Association of STAT3 Gene polymorphism with noise-induced hearing loss(NIHL) in the Chinese population

Objectives: NIHL is a disease with irreversible damage caused by multi-factor interaction, and the STAT3 is an essential protein with signal transduction and transcription functions. This study is aimed to explore the effect of STAT3 polymorphism on individual susceptibility to NIHL individuals, and to further examine the interaction between lifestyles and NIHL in the dominant model . Methods: This study was designed a case-control study and included 609 NIHL cases and 611 healthy hearing controls from the Jiangsu province of China. By collecting the lifestyle and demographic information of the study participants, genotyping the collected blood samples, and performing Real-time Quantitative PCR (qRT-PCR) to analyze and investigate the potential association between STAT3 SNPs and NIHL. Results: Statistical analysis results show that individuals carrying the C allele of rs1053023 and rs1053005 were more likely to develop NIHL than individuals carrying the T allele (OR = 1.367, 95% CI = 1.148-1.628, P = 0.0001 and OR = 1.37, 95% CI = 1.147-1.636, P = 0.001). In the dominant model (TC/CC genotype and TT genotype), stratied analysis of lifestyle, we found that people who smoke regularly are more likely to suffer from NIHL than non-smokers or occasional smokers. Besides, the expression of STAT3 was higher in NIHL cases by real-time quantitative PCR. Conclusion: Gene polymorphisms, rs1053023 and rs1053005, from STAT3, are associated with NIHL and C alleles of rs1053023 and rs1053005 within STAT3 may become biomarkers for workers exposed to noise.

Previous research found that the signi cant mechanisms of NIHL are cell apoptosis and oxidative stress [16].Signal Transducer and Activator of Transcription 3 (STAT3) has both transmission and transcription functions [17] - [18]. STAT3, as a cytoplasmic transcription factor has many vital functions,such as regulating cell growth, differentiation and programmed death, and vascularization, etc [19][20][21]. Animals experiment show that STAT3 can be expressed on inner ear hair cells, and as a downstream molecule of Notch signal, involved in the inner ear hair cells during development [22]. Therefore, STAT3 may be included in the event of NIHL.Wilson.et al. have indicated the crucial role of JAK2/STAT3 signalling pathway on the development of NIHL [23]. However, there is no research on the correlation between the SNPs of STAT3 and NIHL. By inhibiting the function of JAK / STAT3 pathway, the oxidative stress and apoptosis of inner ear hair cells can be appropriately reduced;we speculated that SNPs in STAT3 might be related to the genetic susceptibility of NIHL. This study is the rst report to explore the correlation of multiple SNP sites of STAT3 genes, lifestyle factors with NIHL. So this study is to pursue the potential relevance of the ve SNP sites of STAT3(rs4796793, rs1053023, rs1053005, rs1053004, and rs3744483) and NIHL.

Subjects
The research subjects came from employees who are exposed to noise in automobile, energy, and cotton spinning factories in Jiangsu Province, China. Employees who had been working steadily for more than three years and exposed to > 85dB (A) noise formed our study cohort. They all participated in the physical examination and completed the noise-exposure survey by a face-to-face conversation with a professional questionnaire investigator. Through the questionnaire, we collect information as follows: (1) general demographic information (age, gender, etc.);(2) type of work and working-age; (3) lifestyle factors(smoking, drinking). The variables involved are de ned as follows:: 1) smoking: "yes" means "smoking every day", "ever" means "smoking occasionally", and "never" means "never smoking".; 2) drinking: "yes" representatives have daily alcohol intake, e.g. white wine ≥ 50 g, red wine ≥ 150 g, or beer ≥ 500 g, and last for one year or longer, "ever" means "drinking occasionally", and "never" means "never drinking" .(4) overall health(the history of illness, medication history). This study uses case-control research, and the exclusion criteria included: a) Employees with ear deformities or diseases that may affect hearing thresholds (such as mumps, measles, otitis media, Meniere syndrome, ear canal stenosis, etc.); b) Workers who have taken or are taking ototoxic drugs (such as aspirin, quinolones, aminoglycosides, etc.); c) Workers with a family history of deafness and head trauma. At last, there are 1220 subjects entered the study. All research subjects signed an informed consent form, and the institutional review committee of the Jiangsu Center for Disease Control and Prevention approved the study.

PTA and case de nition
Before the audiometry , each individual was removed from the noise exposure environment for more than 16 hours following the relevant testing requirements according to the provisions of GBZ 49-2014 "Diagnostic Standards for Occupational Noise Deafness", pure tone audiometry (PTA) was performed by otolaryngologists in a room with sound insulation and a noise background of less than 25 dB. The audiometry study subjects binaural hearing thresholds at six frequencies (500, 1000, 2000, 3000, 4000, and 6000 Hz). All measurement results were corrected for age and gender according to standards. The case group was de ned whose pure-tone hearing test binaural high-frequency (3000, 4000, 6000Hz) average hearing threshold ≥ 25 dB (A), otherwise as to the control group.

DNA extractions
EDTA anticoagulation tube was used to collect peripheral blood (> 2 mL) and store blood in a refrigerator at -80℃ before DNA extractions. The DNA extraction kit (Tiangen Biochemical, Beijing) was used to extract the peripheral blood DNA, and using an Implant P360 ultramicro spectrophotometer (Shanghai Boyi Biotechnology Co., Ltd., China) to detect the concentration and purity of the DNA.

SNP selection
Screen unlabeled SNPs according to the following methods: (1) minimum allele frequency (MAF)> 0.05; (2) the literature searched for frequently re-ported SNPS in previous studies; (3) linkage disequilibrium (LD) value r 2 > 0.8. The selected data come from the 1000 Genomes Project (1000 Genomes Project, www.1000genomes.org) and the National Center for Biotechnology Information (NCBI) dB SNP database.

Statistical analysis
SPSS 24.0 software (IBM, NYC, USA) was used for statistical analysis of all data. For normally-distributed continuous variables, the statistic "mean ± standard deviation (SD) " was used in the article and using Student's t-tests or one-way analysis of variance to analyze. The categorical variables were analyzed using the chi-square test and expressed as frequencies (percentage). The representativeness of the sample was tested by Hardy-Weinberg genetic balance . Estimating the Hardy-Weinberg equilibrium (HWE) use the chisquare test. After adjusting the risk factors of age, gender, smoking and drinking, analyze the correlation between the selected SNP and NIHL risk and between the different genotypes and NIHL risk and calculate the odds ratio (OR) and 95% con dence interval (95% CI). P < 0.05 means statistically signi cant and shown in bold. Table 1 shows the general symptoms, including: lifestyle features (age, sex, tobacco, and alcohol consumption habits) and occupational characteristics (working years of noise exposure, noise intensity, and high-frequency hearing threshold) of case group and the control group. No statistically signi cant differences in demographic and lifestyle features between the two groups (P> 0.05) was found. However, the statistically signi cant(p<0.001) of the threshold between cases the control subjects was found, and the hearing threshold in case(37.50±12.39) was higher than control (15.58±4.76).

Linkage disequilibrium (LD)
The LD heatmap of these ve SNPs (rs4796793, rs1053023, rs1053005, rs1053004, and rs3744483) were shown in Figure 1. By calculated correlation coe cient (r 2 ) for each pair of the ve SNPs in 3´untranslated region, we found that SNP rs3744483, rs1053005 and rs1053023 present a state of complete LD (r 2 = 1, using 1000 genome phase 3 data).

Multivariate analysis of STAT3 tag SNPs with the risk of NIHL
The association between genetic models of STAT3 tag SNPs (rs4796793, rs1053023, rs1053005, rs1053004, and rs3744483) in 609 NIHL patients and 611 control groups with NIHL was examined and the results shown in Table 2. After adjusting for gender, age, smoking and drinking, the rs1053023 and rs1053005 codominant and dominant models were demonstrated to be signi cantly related to NIHL. In the dominant model, for loci rs1053023, individuals carrying the TT genotype were detected to be less likely to suffer from NIHL than those with TC / CC genotype (adjusted: OR=1.516, 95% CI = 1.201-1.913, P = 0.0001). Similarly, for loci rs1053005, relative to the TT genotype, research participants who were carrying the TC/CC genotype had a higher risk to be NIHL, the odds ratio (OR) is 1.509 (adjusted: 95% CI = 1.191-1.912, P = 0.001). Furthermore, the proportion of the C alleles of rs1053023 and rs1053005 in NIHL case group was obviously more than that of the control group (adjusted: OR=1.367, 95% CI =1.148-1.628, P =0.001; OR=1.370, 95% CI =1.147-1.636, P =0.002 respectively). This suggests that, among all study subjects, who carrying the T allele at the lower risk to affect NIHL than those carrying the C allele. Thus, We could speculate that allele T may be a protective factor for hearing loss.

Strati ed analysis of rs1053023 and rs1053005 polymorphism and NIHL risk
We further examined the influence of rs1053023 and rs1053005 different genotypes about NIHL on a series of risk characteristics in the dominant model. Table 3 and Table 4 presents the consequences of the analysis.
Compared with women in gender strati cation, the NIHL risk of the TC / CC combination genotype of rs1053023 (adjusted: OR= 1.545, 95% CI = 1.215-1.964, P = 0.001) in men is 1.545 times than the TT genotype. Analogously, the NIHL risk of TC / CC genotype of rs1053005 ( adjusted: OR= 1.531, 95% CI = 1.199-1.955, P = 0.002) in men is 1.531 times than that of TT genotype. In addition ,for loci rs1053023, when smoking and drinking were strati ed, the individual carrying the TC/CC genotype relatively prone to NIHL disease in the regular smoking group (OR = 1.595, 95% CI = 1.163-2.187, P = 0.008), the never-drinking group (OR = 1.467, 95% CI = 1.076-2.002, P = 0.019) and the group drinking occasionally(OR = 10.55, 95% CI = 3.803-29.287, P <0.001). After adjustment for gender, age, within the strata of less than ten years noise exposure, subjects with TC / CC genotype were is more likely to develop into NIHL (OR = 2.448, 95% CI = 1.498-3.998, P = 0.001 ). Similar data features were found in the noise exposure group over 20 years (OR = 1.678, 95% CI = 1.151-2.447, P = 0.031). A similar statistical analysis result was found for loci rs1053005, which is that individuals carrying TC / CC genotypes were likely to be NIHL when they regular smoking, or never drinking, or the year of noise exposure was less than 10(or more than 20).
3.5 Comparison of changes in high-frequency hearing thresholds of different genotypes for rs1053023 and rs1053005.
The results of the comparison of high-frequency hearing threshold shift of rs1053023 and rs1053005 genotypes in all noise-exposed workers were shown in Figure 2. The CC genotype bars were substantially higher than those of TC (P = 0.039) and TT (P= 0.024). Besides, subjects with rs1053005 TT genotype demonstrated a signi cantly lower hearing threshold shift when compared with subjects carrying CC and TC genotype (P = 0.046 and P = 0.033).
3.6 Quantitative Real-time PCR Figure 3 shows the expression level of STAT3 in the population by Quantitative Real-time PCR(qRT-PCR) experiments. We can notice that STAT3 was highly expressed in the case group than the control group. The primer information used in qRT-PCR experiments was shown in Table 5.

Discussion
The most common sensory organ defect in the world is hearing loss [24]. Hearing can be impaired directly by high-intensity and long-term noise exposure, eventually leading to NIHL [25]. The number of people with hearing disability is rising year by year, with 42,000 in 1985, 120 million in 1995, 250million in 2001, and up to 360 million in 2011, speculated by the World Health Organization (WHO) [26]. NIHL, a multifactoral disease, has been the focus of research in recent years, especially in the study of the association with genetic susceptibility. Population epidemiological studies have shown that after noise exposure, 50% of the variation in hearing loss can be explained by genetic factors [27,28].
Single-nucleotide polymorphism (SNP) has become the third generation of polymorphism markers due to the characteristics of the high density of genetic markers, high stability, and secure automation of typing tests.
There are four theories about the mechanism of NIHL: oxidative stress theory, Ca 2 + overload theory, immune in ammation theory, and vascular theory. Previous studies have found that KCNQ4 [29], HSP70 [30], FOXO3 [31], Notch [32] are associated with NIHL. Most of the physiological processes involved in these genes belong to only one of these four theories. However, the STAT3 gene is involved in not only oxidative stress but also immune in ammation [33]. Therefore, to study the potential relationship between STAT3 gene polymorphism and NIHL provides a basis for the future research on the speci c molecular mechanism of hearing loss, and may offer a scienti c basis for the future prevention of NIHL.
STAT3 is an essential signalling protein. It was improved that STAT3 exerts a vital role in a series of reactions in which extracellular signals are transmitted into the nucleus and promote transcription reactions [23]. An animal experiment shows that STAT3 affects the occurrence and development of NIHL. In this noise-exposed mouse experiment, the mice were processed using JSI-124 before the noise exposure, a JAK2/STAT3 signal path-speci c inhibitor, which allowed a signi cant recovery of hearing sensitivity after two weeks of exposure to noise [23]. This indicates that STAT3 may be as a negative regulatory factor in the development of NIHL in the animal model. But the relationship between the SNP in STAT3 and NIHL need to be illuminated.
The ve common SNPs of STAT3 (rs4796793 rs1053004, rs1053005,rs1053023 and rs3744483) were selected in our study. Our results showed that the rs1053023 and rs1053005 loci of STAT3 have statistical signi cance with NIHL. After adjusting for factors other than genotype such as age, gender, smoking and drinking, individuals carrying the C allele were more susceptible to NIHL than those carrying the T allele in the rs1053023 (OR = 1.367, 95% CI = 1.148-1.628, P = 0.0001) and rs1053005 (OR = 1.37, 95% CI = 1.147-1.636, P = 0.001) through Logistic regression analysis. In addition, the correlation analysis of different genotypes and a series of risk factors was conducted, and observed the results of that regardless of the locus rs1053023 or rs1053005, the risk of NIHL carrying TC / CC combination genotype is higher than that of the TT genotype (rs1053023, OR = 1.545, 95% CI = 1.215-1.964, P = 0.001 and rs1053005, OR = 1.531, 95% CI = 1.199-1.955, P = 0.002). When stratifying the working years exposed to noise, it is obvious that in the group with a noise exposure period of fewer than ten years or more than 20 years, compared with people with TT genotypes, those with TC / CC genotypes Subjects had a higher risk to NIHL (adjusted: rs1053023, P = 0.001, P =0.031, respectively;rs1053005, P = 0.003, P =0.027). However, there was no statistical signi cance between the distribution of genotypes and the years of noise exposure within 10~20 years. The reason for this result may be related to complex regulatory functions of STAT3, which may appear as early promotion and late inhibition in immunomodulation [34].
In the dominant model, in analyzing the impact of smoking and NIHL, we found that people who smoke regularly are more likely to suffer from NIHL than non-smokers or occasional smokers. This coincides with the results of the Mofateh et al.'s study [35]. Using the same manner to analyze the relationship between drinking and NIHL, we found that not drinking but has a higher risk of NIHL, which consistent with of the Upile et al.'s found ( hearing loss can be induced by alcohol even eliminating the effects of noise) [36].
The result of real-time quantitative PCR shows that STAT3 expression is higher in NIHL cases group than the control group. This con rmed that some SNPs of STAT3 might be potentially associated with NIHL. Our study is an association study showing that the C allele of the STAT3 gene increases the risk of NIHL in the Chinese population. Being consistent with the previous experiment, the results of this study show that there is a superimposed effect between the risk factors and SNPs.The interaction between drinking, smoking, and rs1053023/ rs1053005 had an association with NIHL.
At present, the mechanism of NIHL's occurrence is not precise. But many scientists agree that, when the workers are exposed to heavy noise, DNA damage of cochlear hair cells severely affect the development of NIHL [16]. When an individual was exposed to noise, the inner ear tissue can be damaged severely. An important potential cause of that is the production of reactive oxygen [16,37,38]. Many scientists have also proposed that apoptosis and necrosis of inner ear cells caused by metabolites or oxidative stress, as well as structural damage directly affecting the structure of the cochlea, maybe the most likely pathogenesis of NIHL [16,39,40].
After engaged a multitude of growth factors and cytokines, STAT3 can lead to diverse biological outcomes, such as cell growth, differentiation, and survival and has a relatively meaningful impact on oxidative stress-mediated tissue injury. More and more experiment research has indicated that the JAK2/STAT3 signalling pathway can effectively regulate damage due to oxidative stress, and the hydrogen peroxide-induced cell death can be effectively reduced through the activity of JAK2/STAT3 signal pathway [41,42]. In animal models, STAT3 tyrosine 705 phosphorylation can signi cantly increase due to noise exposure [23]. Inhibition of JAK2 / STAT3 signalling can reduce noise exposure-induced phosphorylation of STAT3 and the expression of STAT3 target genes. The suppression of the JAK2/STAT3 pathway improves the recovery of loud sound exposure [23]. The pathogenic mechanism of NIHL may be explained by further study of STAT3 in depth.
This study is helpful to the development of NIHL's gene chip. Although this study is a relatively complete correlation study based on a large sample population, it also has some shortcomings. First, the subjects of this study come from just a few noise-exposed textile and automobile manufacturing enterprises from China, not all noise-exposed enterprises. Therefore, the research results cannot be generalized to all individuals.
Furthermore, the noise exposure characteristics of different enterprises and the operation schedule cannot be the same, so the degree of hearing loss of their employees may also differ.

Conclusion
In conclusions, the rs1053023 and rs1053005 polymorphism were found to be associated with NIHL and smoking, and drinking also affect NIHL's susceptibility to some extent. Our results suggest that the rs1053023 and rs1053005 C alleles of STAT3 may play a pivotal role in the occurrence of NIHL and maybe a susceptibility biomarker of NIHL for Chinese workers. These results provide a theoretical basis for NIHL prevention and provide data support for future NIHL genetic testing.   Figure 1 Linkage disequilibrium pattern of the ve SNPs.

Figure 2
Comparison of high-frequency hearing threshold shift of rs1053005 and rs1053023 genotypes in all subjects. Data are presented as mean ± SE and analyzed by ANOVA.