Association between microRNA-146a, -499a and -196a-2 SNPs and non-small cell lung cancer: a case–control study involving 2249 subjects

Abstract MicroRNA (miR) acts as a negative regulator of gene expression. Many literatures have suggested that miRs may be involved in the process of cell proliferation, inflammation, oxidative stress, energy metabolism and epithelial–mesenchymal transition. Thus, miRs may be implicated in the occurrence of non-small cell lung cancer (NSCLC). In the current investigation, we included 2249 subjects (1193 NSCLC patients and 1056 controls) and designed a study to identify the relationship of miR-146a rs2910164 C/G, -499a rs3746444 A/G and -196a-2 rs11614913 T/C with the risk of NSCLC. The risk factors (e.g., body mass index (BMI), sex, smoking, drinking and age) was used to adjust the odds ratios (ORs) and 95% confidence intervals (CIs). After conducting a power value assessment, we did not confirm that the miR-single nucleotide polymorphisms (SNPs) genotypic distributions were different in NSCLC cases and controls. However, the association of miR-196a-2 rs11614913 with a decreased risk of NSCLC was identified in the female subgroup (adjusted P=0.005, power = 0.809 for TC vs. TT, and adjusted P=0.004, power = 0.849 for CC/TC vs. TT). In addition, gene–gene interaction analysis showed that rs11614913 TC/3746444 AA and rs11614913 CC/rs3746444 AA could also reduce the susceptibility to NSCLC (rs11614913 TC/rs3746444 AA vs. rs11614913 TT/rs3746444 AA, P=0.001, power = 0.912 and rs11614913 CC/rs3746444 AA vs. rs11614913 TT/rs3746444 AA, P=0.003, power = 0.836). In conclusion, in overall comparisons, we did not confirm that the rs2910164, rs3746444, and rs11614913 SNPs genotypic distributions were different in NSCLC cases and controls. However, this case–control study demonstrates that miR-196a-2 rs11614913 may be a protective factor for the development of NSCLC among female patients.


Introduction
Lung cancer (LC) caused about 11.6% of all new cancer cases and 18.4% of all cancer-related deaths worldwide [1]. In China, 733.3 thousand new LC patients and 610.2 thousand LC-related deaths were assessed to occur in 2015 [2]. The etiology of LC was unclear. It is reported that a number of genetic and environmental risk factors may cause the development of LC [3,4,5]. Non-small cell lung cancer (NSCLC) is the most common type of LC. The individual's hereditary factor may be implicated in the occurrence of NSCLC.
MicroRNA (miR), a small non-coding RNA, acts as a negative regulator of gene expression. In the nucleus, the Drosha/DiGeorge syndrome critical region 8 complex cleaves pri-miRNAs [6]. Then, in the cytoplasm, Dicer crops these formed pre-miRNAs [7]. Finally, they are incorporated into the Argonaute-containing RNAinduced silencing complexes [8]. Mature miR is composed of about 22 nucleic acids, which is generated from primary miRs and further changed to mature miRs in cytoplasm. The target mRNAs located in 3′-untranslated regions (3′-UTRs). Matured miRs can recognize the 3′-UTRs of mRNA and bind to them, and then result in a weakened expression of target genes. The mechanism of the process is hybridization of seed sequences of matured miRs with 3'-UTRs. An individual miR can bind to masses of targets, and regulate a number of pathways. Many investigations have suggested that miRs may be involved in the process of cell proliferation, inflammation, oxidative stress, energy metabolism and epithelial mesenchymal transition (EMT) [9,10,11,12,13,14,15,16]. Of late, some previous investigations have indicated that miRs have been implicated in the occurrence of NSCLC [17,18]. There are single nucleotide polymorphisms (SNPs) in certain miRs. These SNPs might influence the generation process of miRs or alter target recognition/hybridization. Thus, miR polymorphisms may be implicated in the occurrence and/or progress of cancer [19,20,21,22,23,24,25].
Park et al. reported that miR-146a could restrain EMT progression in NSCLC by repressing the expression of insulin receptor substrate-2 [14]. It was found that miR-146a inhibited migratory capacity, downstream signaling of epidermal growth factor receptor and NSCLC cell growth; however, it could promote the apoptosis process of NSCLC cell lines [13]. Xiong et al. reported that miR-146a rs2910164 C>G locus could affect its maturation in peripheral blood mononuclear cells [26]. A recent study reported that G allele of rs2910164 mgiht increase miR-146a level [27]. A previous study suggested that rs2910164 locus might influence the toxicity in LC chemotherapy [28]. Several reports indicated that rs2910164 polymorphism in miR-146a could decrease the risk to LC [29,30]. However, other case-control studies suggested that rs2910164 might not influence the occurrence of LC [31,32]. These controversial observations may be due to the limited sample sizes. Here, we explored the role of miR-146a rs2910164 SNP with the development of NSCLC and a potential interaction of this SNP with risk factors to identify whether this locus could be used as a biomarker for susceptibility to NSCLC in Chinese populations.
Rs11614913 T>C was widely explored in malignancy as a candidate locus of miR-196a-2 [33,34]. Hu et al. reported that that the rs11614913 T→C variant in miR-196a-2 could affect the binding ability of mature hsa-mir-196a-2-3p binding with its target mRNA [35]. Recently, this polymorphism was thought to alter LC cases' sensitivity to platinum-based chemotherapy [23]. A functional study highlighted that rs11614913 might be involved in the development of LC through altering the secondary structure and the expression of miR-196a-2 [36]. Thus, rs11614913 polymorphism might be implicated in carcinogenesis of LC and could affect an individual's susceptibility of LC. Indeed, several case-control studies have investigated the role of rs11614913 in the occurrence of LC [23,36]. However, the observations were conflicting, even in a same ethnicity. For example, some recent studies indicated a significant relationship between miR-196a-2 rs11614913 and the development of LC [36, 37, 38], whereas others did not confirm the potential correlation [23,32].
A previous investigation reported that miR-499a rs3746444 SNP could affect the process of miR-499-5p maturation and the role of antiapoptosis [39]. The relationship between miR-499a rs3746444 A>G and the susceptibility and progress of LC has been explored. Ge et al. reported that miR-499a rs3746444 AA genotype could inhibit the expression of miR-499a gene and CD200 [40]. And then this SNP could influence the survival of NSCLC cases. Several studies have focused on the role of miR-499a rs3746444 in the development of LC [40,41]. However, recent meta-analyses have reported contradictory findings [42,43,44]. Thus, the correlation of miR-499a rs3746444 with the development of LC was more inconsistent.
In the current investigation, we designed a larger sample size study to identify the correlation of rs3746444, rs2910164 and rs11614913 with the occurrence of NSCLC.

Study population and ethical approval
Each participant donated a peripheral blood sample. NSCLC cases in the current investigation were recruited from the Zhenjiang Medical College of Nanjing Medical University (Jiangsu Province, China) and the Union Medical College of Fujian Medical University (Fujian Province, China) between January 2014 and June 2018. All NSCLC cases were diagnosed via histopathological examination. In this study, the selection criteria were defined as the following: (1) Chinese Han populations, (2) sporadic cases and (3) without any history of other cancer. And the exclusion criteria were summarized as: (1) a patient who has an autoimmune disease, (2) NSCLC patients who have treated with chemoradiotherapy and/or targeted therapy, (3) NSCLC recurrent cases and (4) heterochronous NSCLC. In total, 1,193 NSCLC cases were enrolled. At the same time, 1,056 participants without a history of cancer were included as controls in the Medical Colleges mentioned above. The data of demographics and potential risk factors were collected by a pre-structured questionnaire. During the recruitment, each participant signed a written informed consent. This study was approved by the Ethics Review Committee of Fujian Union Hospital (2018KY023).

Isolation of DNA and genotyping
Using DNA Isolation Kit (Promega, Madison, USA), we extracted genomic DNA. The obtained DNA was kept at -80℃. The quality of DNA sample was assessed by Nanodrop ND-1000 UV. A custom-SNPscan TM Kit (Genesky Biotechnologies Inc., Shanghai, China) was used to analyze the genotypes. Briefly, no less than 120 ng DNA sample was used to conduct a double ligation and multiplex fluorescence polymerase chain reaction (PCR). ABI-3730XL sequencer (PE Applied Biosystems, Foster City, CA, USA) was used to detect the PCR products. The obtained raw data were analyzed by harnessing GeneMapper 4.1 (AppliedBiosystems, USA). To conduct a quality control, ninety samples were randomly chosen and Downloaded from http://portlandpress.com/bioscirep/article-pdf/doi/10.1042/BSR20201158/903897/bsr-2020-1158.pdf by guest on 09 February 2021 repeated genotyped in the same PCR method. The results indicated that 100% concordant results were observed.

Statistical analysis
Hardy-Weinberg equilibrium (HWE) (https://ihg.gsf.de/cgi-bin/hw/hwa1.pl) [45] and SAS 9.4 (SAS Institute, Cary, North Carolina) softwares were harnessed to analyze HWE and genetic data. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to estimate the relationship of rs2910164, rs11614913 and rs3746444 with the risk of NSCLC. We also calculated adjusted ORs and 95% CIs using logistic regression analyses. In the current study, five risk factors [e.g., body mass index (BMI), smoking, drinking, age and gender] were included. Power Calculator (http://biostat.mc.vanderbilt.edu/twiki/bin/view/Main/PowerSampleSize) was used to calculate the power of sample size [19,46]. We also used the false-positive report probability (FPRP) to evaluate the findings [47].

Characteristics of the study population
In the current study, 1,193 cases with NSCLC (mean ± SD age, 58.92±10.44 years) and 1,056 controls (mean ± SD age, 59.36±9.19 years) were collected ( Table 1). In NSCLC group, 642 males and 551 females were included. While in controls, there were 586 males and 470 females. The age and gender were well-mathed (P=0.960 and 0.425, respectively). The distribution of smoking, drinking and BMI were different between two groups (All P<0.001). Raw data of genotypes and characteristics were summarized in Table S1.

Information of rs3746444, rs2910164 and rs11614913 SNPs
The successful ratio of genotyping was more than 99.00%. Table 2 has summarized some vital information for rs2910164, rs11614913 and rs3746444. In controls, these included miR-SNPs genotype distributions met HWE (P>0.05). Table  S1 summarized the detailed information and genotypes for each individual.

Rs3746444, rs2910164 and rs11614913 SNPs and NSCLC susceptibility
The number of miR-146a rs2910164 allele and genotype in NSCLC cases and controls is summarized in Table 3. In this case-control study, for overall comparisons, we identified that the miR-146a genotype frequency was not significantly different among the two groups. As well, we also found that the miR-499a rs3746444 genotypic distribution was not different in NSCLC cases and controls. Table 3 listes the miR-196a-2 rs11614913 genotype distribution in NSCLC cases and controls. It was notable that there was statistically significant in comparison of rs11614913 genotypes in three genetic models among NSCLC cases and controls. The decreased genotype frequencies of rs11614913 TC, CC and TC/CC were found in NSCLC patients. In relation to rs11614913 TT, individuals carrying rs11614913 TC genotypes had a decreased 21% susceptibility to the cocurrence of NSCLC (P=0.014, Table 4). Additionally, compared to rs11614913 TT, rs11614913 CC and TC/CC genotypes were also protective factors for the cocurrence of NSCLC (CC vs. TT: P=0.027 and TC/CC vs. TT: P=0.007, Table 4). When we adjusted for risk factors, the decreased susceptibility for the occurrence of NSCLC was not changed ( Table 4). Table S2 and Table S3 summarized the detailed information and genotypes for squamous cell carcinoma (SCC) and non-SCC cases, respectively. When we conducted a subgroup analysis by type of pathology, for rs11614913 SNP, the decreased susceptibility for the occurrence of NSCLC was also found in non-SCC subgroup (TC vs. TT: adjusted P=0.026 and TC/CC vs. TT: adjusted P=0.015, Table  4). For rs2910164 and rs3746444 polymorphisms, no significant association between these SNPs and NSCLC risk was found ( Table 4).

Stratification analysis of miR-SNPs and NSCLC susceptibility (a) miR-146a rs2910164 C>G locus
When we conducted stratification analyses by risk factors, an increased risk for the occurrence of NSCLC was identified in never drinking subgroup (CG vs. CC: adjusted P=0.043 and GG/CG vs. CC: adjusted P=0.028, Table 5).

(c) miR-196a-2 rs11614913 T>C locus
For miR-196a-2 rs11614913, significant difference in frequency of its genotype was found between NSCLC cases and controls. We identified that rs11614913 polymorphism may be a protective factor for the occurrence of NSCLC ( Table 7).

Gene-gene interaction analysis
We also conducted miR-SNPs combined analysis for three included SNPs. Three potential types (rs11614913/rs2910164, rs11614913/rs3746444, rs2910164/rs3746444 and rs11614913/rs2910164/rs3746444) were combined to explore the gene-gene interaction and their roles on the occurrence of NSCLC.
In analysis of rs11614913/rs2910164 loci combination, we used rs11614913 TT/rs2910164 CC as reference. It was notable that the rs11614913 CC/rs2910164 CC combination was a protective factor for the development of NSCLC (P=0.010, Table  8). In another analysis of rs11614913/rs3746444 loci combination, compared to rs11614913 TT/rs3746444 AA, frequency of rs11614913 TC/rs3746444 AA was lower in NSCLC patients 32.54% (384/1080) than in controls 37.70% (397/1053). When rs11614913 TT/rs3746444 AA was used as a reference, frequency of rs11614913 CC/rs3746444 AA was also lower in NSCLC patients 12.46% (147/1080) than in controls 15.19% (160/1053). When rs11614913 TT/rs2910164 CC/rs3746444 AA was used as a reference, TC/CC/AA, TC/GG/AA and CC/CC/AA genotype combinations might decreased the risk of NSCLC (Table 8).

Study power (α= 0.05) and FPRP mothed
For overall comparisons, these miR-SNPs did not confer a risk to NSCLC. Each power value for overall positive report was less than 0.8 (data not shown). For the comparison of miR-SNPs and NSCLC susceptibility in different type of pathology, we also could not confirm the positive report (data not shown). In stratification analysis of miR-SNPs with NSCLC susceptibility, we only confirmed that rs11614913 polymorphism could be a protective factor for the occurrence of NSCLC in the female subgroup (the power values were 0.809 in TC vs. TT and 0.848 in CC/TC vs. TT). In these miR-SNPs combination analysis, compared to rs11614913 TT/3746444 AA, rs11614913 TC/3746444 AA and rs11614913 CC/rs3746444 AA could decrease the susceptibility of NSCLC (power value: 0.912 and 0.836, respectively). Other power values less than 0.8 were not shown. After assessing power value and FPRP, we highlighted that miR-196a-2 rs11614913 decreased the risk to NSCLC in the female subgroup. As well, gene-gene interaction analysis showed that rs11614913 TC/3746444 AA and rs11614913 CC/rs3746444 AA could also reduce the susceptibility to NSCLC.

Discussion
LC is a common malignancy with 18.4% of overall cancer-related deaths worldwide [1]. The etiology of LC is not well-known. NSCLC is the most common subtype of LC. MiR is a negative regulator of gene expression. It may involve in the development of cancer. Some investigations have focused on the role of miRs on the occurrence and survival of NSCLC [40,48,49]. The individual's hereditary factor may be implicated in the occurrence of NSCLC. In this investigation, we designed a study to identify the correlation of miR-SNPs (rs3746444, rs2910164 and rs11614913) with the risk of NSCLC in Chinese populations. We highlighted that rs11614913, in the female subgroup, could decrease the risk to NSCLC. As well, gene-gene interaction analysis showed that rs11614913 TC/3746444 AA and rs11614913 CC/rs3746444 AA could also reduce the susceptibility to NSCLC.
Rs11614913 locates on the 3p strand region of mature Toraih et al. found that individuals carrying the rs11614913 C allele might be a protective factor of LC, which was associated with miR-196a-2 low-expression in tissue [54]. A recent investigation indicated that the polymorphism of rs11614913, through influencing the level of miR-196a-2 and secondary structure, conferred risk to LC in females [36]. In the current invstigation, we found that the miR-196a-2 rs11614913 could reduce the susceptibility to NSCLC in female. In view of these investigations mentioned above, we might conclude that rs11614913 C allele could be a protective factor to the occurrence of NSCLC though altering the level of miR-196a-2 and secondary structure. It is well known that smoking is a major risk for LC. However, in this study, we did not find the interaction of tobacco using and rs11614913 SNP with the development of NSCLC. In the future, these conclusions should be confirmed by further studies.
Several literatures have focused on the relationship between gene-gene interaction and the occurrence of human diseases [55,56,57]. In this study, we analyzed the combined effect of these miR-SNPs. Gene-gene interaction analyses showed that rs11614913 TC/3746444 AA and rs11614913 CC/rs3746444 AA could also decrease the susceptibility of NSCLC, which suggested that rs11614913 C allele could inhibit the development of NSCLC. We first confirmed that rs11614913 TC/3746444 AA and rs11614913 CC/rs3746444 AA combinations could decrease the risk of NSCLC. However, this combination did not influence the risk of cervical cancer [56]. Therefore, the effect of rs11614913 TC/3746444 AA combination could be different in different cancer. In the future, the possible correlation is needed to verify in other studies.
Several limitations, in this investigation, should be pointed out. Firstly, some vital data were unknown; thus, a more extensively stratified analysis for other risk factors (e.g., vegetable and fruit intake, air pollution, lifestyle and occupational exposure) could not be done. Second, due to the hospital-based study, bias might have happened in our analysis. Third, the number of participants in this study was moderate. Last, we only included three miR-SNPs in this study, and other important miR-SNPs should not be ignored.
In conclusion, this study highlights that miR-196a-2 rs11614913 decreases the risk to NSCLC among female subgroup. Additionally, combined gene-gene analyses suggest that rs11614913 TC/3746444 AA and rs11614913 CC/rs3746444 AA are protective factors for the development of NSCLC. More investigations are needed to validate the potential effect of these miR-SNPs in NSCLC. And more functional studies should also be done.

Acknowledgements:
We appreciate the help/participation of all people who participated in this study.

Disclosure of Potential Conflicts of Interests:
The authors have no potential financial conflicts of interest.  Table S1.