The Study On The Detection Of MicroRNA Expression Profiles Of Human Colorectal Cancer And The Functions Of MiR-27b

BackgroundColorectal cancer (CRC) is one of the most prevalent cancers globally and is one of the leading causes of cancer-related deaths due to therapy resistance and metastasis. CSCs are a subpopulation of cells within a tumor that possess the capacity to self-renew and produce heterogeneous lineages of cancer cells that comprise the tumor. CSCs can drive tumor growth and recurrence and are resistant to many current anticancer treatments. A number of surface markers can be used for CSCs sorting, including CD24, CD44, CD166and CD133.microRNA (miRNA) are18-25nucleotides non-coding endogenous RNA that negatively regulate gene expression at the post transcriptional level by inhibiting mRNA translation or promoting mRNA degradation. miRNAs are deemed to have widespread regulatory activity in a broad range of developmental processes and are implicated in diverse diseases, including cancer. miRNA can act as either an oncogene or a tumor suppressor in cancer, as evidenced by miR-155activate while let-7repress cancer related cell signaling pathway. miR-200suppress cancer invasion and metastasis via regulate epithelial-to-mesenchymal transition (EMT). miR-27b is located on chromosome9and has been shown to function as a tumor suppressor in neuroblastoma via targeting the PPARγ. It has also been reported that miR-27b can act as an angiogenic switch by promoting endothelial tip cell fate and sprouting.CSCs are both structurally and functionally distinct from the other cells within a tumor mass, and are regarded as playing central role in tumor progression. We hypothesize that molecular differences between CSCs and differentiated cancer cells may identify a key molecule in tumor growth and progression, and in this study investigated differences in miRNA expression between CSCs and differentiated CRC cells using miRNA microarrays. We identified27differentially regulated miRNAs and confirmed that miR-27b act as tumor suppressor in CRC.MethodsIn order to screen the key molecule involving in CRC, we assessed miRNA expression profiles in CD133+and CD133-cells using miRNA microarrays. miR-27b was the only miRNA identified repeatedly in these experiments. Real time PCR was used to measured miR-27b expression in CRC tissue samples. The biological functions of miR-27b in CRC were determined by proliferation and colony formation in vitro and tumorigenesis in vivo. We further investigated the anti-tumor effect of miR-27b in vivo in a human CRC-bearing mouse model through hematoxylin and eosin staining and immunofluorescence assays. Several experiments including dual luciferase reporter assay were used to analyze the target of miR-27b. And experiments including methylation-specific polymerase chain reactions (MSP) were used to analyze the mechanisms involved in the regulation of transcription.Results1. The miRNA expression profiles differences between CSCs and differentiated CRC cells.We observed CSCs properties in CD133+CRC cells and assessed miRNA expression profiles in CD133+and CD133-cells to identify miRNAs involved in tumor progression. Microarray analysis detected eight up-regulated and19down-regulated miRNAs in CD133+cells. When these results were combined with previous miRNA microarray data, only miR-27b expression differed. The qPCR data of80paired paraffin-embedded CRC and adjacent normal tissues showed that miR-27b expression decreased in60%CRC compared to15%elevated.2. miR-27b inhibits tumor growth and angiogenesis in CRC.We established both miR-27b and anti-miR-27b SW620stable cell lines to study the biological functions of miR-27b. We found that overexpression of miR-27b repressed cell proliferation. A soft-agar colony assay indicated that increased miR-27b expression significantly prohibited colony. In a tumorigenesis assay, we found that miR-27b could strong suppress tumor growth. We further investigated the anti-tumor effect of miR-27b in vivo in a human CRC-bearing mouse model. All of these findings support a tumor suppressive role for miR-27b in CRC and suggest its potential as an anti-CRC drug.3. VEGFC is a novel target of miR-27b in CRC.miRNA function primarily as mediators of gene silencing. We identified VEGFC mRNA3’UTR contains highly conserved miR-27b binding sites that are responsive to miR-27b in a dual luciferase reporter assay. VEGFC protein levels were also decreased in cells and culture medium upon transfection with an miR-27b mimic. VEGFC-knockdown repressed cell proliferation compared to the NC cells, significantly inhibited colony formation and reduced tumor growth. Collectively, these observations strongly suggest that VEGFC is a functional downstream target of miR-27b in CRC.4. DNA hypermethylation reduces miR-27b expression.We found that the methyltransferase inhibitor5-aza-dC (5AZA) can markedly elevated miR-27b expression in CRC. The predicted promoter site of miR-27b in chromosome9was cloned into a luciferase vector and verified using luciferase assays. MSP results indicated miR-27b CpG island hypermethylation in several CRC cell lines. These results suggest that DNA hypermethylation plays an important role in regulation of miR-27b. Indeed, miR-27b may be modulated by transcription factors such as Evi-1and c-Rel. Both transcriptional and epigenetic pathways regulate gene expression.ConclusionWe demonstrated that miR-27b could significantly repress self-renewal in vitro and tumorigenicity in vivo. Moreover, we identified VEGFC as a functional downstream target of miR-27b using several methods. To our knowledge, this is the first study to report the specific function and a novel functional target of miR-27b in CRC. Malignant cells are dependent on aberrant miRNA expression; these small RNAs provide important opportunities for the development of future miRNA-based therapies. miRNA-mediated promoter hypermethylation has been identified in the majority of tumors. We also found DNA hypermethylation reduced miR-27b expression in CRC. These results not only allow for a better understanding of the mechanisms regulating CRC cells but also facilitate the gradual development of more effective cancer therapies.