凌晓璇，杨磊，纪卫东，宾晓农， 吕嘉春 广州医学院 化学致癌研究所、呼吸疾病国家重点实验室

1. Genetic variant in the seed region of hsa-miR-499-3p increases risk of lung cancer in southern Chinese population 凌晓璇，杨磊，纪卫东，宾晓农，吕嘉春 广州医学院 化学致癌研究所、呼吸疾病国家重点实验室 承国家自然科学基金项目(30200235、30371196、30671813、30872178)资助

2. BACKGROUND • Lung cancer is the leading cause of cancer deaths in the world. In 2009, 219,440 lung cancer incidents and 159,390 deaths in the United States. 483,040 new lung cancer patients and 420,411 deaths occurred in each year of 2004 and 2005 in China. • In Guangzhou, the death rate of lung cancer ranked the No. 1 death cause among tumors. It was 63.8/105 in males, and 22.8/105 in females. In other cities of China, such as Shenyang, Shanghai and Tianjin, the situations of lung cancer are similarly getting more and more serious. • Environment factors: cigarette smoking, occupational exposure, air pollution and nutrients or diets. • Other factors: host (eg, chronic lung diseases,) or genetic factors, susceptibility for individuals to develop lung cancer.

3. BACKGROUND MicroRNA (miRNA) : • Post-transcriptional regulators that bind to complementary sequences in the 3' UTRs (or 5’UTR) of target mRNAs, usually resulting in gene silencing • In release 15 of miRBase, 14197 miRNAs (940 in Homo sapiens ) have been reported. They may target about 60% of mammalian genes and are abundant in many human cell types. Each miRNA may affect hundreds of mRNAs . • miRNA are well conserved in eukaryotic organisms, vital and evolutionarily ancient component of genetic regulation. • Averagely, they are 21-23 nucleotides long. The a 2~8 nucleotides in the 5’ end of miRNA belong to the seed region thatplays important roles in target bindings and the processing of miRNA precursor (pre-miRNAs). • SNP in the seed region of miRNA may alter its function, and thus cancer risk.

4. Dynamic of miRNA: 1. Primary miRNA (Pri-miRNA) 2. Precursor miRNA (Pre-miRNA） 3. mature miRNA

5. microRNA might associate with cancer

6. BACKGROUND • Based on Genebank dbSNP database, we found that 4 miRNAs possessed polymorphisms in their seed region with MAF>5%in Chinese population. One locates in the seed region ofhsa-miR-499-3p (rs3746444), that alter miR-499’s functions. • 4 studies reported this SNP that involved in 2 kinds of cancer. However, the real effect of this genetic variantis still unclear. associationwith breast cancer risk (Hu, Z. etal.Hum Mutat, 2009) non- association with lung cancer risk (Tian, T. etal.CEBP, 2009). non-association with lung cancer survival(Hu, Z. etal.J Clin Invest, 2008) non-association with breast cancer risk (Catucci, I. etal.Hum Mutat, 2010)

7. HYPOTHESIS The genetic variant in the seed region of hsa-miR-499-3p is associated with the risk of lung cancer

8. STUDY DESIGN • The studied SNPs were selected based on the dbSNP database and re-sequencing data. • A hospital-based case-control study, 526 newly diagnosed patients with primary lung cancer were recruited in Guangzhou city. • 526 cancer-free unrelated controls were recruited from healthy subjects in the community health centers. • The cases and controls frequency matched by age (±5 years) and sex. • All subjects were Chinese Han.

9. Gene: hsa-mir-499 (ENSG00000207635) Homo sapiens miR-499 stem-loop: rs7267163: No frequency rs3746444: MAF>5% mark AA GG AA GG AG rs3746444A>G

10. GENOTYPING-PCR-RFLP The hsa-miR-499-3p A>G (rs3746444 )PCR primers 5’-CAA AGT CTT CAC TTC CCT GCC A-3’(FP) 5’-GAT GTT TAA CTC CTC TCC ACG TGA TC-3’(RP) products：146bp Restriction enzyme:Bcl I

11. rs3746444 AA、AG、GG三种基因型（箭头指示）的测序图

12. Table I . Frequency distributions of selected variables in lung cancer cases and controls

13. Histological types and Stages of lung cancer n %

14. Table II. Distributions of polymorphismrs3746444 A→G and results of logistic regression analysis for associations with risk of lung cancer

15. Table III. Stratification analysis of the rs3746444 A>G genotypes by selected variables in lung cancer patients and controls

16. Bioinformatics' analysis: Potential target genes of mir-499 (top15 )

17. hsa-miR-499-3p-A (5’-aacaucacagcaagucugugcu-3’) targets to ADAMTS9 hsa-miR-499-3p-G (5’-aacgucacagcaagucugugcu-3’) targets to ADAMTS9 • From bioinformatics, the lower maximal free energy, the more stable of binding; • rs3746444G allele will bind to ADAMTS9 more effectively than that of A allele.

18. Low Neg ANOVA P = 0.023 Med High Relative protein expression of ADAMTS9 in LC tissues AA AG GG Variants of rs3746444 A→G decreased the protein expression of ADAMTS9 in LC tissues

19. False positive report probability (FPRP) for association between rs3746444 A>G variants and risk of lung cancer OR= 1.62 (1.24-2.11), Power=0.969 null OR= (1.5 or 1/0.67) Prior probability =0.01 FPRP=0.039 → noteworthy at 0.2 level. The finding of this study is unlikely by chance.

20. Limitations • Relatively small study. • Hospital-based, retrospective study. • Lack of functional assay for the SNP • in the seed region of hsa-miR-499-3p

21. CONCLUSION The G variant of rs3746444 in hsa-miR-499-3p seed region is associated with an increased risk of lung cancer in Chinese population. One possible explanation is that the rs3746444G variants decreased the expression of tumor suppress gene ADAMTS9.

23. Study OR [95% Conf. Interval] % Weight -------------------------------------------------------------------------------------------- Hu Z (breast cancer) 1.258 1.039 1.524 20.63 Catucci I (breast cancer) 0.993 0.816 1.209 22.00 Catucci I (breast cancer) 1.081 0.901 1.297 24.68 Tian T (lung cancer) 0.956 0.788 1.161 23.13 Ling X (lung cancer) 1.924 1.494 2.478 9.57 -------------------------------------------------------------------------------------------- M-H pooled OR 1.150 1.052 1.257 100.00 Test of OR=1 : z = 3.08 P = 0.002

24. Acknowledgement Dr. Lu’s lab staff, GZMC Dr. Wei’s lab staff, UT MDACC

25. Thanks for your attention ! 谢谢！