1. Joana Ribeiro BSc in Clinical Analysis and Public Health MSc in Medicine and Molecular Oncology PhD student in Biomedical Sciences _________________________________ Molecular Oncology Group IPO Porto FG EPE Email: joana.cribeiro@hotmail.com MIR-125B EXPRESSION IN HPV INFECTION AND CERVICAL CANCER DEVELOPMENT: A POTENTIAL BIOMARKER

2. Normal HPV + CIN1CIN2/3 InfectionTransformation CIS/ICC Progression Introduction Cervical Carcinogenesis Micro-RNA-125b expression in HPV infection and cervical cancer development EUROGIN 2013

3.  Proliferative infection  Inflammation  Host immune response Normal HPV + CIN1CIN2/3 Iniciatio n InfectionTransformation CIS/ICC Progression Introduction Cervical Carcinogenesis Micro-RNA-125b expression in HPV infection and cervical cancer development EUROGIN 2013

4. CIS/ICC Normal HPV + CIN1CIN2/3 InfectionTransformation Progression  Persistent infection  Viral DNA integration Iniciatio n Introduction Cervical Carcinogenesis Micro-RNA-125b expression in HPV infection and cervical cancer development EUROGIN 2013

5.  P16 expression  mRNA E6/E7 expression  HPV L1 expression  Viral integration  MicroRNAs Introduction Biomarkers Micro-RNA-125b expression in HPV infection and cervical cancer development EUROGIN 2013

6. Introduction Micro-RNAs miRNAtargetsActivityReference miR-9E-cadherinTumor SupressorTan et al, 2010 miR-15-16 cluster Bcl2Oncogene Cimmino et al, 2005 Guo et al, 2009 miR-21 hMSH2 CDC25A Bcl2 Oncogene Zhong et al, 2012 Jin Y, 2011 Si et al, 2008 miR-34a Bcl2 Survivin CDK4 CDK6 Cyclin D1 Cyclin E2 c-Myc E2F3 Tumor Supressor He et al, 2007 Hermking H 2010 miR-124aCDK6Tumor SupressorSilber et al, 2008 miR-125b p53 Bak-1 OncogeneGong et al, 2012 Bcl-2 Bcl-w IL-6R Tumor suppressor Scott et al, 2007 Zhang et al, 2011 Zhao et al, 2012 miR-137CDK6Tumor Supressor Silber et al, 2008 Kozaki et al, 2008 miR-504p53OncogeneHu et al, 2012 Micro-RNA-125b expression in HPV infection and cervical cancer development EUROGIN 2013

7. Introduction micro-RNA 125b  Involved in different cellular processes :  Targets  p53, Bak-1, Bcl-2, Bcl-w, IL-6R..  Carcinogenesis:  its action can be either as oncogene or tumor suppressor  Regultation of HPV L2  decrease of proliferative infection  Underexpressed in cervical cancer tissues Micro-RNA-125b expression in HPV infection and cervical cancer development EUROGIN 2013

8. Aims of the study  To characterize the expression of miR-125b in different cervical lesions and cancer;  To evaluate its impact as predictive/prognostic biomarkers of cervical cancer and HPV infection. Micro-RNA-125b expression in HPV infection and cervical cancer development EUROGIN 2013

9. Materials and Methods Micro-RNA-125b expression in HPV infection and cervical cancer development EUROGIN 2013

10. Materials and Methods Study Population Exfoliated cervical cells collected from 110 patient : 29 normal HPV negative, 20 normal HPV positive, 28 LSIL, 12 HSIL, 7 CIS 5 ICC Methodology miR-125b expression was determined by qRT-PCR methodology Micro-RNA-125b expression in HPV infection and cervical cancer development EUROGIN 2013

11. Results/Discussion Micro-RNA-125b expression in HPV infection and cervical cancer development EUROGIN 2013

12. Results/Discussion miR125bmiR23a Mean Ct ± sd VC Mean Ct ± sd VC No Lesion (no HPV) 28.43 ± 2.48 0.09 34.94 ± 1.72 0.05 No Lesion (HPV+) 28.06 ± 2.20 0.08 35.65 ± 2.58 0.07 LSIL 29.34 ± 2.12 0.07 35.42 ± 2.04 0.06 HSIL 29.06 ± 1.81 0.06 35.21 ± 1.73 0.05 CIS 29.48 ± 2.66 0.09 34.40 ± 1.65 0.05 ICC29.27 ± 2.990.1033.68 ± 0.350.01 Table 1: Data obtained from qPCR Variation coefficient (Cts) - <10% Relative expression was calculated using Livak Method Reference group: women with normal cytology and without HPV infection Normalizer: miR-23a Micro-RNA-125b expression in HPV infection and cervical cancer development EUROGIN 2013

13. Results/Discussion Relative expression of miR-125b Increased miR-125b expression among normal cases with HPV infection  inflammation/ immune response (Nicolete et al, 2012; Witwer et al, 2012) Gradual down-regulation  ↓ p53  ↓ Stimulation of miR-125b trasncription (Le et al, 2009) * ** miR125b 2 -ΔΔCt t-test No Lesion (no HPV) (n=29)Reference No Lesion (HPV+) (n=20)2.11 0.038 LSIL (n=28)0.75 0.320 HSIL (n=21)0.78 0.481 CIS/ICC (n=12)0.29 0.008 CIS (n=7)0.33 0.042 ICC (n=5)0.23 0.028 Micro-RNA-125b expression in HPV infection and cervical cancer development EUROGIN 2013

14. Results/Discussion Micro-RNA-125b expression in HPV infection and cervical cancer development EUROGIN 2013 Pos if greater than or = to: Sensitivity1- Specificity -6,6450,875,517 -6,4500,875,483 -6,2550,875,448 -6,0750,875,414 -5,9600,875,379 -5,9100,875,345 -5,8200,875,310 -5,7450,750,310 -5,7250,750,276 Table 2: Cutoff values to CIS/ICC diagnosis miR-125b levels were able to predict CIS/ICC with 88% sensitivity and 70% specificity.

15. Conclusions miR-125b expression is increased in normal cervix infected by HPV, while the relative expression seems to decrease significantly as lesions progress. These results suggest that miR-125b could be used as useful tool for cervical cancer diagnosis/prognosis using a non-invasive approach, though this observation needs to be complemented with further studies. Cohort study Confirmation the miR-125b expression in tissue samples Micro-RNA-125b expression in HPV infection and cervical cancer development EUROGIN 2013

16. Conclusions miR-125b expression is increased in normal cervix infected by HPV, while the relative expression seems to decrease significantly as lesions progress. These results suggest that miR-125b could be used as useful tool for cervical cancer diagnosis/prognosis using a non-invasive approach, though this observation needs to be complemented with further studies. Cohort study Confirmation the miR-125b expression in tissue samples Micro-RNA-125b expression in HPV infection and cervical cancer development EUROGIN 2013

17. AKNOLEDGMENTS Molecular Oncology Group, IPO-Porto Virology Service, IPO-Porto Pathology Department, IPO-Porto Portuguese League of Cancer (Liga Portuguesa Contra o Cancro, Núcleo Regional do Norte) Micro-RNA-125b expression in HPV infection and cervical cancer development EUROGIN 2013

18. Joana Ribeiro BSc in Clinical Analysis and Public Health MSc in Medicine and Molecular Oncology PhD student in Biomedical Sciences _________________________________ Molecular Oncology Group IPO Porto FG EPE Email: joana.cribeiro@hotmail.com MIR-125B EXPRESSION IN HPV INFECTION AND CERVICAL CANCER DEVELOPMENT: A POTENTIAL BIOMARKER