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

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.

BACKGROUNDMicroRNA (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 that plays 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.

Dynamic of miRNA:

1. Primary miRNA (Pri-miRNA)

2. Precursor miRNA (Pre-miRNA）

3. mature miRNA

microRNA might associate with cancer

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 of hsa-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 variant is still unclear.

association with 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)

HYPOTHESIS

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

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.

Gene: hsa-mir-499 (ENSG00000207635)

rs3746444: MAF>5% rs7267163: No frequency

Homo sapiens miR-499 stem-loop:

rs3746444A>G mark AA GG AA GG AG

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

rs3746444 AA 、 AG 、 GG三种基因型（箭头指示）的测序图

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

VariablesPatients (n = 526) Controls (n = 526)

Pa

N % N %

Age (years)

0.084 60 267 50.8 239 45.4 > 60 259 49.2 287 54.6Sex

0.322 Male 364 69.2 349 66.4 Female 162 30.8 177 33.6Smoking status

0.014 Yes 296 56.3 256 48.7 No 230 43.7 270 51.3Drinking status

0.178 Yes 105 20.0 123 23.4 No 421 80.0 403 76.6Family history of cancer

0.174 Yes 57 10.8 44 8.4 No 469 89.2 482 91.6BMI

<0.0001 <18.0 75 14.3 16 3.0 18.0-24.9 396 75.3 349 66.4 >25.0 55 10.5 161 30.6

Lung cancer (526)

Histological types

Adenocarcinoma 191 36.4

Squamous cell carcinoma 184 34.9

Large cell carcinoma 21 4.1

Small cell lung cancer 64 12.1

Other carcinomas 66 12.5

Stages

I 76 14.5

II 47 8.9

III 166 31.5

IV 237 45.0

Histological types and Stages of lung cancer

n %

Patients Controls Genotypes

n (%) n (%) Pa

Crude

OR (95% CI)

Adjusted

OR (95% CI)b

Total no. of

subjects 526 526

Total no. of alleles 1052 1052

rs3746444 A→G

AA 284(54.0) 349(66.4) 1.00 (ref.) 1.00 (ref.)

AG 131(24.9) 120(22.8) 1.34(1.00-1.80) 1.32(1.01-1.81)

GG 111(21.1) 57(10.8)

<0.0001

2.39(1.68-3.42) 2.22(1.52-3.23)

Trend test P value <0.0001 <0.0001

AG+GG 242(46.0) 177(33.6) 1.68(1.31-2.16) 1.62 (1.24-2.11)

G allele 0.336 0.222 0.0004 aP values for a two-sided 2 test for genotype distribution. bAdjusted in a logistic regression model that included age, sex, smoking status, drinking status .

Table II. Distributions of polymorphism rs3746444 A→G and results of

logistic regression analysis for associations with risk of lung cancer

Patients (n = 526) Controls (n = 526) Crude OR ( 95% CI)

Adjusted OR ( 95% CI)a

AA n (%)

AG+GG n (%)

AA

n (%) AG+GG

n (%) AG+GG vs AA AG+GG vs AA P b

Age (years) 60 137(51.3) 130(48.7) 136(56.9) 103(43.1) 1.25(0.88-1.78) 1.18(0.82-1.70) > 60 147(56.8) 112(43.2) 213(74.2) 74(25.8) 2.19 (1.53-3.15) 2.36(1.60-3.49) 0.0103

Sex Male 196(53.8) 168(46.2) 216(61.9) 133(38.1) 1.39(1.03-1.88) 1.35(0.98-1.85) Female 88(54.3) 74(45.7) 133(75.1) 44(24.9) 2.54(1.60-4.03) 2.49(1.51-4.12) 0.0464

Smoking status

Ever 164(55.4) 132(44.6) 160(62.5) 96(37.5) 1.34(0.95-1.89) 1.28(0.89-1.84) Never 120(52.2) 110(47.8) 189(70.0) 81(30.0) 2.14(1.48-3.09) 2.12(1.43-3.15) 0.0777

Drinking status

Ever 56(53.3) 49(46.7) 75(61.0) 48(39.0) 1.37(0.81-2.32) 1.32(0.73-2.37) Never 228(54.2) 193(45.8) 274(68.0) 129(32.0) 1.80(1.35-2.39) 1.73(1.28-2.34) 0.3747

BMI <18.0 45(60.0) 30(40.0) 12(75.0) 4(25.0) 2.00(0.59-6.79) 2.05(0.50-8.41) 18.0-24.9 208(52.5) 188(47.5) 226(64.8) 123(35.2) 1.66(1.24-2.23) 1.64(1.21-2.21) 0.9949 25.0-28.0 31(56.4) 24(43.6) 111(68.9) 50(31.1) 1.72(0.92-3.22) 1.22(0.61-2.45)

Family history of cancer

Yes 28(49.1) 29(50.9) 29(65.9) 15(34.1) 2.00(0.89-4.51) 1.57(1.19-2.07) 0.6485 No 256(54.6) 213(45.4) 320(66.4) 162(33.6) 1.64(1.26-2.14) 2.41(0.89-6.56)

aORs were adjusted for age, sex, smoking status, drinking status, BMI and family history of cancer in a logistic regression model. bP value of test for the multiplicative interaction between rs3746444 A→G and selected variables on cancer risk were calculated using standard unconditional

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

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

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;

rs3746444 G allele will bind to ADAMTS9 more effectively than that of A allele.

Neg Low

Med High

Rel

ati

ve

pro

tein

exp

res

sio

n

of

AD

AM

TS

9 in

LC

tis

sues

AA GGAG

ANOVA P = 0.023

Variants of rs3746444 A→G decreased the protein expression of ADAMTS9 in LC tissues

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.

Limitations

• Relatively small study.

• Hospital-based, retrospective study.

• Lack of functional assay for the SNP in the seed region of hsa-miR-499-3p

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.

Meta-analysis

0.1 1 10

Odds ratio

Hu Z(breast cancer in Chinese)

Catucci I(breast cancer in Germany)

Catucci I(breast cancer in Italy)

Tian T(lung cancer in Chinese)

Ling X(lung cancer in Chinese)

Total (fixed effects)

Total (random effects)

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

Dr. Lu’s lab staff, GZMC

Dr. Wei’s lab staff, UT MDACC

AcknowledgementAcknowledgement

Thanks for your attention !

谢谢！