MiRNA Expression Profiling Studies Of Stem Cells In Liver Cancer And Liver Cancer Cells Different

Background&ObjectiveThe Cancer Stem Cell Hypothesis postulates that there is a rare,phenotypically distinct subset of cells among tumor cells, which has thecapacity to form new tumors and has character of stem cells. Thissubgroup is considered as cancer stem cells and the roots of maintainingtumor growth, metastasis and recurrence. So, the eradication of cancerstem cells would be a new way of cancer therapy and prevention ofcancer recurrence. Recently, some researchers found that miRNAsextensively regulate the proliferation, differentiation, apoptosis of cells inbody. Moreover, abnormal expression of miRNA could attribute to tumoroccurrence and development. The aim of our study was to exploredifferences of miRNA expression between cancer cells andcorrespondingly derived cancer stem cells with gene chip technology.Using non-adherent culture system，we enriched liver cancer stem cellsfrom liver cancer cell lines, and based on the results of gene chip,we willexplore the role and regulatory mechanism of miRNA in liver cancerstem cell differentiation in future study.Methods1. Liver cancer stem-like cells were enriched and expanded thenon-adherent culture system. Then they were identified by several functional assays, including the limited dilution assay, toluidine bluestaining, colony formation assay, detection of stem cell markers byflow cytometry and tumorigenecity assay in nude Mice.2. Total RNA was extracted from liver cancer stem cells and cancercells. The quality of RNA was detected by using UVspectrophotometer and agarose gel electrophoresis. RNA hybridizeswith gene expression profile of Exiqon miRNA after fluorescentlabeled. Then, expression differences of miRNA were analyzed fromthe scanned microarray.Results1. The liver cancer stem-like cells were enriched from hepatoma cell lineHuh7in the non-adherent culture system and expanded in vitro. Singlecell in limited dilution assay confirmed that cancer stem cell could beself-renewal, instead of multiple cell aggregation.2. The ability of in vitro colony-forming of cancer stem cells derivedfrom huh7cells was stronger than Huh7cells.3. Cancer stem cells have more toluidine blue pale cell subpopulationsthan huh7cells.4. CD326marker expression of cancer stem cell derived from huh7cellswas significantly increased compared with the almost no expression inadhesive parental cancer cells. 5. Liver cancer stem cells and adhesive monolayer liver cancer cells wereinoculated into immunodeficiency mice shows that5000liver cancerstem cells generated tumors compared a minimum100,000liver cancercells required for generate in immunodeficiency mice.6. The total RNA of Huh7cells and spherical cells were extracted byusing Trizol method. The OD280/260value of RNA was respectively2.04and2.07by using ultraviolet spectroscopy. Agarosegelelectrophoresis of the28s,18S rRNA was with bright and clear, thebrightness ratio is about2:1, in line with chip testing requirements.7. We use image scanning analysis with Gene Pix4000B whenmiRNA microarray was hybridized to RNA sample after marked withHy3^TM. Once image scanning signals change into digital information,Data analysis would be proceeded by software of Genepix Pro6.0.8. MiRNA microarray analysis shows that there are110up-regulated and283down-regulated miRNAs among total383differential expressedmiRNAs. Moreover，Some miRNAs are associated with liver cancer stemcell differentiation.Conclusion：Our study here found that a subset of tumor stem cells derived fromHuh7liver cancer cells possess the character of both tumor cells and stemcells. We further explored that the microRNA expression profile of Huh7 cancer stem cells was different from the Huh7cancer cells by miRNAarray analysis. Many miRNA expressed much highly in cancer stem cellsthan those in cancer cells while some miRNA expressed lower in cancerstem cells than those in cancer cells. Our study provides new clues toidentify the role of these miRNA in the development and differentiationof cancer stem cells.