison alone, suggesting it may play a role in the normal differentiation process. Previous studies have indicated that miR-145 may inhibit cell proliferation in ESCC and act as a tumor suppressor [13
]. However, our data indicate that miR-145 expression was down-regulated in the NB dissections and not in the tumor population (). Recently, Shi et al
presented data that indicates an association between miR-145 and the insulin receptor substrate-1 (IRS1) in colon cancer cell lines [54
]. In their study, over-expression of miR-145 prevented cell proliferation via IRS1 down-regulation [54
]. Our data indicate that in the NB vs. ND comparison, the opposite is occurring as IRS1 is up-regulated and miR-145 is down-regulated (). Therefore, the normal basal epithelial layer may express cell proliferation factors with the down-regulation of miR-145.
Four other microRNAs (miR-25, miR-106b, miR-21, and miR-203) were differentially expressed in one or more tumor comparisons (). miR-25, miR-106b and miR-21 were up-regulated in the T vs. NB and T vs. ND comparisons, while miR-203 was down-regulated in the T vs. ND comparison. These data implicate miR-25, miR-21, miR-106b, and miR-203 as involved in ESCC development.
Consistent with these findings, other studies have shown that miR-25 is up-regulated in tumors, including; lung cancer, hepatocellular carcinoma, pediatric brain tumors, acute myeloid leukemia, prostate cancer, and gastric cancer [55
]. Interestingly, a recent study by Kuhn et al
correlated the increased expression of the tumor suppressor, KLF4, with inhibition of miR-25 in airway smooth muscle cells of the lung [44
]. A similar relationship was observed in ESCC as KLF4 was down-regulated and miR-25 up-regulated in both the T vs. NB and T vs. ND comparisons ().
Furthermore, miR-25 and miR-106b have been shown to be overexpressed as a polycistron in a number of tumor tissues [12
]. Both miR-25 and miR-106b exhibited increased expression in the ESCC tumor samples studied, although at different levels (). As expected, the miR-106b-25 host gene, minichromosome maintenance protein 7 (MCM7) was also upregulated in the T vs. ND comparison of our mRNA study () [22
]. The miR-106b-25 polycistron has been shown to play a key role in the development of esophageal ade-nocarcinoma via degradation of the p21 mRNA [12
]. However, the predicted down-regulation of p21 was not observed in our mRNA study, indicating a potentially novel role of the miR-106b-25 polycistron in ESCC.
The widely studied microRNA, miR-21, is over-expressed in the majority of tumors [23
]. ESCC is no exception and our study showed this over-expression in the T vs. NB and T vs. ND comparisons (). Interestingly, the analysis of microRNA-mRNA associations indicates that several mRNA are uniquely expressed in either the normal basal (SPRY1) or normal differentiated (DNMT1, JAG1, PTEN, and LRRFIP1) epithelial layers when compared to tumor (). The Sprouty1 (SPRY1) and miR-21 relationship has been demonstrated in cardiovascular disease, but this is the first report of a possible association in ESCC [28
Unlike miR-25, miR-106b, and miR-21, miR-203 is down-regulated in the majority of tumors [6
]. The present study is consistent with those findings, as miR-203 was down-regulated by greater than seventeen fold in the T vs. ND comparison and more than 5 fold in the NB vs. ND comparison (). The effect of miR-203 down-regulation in the NB vs. ND comparison is unclear as no correlating mRNA were found to be differentially expressed in the related mRNA dataset. However, two miR -203-associated mRNA, JunB and ABCE1, were differentially expressed in the T vs. ND comparison (). A direct relationship between miR-203 and ABCE1, a known RNase L inhibitor [84
] is possible since they show opposite directionality in their expression changes and related microRNAs and mRNA are typically inversely correlated.
In summary, the current study presents a technical advance for quantitatively measuring microRNA levels in defined cell populations using laser capture microdissection, and provides the first analysis of tumor-associated microRNA levels in sub-populations of normal squamous epithelium and matched esophageal tumors. The investigation into the correlation of consistently differentiated microRNA and their related mRNA identified relationships unique to the microdissected subpopulations of ESCC. miR-25, miR-106b, miR-21, miR-203 and miR-145 were identified as key dysregulated microRNAs in ESCC and warrant additional study in this cancer type.