Screening Of Small Intestinal Epithelial Cells Differentiation-related MicroRNAs

In the small intestine, the epithelial lining invaginates to form numerous crypts and larger, finger-shaped projections called villi. In the colon, there are again numerous crypts, with regional variations in size and cell types. The intestinal epithelial system is a paradigm for the production of distinct cell lineages from multipotent progenitors. The molecular mechanism by which the continuous generation and differentiation of intestinal epithelial cells has been well studied, as it would be involved in the intestinal diseases such as cancer. Several pathways including Notch and Wnt signaling pathways were identified to play critical roles in the development of intestinal epithelium, while clearly indicating that the committed differentiation of intestinal epithelial cells is under control of dynamic gene regulatory networks.MicroRNAs represent a class of small (,20–25 nucleotides), noncoding RNAs that function as post-transcriptional regulators of gene expression in various species. miRNAs recognize their target(s) with the partially complementary sequences and repress their translation or modify their stability. microRNAs are key regulators of many cellular events, including the balance between proliferation and differentiation during tumorigenesis and organ development. In this study, we aim to screen the differentiation-related miRNA in small intestinal epithelium based an improved method of intestinal epithelial isolation.Objectives:1. To develop the method for mouse intestinal epithelial cell (SIEC) isolation.2. To obtain SIEC differentiation-related miRNAs by using microarray and northern blot.Materials and Methods:1. the small intestinal villus units, crypt units and colon crypt units were isolated using chelating agents at low temperature. The well-preserved cell-cell junctions and clear dissociation of epithelial cells with base membrane were determined by HE sections. The contamination of hematopoietic system derived cells were identified by CD45 immunostaining.2. Screening for SIEC differentiation-related miRNAsAfter isolating small intestinal villus and crypt cells, we extracted total RNA. Then, 18-100nt small RNAs were purified. After fluorescence–labled, the small RNAs were hybridized with microarrays to get miRNAs expression profiles of villus and crypt cells, respectively. As a result, SIEC differentiation-related miRNAs were then obtained by comparison with the other.3. Verifying the microarray results by Northern blotExpression of six differentially expressed miRNAs was confirmed by Northern blot. Results:1. The highly purified small intestinal villus units, crypt units and colonic crypt units were obtained.The small intestinal villus epithelial cells, crypt epithelial cells and colonic crypt epithelial cells with intact anatomic arrangement were isolated and purified at low temperature. The integrality of lamina propria was maintained during this process. And tiny CD45+ cell, contaminated cells from lamina propria in epithelial enrichments is visible.2. Screening of SIEC-development correlative miRNAs Compared with crypt epithelial cells, 35 miRNAs differentially expressed in Villus cells were screened, including 16 upexpressed miRNAs (Fold change >2.0 ) and 19 down regulated miRNA (Fold change <0.7 ). Then the hierarchical tree was generated by cluster software.3. Verification of microarray results by Northern blot. The integrality of RNA samples from small intestine tract, colon tract, deepithialized intestinal tissue, villus epithelial cells, crypt epithelial cells and colonic epithelial cells were maintained during the isolation process.Six miRNAs differentially expressed in villus and crypt cells were selected for Northern blot verification. 3 of 6 real time RT-PCR data were concordant with the microarray data, i.e., villus cells overexpressed hsa-miR-143, hsa-miR-31, and underexpressed has-miR19b.Conclusion:1. We successfully improved the method for isolating small intestinal villus and crypt epithelial cells and colonic crypt epithelial cells.2. We obtained a series of miRNAs related to the differentiation of intestinal epithelial cells.3. We identified the expression pattern of miR143, miR31, miR22, miR183, miR17-5p and miR19b in small intestine and colon.