Tumor Biol. DOI 10.1007/s13277-014-2957-y
RESEARCH ARTICLE
A functional polymorphism in microRNA-196a2 is associated with increased susceptibility to non-Hodgkin lymphoma Tao Li & Lijuan Niu & Lili Wu & Xia Gao & Man Li & Wenxuan Liu & Lei Yang & Dianwu Liu
Received: 15 October 2014 / Accepted: 5 December 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Aberrant expression and structural alterations of microRNAs (miRNAs) play important roles in tumorigenesis. The miRNA-196a2 polymorphism is associated with tumorigenesis, but its association with non-Hodgkin lymphoma (NHL) remains unexplored. We evaluated the association between the miRNA-196a2 T>C polymorphism (rs11614913) and NHL risk in a case–control study of 318 NHL cases and 320 healthy controls. We also examined miRNA-196a expression in tissue samples from NHL patients (n=59). The TC and CC genotypes were associated with cancer risk in NHL [odds ratio (OR)=1.384, confidence interval (CI)=1.010–1.898 for TC vs. TT, and OR=1.822, 95 % CI=1.163–2.853 for CC vs. TT]. Analysis of the association between this polymorphism and the clinicopathology of NHL showed that the combined TC/CC genotypes were associated with Ann Arbor stage (OR=1.852, 95 % CI=1.139–3.010), bone marrow invasion (OR=1.850, 95 % CI=1.062–3.223), and B symptoms (OR=1.852, 95 % CI=1.154–2.972), but not with immunohistological subtype, lymph node size, age, or gender. In addition, the CC or CC/TC genotypes were associated with significantly higher levels of mature miR-196a (p=0.002 or 0.008) in a genotype–phenotype correlation analysis. Our findings suggest that the miR-196a2 polymorphism
T. Li : X. Gao : M. Li : W. Liu : L. Yang : D. Liu (*) Department of Epidemiology and Health Statistics, School of Public Health, Hebei Medical University, Shijiazhuang 050000, Hebei Province, China e-mail: [email protected] L. Niu The Third Hospital of Shijiazhuang, Shijiazhuang, Hebei Province, China L. Wu The Fourth Affiliated Hospital of Hebei Medical University, Shijiazhuang, Hebei Province, China
may increase the risk of NHL by altering the expression of mature miR-196a. Keywords Non-Hodgkin lymphoma . microRNA . miRNA-196a2 . rs11614913 polymorphism . Susceptibility
Introduction The etiology of non-Hodgkin lymphoma (NHL), as well as its dramatic rise in incidence worldwide in recent decades, surpassed only by lung cancer in women and malignant melanoma in both sexes, remains largely unexplained [1, 2]. In China, the incidence rate of NHL is 3.5/100,000 and the number of newly diagnosed cases is 45,000 every year, leading to more than 20,000 deaths. These figures have resulted in a change in the ranking of NHL from the 10th to the 9th position in terms of malignant tumor incidence [3, 4]. NHLs encompass a heterogeneous group of cancers and have a wide range of histological appearances and clinical features at presentation [5]. The most common NHL subtypes are diffuse large B cell lymphoma (DLBCL), T cell lymphoma (TCL), and follicular lymphoma (FL) [6]. The past decade has heralded a new era in the understanding of gene regulation in NHL, particularly after non-coding microRNAs (miRNAs) were discovered. miRNAs are a group of naturally occurring, evolutionarily conserved, single-stranded RNAs of 21–24 nucleotides that play a role in a wide range of physiologic and pathologic processes, including development, cell differentiation, proliferation, apoptosis, and carcinogenesis in various eukaryotic organisms [7, 8]. miRNAs bind to the 3′untranslated region of target messenger RNA (mRNA), leading to their degradation or translational suppression and thereby regulating the expression of at least 30 % of protein-coding genes at the post-transcriptional level [9].
Tumor Biol.
Single nucleotide polymorphisms (SNPs) are the most common type of variation in the human genome, affecting sequence coding and splicing, which can influence population diversity, disease susceptibility, and individual responses to medicines [10]. SNPs in miRNA genes may alter miRNA expression and/or function, because miRNA binding to its mRNA target is dependent on sequence complementarity, and its influence on cancer susceptibility and progression has attracted much attention [11]. The rs11614913 SNP, located in hsa-miRNA-196a2, has been associated with susceptibility to colorectal cancer [12, 13], liver cancer [14], breast cancer [15], lung cancer [16], gastric cancer [17], gallbladder cancer [18], esophageal cancer [19], and acute lymphoblastic leukemia [20]. Thus, the miR-196a2 polymorphism may play an important role in carcinogenesis. However, the role of miRNA196a2 genetic variation in NHL susceptibility remains unknown. Therefore, we assessed the association between this functional polymorphism and susceptibility and progression of NHL in a Chinese population.
Genomic DNA extraction and miR-196a2 genotyping Blood samples were collected in EDTA tubes. Genomic DNA was extracted from white blood cell fractions by using the Qiagen Blood Kit (Qiagen, Chatsworth, CA). miR-196a2 genotyping was performed using polymerase chain reaction– restriction fragment length polymorphism (PCR-RFLP) [22] with the following primers: forward 5′-CCCCTTCCCTTCTC CTCCAGATA-3′ and reverse 5′-CGAAAACCGACTGATG TAACTCCG-3′. The 149-bp PCR product was digested overnight with 10 units of MspI at 37 °C, separated by electrophoresis in 2.5 % agarose gel, and visualized by staining with ethidium bromide. MspI digestion yielded 24- and 125-bp fragments for the C allele and a single 149-bp fragment for the T allele. Heterozygotes produced 24-, 125-, and 149-bp fragments. To ensure accuracy, all analyses were performed blindly without knowledge of the case or control status and a 15 % random sample of cases and controls was genotyped twice by different persons; reproducibility was 100 %. Tissue samples and quantitative RT-PCR
Materials and methods Study participants Between January 2010 and June 2014, 318 patients diagnosed with NHL at Shijiazhuang Third People’s Hospital and the Fourth Affiliated Hospital of Hebei Medical University were consecutively enrolled in the study. All patients were genetically unrelated ethnic Han Chinese and were from Shijiazhuang City and the surrounding regions in Hebei Province of North China. Some similar resources that were not included in this study exist in our department. The overall participation rate was 75 %. The histological types of NHL were confirmed according to WHO tumor classification guidelines [21]. All patients were evaluated by clinical status, computerized tomography (CT) of the chest and the abdomen, and bone marrow puncture or biopsy. Clinical staging was assessed according to the Ann Arbor classification system. In total, 320 controls were chosen randomly from local residents during the same period; all controls underwent a routine health check at one of the two hospitals and were free of any known major diseases. A sample of approximately 3–5 mL of venous blood was collected from each participant. In addition, NHL tissues were obtained from 59 of the NHL patients for measurement of miR-196a2 expression. The study was approved by the institutional review board of Hebei Medical University, and informed consent was obtained from all individuals before their participation in the study.
In order to detect miR-196a expression levels, we collected tissue from the tumors of 59 NHL patients who had undergone biopsy. TaqMan miRNA Assays (Applied Biosystems, Foster City, CA) were used to quantify mature miR-196a and the small nuclear RNA U6. Complementary DNA was synthesized by priming with genespecific looped primers. All real-time reactions, including no-template controls and non-reverse-transcribed controls, were run in triplicate on an ABI7300 Real-Time PCR System (Applied Biosystems, USA). Relative expression was calculated using the Ct values provided by the manufacturer. Statistical analysis Data analysis was performed with Statistical Package for Social Sciences (SPSS, Inc., Chicago, IL, USA) for Windows (version 13.0). The Hardy–Weinberg equilibrium was tested by the chi-square (χ2) test in the control group. The χ2 test was used to test differences in categorical variables and genotype frequencies between cases and controls; the Student’s t test was used for continuous variables. Unconditional logistic regression was used to estimate odds ratios (ORs) and 95 % confidence intervals (CIs) for NHL risk in relation to miR196a2 polymorphisms, with adjustment for age and gender. The wild-type genotype TT served as the reference for analysis. Expression levels of miR-196a were calculated using the equation 2−(CT196a−CTU6). A p value of less than 0.05 was considered statistically significant, and all statistical tests were two sided.
Tumor Biol.
Results
miR-196a2 polymorphisms and risk of NHL
Patient characteristics
As shown in Table 2, genotype frequencies were 19.18 % (CC), 45.91 % (TC), and 34.91 % (TT) among the cases, and 13.12 % (CC), 41.88 % (TC), and 45.00 % (TT) among the controls. Likewise, the percentage of CC/TC genotypes was lower in controls than in cases. After being adjusted for age, gender, smoking status, and drinking status, logistic regression analysis revealed that the TC and CC genotypes were associated with a significantly increased risk of NHL, compared with the TT genotype (OR=1.384, 95 % CI=1.010–1.898 for TC vs. TT, and OR =1.822, 95 % CI= 1.163–2.853 for CC vs. TT). A significant increased risk of NHL was found in the combined genotypes TC/CC versus the TT genotype (OR= 1.514, 95 % CI=1.104–2.075). The genotype distributions of SNP rs11614913 were in agreement with the Hardy– Weinberg equilibrium in the controls (χ2 = 1.4704, p = 0.479).
A total of 318 NHL patients, including 147 diagnosed with diffuse large B cell lymphoma, 90 with T cell lymphoma (TCL), 9 with follicular lymphoma (FL), and 72 with other subtypes, were enrolled. The clinical characteristics of the NHL patients and control subjects are summarized in Table 1. As expected, no significant difference was found between the cases and controls with regard to age, gender, smoking status, or drinking status (p=0.837, 0.167, 0.334, and 0.758, respectively), which suggested that these variables were matched adequately. In patients with NHLs, lymph node diameter was measured by computed tomography (CT); 93 patients were ≤5 cm, 225 patients were >5 cm. Ann Arbor staging showed 103 cases in stages I–II and 215 cases in stages III–IV. Bone marrow invasion and the presence of B symptoms was found in 86 and 163 cases, respectively.
Table 1 Characteristics of the patients and controls Age (years) median (range) Gender n (%) Male Female Smoking status n (%) Yes No Drinking status n (%) Yes No Ann Arbor stage n (%) I/II III/IV Bone marrow invasion n (%) Negative Positive Lymph node size (cm) n (%) ≤5 >5 B symptoms n (%) No Yes Histological subtype n (%) DLBCL TCL FL Other
NHL patients (n=318)
Healthy controls (n=320)
p value
53.00 (21–82)
55.50 (20–83)
0.837
196 (61.64) 122 (38.36)
180 (56.25) 140 (43.75)
0.167
121 (38.05) 197 (61.95)
110 (34.38) 210 (65.62)
0.334
105 (33.02) 213 (66.98)
102 (31.88) 218 (68.12)
0.758
103 (32.39) 215 (67.61) 232 (72.96) 86 (27.04) 93 (29.25) 225 (70.75) 155 (48.74) 163 (51.26) 147 (46.23) 90 (28.30) 9 (2.83) 72 (22.64)
Tumor Biol. Table 2
Genotype frequencies of miR-196a2 in NHL and controls
miR-196a2 polymorphism
Patients (n=318)
Controls (n=320)
Crude OR (95 % CI)
p value
Adjusted OR (95 % CI)a
p value a
TT TC CC TC+CC
111 (34.91) 146 (45.91) 61 (19.18) 207 (65.09)
144 (45.00) 134 (41.88) 42 (13.12) 176 (55.00)
1 1.413 (1.005–1.988) 1.884 (1.184–2.998) 1.526 (1.109–2.099)
0.046 0.007 0.009
1 1.384 (1.010–1.898) 1.822 (1.163–2.853) 1.514 (1.104–2.075)
0.043 0.009 0.010
a
Adjusted for gender, age, smoking status, and drinking status
miR-196a2 polymorphisms and clinicopathological characteristics The effect of miR-196a2 SNP rs11614913 on the clinicopathological characteristics of NHL was evaluated (Table 3). The combined TC/CC genotypes showed a significant association with Ann Arbor stage (adjusted OR=1.852, 95 % CI=1.139– 3.010, p=0.013), bone marrow invasion (adjusted OR=1.850, 95 % CI=1.062–3.223, p=0.030), and B symptoms (adjusted OR=1.852, 95 % CI=1.154–2.972, p=0.011) compared with the TT genotype. However, no significant associations were found between rs11614913 and other clinical characteristics, including immunohistological subtype and lymph node size. miR-196a expression in NHL patients Using real-time PCR, we confirmed that the rs11614913 variant genotype affects the expression of mature miR-196a. The relative expression of miR-196a was 3.20 for TT, 4.48 for TC, and 5.23 for the CC genotype in tumor tissues (Table 4). Table 3 Variable
Discussion In this case–control study of NHL in Chinese subjects, we found that hsa-miR-196a2 rs11614913 T>C polymorphism was associated with significantly increased risks of NHL in the variant TC heterozygote and the combined CC/TC genotype as opposed to the TT wild-type homozygote, including 318 NHL cases and 320 controls. The study provides the first evidence that common SNPs in miRNAs may be used as candidate biomarkers for NHL susceptibility. Several studies have reported a significant association between miR-196a2 T>C polymorphisms and the risk of several cancers [12–20]. Tong et al. reported that the CC genotype of miR-196a2 T>C was associated with an increased risk of acute lymphoblastic
Clinicopathological characteristics and miR-196a2 rs11614913 genotype frequencies in NHL TC+CC/TT
Ann Arbor stage n I/II 57/46 III/IV 150/65 Bone marrow invasion n Negative 143/89 Positive 65/21 Lymph node size (cm) n ≤5 53/40 >5 154/71 B symptoms n No 90/65 Yes 117/46 Histological subtype n DLBCL 95/52 TCL 59/31 FL 6/3 Other 47/25 a
miR-196a expression of the miR-196a CC genotype was significantly higher than in carriers of one C allele or the TT genotype (p=0.002 or 0.008).
Crude OR (95 % CI)
p value
Adjusted OR (95 % CI)a
p valuea
1 1.862 (1.146–3.026)
0.012
1 1.852 (1.139–3.010)
0.013
1 1.926 (1.102–3.368)
0.020
1 1.850 (1.062–3.223)
0.030
1 1.637 (0.995–2.692)
0.051
1 1.627 (0.989–2.679)
0.056
1 1.837 (1.152–2.930)
0.010
1 1.852 (1.154–2.972)
0.011
1 0.960 (0.553–1.665) 0.913 (0.219–3.804) 0.972 (0.538–1.755)
0.884 0.901 0.924
1.027 (0.591–1.786) 1.064 (0.255–4.449) 1.013 (0.559–1.837)
0.924 0.932 0.967
Adjusted for gender, age, smoking status, and drinking status
Tumor Biol. Table 4
Genotype–phenotype correlation for rs11614913 and miR-196a −3
miR-196a2 polymorphism Relative expression (1×10 ) (n=59) n
Value
21 27 11 38
3.20 4.48 5.23 4.88
p value
Genotypes TT TCa CCa TC+CCa a
– 0.041 0.002 0.008
Compared with rs11614913 genotype TT; pG, miR-149T>C, and miR-196a2T>C polymorphisms with gastric cancer risk and survival in the Greek population. Mol Biol Rep. 2014;41:1075–80. 24. Wang PY, Gao ZH, Jiang ZH, Li XX, Jiang BF, Xie SY. The associations of single nucleotide polymorphisms in miR-146a, miR-196a and miR-499 with breast cancer susceptibility. PLoS One. 2013;8:e70656. 25. Chen H, Sun LY, Chen LL, Zheng HQ, Zhang QF. A variant in microRNA-196a2 is not associated with susceptibility to and progression of colorectal cancer in Chinese. Intern Med J. 2012;42: e115–9. 26. Kang ZJ, Li YH, He XK, Jiu T, Wei JX, Tian FY, et al. Quantitative assessment of the association between miR-196a2 rs11614913 polymorphism and cancer risk: evidence based on 45,816 subjects. Tumor Biol. 2014;35:6271–82. 27. Diao LP, Ma H, Wei GC, Li T, Liu HS, Liu LH, et al. Matrix metallopro teinase-2 promoter and tissue inhibitor of metalloproteinase-2 gene polymorphisms in non-Hodgkin’s lymphoma. Int J Cancer. 2012;131:1095–103. 28. Yu ZF, Kim J, He L, Creighton CJ, Gunaratne PH, Hawkins SM, et al. Functional analysis of miR-34c as a putative tumor suppressor in high grade serous ovarian cancer. Biol Reprod. 2014. doi:10.1095/ biolreprod.114.121988. 29. Li XD, Li ZG, Song XX, Liu CF. A variant in microRNA-196a2 is associated with susceptibility to hepatocellular carcinoma in Chinese patients with cirrhosis. Pathology. 2010;42:669–73. 30. Hoffman AE, Zheng T, Yi C, Leaderer D, Weidhaas J, Slack F, et al. microRNA miR-196a-2 and breast cancer: a genetic and epigenetic association study and functional analysis. Cancer Res. 2009;69: 5970–7.
RESEARCH ARTICLE
A functional polymorphism in microRNA-196a2 is associated with increased susceptibility to non-Hodgkin lymphoma Tao Li & Lijuan Niu & Lili Wu & Xia Gao & Man Li & Wenxuan Liu & Lei Yang & Dianwu Liu
Received: 15 October 2014 / Accepted: 5 December 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Aberrant expression and structural alterations of microRNAs (miRNAs) play important roles in tumorigenesis. The miRNA-196a2 polymorphism is associated with tumorigenesis, but its association with non-Hodgkin lymphoma (NHL) remains unexplored. We evaluated the association between the miRNA-196a2 T>C polymorphism (rs11614913) and NHL risk in a case–control study of 318 NHL cases and 320 healthy controls. We also examined miRNA-196a expression in tissue samples from NHL patients (n=59). The TC and CC genotypes were associated with cancer risk in NHL [odds ratio (OR)=1.384, confidence interval (CI)=1.010–1.898 for TC vs. TT, and OR=1.822, 95 % CI=1.163–2.853 for CC vs. TT]. Analysis of the association between this polymorphism and the clinicopathology of NHL showed that the combined TC/CC genotypes were associated with Ann Arbor stage (OR=1.852, 95 % CI=1.139–3.010), bone marrow invasion (OR=1.850, 95 % CI=1.062–3.223), and B symptoms (OR=1.852, 95 % CI=1.154–2.972), but not with immunohistological subtype, lymph node size, age, or gender. In addition, the CC or CC/TC genotypes were associated with significantly higher levels of mature miR-196a (p=0.002 or 0.008) in a genotype–phenotype correlation analysis. Our findings suggest that the miR-196a2 polymorphism
T. Li : X. Gao : M. Li : W. Liu : L. Yang : D. Liu (*) Department of Epidemiology and Health Statistics, School of Public Health, Hebei Medical University, Shijiazhuang 050000, Hebei Province, China e-mail: [email protected] L. Niu The Third Hospital of Shijiazhuang, Shijiazhuang, Hebei Province, China L. Wu The Fourth Affiliated Hospital of Hebei Medical University, Shijiazhuang, Hebei Province, China
may increase the risk of NHL by altering the expression of mature miR-196a. Keywords Non-Hodgkin lymphoma . microRNA . miRNA-196a2 . rs11614913 polymorphism . Susceptibility
Introduction The etiology of non-Hodgkin lymphoma (NHL), as well as its dramatic rise in incidence worldwide in recent decades, surpassed only by lung cancer in women and malignant melanoma in both sexes, remains largely unexplained [1, 2]. In China, the incidence rate of NHL is 3.5/100,000 and the number of newly diagnosed cases is 45,000 every year, leading to more than 20,000 deaths. These figures have resulted in a change in the ranking of NHL from the 10th to the 9th position in terms of malignant tumor incidence [3, 4]. NHLs encompass a heterogeneous group of cancers and have a wide range of histological appearances and clinical features at presentation [5]. The most common NHL subtypes are diffuse large B cell lymphoma (DLBCL), T cell lymphoma (TCL), and follicular lymphoma (FL) [6]. The past decade has heralded a new era in the understanding of gene regulation in NHL, particularly after non-coding microRNAs (miRNAs) were discovered. miRNAs are a group of naturally occurring, evolutionarily conserved, single-stranded RNAs of 21–24 nucleotides that play a role in a wide range of physiologic and pathologic processes, including development, cell differentiation, proliferation, apoptosis, and carcinogenesis in various eukaryotic organisms [7, 8]. miRNAs bind to the 3′untranslated region of target messenger RNA (mRNA), leading to their degradation or translational suppression and thereby regulating the expression of at least 30 % of protein-coding genes at the post-transcriptional level [9].
Tumor Biol.
Single nucleotide polymorphisms (SNPs) are the most common type of variation in the human genome, affecting sequence coding and splicing, which can influence population diversity, disease susceptibility, and individual responses to medicines [10]. SNPs in miRNA genes may alter miRNA expression and/or function, because miRNA binding to its mRNA target is dependent on sequence complementarity, and its influence on cancer susceptibility and progression has attracted much attention [11]. The rs11614913 SNP, located in hsa-miRNA-196a2, has been associated with susceptibility to colorectal cancer [12, 13], liver cancer [14], breast cancer [15], lung cancer [16], gastric cancer [17], gallbladder cancer [18], esophageal cancer [19], and acute lymphoblastic leukemia [20]. Thus, the miR-196a2 polymorphism may play an important role in carcinogenesis. However, the role of miRNA196a2 genetic variation in NHL susceptibility remains unknown. Therefore, we assessed the association between this functional polymorphism and susceptibility and progression of NHL in a Chinese population.
Genomic DNA extraction and miR-196a2 genotyping Blood samples were collected in EDTA tubes. Genomic DNA was extracted from white blood cell fractions by using the Qiagen Blood Kit (Qiagen, Chatsworth, CA). miR-196a2 genotyping was performed using polymerase chain reaction– restriction fragment length polymorphism (PCR-RFLP) [22] with the following primers: forward 5′-CCCCTTCCCTTCTC CTCCAGATA-3′ and reverse 5′-CGAAAACCGACTGATG TAACTCCG-3′. The 149-bp PCR product was digested overnight with 10 units of MspI at 37 °C, separated by electrophoresis in 2.5 % agarose gel, and visualized by staining with ethidium bromide. MspI digestion yielded 24- and 125-bp fragments for the C allele and a single 149-bp fragment for the T allele. Heterozygotes produced 24-, 125-, and 149-bp fragments. To ensure accuracy, all analyses were performed blindly without knowledge of the case or control status and a 15 % random sample of cases and controls was genotyped twice by different persons; reproducibility was 100 %. Tissue samples and quantitative RT-PCR
Materials and methods Study participants Between January 2010 and June 2014, 318 patients diagnosed with NHL at Shijiazhuang Third People’s Hospital and the Fourth Affiliated Hospital of Hebei Medical University were consecutively enrolled in the study. All patients were genetically unrelated ethnic Han Chinese and were from Shijiazhuang City and the surrounding regions in Hebei Province of North China. Some similar resources that were not included in this study exist in our department. The overall participation rate was 75 %. The histological types of NHL were confirmed according to WHO tumor classification guidelines [21]. All patients were evaluated by clinical status, computerized tomography (CT) of the chest and the abdomen, and bone marrow puncture or biopsy. Clinical staging was assessed according to the Ann Arbor classification system. In total, 320 controls were chosen randomly from local residents during the same period; all controls underwent a routine health check at one of the two hospitals and were free of any known major diseases. A sample of approximately 3–5 mL of venous blood was collected from each participant. In addition, NHL tissues were obtained from 59 of the NHL patients for measurement of miR-196a2 expression. The study was approved by the institutional review board of Hebei Medical University, and informed consent was obtained from all individuals before their participation in the study.
In order to detect miR-196a expression levels, we collected tissue from the tumors of 59 NHL patients who had undergone biopsy. TaqMan miRNA Assays (Applied Biosystems, Foster City, CA) were used to quantify mature miR-196a and the small nuclear RNA U6. Complementary DNA was synthesized by priming with genespecific looped primers. All real-time reactions, including no-template controls and non-reverse-transcribed controls, were run in triplicate on an ABI7300 Real-Time PCR System (Applied Biosystems, USA). Relative expression was calculated using the Ct values provided by the manufacturer. Statistical analysis Data analysis was performed with Statistical Package for Social Sciences (SPSS, Inc., Chicago, IL, USA) for Windows (version 13.0). The Hardy–Weinberg equilibrium was tested by the chi-square (χ2) test in the control group. The χ2 test was used to test differences in categorical variables and genotype frequencies between cases and controls; the Student’s t test was used for continuous variables. Unconditional logistic regression was used to estimate odds ratios (ORs) and 95 % confidence intervals (CIs) for NHL risk in relation to miR196a2 polymorphisms, with adjustment for age and gender. The wild-type genotype TT served as the reference for analysis. Expression levels of miR-196a were calculated using the equation 2−(CT196a−CTU6). A p value of less than 0.05 was considered statistically significant, and all statistical tests were two sided.
Tumor Biol.
Results
miR-196a2 polymorphisms and risk of NHL
Patient characteristics
As shown in Table 2, genotype frequencies were 19.18 % (CC), 45.91 % (TC), and 34.91 % (TT) among the cases, and 13.12 % (CC), 41.88 % (TC), and 45.00 % (TT) among the controls. Likewise, the percentage of CC/TC genotypes was lower in controls than in cases. After being adjusted for age, gender, smoking status, and drinking status, logistic regression analysis revealed that the TC and CC genotypes were associated with a significantly increased risk of NHL, compared with the TT genotype (OR=1.384, 95 % CI=1.010–1.898 for TC vs. TT, and OR =1.822, 95 % CI= 1.163–2.853 for CC vs. TT). A significant increased risk of NHL was found in the combined genotypes TC/CC versus the TT genotype (OR= 1.514, 95 % CI=1.104–2.075). The genotype distributions of SNP rs11614913 were in agreement with the Hardy– Weinberg equilibrium in the controls (χ2 = 1.4704, p = 0.479).
A total of 318 NHL patients, including 147 diagnosed with diffuse large B cell lymphoma, 90 with T cell lymphoma (TCL), 9 with follicular lymphoma (FL), and 72 with other subtypes, were enrolled. The clinical characteristics of the NHL patients and control subjects are summarized in Table 1. As expected, no significant difference was found between the cases and controls with regard to age, gender, smoking status, or drinking status (p=0.837, 0.167, 0.334, and 0.758, respectively), which suggested that these variables were matched adequately. In patients with NHLs, lymph node diameter was measured by computed tomography (CT); 93 patients were ≤5 cm, 225 patients were >5 cm. Ann Arbor staging showed 103 cases in stages I–II and 215 cases in stages III–IV. Bone marrow invasion and the presence of B symptoms was found in 86 and 163 cases, respectively.
Table 1 Characteristics of the patients and controls Age (years) median (range) Gender n (%) Male Female Smoking status n (%) Yes No Drinking status n (%) Yes No Ann Arbor stage n (%) I/II III/IV Bone marrow invasion n (%) Negative Positive Lymph node size (cm) n (%) ≤5 >5 B symptoms n (%) No Yes Histological subtype n (%) DLBCL TCL FL Other
NHL patients (n=318)
Healthy controls (n=320)
p value
53.00 (21–82)
55.50 (20–83)
0.837
196 (61.64) 122 (38.36)
180 (56.25) 140 (43.75)
0.167
121 (38.05) 197 (61.95)
110 (34.38) 210 (65.62)
0.334
105 (33.02) 213 (66.98)
102 (31.88) 218 (68.12)
0.758
103 (32.39) 215 (67.61) 232 (72.96) 86 (27.04) 93 (29.25) 225 (70.75) 155 (48.74) 163 (51.26) 147 (46.23) 90 (28.30) 9 (2.83) 72 (22.64)
Tumor Biol. Table 2
Genotype frequencies of miR-196a2 in NHL and controls
miR-196a2 polymorphism
Patients (n=318)
Controls (n=320)
Crude OR (95 % CI)
p value
Adjusted OR (95 % CI)a
p value a
TT TC CC TC+CC
111 (34.91) 146 (45.91) 61 (19.18) 207 (65.09)
144 (45.00) 134 (41.88) 42 (13.12) 176 (55.00)
1 1.413 (1.005–1.988) 1.884 (1.184–2.998) 1.526 (1.109–2.099)
0.046 0.007 0.009
1 1.384 (1.010–1.898) 1.822 (1.163–2.853) 1.514 (1.104–2.075)
0.043 0.009 0.010
a
Adjusted for gender, age, smoking status, and drinking status
miR-196a2 polymorphisms and clinicopathological characteristics The effect of miR-196a2 SNP rs11614913 on the clinicopathological characteristics of NHL was evaluated (Table 3). The combined TC/CC genotypes showed a significant association with Ann Arbor stage (adjusted OR=1.852, 95 % CI=1.139– 3.010, p=0.013), bone marrow invasion (adjusted OR=1.850, 95 % CI=1.062–3.223, p=0.030), and B symptoms (adjusted OR=1.852, 95 % CI=1.154–2.972, p=0.011) compared with the TT genotype. However, no significant associations were found between rs11614913 and other clinical characteristics, including immunohistological subtype and lymph node size. miR-196a expression in NHL patients Using real-time PCR, we confirmed that the rs11614913 variant genotype affects the expression of mature miR-196a. The relative expression of miR-196a was 3.20 for TT, 4.48 for TC, and 5.23 for the CC genotype in tumor tissues (Table 4). Table 3 Variable
Discussion In this case–control study of NHL in Chinese subjects, we found that hsa-miR-196a2 rs11614913 T>C polymorphism was associated with significantly increased risks of NHL in the variant TC heterozygote and the combined CC/TC genotype as opposed to the TT wild-type homozygote, including 318 NHL cases and 320 controls. The study provides the first evidence that common SNPs in miRNAs may be used as candidate biomarkers for NHL susceptibility. Several studies have reported a significant association between miR-196a2 T>C polymorphisms and the risk of several cancers [12–20]. Tong et al. reported that the CC genotype of miR-196a2 T>C was associated with an increased risk of acute lymphoblastic
Clinicopathological characteristics and miR-196a2 rs11614913 genotype frequencies in NHL TC+CC/TT
Ann Arbor stage n I/II 57/46 III/IV 150/65 Bone marrow invasion n Negative 143/89 Positive 65/21 Lymph node size (cm) n ≤5 53/40 >5 154/71 B symptoms n No 90/65 Yes 117/46 Histological subtype n DLBCL 95/52 TCL 59/31 FL 6/3 Other 47/25 a
miR-196a expression of the miR-196a CC genotype was significantly higher than in carriers of one C allele or the TT genotype (p=0.002 or 0.008).
Crude OR (95 % CI)
p value
Adjusted OR (95 % CI)a
p valuea
1 1.862 (1.146–3.026)
0.012
1 1.852 (1.139–3.010)
0.013
1 1.926 (1.102–3.368)
0.020
1 1.850 (1.062–3.223)
0.030
1 1.637 (0.995–2.692)
0.051
1 1.627 (0.989–2.679)
0.056
1 1.837 (1.152–2.930)
0.010
1 1.852 (1.154–2.972)
0.011
1 0.960 (0.553–1.665) 0.913 (0.219–3.804) 0.972 (0.538–1.755)
0.884 0.901 0.924
1.027 (0.591–1.786) 1.064 (0.255–4.449) 1.013 (0.559–1.837)
0.924 0.932 0.967
Adjusted for gender, age, smoking status, and drinking status
Tumor Biol. Table 4
Genotype–phenotype correlation for rs11614913 and miR-196a −3
miR-196a2 polymorphism Relative expression (1×10 ) (n=59) n
Value
21 27 11 38
3.20 4.48 5.23 4.88
p value
Genotypes TT TCa CCa TC+CCa a
– 0.041 0.002 0.008
Compared with rs11614913 genotype TT; pG, miR-149T>C, and miR-196a2T>C polymorphisms with gastric cancer risk and survival in the Greek population. Mol Biol Rep. 2014;41:1075–80. 24. Wang PY, Gao ZH, Jiang ZH, Li XX, Jiang BF, Xie SY. The associations of single nucleotide polymorphisms in miR-146a, miR-196a and miR-499 with breast cancer susceptibility. PLoS One. 2013;8:e70656. 25. Chen H, Sun LY, Chen LL, Zheng HQ, Zhang QF. A variant in microRNA-196a2 is not associated with susceptibility to and progression of colorectal cancer in Chinese. Intern Med J. 2012;42: e115–9. 26. Kang ZJ, Li YH, He XK, Jiu T, Wei JX, Tian FY, et al. Quantitative assessment of the association between miR-196a2 rs11614913 polymorphism and cancer risk: evidence based on 45,816 subjects. Tumor Biol. 2014;35:6271–82. 27. Diao LP, Ma H, Wei GC, Li T, Liu HS, Liu LH, et al. Matrix metallopro teinase-2 promoter and tissue inhibitor of metalloproteinase-2 gene polymorphisms in non-Hodgkin’s lymphoma. Int J Cancer. 2012;131:1095–103. 28. Yu ZF, Kim J, He L, Creighton CJ, Gunaratne PH, Hawkins SM, et al. Functional analysis of miR-34c as a putative tumor suppressor in high grade serous ovarian cancer. Biol Reprod. 2014. doi:10.1095/ biolreprod.114.121988. 29. Li XD, Li ZG, Song XX, Liu CF. A variant in microRNA-196a2 is associated with susceptibility to hepatocellular carcinoma in Chinese patients with cirrhosis. Pathology. 2010;42:669–73. 30. Hoffman AE, Zheng T, Yi C, Leaderer D, Weidhaas J, Slack F, et al. microRNA miR-196a-2 and breast cancer: a genetic and epigenetic association study and functional analysis. Cancer Res. 2009;69: 5970–7.
