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A genetic variant in pre-miR-27a is associated with a reduced cervical cancer risk in southern Chinese women
Gynecologic Oncology 132 (2014) 450–454
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A genetic variant in pre-miR-27a is associated with a reduced cervical cancer risk in southern Chinese women Xing-Dong Xiong a,b,c,d,⁎, Xi-Ping Luo e, Jie Cheng a,b,c, Xinguang Liu a,b,c, En-Min Li d,f, Li-Qin Zeng e a
Institute of Aging Research, Guangdong Medical College, Dongguan 523808, China Institute of Biochemistry & Molecular Biology, Guangdong Medical College, Zhanjiang 524023, China Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, Dongguan 523808, China d Key Laboratory of Molecular Biology in High Cancer Incidence Coastal Chaoshan Area of Guangdong Higher Education Institutes, Medical College of Shantou University, Shantou 515041, China e Department of Gynecology, Guangdong Women and Children Hospital and Health Institute, Guangzhou 510010, China f Department of Biochemistry and Molecular Biology, Medical College of Shantou University, Shantou 515041, China b c
H I G H L I G H T S • The miR-27a rs895819 polymorphism is associated with a decreased risk of cervical cancer in southern Chinese women. • No association between rs7372209/rs1834306 polymorphism and cervical cancer risk was detected among southern Chinese women.
a r t i c l e
i n f o
Article history: Received 4 September 2013 Accepted 19 December 2013 Available online 28 December 2013 Keywords: miRNA Single nucleotide polymorphism Cervical cancer Risk
a b s t r a c t Objective. MicroRNAs (miRNAs) play critical roles in cervical carcinogenesis. Common single nucleotide polymorphisms (SNPs) in pre/pri-miRNAs may change their property through altering miRNAs expression and/or maturation. Here we aimed to investigate the influence of three common SNPs in pre/pri-miRNAs (pri-miR-26a1 rs7372209, pre-miR-27a rs895819 and pri-miR-100 rs1834306) on individual susceptibility to cervical cancer. Methods. We genotyped these three polymorphisms in 103 cervical cancer cases and 417 cancer-free female subjects using polymerase chain reaction–ligation detection reaction (PCR–LDR) method. Unconditional logistic regression analysis was utilized to estimate the association between these polymorphisms and the risk of cervical cancer. Results. In a logistic regression analysis, we found that the rs895819 polymorphism in pre-miR-27a exhibited a significant effect on cervical cancer risk; T allele (OR = 0.68, 95% CI = 0.49–0.95, P = 0.025), and CT (OR = 0.33, 95% CI = 0.15–0.74, P = 0.007) or TT (OR = 0.33, 95% CI = 0.15–0.72, P = 0.006) genotype were associated with the decreased risk, compared to C and CC respectively. As we used further genotype association models, we found a similar trend of the association in additive (OR = 0.70, P = 0.041) and recessive model (OR = 0.33, P = 0.004). We did not detect any association of the other two SNPs in pri-miR-26a-1 (rs7372209) and pri-miR100 (rs1834306) with cervical cancer risk. Conclusion. Our study provides the first evidence that the miR-27a rs895819 polymorphism is associated with a decreased risk of cervical cancer in southern Chinese women. © 2013 Elsevier Inc. All rights reserved.
Introduction Cervical cancer is the second most common cancer following breast cancer in women worldwide, especially in developing countries [1]. It is well-recognized that specific oncogenic human papillomaviruses (HPVs)
Abbreviations: miRNA, microRNA; pre-miRNA, precursor microRNA; pri-miRNA, primary microRNA; SNP, single nucleotide polymorphism; HPV, human papillomavirus; OR, odds ratio; CI, confidence interval. ⁎ Corresponding author at: Institute of Aging Research, Guangdong Medical College, Dongguan 523808, China. Fax: +86 769 22896425. E-mail address: [email protected] (X.-D. Xiong). 0090-8258/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ygyno.2013.12.030
are primary etiologic factors in cervical cancer, while the majority of infected women do not develop the cancer during their lifetime, indicating that other factors contribute to the progression to cervical cancer [2]. Accumulating evidence supports that host genetic variations, apart from HPV infection, play an important role in the cervical carcinogenesis [3]. MicroRNAs (miRNAs) are a class of endogenous, small and noncoding RNAs (~ 22 nt), which are initially transcribed from genomic DNA to long primary transcripts (pri-miRNAs) and then are cleaved by nuclear Drosha into 60–70 nt hairpin-shaped precursor RNAs (premiRNAs) [4]. Pre-miRNAs are exported to the cytoplasm by Exportin-5 and are further processed into ~ 22 nt mature miRNA duplexes by the cleavage of Dicer [5,6]. In association with RNA-induced silencing
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complex (RISC), one strand of the miRNA duplex matches target mRNA sequence in the 3′-untranslated regions. This binding finally leads to mRNA degradation or translational repression and consequently down-regulates the expression of the protein [7]. To date, it has been estimated that miRNAs modulate the expression of approximately 30% of human genes [8]. MiRNAs are involved in a wide range of biological processes including cell proliferation, differentiation, development, and apoptosis [8]. Elevated or decreased expression of miRNAs has been found in various tumor types, including cervical cancer. Recent studies have demonstrated that miR-27a, miR-26a-1 and miR-100 were up-regulated or down-regulated in cervical cancer and were considered to play a role in cervical carcinogenesis [9–15]. SNPs in miRNA genes are a novel class of genetic variations in the human genome which are currently being identified and investigated in human cancers [16]. SNPs, or mutations within miRNA genes, including pri-miRNAs, pre-miRNAs and mature miRNAs, potentially influence the processing and/or target selection of miRNAs and thus contribute to cancer risk [16,17]. Because each miRNA potentially regulates hundreds of genes, identifying the risk factors associated with miRNA genes could serve to elucidate novel pathways and mechanisms of pathogenesis. However, the exact role played by genetic variants in miRNA genes concerning cervical cancer susceptibility remains largely unknown [18–20]. Recent studies have shown that three potentially functional polymorphisms (rs895819, rs7372209, and rs1834306) in pre-miRNA or pri-miRNAs (pre-miR-27a, pri-miR-26a-1, and pri-miR-100, respectively) were associated with the risk and clinical outcome for cancers [17,21–26]. Based on their roles of these miRNAs on cervical cancer [9–15], we speculated that polymorphisms in these miRNAs may have an impact on miRNA processing, and so they modulate the susceptibility to cervical cancer. Therefore, here we conducted a case–control study to elucidate the association of these polymorphisms with the risk of cervical cancer. Our analysis revealed that the T allele of SNP rs895819, located in pre-miR-27a, has a significant association with a reduced cervical cancer risk in southern Chinese women. Materials and methods Study population This case–control study included 103 cervical cancer patients and 417 cancer-free control subjects. The method used for subject enrollment was the same as for our previous studies [27]. In brief, patients with cervical cancer were recruited at Guangdong Women and Children Hospital and Health Institute, Guangzhou, PR China from February 2009 to July 2012. The diagnosis of cervical cancer was confirmed in all cases by histological examination of tissue from biopsy or resected specimens. All clinicopathological data were obtained from clinical and pathologic records. Controls (n = 417) were randomly selected from healthy women volunteers who took part in the physical examination during the time period of case collection in the same hospital. The criteria for the selection of controls included no clinical detection for cervical lesions, no gynecological tumor, no endometriosis and no history of cancer. All study subjects enrolled in this case–control study were unrelated ethnic Han Chinese women who lived in southern China. At recruitment, informed consent was obtained from each subject who was then interviewed to collect detailed information. This study was approved by the Medical Ethics Committee at Guangdong Women and Children Hospital and Health Institute. DNA extraction Genomic DNA was extracted from peripheral whole blood by TIANamp blood DNA extraction kit (TianGen Biotech, Beijing, China) according to the manufacturer's instructions. All DNA samples were dissolved in water and stored at −20 °C until use.
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Genotyping Genotypes were determined by the polymerase chain reaction– ligation detection reaction (PCR–LDR) method (Shanghai Biowing Applied Biotechnology Company). The sequence of primers and probes is summarized in Table S1. The PCR was carried out on the ABI 9600 (Applied Biosystems, USA) in a total volume of 20 μl including 50 ng genomic DNA, 1× PCR buffer, 2.5 mM MgCl2, 0.2 mM dNTPs, 0.5 μM each primer, and 1 U hot-start Taq DNA polymerase (Qiagen). Cycling parameters were as follows: 95 °C for 15 min; 35 cycles at 94 °C for 30 s, 59 °C for 1 min, and 72 °C for 1 min; and a final extension step at 72 °C for 10 min. The ligation reaction for each PCR product was carried out with a final volume of 10 μl containing 1 μl 10× ligation buffer, 1 μl of PCR product, 1 pmol of each discriminating probe, and 2 U Taq DNA ligase (New England Biolabs, USA). The LDR parameters were as follows: 95 °C for 2 min, 30 cycles at 94 °C for 15 s and 50 °C for 25 s. Following the LDR reaction, 1 μl LDR reaction product was mixed with 1 μl ROX and 1 μl loading buffer. The mixture was then analyzed by the ABI Prism 377 DNA Sequencer (Applied Biosystems, USA). About 10% of the samples were randomly selected to perform the repeated assays and the results were 100% concordant. HPV detection Cervical scrape specimens were collected using the cervix brush and then processed to isolate DNA for HPV detection assays. HPV infection was detected using HPV GenoArray test kit (Hybribio Inc., China) according to the manufacturer's instructions. This assay kit can detect 15 high risk type HPV (HR-HPV, 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, and 68) and six low risk type HPV (LR-HPV, 6, 11, 42, 43, 44, and CP8304). Statistical analysis A statistical power analysis was performed using a program for power and sample size computations (known as PS program) [28]. Genotype frequencies in control group were checked for the Hardy– Weinberg equilibrium by using goodness-of-fit χ2-test. Pearson chisquare test was used to examine distribution difference of genotypes between cases and controls. Association between the polymorphism and the susceptibility of cervical cancer was evaluated using unconditional logistic regression analysis, adjusted by age, age at the first sexual intercourse, number of sexual partners, number of vaginal delivery and smoking status. These statistical analyses were done with the SPSS software program (version 13.0, SPSS Inc., Chicago, IL, USA). All statistical tests were two-sided and a P value of less than 0.05 was used as the criterion of statistical significance. Results Characteristics of the study population One hundred three cervical cancer cases and 417 control subjects were included in the present study. The frequency distributions of selected demographic characteristics of the cases and controls are shown in Table 1. There was no significant difference in the distributions of age and smoking status between the cases and controls. However, the incidence of HR-HPV infection in the patients (82.7%) was much higher than that in the control subjects (16.0%, χ2 = 127.45, P b 0.001, Table 1), implying HR-HPV infection as a critical risk factor for cervical cancer development in our study population. Among the subtypes of HR-HPV, 87.7% (71/81) were HPV16/18 positive in the cases and 14.7% (5/34) were HPV16/18 positive in the controls. In addition, compared with controls, the cervical cancer cases were significantly earlier age at first intercourse, and had more sexual partners and number of vaginal delivery (all P b 0.05). Of the 103 cases, 81 (78.7%) had
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Table 1 Characteristics of cervical cancer cases and normal controls. Variable
Age, years b45 45–55 N55 Age at the first sexual intercourse, years ≤20 N20 Number of sexual partners, n (%) ≤1 N1 Number of vaginal delivery ≤1 N1 Smoking status Nonsmoker Smoker HR-HPV infection Positive Negative Total Type of HPV DNAc HPV16 or 18 Other HPV types Histologic types Squamous cell carcinoma Adenocarcinoma Adenosquamous carcinoma Others Stage I II III IV a b c
Controls (n = 417)
Cases (n = 103)
No. (%)
No. (%)
173 (41.5) 164 (39.3) 80 (19.2)
56 (54.3) 32 (31.1) 15 (14.6)
Pa
0.062
0.025b 156 (37.4) 261 (62.6)
51 (49.5) 52 (50.5)
311 (74.6) 106 (25.4)
46 (44.7) 57 (55.3)
141 (33.8) 276 (66.2)
21 (20.4) 82 (79.6)
395 (94.7) 22 (5.3)
101 (98.1) 2 (1.9)
34 (16.0) 178 (84.0) 212
81 (82.7) 17 (17.3) 98
5 (14.7) 31 (85.3)
71 (87.7) 10 (12.3)
b0.001
0.008
0.149
b0.001
b0.001
81 (78.7) 9 (8.7) 4 (3.9) 9 (8.7) 61 (59.2) 38 (36.9) 3 (2.9) 1 (1.0)
Two-sided chi-square test. P values under 0.05 were indicated in bold font. Among subjects with positive HR-HPV DNA.
squamous cell carcinomas. Other patients who had adenocarcinoma, adenosquamous carcinoma and unclassified were counted twentytwo (21.3%). In all the 103 cases, 59.2% of patients were in stage I; 36.9%, 2.9%, and 1.0% was found to be in stage II, III, and IV, respectively (Table 1). Associations of miRNA polymorphisms with risk of cervical cancer The primary information for miR-26a-1 rs7372209 C N T, miR-27a rs895819 T N C and miR-100 rs1834306 T N C polymorphisms was in Table S2. Minor allele frequency (MAF) in our controls was similar to MAF for Chinese in HapMap database for all three SNPs (Table S2). Genotype distributions of three SNPs in our control subjects were in agreement with that expected under the Hardy–Weinberg equilibrium (rs7372209, P = 0.737; rs895819, P = 0.254; rs1834306, P = 0.723, Table S2), providing no evidence of population stratification within the dataset. The allele and genotype distributions of three polymorphisms in the cases and the controls are shown in Table 2. From the allelic association analysis, we found only rs895819 in pre-miR-27a showed statistical significance (P = 0.025, Table 2). Unconditional logistic regression analysis revealed that the risk of cervical cancer was significantly decreased in both CT heterozygotes [odds ratio (OR) = 0.33, 95% confidence interval (CI) = 0.15–0.74, P = 0.007] and TT homozygotes (OR = 0.33, 95% CI = 0.15–0.72, P = 0.006), compared with CC homozygotes (Table 2). In addition, a similar trend of the decreased risk of cervical cancer was detected in additive and recessive model in which CT and TT genotypes were combined (OR = 0.70, 95% CI = 0.49–0.98, P = 0.041, and OR = 0.33, 95% CI = 0.16–0.70, P = 0.004, respectively, Table 2). These data indicate
that pre-miR-27a rs895819 polymorphism may be associated with risk of cervical cancer and that individuals carrying T allele may have significantly decreased cervical cancer susceptibility. However, we did not find any association between the pri-miR-26a-1 rs7372209 or primiR-100 rs1834306 and the risk of cervical cancer in allelic or genotypic analyses (Table 2). In addition, no more evident association between pre-miR-27a rs895819 polymorphism and risk of cervical cancer was observed among subgroups by age at first intercourse, status of smoking or HPV (data not shown). We further conducted the analysis in the patients with early-stage cancer and found that a more pronounced reduction in cervical cancer risk was observed among stage I patients carrying rs895819 CT or TT genotypes (Table 3, OR = 0.24, 95% CI = 0.10–0.60, P = 0.002 and OR = 0.28, 95% CI = 0.12–0.68, P = 0.005, respectively). Significantly decreased cervical cancer risk was also found in recessive model (Table 3, OR = 0.26, 95% CI = 0.11–0.61, P = 0.002, respectively). Out of the 103 cervical cancer cases, 81 (78.7%) were squamous cell carcinoma, 9 (8.7%) were adenocarcinoma, 4 (3.9%) were adenosquamous carcinoma, and 9 (8.7%) was unknown. We also found that a more pronounced reduction in cervical cancer risk was observed among squamous cell carcinoma patients carrying rs895819 CT or TT genotypes (Table 3, OR = 0.31, 95% CI = 0.13–0.72, P = 0.006 and OR = 0.30, 95% CI = 0.13–0.68, P = 0.004, respectively), and in the additive and recessive model (Table 3, OR = 0.66, 95% CI = 0.45–0.97, P = 0.035 and OR = 0.30, 95% CI = 0.14–0.66, P = 0.003, respectively). Discussion Increasing evidences suggest that miRNAs are crucial factors in cancer progress as oncogenes or tumor suppressors by degradation of the target mRNAs and/or inhibition of their translation [29]. MicroRNAs are also considered to be potential biomarkers and therapeutic targets in human cancers [30]. Variations in expression of miRNAs have been reported to be related to many tumors including cervical cancer [31–33]. Differential miRNA expression could be caused by sequence variations, such as mutations and SNPs [34]. Although SNPs in miRNAs have been widely studied, the association between SNPs in miRNAs and cervical cancer risk is still largely unknown. Therefore, we conducted a case–control population study to investigate the influence of three common SNPs in pre/pri-miRNAs (pri-miR-26a-1 rs7372209 C N T, premiR-27a rs895819 T N C and pri-miR-100 rs1834306 T N C) on individual susceptibility to cervical cancer. As a result, we found that the common T N C polymorphism within pre-miR-27a (rs895819) exhibited a significant effect on cervical cancer risk. Compared with CC homozygotes, subjects who were CT heterozygotes or TT homozygotes exhibited a decreased risk of cervical cancer (Table 2). In addition, the other two SNPs (rs7372209 and rs1834306) located in the pri-miRNAs did not show a significant association with cervical cancer. Our study suggested that miR-27a rs895819 polymorphism played a role in cervical carcinogenesis. The oncogenic activity of miR-27a has been recently reported in gastric, breast and pancreatic cancer [35–37]. However, a recent study reported that miR-27a had significantly decreased expression in head and neck squamous cell carcinoma (HNSCC) as compared to normal tissues [38]. Likewise, Wang et al. found that miR-27a was downregulated in cervical cancer tissues and cancer cell lines when compared with normal cervical tissue, which suggested that miR-27a functions as tumor suppressor in the development of cervical cancer [9]. SNP rs895819 was found in pre-miRNA regions of hsa-miR-27a, which was in chromosome 19, and it was located at position 40 relative to the first nucleotide [39]. Recently, a Chinese study found that rs895819 TT genotype was associated with a decreased risk of gastric cancer [22,39]. Although the binding of the mature miRNA to target mRNAs was not influenced by the SNP rs895819, some studies had demonstrated that polymorphisms in pre-miRNAs could influence the expression of their mature forms, and these were as well involved in the binding of some nuclear factors
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Table 2 Association between SNPs in miRNAs and risk of cervical cancer.
Pri-miR-26a-1 rs7372209 Allele Genotype
Additive Dominant Recessive
Pre-miR-27a rs895819 Allele Genotype
Additive Dominant Recessive
Pri-miR-100 rs1834306 Allele Genotype
Additive Dominant Recessive a b
Controls (n = 417)
Cases (n = 103)
No. (%)
No. (%)
609 (73.0) 225 (27.0) 221 (53.0) 167 (40.0) 29 (7.0)
150 (72.8) 56 (27.2) 57 (55.3) 36 (35.0) 10 (9.7)
196 (47.0) 221 (53.0) 388 (93.0) 29 (7.0)
46 (44.7) 57 (55.3) 93 (90.3) 10 (9.7)
C T CC CT TT – CT + CC TT CC CT + TT
218 (26.1) 616 (73.9) 24 (5.7) 170 (40.8) 223 (53.5)
70 (34.0) 136 (66.0) 15 (14.6) 40 (38.8) 48 (46.6)
194 (46.5) 223 (53.5) 24 (5.7) 393 (94.3)
55 (53.4) 48 (46.6) 15 (14.6) 88 (85.4)
C T CC CT TT – CT + TT CC CT + CC TT
457 (54.8) 377 (45.2) 127 (30.5) 203 (48.7) 87 (20.9)
114 (55.3) 92 (44.7) 38 (36.9) 38 (36.9) 27 (26.2)
290 (69.5) 127 (30.5) 330 (79.1) 87 (20.9)
65 (63.1) 38 (36.9) 76 (73.8) 27 (26.2)
C T CC CT TT – CT + TT CC CT + CC TT
OR (95% CI)a
P valuea
1.00 1.01 (0.72–1.42) 1.00 0.76 (0.46–1.24) 1.10 (0.47–2.55) 0.92 (0.64–1.32) 1.00 1.23 (0.78–1.96) 1.00 1.22 (0.54–2.79)
1.00 0.68 (0.49–0.95) 1.00 0.33 (0.15–0.74) 0.33 (0.15–0.72) 0.70 (0.49–0.98) 1.00 0.82 (0.51–1.29) 1.00 0.33 (0.16–0.70)
1.00 1.02 (0.76–1.37) 1.00 0.61 (0.36–1.03) 1.05 (0.58–1.91) 0.96 (0.70–1.31) 1.00 1.41 (0.87–2.28) 1.00 1.35 (0.79–2.30)
0.953 0.275 0.828 0.632 0.379 0.623
0.025b 0.007 0.006 0.041 0.382 0.004
0.892 0.066 0.865 0.785 0.161 0.272
Adjusted for age, age at the first sexual intercourse, number of sexual partners, number of vaginal delivery and smoking status. P values under 0.05 were indicated in bold font.
in miRNA processing [40,41]. The present study showed that the rs895819 T allele may be associated with a lower risk of cervical cancer, indicating that the individuals with rs895819 T allele may have a higher expression level of miR-27a and therefore are in reduced risk of cervical cancer. Further studies are needed to clarify the underlying mechanism of how the genetic variant in pre-miR-27a affects cervical carcinogenesis. Several limitations need to be addressed in this case–control study. First, the patients and controls were enrolled from hospitals and may not represent the general population. Nonetheless, the genotype distribution of the controls was in Hardy–Weinberg equilibrium. Second, the sample size of the present study was relatively small, particularly for stratified analyses. Third, HPV infection is one of the independent risk
factors of cervical cancer. We did not have enough information on HPV status due to lack of the cervical scrape specimens from all study subjects. Finally, further studies in different population could help to establish the true significance of the association between this SNP and the risk of cervical cancer. However, our observations provided valuable insights and interesting information and might serve to guide future studies in this area. In conclusion, our study provides the first evidence that the miR-27a rs895819 polymorphism, but not miR-26a-1 rs7372209 and miR-100 rs1834306, is associated with a decreased risk of cervical cancer in southern Chinese women. This finding suggests that the common genetic polymorphism in pre-miR-27a may play a role in the development of cervical cancer, although additional studies with larger sample size,
Table 3 Association between the pre-miR-27a rs895819 polymorphism and risk of cervical squamous cell carcinoma and stage I cervical cancer.
Allele Genotype
Additive Dominant Recessive a b
C T CC CT TT – CT + CC TT CC CT + TT
Controls (n = 417)
Squamous cell carcinoma (n = 81)
No. (%)
No. (%)
218 (26.1) 616 (73.9) 24 (5.8) 170 (40.8) 223 (53.5)
57 (35.2) 105 (64.8) 13 (16.0) 31 (38.3) 37 (45.7)
194 (46.5) 223 (53.5) 24 (5.8) 393 (94.2)
44 (54.3) 37 (45.7) 13 (16.0) 68 (84.0)
OR (95% CI)a
P valuea
Stage I n = 61)
OR (95% CI)a
P valuea
No. (%) 1.00 0.65 (0.43–0.97) 1.00 0.31 (0.13–0.72) 0.30 (0.13–0.68) 0.66 (0.45–0.97) 1.00 0.77 (0.46–1.27) 1.00 0.30 (0.14–0.66)
0.036b 0.006 0.004 0.035 0.303 0.003
Adjusted for age, age at first intercourse, number of sexual partners, number of vaginal delivery and smoking status. P values under 0.05 were indicated in bold font.
43 (35.2) 79 (64.8) 11 (18.0) 21 (34.5) 29 (47.5) 32 (52.5) 29 (47.5) 11 (18.0) 50 (82.0)
1.00 0.65 (0.43–0.97) 1.00 0.24 (0.10–0.60) 0.28 (0.12–0.68) 0.68 (0.44–1.04) 1.00 0.86 (0.49–1.51) 1.00 0.26 (0.11–0.61)
0.036 0.002 0.005 0.073 0.559 0.002
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detailed HPV infection data and in diverse ethnic populations are required to confirm the general validity of our findings. In addition, further functional studies on this polymorphism are also needed to elucidate the underlying molecular mechanisms of the observed association. Conflicts of interest statement The authors declare that there are no conflicts of interest.
Acknowledgements We thank all individuals who participated to this study. This work was supported by grants from the National Natural Science Foundation of China (81000143, 81370456, 81170327), the Natural Science Foundation of Guangdong Province (S2012010008219, S2011010002922), the Medical Scientific Research Foundation of Guangdong Province (B2009191, A2011431), the Science and Technology Planning Project for University Research Institutions and Medical and Health Organizations of Dongguan City (2011105102007, 200910815256), the Guangdong University Students Innovative Pilot Program (KY1023, KY1232) and the Science & Technology Innovation Fund of Guangdong Medical College (STIF201102). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ygyno.2013.12.030. References [1] Karimi Zarchi M, Behtash N, Chiti Z, Kargar S. Cervical cancer and HPV vaccines in developing countries. Asian Pac J Cancer Prev 2009;10:969–74. [2] Magnusson PK, Sparen P, Gyllensten UB. Genetic link to cervical tumours. Nature 1999;400:29–30. [3] de Freitas AC, Gurgel AP, Chagas BS, Coimbra EC. do Amaral CM. Susceptibility to cervical cancer: an overview. Gynecol Oncol 2012;126:304–11. [4] Lee Y, Jeon K, Lee JT, Kim S, Kim VN. MicroRNA maturation: stepwise processing and subcellular localization. EMBO J 2002;21:4663–70. [5] Hutvagner G, McLachlan J, Pasquinelli AE, Balint E, Tuschl T, Zamore PD. A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA. Science 2001;293:834–8. [6] Lund E, Guttinger S, Calado A, Dahlberg JE, Kutay U. Nuclear export of microRNA precursors. Science 2004;303:95–8. [7] Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004;116:281–97. [8] Ambros V. MicroRNA pathways in flies and worms: growth, death, fat, stress, and timing. Cell 2003;113:673–6. [9] Wang X, Tang S, Le SY, Lu R, Rader JS, Meyers C, et al. Aberrant expression of oncogenic and tumor-suppressive microRNAs in cervical cancer is required for cancer cell growth. PLoS One 2008;3:e2557. [10] Shen Y, Li Y, Ye F, Wang F, Wan X, Lu W, et al. Identification of miR-23a as a novel microRNA normalizer for relative quantification in human uterine cervical tissues. Exp Mol Med 2011;43:358–66. [11] Lee JW, Choi CH, Choi JJ, Park YA, Kim SJ, Hwang SY, et al. Altered microRNA expression in cervical carcinomas. Clin Cancer Res 2008;14:2535–42. [12] Wilting SM, Snijders PJ, Verlaat W, Jaspers A, van de Wiel MA, van Wieringen WN, et al. Altered microRNA expression associated with chromosomal changes contributes to cervical carcinogenesis. Oncogene 2013;32:106–16. [13] Rao Q, Shen Q, Zhou H, Peng Y, Li J, Lin Z. Aberrant microRNA expression in human cervical carcinomas. Med Oncol 2012;29:1242–8.
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Gynecologic Oncology journal homepage: www.elsevier.com/locate/ygyno
A genetic variant in pre-miR-27a is associated with a reduced cervical cancer risk in southern Chinese women Xing-Dong Xiong a,b,c,d,⁎, Xi-Ping Luo e, Jie Cheng a,b,c, Xinguang Liu a,b,c, En-Min Li d,f, Li-Qin Zeng e a
Institute of Aging Research, Guangdong Medical College, Dongguan 523808, China Institute of Biochemistry & Molecular Biology, Guangdong Medical College, Zhanjiang 524023, China Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, Dongguan 523808, China d Key Laboratory of Molecular Biology in High Cancer Incidence Coastal Chaoshan Area of Guangdong Higher Education Institutes, Medical College of Shantou University, Shantou 515041, China e Department of Gynecology, Guangdong Women and Children Hospital and Health Institute, Guangzhou 510010, China f Department of Biochemistry and Molecular Biology, Medical College of Shantou University, Shantou 515041, China b c
H I G H L I G H T S • The miR-27a rs895819 polymorphism is associated with a decreased risk of cervical cancer in southern Chinese women. • No association between rs7372209/rs1834306 polymorphism and cervical cancer risk was detected among southern Chinese women.
a r t i c l e
i n f o
Article history: Received 4 September 2013 Accepted 19 December 2013 Available online 28 December 2013 Keywords: miRNA Single nucleotide polymorphism Cervical cancer Risk
a b s t r a c t Objective. MicroRNAs (miRNAs) play critical roles in cervical carcinogenesis. Common single nucleotide polymorphisms (SNPs) in pre/pri-miRNAs may change their property through altering miRNAs expression and/or maturation. Here we aimed to investigate the influence of three common SNPs in pre/pri-miRNAs (pri-miR-26a1 rs7372209, pre-miR-27a rs895819 and pri-miR-100 rs1834306) on individual susceptibility to cervical cancer. Methods. We genotyped these three polymorphisms in 103 cervical cancer cases and 417 cancer-free female subjects using polymerase chain reaction–ligation detection reaction (PCR–LDR) method. Unconditional logistic regression analysis was utilized to estimate the association between these polymorphisms and the risk of cervical cancer. Results. In a logistic regression analysis, we found that the rs895819 polymorphism in pre-miR-27a exhibited a significant effect on cervical cancer risk; T allele (OR = 0.68, 95% CI = 0.49–0.95, P = 0.025), and CT (OR = 0.33, 95% CI = 0.15–0.74, P = 0.007) or TT (OR = 0.33, 95% CI = 0.15–0.72, P = 0.006) genotype were associated with the decreased risk, compared to C and CC respectively. As we used further genotype association models, we found a similar trend of the association in additive (OR = 0.70, P = 0.041) and recessive model (OR = 0.33, P = 0.004). We did not detect any association of the other two SNPs in pri-miR-26a-1 (rs7372209) and pri-miR100 (rs1834306) with cervical cancer risk. Conclusion. Our study provides the first evidence that the miR-27a rs895819 polymorphism is associated with a decreased risk of cervical cancer in southern Chinese women. © 2013 Elsevier Inc. All rights reserved.
Introduction Cervical cancer is the second most common cancer following breast cancer in women worldwide, especially in developing countries [1]. It is well-recognized that specific oncogenic human papillomaviruses (HPVs)
Abbreviations: miRNA, microRNA; pre-miRNA, precursor microRNA; pri-miRNA, primary microRNA; SNP, single nucleotide polymorphism; HPV, human papillomavirus; OR, odds ratio; CI, confidence interval. ⁎ Corresponding author at: Institute of Aging Research, Guangdong Medical College, Dongguan 523808, China. Fax: +86 769 22896425. E-mail address: [email protected] (X.-D. Xiong). 0090-8258/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ygyno.2013.12.030
are primary etiologic factors in cervical cancer, while the majority of infected women do not develop the cancer during their lifetime, indicating that other factors contribute to the progression to cervical cancer [2]. Accumulating evidence supports that host genetic variations, apart from HPV infection, play an important role in the cervical carcinogenesis [3]. MicroRNAs (miRNAs) are a class of endogenous, small and noncoding RNAs (~ 22 nt), which are initially transcribed from genomic DNA to long primary transcripts (pri-miRNAs) and then are cleaved by nuclear Drosha into 60–70 nt hairpin-shaped precursor RNAs (premiRNAs) [4]. Pre-miRNAs are exported to the cytoplasm by Exportin-5 and are further processed into ~ 22 nt mature miRNA duplexes by the cleavage of Dicer [5,6]. In association with RNA-induced silencing
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complex (RISC), one strand of the miRNA duplex matches target mRNA sequence in the 3′-untranslated regions. This binding finally leads to mRNA degradation or translational repression and consequently down-regulates the expression of the protein [7]. To date, it has been estimated that miRNAs modulate the expression of approximately 30% of human genes [8]. MiRNAs are involved in a wide range of biological processes including cell proliferation, differentiation, development, and apoptosis [8]. Elevated or decreased expression of miRNAs has been found in various tumor types, including cervical cancer. Recent studies have demonstrated that miR-27a, miR-26a-1 and miR-100 were up-regulated or down-regulated in cervical cancer and were considered to play a role in cervical carcinogenesis [9–15]. SNPs in miRNA genes are a novel class of genetic variations in the human genome which are currently being identified and investigated in human cancers [16]. SNPs, or mutations within miRNA genes, including pri-miRNAs, pre-miRNAs and mature miRNAs, potentially influence the processing and/or target selection of miRNAs and thus contribute to cancer risk [16,17]. Because each miRNA potentially regulates hundreds of genes, identifying the risk factors associated with miRNA genes could serve to elucidate novel pathways and mechanisms of pathogenesis. However, the exact role played by genetic variants in miRNA genes concerning cervical cancer susceptibility remains largely unknown [18–20]. Recent studies have shown that three potentially functional polymorphisms (rs895819, rs7372209, and rs1834306) in pre-miRNA or pri-miRNAs (pre-miR-27a, pri-miR-26a-1, and pri-miR-100, respectively) were associated with the risk and clinical outcome for cancers [17,21–26]. Based on their roles of these miRNAs on cervical cancer [9–15], we speculated that polymorphisms in these miRNAs may have an impact on miRNA processing, and so they modulate the susceptibility to cervical cancer. Therefore, here we conducted a case–control study to elucidate the association of these polymorphisms with the risk of cervical cancer. Our analysis revealed that the T allele of SNP rs895819, located in pre-miR-27a, has a significant association with a reduced cervical cancer risk in southern Chinese women. Materials and methods Study population This case–control study included 103 cervical cancer patients and 417 cancer-free control subjects. The method used for subject enrollment was the same as for our previous studies [27]. In brief, patients with cervical cancer were recruited at Guangdong Women and Children Hospital and Health Institute, Guangzhou, PR China from February 2009 to July 2012. The diagnosis of cervical cancer was confirmed in all cases by histological examination of tissue from biopsy or resected specimens. All clinicopathological data were obtained from clinical and pathologic records. Controls (n = 417) were randomly selected from healthy women volunteers who took part in the physical examination during the time period of case collection in the same hospital. The criteria for the selection of controls included no clinical detection for cervical lesions, no gynecological tumor, no endometriosis and no history of cancer. All study subjects enrolled in this case–control study were unrelated ethnic Han Chinese women who lived in southern China. At recruitment, informed consent was obtained from each subject who was then interviewed to collect detailed information. This study was approved by the Medical Ethics Committee at Guangdong Women and Children Hospital and Health Institute. DNA extraction Genomic DNA was extracted from peripheral whole blood by TIANamp blood DNA extraction kit (TianGen Biotech, Beijing, China) according to the manufacturer's instructions. All DNA samples were dissolved in water and stored at −20 °C until use.
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Genotyping Genotypes were determined by the polymerase chain reaction– ligation detection reaction (PCR–LDR) method (Shanghai Biowing Applied Biotechnology Company). The sequence of primers and probes is summarized in Table S1. The PCR was carried out on the ABI 9600 (Applied Biosystems, USA) in a total volume of 20 μl including 50 ng genomic DNA, 1× PCR buffer, 2.5 mM MgCl2, 0.2 mM dNTPs, 0.5 μM each primer, and 1 U hot-start Taq DNA polymerase (Qiagen). Cycling parameters were as follows: 95 °C for 15 min; 35 cycles at 94 °C for 30 s, 59 °C for 1 min, and 72 °C for 1 min; and a final extension step at 72 °C for 10 min. The ligation reaction for each PCR product was carried out with a final volume of 10 μl containing 1 μl 10× ligation buffer, 1 μl of PCR product, 1 pmol of each discriminating probe, and 2 U Taq DNA ligase (New England Biolabs, USA). The LDR parameters were as follows: 95 °C for 2 min, 30 cycles at 94 °C for 15 s and 50 °C for 25 s. Following the LDR reaction, 1 μl LDR reaction product was mixed with 1 μl ROX and 1 μl loading buffer. The mixture was then analyzed by the ABI Prism 377 DNA Sequencer (Applied Biosystems, USA). About 10% of the samples were randomly selected to perform the repeated assays and the results were 100% concordant. HPV detection Cervical scrape specimens were collected using the cervix brush and then processed to isolate DNA for HPV detection assays. HPV infection was detected using HPV GenoArray test kit (Hybribio Inc., China) according to the manufacturer's instructions. This assay kit can detect 15 high risk type HPV (HR-HPV, 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, and 68) and six low risk type HPV (LR-HPV, 6, 11, 42, 43, 44, and CP8304). Statistical analysis A statistical power analysis was performed using a program for power and sample size computations (known as PS program) [28]. Genotype frequencies in control group were checked for the Hardy– Weinberg equilibrium by using goodness-of-fit χ2-test. Pearson chisquare test was used to examine distribution difference of genotypes between cases and controls. Association between the polymorphism and the susceptibility of cervical cancer was evaluated using unconditional logistic regression analysis, adjusted by age, age at the first sexual intercourse, number of sexual partners, number of vaginal delivery and smoking status. These statistical analyses were done with the SPSS software program (version 13.0, SPSS Inc., Chicago, IL, USA). All statistical tests were two-sided and a P value of less than 0.05 was used as the criterion of statistical significance. Results Characteristics of the study population One hundred three cervical cancer cases and 417 control subjects were included in the present study. The frequency distributions of selected demographic characteristics of the cases and controls are shown in Table 1. There was no significant difference in the distributions of age and smoking status between the cases and controls. However, the incidence of HR-HPV infection in the patients (82.7%) was much higher than that in the control subjects (16.0%, χ2 = 127.45, P b 0.001, Table 1), implying HR-HPV infection as a critical risk factor for cervical cancer development in our study population. Among the subtypes of HR-HPV, 87.7% (71/81) were HPV16/18 positive in the cases and 14.7% (5/34) were HPV16/18 positive in the controls. In addition, compared with controls, the cervical cancer cases were significantly earlier age at first intercourse, and had more sexual partners and number of vaginal delivery (all P b 0.05). Of the 103 cases, 81 (78.7%) had
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Table 1 Characteristics of cervical cancer cases and normal controls. Variable
Age, years b45 45–55 N55 Age at the first sexual intercourse, years ≤20 N20 Number of sexual partners, n (%) ≤1 N1 Number of vaginal delivery ≤1 N1 Smoking status Nonsmoker Smoker HR-HPV infection Positive Negative Total Type of HPV DNAc HPV16 or 18 Other HPV types Histologic types Squamous cell carcinoma Adenocarcinoma Adenosquamous carcinoma Others Stage I II III IV a b c
Controls (n = 417)
Cases (n = 103)
No. (%)
No. (%)
173 (41.5) 164 (39.3) 80 (19.2)
56 (54.3) 32 (31.1) 15 (14.6)
Pa
0.062
0.025b 156 (37.4) 261 (62.6)
51 (49.5) 52 (50.5)
311 (74.6) 106 (25.4)
46 (44.7) 57 (55.3)
141 (33.8) 276 (66.2)
21 (20.4) 82 (79.6)
395 (94.7) 22 (5.3)
101 (98.1) 2 (1.9)
34 (16.0) 178 (84.0) 212
81 (82.7) 17 (17.3) 98
5 (14.7) 31 (85.3)
71 (87.7) 10 (12.3)
b0.001
0.008
0.149
b0.001
b0.001
81 (78.7) 9 (8.7) 4 (3.9) 9 (8.7) 61 (59.2) 38 (36.9) 3 (2.9) 1 (1.0)
Two-sided chi-square test. P values under 0.05 were indicated in bold font. Among subjects with positive HR-HPV DNA.
squamous cell carcinomas. Other patients who had adenocarcinoma, adenosquamous carcinoma and unclassified were counted twentytwo (21.3%). In all the 103 cases, 59.2% of patients were in stage I; 36.9%, 2.9%, and 1.0% was found to be in stage II, III, and IV, respectively (Table 1). Associations of miRNA polymorphisms with risk of cervical cancer The primary information for miR-26a-1 rs7372209 C N T, miR-27a rs895819 T N C and miR-100 rs1834306 T N C polymorphisms was in Table S2. Minor allele frequency (MAF) in our controls was similar to MAF for Chinese in HapMap database for all three SNPs (Table S2). Genotype distributions of three SNPs in our control subjects were in agreement with that expected under the Hardy–Weinberg equilibrium (rs7372209, P = 0.737; rs895819, P = 0.254; rs1834306, P = 0.723, Table S2), providing no evidence of population stratification within the dataset. The allele and genotype distributions of three polymorphisms in the cases and the controls are shown in Table 2. From the allelic association analysis, we found only rs895819 in pre-miR-27a showed statistical significance (P = 0.025, Table 2). Unconditional logistic regression analysis revealed that the risk of cervical cancer was significantly decreased in both CT heterozygotes [odds ratio (OR) = 0.33, 95% confidence interval (CI) = 0.15–0.74, P = 0.007] and TT homozygotes (OR = 0.33, 95% CI = 0.15–0.72, P = 0.006), compared with CC homozygotes (Table 2). In addition, a similar trend of the decreased risk of cervical cancer was detected in additive and recessive model in which CT and TT genotypes were combined (OR = 0.70, 95% CI = 0.49–0.98, P = 0.041, and OR = 0.33, 95% CI = 0.16–0.70, P = 0.004, respectively, Table 2). These data indicate
that pre-miR-27a rs895819 polymorphism may be associated with risk of cervical cancer and that individuals carrying T allele may have significantly decreased cervical cancer susceptibility. However, we did not find any association between the pri-miR-26a-1 rs7372209 or primiR-100 rs1834306 and the risk of cervical cancer in allelic or genotypic analyses (Table 2). In addition, no more evident association between pre-miR-27a rs895819 polymorphism and risk of cervical cancer was observed among subgroups by age at first intercourse, status of smoking or HPV (data not shown). We further conducted the analysis in the patients with early-stage cancer and found that a more pronounced reduction in cervical cancer risk was observed among stage I patients carrying rs895819 CT or TT genotypes (Table 3, OR = 0.24, 95% CI = 0.10–0.60, P = 0.002 and OR = 0.28, 95% CI = 0.12–0.68, P = 0.005, respectively). Significantly decreased cervical cancer risk was also found in recessive model (Table 3, OR = 0.26, 95% CI = 0.11–0.61, P = 0.002, respectively). Out of the 103 cervical cancer cases, 81 (78.7%) were squamous cell carcinoma, 9 (8.7%) were adenocarcinoma, 4 (3.9%) were adenosquamous carcinoma, and 9 (8.7%) was unknown. We also found that a more pronounced reduction in cervical cancer risk was observed among squamous cell carcinoma patients carrying rs895819 CT or TT genotypes (Table 3, OR = 0.31, 95% CI = 0.13–0.72, P = 0.006 and OR = 0.30, 95% CI = 0.13–0.68, P = 0.004, respectively), and in the additive and recessive model (Table 3, OR = 0.66, 95% CI = 0.45–0.97, P = 0.035 and OR = 0.30, 95% CI = 0.14–0.66, P = 0.003, respectively). Discussion Increasing evidences suggest that miRNAs are crucial factors in cancer progress as oncogenes or tumor suppressors by degradation of the target mRNAs and/or inhibition of their translation [29]. MicroRNAs are also considered to be potential biomarkers and therapeutic targets in human cancers [30]. Variations in expression of miRNAs have been reported to be related to many tumors including cervical cancer [31–33]. Differential miRNA expression could be caused by sequence variations, such as mutations and SNPs [34]. Although SNPs in miRNAs have been widely studied, the association between SNPs in miRNAs and cervical cancer risk is still largely unknown. Therefore, we conducted a case–control population study to investigate the influence of three common SNPs in pre/pri-miRNAs (pri-miR-26a-1 rs7372209 C N T, premiR-27a rs895819 T N C and pri-miR-100 rs1834306 T N C) on individual susceptibility to cervical cancer. As a result, we found that the common T N C polymorphism within pre-miR-27a (rs895819) exhibited a significant effect on cervical cancer risk. Compared with CC homozygotes, subjects who were CT heterozygotes or TT homozygotes exhibited a decreased risk of cervical cancer (Table 2). In addition, the other two SNPs (rs7372209 and rs1834306) located in the pri-miRNAs did not show a significant association with cervical cancer. Our study suggested that miR-27a rs895819 polymorphism played a role in cervical carcinogenesis. The oncogenic activity of miR-27a has been recently reported in gastric, breast and pancreatic cancer [35–37]. However, a recent study reported that miR-27a had significantly decreased expression in head and neck squamous cell carcinoma (HNSCC) as compared to normal tissues [38]. Likewise, Wang et al. found that miR-27a was downregulated in cervical cancer tissues and cancer cell lines when compared with normal cervical tissue, which suggested that miR-27a functions as tumor suppressor in the development of cervical cancer [9]. SNP rs895819 was found in pre-miRNA regions of hsa-miR-27a, which was in chromosome 19, and it was located at position 40 relative to the first nucleotide [39]. Recently, a Chinese study found that rs895819 TT genotype was associated with a decreased risk of gastric cancer [22,39]. Although the binding of the mature miRNA to target mRNAs was not influenced by the SNP rs895819, some studies had demonstrated that polymorphisms in pre-miRNAs could influence the expression of their mature forms, and these were as well involved in the binding of some nuclear factors
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Table 2 Association between SNPs in miRNAs and risk of cervical cancer.
Pri-miR-26a-1 rs7372209 Allele Genotype
Additive Dominant Recessive
Pre-miR-27a rs895819 Allele Genotype
Additive Dominant Recessive
Pri-miR-100 rs1834306 Allele Genotype
Additive Dominant Recessive a b
Controls (n = 417)
Cases (n = 103)
No. (%)
No. (%)
609 (73.0) 225 (27.0) 221 (53.0) 167 (40.0) 29 (7.0)
150 (72.8) 56 (27.2) 57 (55.3) 36 (35.0) 10 (9.7)
196 (47.0) 221 (53.0) 388 (93.0) 29 (7.0)
46 (44.7) 57 (55.3) 93 (90.3) 10 (9.7)
C T CC CT TT – CT + CC TT CC CT + TT
218 (26.1) 616 (73.9) 24 (5.7) 170 (40.8) 223 (53.5)
70 (34.0) 136 (66.0) 15 (14.6) 40 (38.8) 48 (46.6)
194 (46.5) 223 (53.5) 24 (5.7) 393 (94.3)
55 (53.4) 48 (46.6) 15 (14.6) 88 (85.4)
C T CC CT TT – CT + TT CC CT + CC TT
457 (54.8) 377 (45.2) 127 (30.5) 203 (48.7) 87 (20.9)
114 (55.3) 92 (44.7) 38 (36.9) 38 (36.9) 27 (26.2)
290 (69.5) 127 (30.5) 330 (79.1) 87 (20.9)
65 (63.1) 38 (36.9) 76 (73.8) 27 (26.2)
C T CC CT TT – CT + TT CC CT + CC TT
OR (95% CI)a
P valuea
1.00 1.01 (0.72–1.42) 1.00 0.76 (0.46–1.24) 1.10 (0.47–2.55) 0.92 (0.64–1.32) 1.00 1.23 (0.78–1.96) 1.00 1.22 (0.54–2.79)
1.00 0.68 (0.49–0.95) 1.00 0.33 (0.15–0.74) 0.33 (0.15–0.72) 0.70 (0.49–0.98) 1.00 0.82 (0.51–1.29) 1.00 0.33 (0.16–0.70)
1.00 1.02 (0.76–1.37) 1.00 0.61 (0.36–1.03) 1.05 (0.58–1.91) 0.96 (0.70–1.31) 1.00 1.41 (0.87–2.28) 1.00 1.35 (0.79–2.30)
0.953 0.275 0.828 0.632 0.379 0.623
0.025b 0.007 0.006 0.041 0.382 0.004
0.892 0.066 0.865 0.785 0.161 0.272
Adjusted for age, age at the first sexual intercourse, number of sexual partners, number of vaginal delivery and smoking status. P values under 0.05 were indicated in bold font.
in miRNA processing [40,41]. The present study showed that the rs895819 T allele may be associated with a lower risk of cervical cancer, indicating that the individuals with rs895819 T allele may have a higher expression level of miR-27a and therefore are in reduced risk of cervical cancer. Further studies are needed to clarify the underlying mechanism of how the genetic variant in pre-miR-27a affects cervical carcinogenesis. Several limitations need to be addressed in this case–control study. First, the patients and controls were enrolled from hospitals and may not represent the general population. Nonetheless, the genotype distribution of the controls was in Hardy–Weinberg equilibrium. Second, the sample size of the present study was relatively small, particularly for stratified analyses. Third, HPV infection is one of the independent risk
factors of cervical cancer. We did not have enough information on HPV status due to lack of the cervical scrape specimens from all study subjects. Finally, further studies in different population could help to establish the true significance of the association between this SNP and the risk of cervical cancer. However, our observations provided valuable insights and interesting information and might serve to guide future studies in this area. In conclusion, our study provides the first evidence that the miR-27a rs895819 polymorphism, but not miR-26a-1 rs7372209 and miR-100 rs1834306, is associated with a decreased risk of cervical cancer in southern Chinese women. This finding suggests that the common genetic polymorphism in pre-miR-27a may play a role in the development of cervical cancer, although additional studies with larger sample size,
Table 3 Association between the pre-miR-27a rs895819 polymorphism and risk of cervical squamous cell carcinoma and stage I cervical cancer.
Allele Genotype
Additive Dominant Recessive a b
C T CC CT TT – CT + CC TT CC CT + TT
Controls (n = 417)
Squamous cell carcinoma (n = 81)
No. (%)
No. (%)
218 (26.1) 616 (73.9) 24 (5.8) 170 (40.8) 223 (53.5)
57 (35.2) 105 (64.8) 13 (16.0) 31 (38.3) 37 (45.7)
194 (46.5) 223 (53.5) 24 (5.8) 393 (94.2)
44 (54.3) 37 (45.7) 13 (16.0) 68 (84.0)
OR (95% CI)a
P valuea
Stage I n = 61)
OR (95% CI)a
P valuea
No. (%) 1.00 0.65 (0.43–0.97) 1.00 0.31 (0.13–0.72) 0.30 (0.13–0.68) 0.66 (0.45–0.97) 1.00 0.77 (0.46–1.27) 1.00 0.30 (0.14–0.66)
0.036b 0.006 0.004 0.035 0.303 0.003
Adjusted for age, age at first intercourse, number of sexual partners, number of vaginal delivery and smoking status. P values under 0.05 were indicated in bold font.
43 (35.2) 79 (64.8) 11 (18.0) 21 (34.5) 29 (47.5) 32 (52.5) 29 (47.5) 11 (18.0) 50 (82.0)
1.00 0.65 (0.43–0.97) 1.00 0.24 (0.10–0.60) 0.28 (0.12–0.68) 0.68 (0.44–1.04) 1.00 0.86 (0.49–1.51) 1.00 0.26 (0.11–0.61)
0.036 0.002 0.005 0.073 0.559 0.002
454
X.-D. Xiong et al. / Gynecologic Oncology 132 (2014) 450–454
detailed HPV infection data and in diverse ethnic populations are required to confirm the general validity of our findings. In addition, further functional studies on this polymorphism are also needed to elucidate the underlying molecular mechanisms of the observed association. Conflicts of interest statement The authors declare that there are no conflicts of interest.
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