UHRF1 is regulated by miR-9 in colorectal cancer Feng Yan Department of Clinical Laboratory, Nanjing Medical University Cancer Hospital, Nanjing, 210009, China Email: yanfeng1895@163.com

SEER Stat Fact Sheets: Colon and Rectum Cancer http://seer.cancer.gov/statfacts/html/stomach.html

Expected Cancer Incidence and Mortality in 2015, China

UHRF1(Ubiquitin-like with PHD and ring finger domains) UHRF1 has been identified as a novel oncogene. known as ICBP90 or Np95,located on the short (p) arm of chromosome 19 at position 13.3 (Gene ID: 29128). in vertebrates, the SRA domain is restricted to the UHRF family. Biochemical Pharmacology 86 (2013)1643–1649

UHRF1 and cancer Biochemical Pharmacology 86 (2013)1643–1649

miR-9 and CRC In CRC, miR-9 serves as a tumor suppressor miR-9 is associated with the development of CRC and could be utilized for CRC molecular targeted therapies

Samples collection Cells: HT29 and HCT116 pairs of CRC tissues: 38 cancer tissues and adjacent normal tissues average age of 69 years (ranging from 45 to 85 years)

Strong staining was found mainly in nucleus UHRF1 expression in tissues Strong staining was found mainly in nucleus 36.8%(14/38) , positive 23.6%(9/38), strong expression 13.2%(5/38), moderate expression

in situ hybridization results MiR-9 expression is downregulated in CRC tissues in situ hybridization results miR-9 reduced in CRC tissues U6 snRNA expression levels remained unchanged

the expression levels of miR-9 RT-PCR results significantly lower in the CRC specimens than those in the adjacent non-cancerous specimens

The correlation between the miR-9 expression and clinicopathologic features of CRC Characteristics Total miR-9 expression p High Low Age 0.75 ≤69 18 5 13 ＞69 20 7 Gender Male 11 9 Female T stage <0.01 1-2 3 1 2 29 15 14 4 6 Regional lymph nodes 0.19 23 Distant metastasis 35 17 Dukes’ stage 10

Kaplan–Meier survival analysis low miR-9 expression associated with a poor clinical outcome Multivariate survival analysis：miR-9 expression level and distant metastasis were independent prognostic factors for outcome in patients with CRC.

Establish stable HCT-116 and HT29 cell lines expressing lenti-miR-9 After transfection for 72 h in cells, the expression levels of miR-9 were substantially increased about 57-fold and 45-fold, respectively, compared to pre-miR-control.

cell proliferation assays over expression of miR-9 can significantly inhibit CRC cell proliferation (P < 0.05)

colony formation assay colony formation was significantly reduced in the miR-9 upregulated cells

miR-9 promotes CRC cells apoptosis in vitro After transfection with miR-9 for 72 h, the proportion of apoptotic cells was significantly increased in HCT-116 and HT29

UHRF1 is a direct target of miR-9 the expression of UHRF1 significantly decreased in stable Pre-miR-9-transfection cells, whereas UHRF1 was upregulated after inhibition of miR-9 in both cell lines

the relationship between miR-9 and UHRF1 miR-9 could target 3’UTR region of UHRF1. To validate the prediction, the wild-type or mutation 3’UTRs of UHRF1 was cloned into luciferase reporter vector

the relationship between miR-9 and UHRF1 Dual-Luciferase reporter assay showed that compared to miRNA cont, miR-9 leaded to a significant relative luciferase activity reduction in the wild-type UHRF1 3’UTR plasmid. While the luciferase activity was not reduced in the 3’UTR with mutant binding sites

RT-PCR to analyze the expression of UHRF1 UHRF1 mRNA expression (r=-0.606, p=0.0004) Spearman’s correlated

Summary 1.UHRF1 is regulated by miR-9 in CRC 2.miR-9 was underexpressed in CRC tissues 3. miR-9 was inversely correlated with UHRF1 4. overexpression of miR-9 could suppress the expression of UHRF1. 5. Cell proliferation was also suppressed in pre-miR-9 transfected cells. 6. restoration of miR-9 expression significantly induced cell apoptosis. 7.more researches on a larger population are needed to confirm these results

Conclusion UHRF1 is upregulated in CRC. miR-9 could function as a tumor-suppressive miRNA by repressing UHRF1 expression. not only increase our understanding of CRC tumorigenesis, but also allow the development of a novel therapeutic strategy based on upregulation of miR-9. Conclusion