1. UHRF1 is regulated by miR-9 in colorectal cancer
Feng Yan
Department of Clinical Laboratory,
Nanjing Medical University Cancer Hospital,
Nanjing, , China

2. SEER Stat Fact Sheets: Colon and Rectum Cancer

3. Expected Cancer Incidence and Mortality in 2015, China

4. 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

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

6. 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

7. 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)

8. 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

9. 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

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

11. 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

12. 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.

13. 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.

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

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

16. 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

17. 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

18. 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

19. 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

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

21. 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

22. 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